Methods in Microbiology, Volume 3A

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METHODS in MICROBIOLOGY

This Page Intentionally Left Blank

METHODS in MICROBIOLOGY Edited by J. R. NORRIS Milstead Laboratory of Chemical Enzymology, Sittingbourne, Kent, England

D. W. RIBBONS Department of Biochemistry, University of Miami School of Medicine, and Howard Hughes Medical Institute, Miami, Florida, U.S.A .

Volume 3A

ACADEMIC PRESS London and New York

ACADEMIC PRESS INC. (LONDON) L T D Berkeley Square House Berkeley Square London, W1X 6BA

US. Edition published by ACADEMIC PRESS INC. 11 1 Fifth Avenue New York, New York 10003

Copyright

0 1970 By ACADEMIC PRESS INC.

(LONDON) L T D

All Rights Reserved No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers Library of Congress Catalog Card Number: 68-57745 SBN: 12-521503-7

PRINTED IN GREAT BRITAIN BY ADLARDAND SONLIMITED DORKING, SURREY

LIST OF CONTRIBUTORS A. BRECKER, Oxoid Limited, London, England E. Y . BRIDSON, Oxoid Limited, London, England C. T . CALAM,Imperial Chemical Industries Ltd, Pharmaceuticals Division, Alderley Park, Macclesfeld, Cheshire, England DAVIDA. HOPWOOD, John Innes Institute, Norwich, Norfolk, England S . P. LAPAGE, National Collection of Type Cultures, Central Public Health Laboratory, London, England A. R. MACKENZIE, National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland 7’.G. MITCHELL, Research and Development Establishment, British-American Tobacco Co. Ltd, Southampton, Hants., England D. W . RIBBONS, Department of Biochemistry, University of Miami School of Medicine, and Howard Hughes Medical Institute, Miami, Florida, U.S.A. JEAN E. SHELTON, National Collection of Type Cultures, Central Public Health Laboratory, London, England H . VELDKAMP, Microbiological Laboratory, State University, Groningen, The Netherlands

ACKNOWLEDGMENTS For permission to reproduce, in whole or in part, certain figures and diagrams we are grateful to the following publishersCambridge University Press ; Edwards High Vacuum Ltd; Gustav Fischer Verlag, Stuttgart ; Society for Applied Bacteriology; Springer Verlag, Heidelberg. Detailed acknowledgments are given in the legends to figures.

Vi

PREFACE Volume 3 of “Methods in Microbiology” is concerned with the techniques used for isolating, growing and preserving micro-organisms. We considered that information on these themes was required in two distinct forms: a comprehensive list of growth media which would provide the reader with easy access to formulae and growth conditions for a wide range of microorganisms, and detailed descriptions of the special methods used for certain selected groups of micro-organisms. In addition general articles describing the principles involved in enrichment techniques for different types of micro-organisms and for the isolation of mutants and the design of mutation/selection programmes are also relevant to the main theme. As the contributions to Volume 3 took shape it became apparent that the amount of material involved was too much for inclusion in one volume and the material split relatively easily into two sub-volumes which are called Volumes 3A and 3B. Volume 3A contains Chapters concerned with the composition of growth media and media tables. Tabulated information about the preservation of micro-organisms and general articles concerned with enrichment, mutation and strain selection procedures are also provided. Volume 3B deals entirely with selected groups of micro-organisms, the emphasis being on methods of isolation, growth and handling in the 1aboratory, and preservation of cultures. In selecting the particular groups described we have been concerned to choose organisms which are not well described in other publications or which involve, because of their unusual physiology, special techniques. The actual treatment of the material we have left very largely to the choice of the individual authors. Our aim throughout has been to provide a useful treatment of important topics which are not well covered elsewhere while at the same time avoiding pointless repetition of readily available information.

J. R. NORRIS D. W. RIBBONS December, 1969

vii


CONTENTS

LISTOF CONTRIBUTORS.

.

v

ACKNOWLEDGMENTS .

vi

PREFACE

vii

CONTENTS OF PUBLISHED VOLUMES

.

X

Chapter I. Media for the Maintenance and Preservation of BacteriaS. P. LAPAGE, JEANE. SHELTON AND T. G. MITCHELL .

1

Chapter 11. Culture Collections and the Preservation of BacteriaS. P. LAPAGE, JEANE. SHELTON, T. G. MITCHELLAND A. R. MACKENZIE .

135

Chapter 111. Design and Formulation of Microbial Culture MediaE. Y. BRIDSON AND A. BRECKER .

229

Chapter IV. Quantitative Relationships Between Growth Media W. Constituents and Cellular Yields and Composition-D. 297 RIBBONS . Chapter V. Enrichment Cultures of Prokaryotic OrganismsH. VELDKAMP . Chapter VI. The Isolation of Mutants-DAVID

A. HOPWOOD

305

.

363

Chapter VII. Improvement of Micro-organisms by Mutation, 435 Hybridization and Selection-C. T. CALM . AUTHOR INDEX

SUBJECT INDEX

461 473

.

1x

CONTENTS OF PUBLISHED VOLUMES Volume 1

E. C. ELLIOTTAND D. L. GEORGALA. Sources, Handling and Storage of Media and Equipment R. BROOKES.Properties of Materials Suitable for the Cultivation and Handling of Micro-organisms G. SYKES.Methods and Equipment for Sterilization of Laboratory Apparatus and Media R. ELSWORTH. Treatment of Process Air for Deep Culture J. J. MCDADE, G. B. PHILLIPS, H. D. SIVINSKI AND W. J. WHITFIELD. Principles and Applications of Laminar-flow Devices H. M. DARLOW. Safety in the Microbiological Laboratory J. G. MULVANY. Membrane Filter Techniques in Microbiology C. T. CALAM. The Culture of Micro-organisms in Liquid Medium CHARLES E. HELMSTETTER. Methods for Studying the Microbial Division Cycle LOUISB. QUESNEL. Methods of Microculture R. C. CODNER. Solid and Solidified Growth Media in Microbiology K. I. JOHNSTONE. The Isolation and Cultivation of Single Organisms N. BLAKEBROUGH. Design of Laboratory Fermenters K. SARGEANT. The Deep Culture of Bacteriophage M. F. MALLETTE. Evaluation of Growth by Physical and Chemical Means C. T. CALAM.The Evaluation of Mycelial Growth H. E. KUBITSCHEK. Counting and Sizing Micro-organisms with the Coulter Counter Viable Counts and Viability J. R. POSTGATE. A. H. STOUTHAMER. Determination and Significance of Molar Growth Yields Volume 2

D. G. MACLENNAN. Principles of Automatic Measurement and Control of Fermentation Growth Parameters J. W. PATCHING AND A. H. ROSE.The Effects and Control of Temperature A. L. S. MUNRO.Measurement and Control of pH Values

H.-E. JACOB. Redox Potential D. E. BROWN.Aeration in the Submerged Culture of Micro-organisms D. FREEDMAN. The Shaker in Bioengineering J. BRYANT. Anti-foam Agents X

CONTENTS OF PUBLISHED VOLUMES

xi

N. G. CARR.Production and Measurement of Photosynthetically Useable Light R. ELSWORTH. The Measurement of Oxygen Absorption and Carbon Dioxide Evolution in Stirred Deep Cultures G. A. PLATON. Flow Measurement and Control. RICHARD Y. MORITA.Application of Hydrostatic Pressure to Microbial Cultures D. W. TEMPEST. The Continuous Cultivation of Micro-organisms: 1. Theory of the Chemostat AND D. W. TEMPEST. The Continuous Cultivation C. G. T. EVANS,D. HERBERT of Micro-organisms: 2. Construction of a Chemostat J. ~ E I C A Multi-stage . Systems R. J. MUNSON. Turbidostats R. 0. THOMSON AND W. H. FOSTER. Harvesting and Clarification of CulturesStorage of Harvest

Volume 3B VERAG. COLLINS.Isolation, Cultivation and Maintenance of Autotrophs N. G. CARR.Growth of Phototrophic Bacteria and Blue-Green Algae A. T. WILLIS.Techniques for the Study of Anaerobic, Spore-forming Bacteria R. E. HUNGATE. A Roll Tube Method for Cultivation of Strict Anaerobes Rumen Bacteria P. N. HOBSON. ELLAM. BARNES. Methods for the Gram-negative Non-sporing Anaerobes T. D. BROCK AND A. H. ROSE.Psychrophiles and Thermophiles N. E. GIBBONS. Isolation, Growth and Requirements of Halophilic Bacteria JOHNE. PETERSON. Isolation, Cultivation and Maintenance of the Myxobacteria AND P. WHITTLESTONE. Isolation, Cultivation and Maintenance of R. J. FALLON Mycoplasmas M. R. DROOP.Algae EVEBILLING.Isolation, Growth and Preservation of Bacteriophages


CHAPTER I

Media for the Maintenance and Preservation of Bacteria S. P. LAPAGE, JEAN E. SHELTON National Collection of Type Cultures, Central Public Health Laboratory, London, England AND

T. G. MITCHELL*

National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland I. Introduction

.

11. Genera Covered

1 3

.

111. Media Described . A. Growth media B. Suspendingfluids IV.

.

Maintenance and Preservation Methods by Genus and Species A. Explanation . B. Symbols used in the list . C. List of species .

V.

Formulae of Media . A. Growthmedia B. Suspending fluids

. .

VI.

Index of Manufacturers

.

References

4 4

.

.

6 6 6

.

9 10

.

. .

93 93 130

.

131

.

132

I. INTRODUCTION The methods and media for the maintenance and preservation of bacteria given in this Chapter have been derived from the experience of the National Collection of Industrial Bacteria (NCIB), National Collection of Marine

* Present address : Research and Development Establishment, British-American Tobacco Co. Ltd., Southampton, Hants, England. 2

2

S. P. LAPAGE, J. E. SHELTON AND T. G. MITCHELL

Bacteria (NCMB) and the National Collection of Type Cultures (NCTC) This experience has been built up over the years since the inception of these three Culture Collections in 1950, 1957 and 1920, respectively. The recommended procedures have been derived from experience of over 10,000 bacterial cultures that have had regular checks for the maintenance of their viability. The authors wish to point out that this summary represents the work of all the staff, both past and present, of these Collections, and is not merely a statement of the authors’ opinions. Full acknowledgment is therefore due to the staff who work or have worked in these Collections. Naturally, in the operation of a large culture collection, optimal conditions for preservation cannot be determined and used for each culture, as some system has to be maintained in the routine preservation of 3000 to 5000 cultures. Therefore, the methods given have proved satisfactory for routine preservation, but are not necessarily the optimal conditions for a particular strain. For example, the use of carbohydrates other than glucose or sodium glutamate or polyvinyl pyrollidone in the fluid in which the bacteria are suspended is not mentioned. A worker wishing to preserve a particular strain or group of bacteria may well find a more satisfactory method by investigation. In operating a culture collection, if a method has proved satisfactory, the advantages of a new method may be offset by the disadvantage of making long-term records no longer comparable. Cost may preclude the use of otherwise preferable methods or media, as, for example, bovine albumin in the suspending fluid. Time and staff may not be available for freeze-drying research and for large-scale comparison of methods, for exampleto freeze-dry a collection in duplicate to compare sucrose and glucose in the suspending fluid. It should also be pointed out that many of the strains preserved are received after a longer or shorter period of maintenance in sub-culture, and may have been adapted to a greater or lesser degree to growth and preservation under artificial conditions. Preservation of strains directly from isolation in the field may require investigation. Although the list and media are arranged alphabetically, an alphabetical list of the genera and media are given in Sections I1 and 111. This is for convenience; if an organism cannot be found in the Tables, the relatively short list of genera can be searched for a synonym or a related genus. Similarly a suitable medium can be chosen or a given medium found; this is of particular use as the alphabetical arrangement and order of words in the names of media is somewhat arbitrary. Cultures dealt with by the NCIB and NCMB have been marked with an asterisk (*) throughout, while those dealt with by the NCTC have not been marked; this will also help to prevent requests for organisms being sent to the wrong Culture Collection if this list is used as a catalogue.

I. MEDIA TABLES

3

11. GENERA COVERED+ Common synonyms of some of the genera are given in italic type. "Acetobacter, "Acetomonas, ""Achromobacter, "'Acinetobacter, Actinobacillus, "Actinobifida, Actinomyces, "Actinoplanes, Aerobacter, Aerococcus, Aeromonas, "Agrobacterium, ""Alcaligenes, Alkalescens Dispar, Alysiella, "Amorphosporangium, "Ampullariella, Anaerobic cocci, Arizona, "Arthrobacter, "Asticacaulis, "hotobacter, "hotomonas. ""Bacillus, Bacterionema, Bacteroides, "Bdellovibrio, "Beggiatoa, "Beijerinckia, Bethesda Ballerup, BiJTdobacterium, Bordetella, "Brevibacterium, Brucella, ""Butyribacterium. Campylobacter, Cardiobacterium, "Caryophanon, "Caulobacter, "Cellulomonas, "Cellvibrio, "Chlorobium, "Chloropseudomonas, "Chondrococcus, "Chromatium, ""Chromobacterium, Citrobacter, Cloaca, "'Clostridium, Cocci-anaerobic, Comamonas, ""Corynebacterium, "Cytophaga. "Dactylosporangium, Dermatophilus, "Derxia, "Desulfotomaculum, "Desulfovibrio, Diplococcus. Edwardsiella, "Elytrosporangium, Enterobacter, "Erwinia, Erysipelothrix, Escherichia. Ferrobacillus, ""Flavobacterium. Gaflkya, Gemella. Haemophilus, Hafnia, "Halobacterium, "Hydrogenomonas, "Hyphomicrobium. Jensenia. Klebsiella, Kurthia. ""Lactobacillus, "Lampropedia, Leptotrichia, "Leuconostoc, "Leucothrix, Lineola, Listeria, Loefflerella, Lophomonas. Macrospora, "Metallogenium, "Methanobacillus, "Methanococcus, "Methanosarcina, "Methylococcus, "Microbacterium, "Microbispora, WPMicrococcus, "Microcyclus, "Microechinospora, "Microellobosporia, "Micromonospora, "Micropolyspora, Moraxella, Morganella, ""Mycobacterium, M~COCOCCUS, "Mycoplana, Mycoplasma, 'Myxococcus. Neisseria, "Nitrobacter, "Nitrocystis, "Nitrosomonas, @#Nocardia. .i-

Italic type = synonym; these are not marked to indicate which Collection has covered them. Unmarked genera = covered by NCTC * = covered by NCIB or NCMB ** = covered by either the NCIB or NCMB together with the

NCTC.

4


Obesumbacterium . Pasteurella, "Pediococcus, "Pelodictyon, "Peptococcus, Peptostreptococcus, Pfezflerella, "Photobacterium, "Pilimelia, Plesiomonas, "Polyangium, Polysepta, ""Propionibacterium, "Protaminobacter, Proteus, Providencia, ""Pseudomonas, "Pseudonocardia. Ramibacterium, Rettgerella, "Rhizobium, "Rhodomicrobium, "Rhodopseudomonas, "Rhodospirillum. Salmonella, "Saprospira, "Sarcina, Serratia, Shigella, Simonsiella, "Sorangium, Sphaerophorus, Wphaerotilus, "Spirillospora, "Spirillum, "Sporocytophaga, "Sporolactobacillus, "Sporosarcina, Staphylococcus, Streptobacillus, Streptococcus, ""Streptomyces, *Streptosporangium. "Thermomonospora, "Thiobacillus, "Thiococcus, "Thiopedia, "Thiospirillum, "Thiothrix, "Treponema. Veillonella, ""Vibrio, "Vitreoscilla.

Waksmania. Yersinia. Zoogloea, Zopfius, "Zymobacterium, "Zymomonas.

111. MEDIA DESCRIBED

A. Growth media Two formulae are given for nutrient agar, one used in the NCIB and one in the NCTC. With certain species covered by the NCIB it is important that the NCIB formula is used, e.g., Fluwobacterium. In many cases it would make little difference which was used, but growth may be slower on the NCIB formula. It may be noted that the recovery of some organisms, e.g., pseudomonads and vibrios, is better on blood agar than on nutrient agar prepared by the NCTC formula. 13. CCT medium 1. Acetate agar 14. Charcoal blood agar 2. Alcaligenes tolerans agar 15. Chloropseudomonas medium 3. Bdellovibrio medium 16. Chocolate blood agar 4. Beggiatoa medium 17. Clostridium aceticum medium 5 . Blood agar (with or without 18. Clostridium chauvoei medium glucose) 19. Clostridium kluyveri medium 6. 4% Blood agar 20. Clostridium sticklandii 7. Bordet-Gengou agar medium 8. Butyribacterium medium 21, Clostridium tetanomorphum 9. Casein agar medium 10. Casitone agar 22. Clostridium thermoaceticum 11. Casman's blood agar medium 12. Caulobacter medium

I. MEDIA TABLES

23. Clostridium thermosaccharolyticum medium 24. Cooked meat medium (with or without glucose) 25. Cytophaga agar No. 1 26. Cytophaga agar No. 2 27. Cytophaga medium No. 3 28. Cytophaga medium No. 4 29. Czapek’s peptone agar 30. Dorset’s egg medium 3 1. Dubos’ salts solution 32. Ethanol malt agar 33. Ferrooxydans medium 34. Flavobacterium heparinum medium 35. Freshwater flexibacteria medium 36. Galactose agar 37. Glucose agar 38. Glucose broth 39. Glucose yeast extract agar 40. Glycerol asparagine agar 41. Glycerol casitone agar 42. Gyorgy and Rose medium 43. Halophile medium 44.Horse serum agar 45. Horse serum broth 46. Hyphomicrobium medium 47. Klebsiella agar 48. Krebs’ yeast lactate medium 49. Leuconostoc oenos medium 50. Leucothrix mucor medium 51. Maize mash 52. Manganous acetate agar 53. Marine agar 54. Marine flexibacteria medium 55. Marine Spirillum medium 56. Methanobacillus medium 57. Methanococcus medium 58. Methanol salts medium 59. Methanosarcina barkeri medium 60. Methylococcus medium 61. MRS medium 62. Mycobacterium johnei medium 63. Mycoplasma suipneumoniae agar 64. Mycoplasma suipneumoniae broth

65. 66. 67. 68.

5

Nitrobacter agilis medium Nitrogen-free agar Nitrosomonas europaea medium Nutrient agar (a) NCIB formula (b) NCTC formula 69. 4% Nutrient agar 70. Nutrient agar, pH 8.0 71. Nutrient broth 72. Oatmeal agar 73. Peptone ferric citrate agar 74. Peptone saline agar 75. Peptone yeast glutamate 76. Pfennig’s medium 77. Postgate’s medium 78. Pseudomonas methanica medium 79. Pseudomonas saccharophila medium 80. Purple milk 81. Rhizobium medium 1 82. Rhizobium medium 2 83. Sarcina maxima medium 84. Sarcina ventriculi medium 85. Sea water agar 86. Sodium caseinate agar 87. Soil extract agar 88. Soil extract agar, pH 8.0 89. Spirillum medium 90. Spirillum volutans medium 91. Starch salts agar 92. Sulphur medium 93. Thiobacillus denitrificans medium 94. Thiobacillus perometabolis medium 95. Thiosulphate agar No. 1 96. Thiosulphate agar No. 2 97. Thiothrix medium 98. Tomato juice agar 99. Treponema zuelzerae medium 100. Tryptone glycine medium 101. Urea nutrient agar 102. Urea soil extract agar 103. Uric acid medium 1 104. Uric acid medium 2 105. V 17 medium

6


106. Yeast agar 107. Yeast malate medium

108. Yeast malt agar B. Suspending fluids 112. 7.5% Glucose broth

109. Yeast peptone broth 110. Zymobacterium medium 111. Zymomonas medium

113. 7.5% Glucose serum 114. Mist. desiccans

IV. MAINTENANCE AND PRESERVATION METHODS BY GENUS AND SPECIES

A. Explanation The list is given in a set of columns for convenience. 1. Column 1 The names of the organisms. These are arranged alphabetically by genus and species. It is not practicable to give an excessive number of synonyms; nevertheless, some common synonyms for species and genera are included in the list. These were selected partly from names used in requesting cultures from the Collections. Older names are not given, since their current synonyms can be obtained from any standard textbook of bacteriology. In some cases, the methods of the NCIB and NCTC are given where they differ from each other. This may be convenient as some media or conditions of incubation may not be available in a given laboratory. This may apply particularly when a special medium is used by one of the Collections, but the species will freeze-dry satisfactorily from a medium in more general use.

2 . Column 2 Maintenance media. These have been chosen as suitable for maintenance by sub-culturing. Where there is no entry in this column it means that no experience was available in the Collection concerned, because the cultures have been maintained in a freeze-dried state.

3 . Column 3 Pre-drying medium. This is the medium on which the organism is grown and the resulting growth is then emulsified in the suspending fluid for freezedrying. The entry “Same” in this column indicates that the pre-drying medium is the same as the maintenance medium. For some of the entries alternatives are given, since experience has grown up over the years and different media have proved satisfactory. In the case of Bacillus, soil extract agar has been widely used for growing the cultures before freeze-drying, with the intention of obtaining well sporulated cultures.

I. MEDIA TABLES

7

If there is no entry in this column, we have had no experience of the ability of the organism to survive freeze-drying or drying by the method of Annear (1961), and the organism is maintained in the active state.

4. Column 4 Pre-drying culture requirements, i.e., the most favourable conditions we have found under which to grow the culture. These are divided into(a) Temperature of incubation. This is given in degrees centigrade. If an incubator at a given temperature is not available, then a related temperature may prove satisfactory. In some cases different temperatures are given, because experience over the years has shown different temperatures to be equally successful. However in some cases different temperatures are shown because particular strains have different temperature requirements, and this is indicated in the “Remarks” column (Column 6). (b) Gaseous conditions. These are specified for each organism. Carbon dioxide is added for the growth of many aerobic organisms, although it is not obligatory for all of these, but it may improve growth in many cases. Critical experiments have not been made to determine whether the humidity of the jar or the added C02 contribute to the improvement in growth. The amount of C02 added is not exact, but is about 4-5%; this is represented in the column by “C02”. “C02 only” in this column indicates that the organisms are incubated in an atmosphere consisting entirely of COz. Anaerobic conditions. The NCTC uses 95% H2 and 5% COz for the routine cultivation of anaerobes, while the NCIB uses an atmosphere of Hz only, except in the case of Thiococcus and Thiospirillum where 95% Hz and 5% C02 are used. For certain groups, such as the photosynthetic non-sulphur organisms, critical anaerobic conditions are not required, and for these prior boiling of the medium and the use of completely filled bottles is sufficient to ensure good growth. With the photosynthetic sulphur organisms, active cultures can be successfully sub-cultured without anaerobic atmospheres of special gases if care is taken to use fresh medium and completely filled bottles. The latter group are generally more difficult to revive from the dried condition and conditions become more critical. For this purpose, the use of pyrogallol plugs with saturated Na2C03 or K2C03 is recommended. (c) Incubation time. Given in hours unless otherwise specified. I n general, the length of incubation used by the NCIB is longer than that used by the NCTC. However, it is important to have a high initial count, i.e., good growth on the pre-drying medium before freeze-drying, and in many cases the NCTC incubation times would be the minimum time for sub-culture.

S


In addition, many of the organisms kept in the NCTC grow more quickly than some of those kept in the NCIB. On some of the media used by the NCTC quicker growth would be expected than on the corresponding NCIB media, e.g., on the NCTC nutrient agar compared to the NCIB formula (media No. 68 a and b). I n the case of the sporulating actinomycetes, e.g., Streptomyces, a wide variation in incubation time may be experienced between strains in order to obtain a well sporulated growth for drying purposes. The figures in the Tables are therefore only a rough guide. Care is necessary with some strains, e.g., Streptomyces xantholiticus, where the aerial mycelium becomes deliquescent if the time of incubation is too long.

5 . Column 5 SuspendingJEuids. These are specified in this column. The NCIB routinely use Mist. desiccans (medium No. 114) which contains glucose, broth and serum. In the past the NCTC also used Mist. desiccans, but have in recent years found 7.5% glucose serum (medium No. 113) to be satisfactory. It would probably make little difference in most cases which suspending fluid is used. The NCTC has traditionally used 7.5% glucose broth (medium No. 112) for suspending enterobacteria, in order to avoid any possibility of antigenic changes due to antibodies in horse serum. For species dealt with by the NCIB or NCMB, where a suspending fluid is shown, the preferred method of long-term maintenance is freeze-drying when there is no contrary indication in the “Remarks” column. For certain organisms where the method of Annear (1961), involving drying from the liquid state, has been shown to be superior to freeze-drying this is indicated in the “Remarks” column by the comment “L-dry”. Where no suspending fluid is shown, this indicates that either no results are available with the particular organism for freeze- or L-drying, e.g., Pilimelia, or in some cases one or both methods have been tried, but have proved unsatisfactory, e.g., most fruiting myxobacteria.

6 . Column 6 Remarks are given in this column. If no comment appears in this column, then the recovery medium can be assumed to be the same as the pre-drying medium, and if there are differences these are specified. Various media have been tried for recovery in the past and sometimes alternatives are mentioned ; if one medium has been found to be better than the others, this is stated. In the case of those species of Proteus and Clostridium which swarm, we have found agar at a concentration of 4% in the medium inhibits swarming and enables us to check purity and carry out bacterial counts.

I. MEDIA TABLES

9

B. Symbols used in the list Column 1: Organisms

* = NCIB, or NCWIB information Unmarked = NCTC information Italic type = Synonym. These are not marked to indicate whether information was provided by the NCIB, NCMB or NCTC. Column 2: Maintenance medium No entry = No available experience of maintenance by sub-culture. Nutrient agar: for species marked * (NCIB or NCMB), Nutrient agar NCIB formula (medium No. 68a) is used; for unmarked species (NCTC), Nutrient agar NCTC formula (medium No, 68b) is used.

Column 3 :Pre-drying medium Same = Same medium used as in Column 2.

Column 4: Pre-drying culture requirements = Temperature in degrees centigrade =

atmosphere of hydrogen

= atmosphere of 95% hydrogen and 5oj, carbon dioxide

Gaseous :

= atmosphere of air and 6 5 % carbon dioxide COz only = atmosphere of carbon dioxide only = atmosphere of methane = atmosphere of nitrogen = Time of incubation is given in hours unless otherwise Time stated.

12

Column 5 :Suspending JEuidfor drying If no suspending fluid is given, see Section IVA.5 p. 8. Column 6: Remarks L-dry

=

Drying by the method of Annear (1961).

L

C. List of species

0

Pre-drying culture requirements Organism

Maintenance medium

Pre-drying medium

Same Glucose yeast extract agar Acetobacter acetigenum-see A. xylinum Acetobacter acetosum-see A. rancens Acetobacter acidum-mucosum-see A. rancens Acetobacter alcoholophilus--see A. rancens "Acetobacter Ethanol malt Same ascendens agar Acetobacter capsulatumsee Acetomonas oxydans "Acetobacter Ethanol malt Same estunense agar Acetobacter gluconicus-see Acetomonas oxydans Acetobacter kuetzingianum-see A. rancens "Acetobacter Ethanol malt Same lovaniense agar Acetobacter melanogmum-see Acetomonas oxydans 'Acetobacter Glucose yeast Same mesoxydans extract agar Acetobacter mobile-see A. rancens Acetobacter orleanense--see A. rancens 'Acetobacter Ethanol malt Same paradoxum agar eAcetobacter Ethanol malt Same pasteurianum agar *Acetobacter aceti

A

f

T, "C 30

Gaseous Air

Time

72

, Suspending fluid for drying Mist. desiccans

Remarks

m

cd Y

Y m

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

*Acetobacter Ethanol malt Same peroxydans agar *Acetobacterrancens Ethanol malt Same agar Acetobacter suboxydans-see Acetomonas oxydans Acetobacter transcapsdatum-see A. xylinum *Acetobacter Glucose yeast Same xylinum extract agar *Acetomonas Glucose yeast Same oxydans extract agar Achromobacter anitraturn-see Acinetobacter anitratus Same *Achromobacter Nutrient agar arsenoxydans Same *Achromobacter Nutrient agar butyri Same Achromobacter Nutrient agar citroalcaligenes Same Achromobacter Nutrient agar conjunctivae Same XAchromobacter Nutrient agar cystinovorum Same *Achromobacter Nutrient agar delictatum Same *Achromobacter Nutrient agar faecalis Same *Achromobacter Nutrient agar

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

37

Air

24

37

Air

24

30

Air

72

7.5% Glucose serum 7 5 % Glucose serum Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

37

Air

24

7.5% Glucose serum

~~

2

r v1 m

gUttatUS

Achromobacter Nutrient agar haemolyticus subsp. alcaligenes For explanation, see pp. 6-9.

Same

CL L

L

List of species-Continued

tQ


Maintenance medium

Pre-drying medium

Achromobacter Nutrient agar Same haemolyticus subsp. haemolvticus +Achromobacter Nutrient agar Same hartlebii +Achromobacter Nutrient agar Same lacticum Achromobacter lwofi-ee Acinetobacter lwoffii 'Achromobacter Nutrient agar Same marshallii Achromobacter Nutrient agar Same metalcaligenes Achromobacter Nutrient agar Same mucosus +Achromobacter Blood agar Same nematophilus +Achromobacter Nutrient agar Same parvulus +Achromobacter Nutrient agar Same venenosum *Achromobacter Nutrient agar Same viscosum

r

A

T , "C

37

Gaseous Air

Time

24

,

Suspending fluid for drying

7.5% Glucose serum

Remarks

rn

a

r > d

>

"2 30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

37

Air

24

37

Air

24

30

Air

72

7.5% Glucose serum 7.5% Glucose serum Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

? m

5

0

*Achromobacter winogradskyi *Achromobacter xerosis *Acinetobacter anitratus Acinetobacter anitratus *Acinetobacter anitratus var. saponophilus Acinetobacter lwofFii Actinobacillus actinomycetemcomitans Actinobacillus equuli Actinobacillus lignieresii *Actinobifida dichotomica Actinomyces bovis

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

37

Air

24

Nutrient agar

Same

30

Air

72

7.5% Glucose serum Mist. desiccans

Nutrient agar

Same

37

Air

24

7.5% Glucose serum

Blood agar

37

coz

24-48

7.5% Glucose serum

Blood agar

Same or Chocolate blood agar Same

37

Air

24

Blood agar

Same

37

Air

24

7.5% Glucose serum 7.5% Glucose serum

Starch salts agar

Same

45

Air

5 days

Mist. desiccans

Cooked meat medium

Blood agar or Glucose agar

37

Hz + C02

2-5 days

7.5% Glucose serum

Blood agar

37

Hz

48

7-5% Glucose serum

Actinomycesisraelii Cooked meat medium For explanation, see pp. 6-9.

+ CO2

Recovery on Blood agar invariably better Recovery also on Glucose agar L

w


L

4

Pre-drying culture requirements Organism Actinomyces naeslundii

Maintenance medium Cooked meat medium

Actinomyces Cooked meat odontolyticus medium +Acthoplanes Oatmeal agar missouriensis *Acthoplanes Glycerol philippinensis asparagine agar *Actinoplanes Oatmeal agar utahensis Aerobacter-see Enterobacter Aerococcus viridans Blood agar Aeromonas formicans Aeromonas hydrophila Aeromonas liquefaciens Aeromonas salmonicida

Pre-drying medium Glucose agar

Time

, Suspending fluid for drying

48

7.5% Glucose

A

I

T,"C 37

Gaseous Air

serum Blood agar or Glucose agar Same

37

Hz + COa

48-72

25

Air

21 days


Same

25

Air

21 days

Mist. desiccans

Same

25

Air

21 days

Mist. desiccans

Same

37

Air

24-48

7.5% Glucose

Nutrient agar

Same

37

Air

24

Nutrient agar

Same

37

Air

24

Nutrient agar

Same

37

Air

24

Blood agar

Same

22 or 30

Air

24

serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum

Remarks Recovery usually better on Blood agar Counts on Blood agar often better

M

0

Also recovered on Nutrient agar. Strains require different temperatures

Same

37

Air

24

Same

30

Air

72


Same

30

Air

72

Mist. desiccans

Same

30

Air

72

Mist. desiccans

Same Same

30 30

Air Air

72 72

Mist. desiccans Mist. desiccans

Nutrient agar

Same

37

Air

24

7.5% Glucose

Nutrient agar

Same

37

Air

24

Alcaligenes odorans Nutrient agar

Same

37

Air

24

Alcaligenes odorans Nutrient agar var. viridans *Alcaligenes tolerans Alcaligenes tolerans agar Alcaligenes viscosus Nutrient agar

Same

37

Air

24

Same

30

Air

72

Same

37

Air

24

7.5% Glucose serum

Same

37

Air

48

7.5% Glucose serum

Aeromonas Nutrient agar shigelloides *Agrobacterium Nutrient agar gypsophilae *Agrobacterium Nutrient agar radiobacter 'Agrobacterium Nutrient agar rhizogenes QAgrobacteriumrubi Nutrient agar *Agrobacterium Nutrient agar tumefaciens Alcaligenes denitrificans Alcaligenes faecalis

serum 7.5% Glucose serum 7.5% Glucose serum 7.574 Glucose serum Mist. desiccans

Alkalescens dispar-see Escherichia Alysiella filiormis

Blood agar

For explanation see pp. 6-9.

List of species-Continued Pre-drying culture requirements Organism

Maintenance medium

Pre-drying medium

I

A

T,"C

Gaseous

Time


*Amorphosporangium auranticolor

Czapek's peptone agar

Same

25

AiI

28 days

Mist. desiccans

*Ampullariella campanulata *Ampullariella

Czapek's peptone agar Czapek's peptone agar Czapek's peptone agar Czapek's peptone agar

Same

25

Air

28 days


digitata

*Ampullariella lobata *Ampullariella regularis

?

Same

25

Air

28 days

Same

25

Air

28 days

Mist. desiccans

Same

25

Air

28 days

Mist. desiccans

37

Arizona arizonae

37

Same

P

U

Anaerobic cocci-see also Peptococcus, Veillonella Anaerobic cocci Cooked meat Blood agar medium Nutrient agar

Remarks

H2

+ C02

48

7.5% Glucose serum

Air

24

ri

n

7.5% Glucose broth

*Arthrobacter atrocyaneus *Arthrobacter aurescens LArthrobactercitreus 'Arthrobacter crystallopoites

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar Nutrient agar

Same Same

30 30

Air Air

72 72


i.

*Arthrobacter duodecadis *Arthrobacter flavescens *Arthrobacter globiformis *Arthrobacter histidinolovorans *Arthrobacter nicotianae *Arthrobacter oxydans *Arthrobacter pascens XArthrobacter ramosus *Arthrobacter simplex 'Arthrobacter terregens *Arthrobacter ureafaciens 'Arthrobacter variabilis 'Arthrobacter viscOsus

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Soil extract agar Same

30

Air

5 days

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Yeast malt agar

Same

25

Air

5 days

Mist. desiccans

*Asticacaulis excentricus

Caulobacter medium

Same

25

Air

5 days

Mist. desiccans

*Azotobacter agilis

Nitrogen-free agar

Same

25

Air

72

Mist. desiccans

For explanation, see pp. 6-9.

c1

1

&

c

L-dry

c.

List of species-Contimred

00


Maintenance medium

Pre-drying medium

*Azotobacter Nitrogen-free Same beijerinckii agar *Azotobacter Nitrogen-free Same chroococcum agar Azotobacter indicum- -see Beijerinckia indica * h t o b a c t e r insigne Nitrogen-free Same agar *Azotobacter Nitrogen-free Same macrocytogenes agar *Azotobacter Nitrogen-free Same vinelandii agar *Azotomonas insolita Nutrient agar Same

I

A

T,"C

Gaseous

Time

,


Remarks

v, cd

25

Air

72

Mist. desiccans

25

Air

72

Mist. desiccans

25

Air

72

Mist. desiccans

L-dry

25

Air

72

Mist. desiccans

L-dry

25

Air

72

Mist. desiccans

25

Air

72

Mist. desiccans

Air

4 days

7.5% Glucose

Y

Y m

cl

Bacillus alcalophilus Nutrient agar, pH 8.0

Soil extract agar, pH 8.0

30

Bacillus alvei

Soil extract agar

30 or 37

Nutrient agar

serum Air

4 days

7.5% Glucose serum

Bacillus anthracis

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

*Bacillus aporrhoeus Nutrient agar

Same

30

Air

72

Mist. desiccans

Recovery on Nutrient agar, pH 8-0 Recovery medium Nutrient agar, or 4% Nutrient agar Recovery medium Nutrient agar

0

Bacillus badius

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus brevis

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus carotarum

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus cereus

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus circulans

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus coagulans

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

*Bacillus fastidiosus

Uric acid medium 1 Nutrient agar

Same

30

Air

48

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus freudenreichii

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus laterosporus

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus lentus

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus firmus


Mist. desiccans

Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium

I4

5

Nutrient agar

H 2

Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar

,-

bi

\o


Maintenance medium

Pre-dryhg medium

A

I

T,"C

Gaseous

Time


Remarks

-

Bacillus lichenifonnis

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus loehnisii

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus macerans

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

*Bacillus macroides Bacillus megaterium

Acetate agar Nutrient agar

Same Soil extract agar

30 30 or 37

Air Air

72 4 days

Mist. desiccans 7.5% Glucose serum

Bacillus mycoides

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

'Bacillus palustris *Bacillus palustris var. gelatiBacillus pantothenticus


Same Same

30 30

Air Air

72 72


Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose

Bacillus pasteurii

Urea nutrient agar

serum Same or Urea soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar

Recovery medium Nutrient agar Recovery medium Urea nutrient agar

Bacillus polymyxa

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus pumilus

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus sphaericus

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Bacillus stearothermophilus Bacillus subtilis

Nutrient agar

Soil extract agar

56 or 60

Air

4 days

7.5% Glucose serum

Nutrient agar

Soil extract agar

30 or 37

Air

4 days

7.5% Glucose serum

Nutrient agar

Same

30

Air

48

Mist. desiccans

Bacterionema matruchotii

Glucose agar or Blood agar

Glucose agar

37

Air

48

7.5% Glucose serum

Also recovered on Blood agar

Bacteroides fragilis

Cooked meat medium

Blood agar or Chocolate blood agar Blood agar

37

Hz C02

1-3 days

7.5% Glucose

Also recovered on Glucose agar

Blood agar

37

*Bacillus thuringiensis

Bacteroides melaninogenicum Bacteroides necrophorus

Cooked meat medium Cooked meat medium


+

serum

37

+ H2 + COz

H2 C02

2-3 days 2-3 days


Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar Recovery medium Nutrient agar

?

U

N

List of s p e c i e s 4 o n t i n u e d

lu


Maintenance medium

T,"C


Time

30

Air

5 days

Mist. desiccans

25

Air

Same

30

Air

72

Mist. desiccans

Same

30

Air

72

Mist. desiccans

Same

30

Air

72

Mist. desiccans

Same

30

Air

72

Mist. desiccans

Same

30

Air

72

Mist. desiccans

Same

30

Air

72

Mist. desiccans

Lactobacillus bifidus Blood agar or Same Nutrient agar Blood agar Same

37

Air

24

37

Air

48


Bdellovibrio medium

XBeggiatoa

Beggiatoa medium

*Beijerinckia congensis 'Beijerinckia derxii

Nitrogen-free agar Nitrogen-free agar 'Beijerinckia Nitrogen-free fluminensis agar aBeijerinckia indica Nitrogen-free agar 'Beijerinckia Nitrogen-free lacticogenes agar 'Beijerinckia mobilis Nitrogen-free agar Bifdobacteriu-ee Bordetella bronchiseptica Bordetella parapertussis

A

I

Gaseous

XBdellovibrio bacteriovorus

Bethesda Ballerup-see

Pre-drying medium Same

Remarks

Citrobacter

Also recovered on BordetGengou agar

v1

Bordetella pertussis

eBrevibacterium acetylicum *Brevibacterium ammoniagenes 'Brevibacterium divaricatum *Brevibacterium fermentans YBrevibacterium flavum 'Brevibacterium fuscum *Brevibacterium immariophilum +Brevibacterium impenale YBrevibacterium incertum +Brevibacterium lactofermentum +Brevibacterium leucinophagum *Brevibacterium linens +Brevibacterium liquefaciens

Bordet-Gengou agar

Same

37

Air

48-72

7.5 % Glucose serum

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Blood agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans


Also recovered on Charcoal blood agar

p3

w

List of s p e c i e s x m t i n u e d Pre-drying culture requirements Organism

Maintenance medium

Pre-drying medium

I

A

T,"C

Gaseous

Time

,


Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Sea water agar

Same

25

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Brucella abortus

Blood agar

Same

37

coz

24-48

7.5% Glucose serum

Brucella melitensis

Blood agar

Same

37

with or without CO2

48

7.5% Glucose serum

'Brevibacteriun oxydans 'Brevibacterium roseum 'Brevibacterium saccharolyticum 'Brevibacterium sulphureum 'Brevibacterium vitarumen

Remarks

To assure survival on laboratory media, subculture is advisable every 2-3 weeks T o assure survival on laboratory media, subculture is advisable every 2-3 weeks

00

d

k

? I\

m

ab I

m (u

M

k

'go

I. MEDIA TABLES

1 (u

k

a

2

m k 0

a

z o

2

.r(

E

m

a

00

d

25

2d a

0

P

N


m


Maintenance medium

Varyophanon latum Acetate agar Waryophanon tenue Acetate agar +Caulobacter bacteroides *Caulobacter crescentus "Caulobacter fusiformis *Caulobacter halobacteroides *Caulobacter henricii Waulobacter intermedius XCaulobacter leidyi *Caulobacter maris Waulobacter subvibrioides Waulobacter variabilis

Caulobacter medium Caulobacter medium Caulobacter medium Caulobacter medium 2.5 % NaCl Caulobacter medium Caulobacter medium Caulobacter medium Caulobacter medium 2-594 NaCl Caulobacter medium Caulobacter medium

+

+

Pre-drying medium

I

A

T, "C

Gaseous

Time


Same Same

25 25

Air Air

5 days 5 days


Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Remarks

r

*Caulobacter vibrioides Vellulomonas biazotea Tellulomonas bibula ‘Cellulomonas cellasea ‘Cellulomonas fimi XCellulomonas flavigena Tellulomonas gelida *Cellulomonas rossica *Cellulomonas subalbus ‘Cellulomonas uda

Caulobacter medium Dubos’ salts solution Dubos’ salts solution Dubos’ salts solution Nutrient agar Dubos’ salts solution Dubos’ salts solution Dubos’ salts solution Dubos’ salts solution Dubos’ salts solution

Same

25

Air

5 days

Mist. desiccans

Nutrient agar

30

Air

5 days

Mist. desiccans

Nutrient agar

30

Air

5 days

Mist. desiccans

Nutrient agar

30

Air

5 days

Mist. desiccans


30 30

Air Air

5 days 5 days


Nutrient agar

30

Air

5 days

Mist. desiccans

Nutrient agar

30

Air

5 days

Mist. desiccans

Nutrient agar

30

Air

5 days

Mist. desiccans

Nutrient agar

30

Air

5 days

Mist. desiccans

Wellvibrio fulvus

Dubos’ salts solution Dubos’ salts solution

Nutrient agar

30

Air

5 days

Mist. desiccans

Nutrient agar

30

Air

5 days

Mist. desiccans

CCT medium

Same

30

coz only

7 days

Mist. desiccans

CCT medium

Same

30

COZonly

7 days

Mist. desiccans

30

COZonly

‘Cellvibrio vulgaris *Chlorobium limicola *Chlorobium thiosulfatophilum ‘Chloropseudomonas ethylicum

Chloropseudomonas medium


EC

51

Incubate in light Incubate in light Incubate in light

h)


00


Maintenance medium

'Chondrococcus columnaris 'Chondrococcus coralloides Thondrococcus lucifugans

Cytophaga agar No. 2 Klebsiella agar

Thromatium, small celled 'Chromatium okenii

CCT medium

'Chromatium vinosum 'Chromatium warmingii

Chromobacterium amethystinum

*Chromobacterium lividum


Casitone agar

f

A

T, "C

Gaseous

20

Air

25

Air

25

Air

Time

5 days

, Suspendingfluid for drying

Remarks

Mist. desiccans

Y

m

Same

Pfennig's medium Pfennig's medium Pfennig's medium

Blood agar

Nutrient agar

30

C02 only

30

coz only

30

c02 only

30

CO2 only

300r 22

Air

7 days

Mist. desiccans

Incubate in light Incubate in light Incubate in light Incubate in light

24-48

7.5% Glucose

Recovery on Blood agar better than on Nutrient agar

serum

Nutrient agar

Same

20

Air

5 days

Mist. desiccans

i.

Chromobacterium lividum

*Chromobacterium maris-mortui Chromobacterium typhiflavum +Chromobacterium violaceum Chromobacterium violaceum

Blood agar

Same

22

Air

2448

7.5744 Glucose serum

Sea water agar

Same

20

Air

5 days

Mist. desiccans

Nutrient agar

Same

37

Air

24

Nutrient agar

Same

20

Air

5 days

7 5 % Glucose serum Mist. desiccans

Blood agar

Same

30

Air

24-48

Nutrient agar Citrobacter ballerupensis Citrobactir freundii Nutrient agar

7.5% Glucose serum

Same

37

Air

24

7.5% Glucose broth 7.5% Glucose broth

Same

37

Air

24

Same

37

H2

48

Mist. desiccans

Same

37

H2

72

Mist. desiccans

Cloaca-see Enterobacter cloacae 'Clostridium aceticum *Clostridium acetobutylicum

Clostridium aceticum medium Maize mash


Recovery on Nutrient agar not usually as good as on Blood agar

Recovery on Blood agar often better than on Nutrient agar

I

'

s9 -I

z E


Maintenance medium

Pre-drying medium

r

A

T,"C

Gaseous

Time


72

Mist. desiccans

2448

7.5% Glucose serum

72

Mist. desiccans

+ C02

48

7.5% Glucose serum

+ C02

48

7.5 yo Glucose serum

72

Mist. desiccans

24-48

7.5% Glucose serum

Same

30

€32

Blood agar

37

H2


Same

30

H2

Blood agar

37

Hz

Clostridium botulinum

Cooked meat medium

Blood agar

37

H2

Wlostridium butylicum Clostridium butyricum

Maize mash

Same

37

H2

Cooked meat medium

Blood agar or Blood agar 1% glucose

37

H2

*Clostridium caloritolerans 'Clostridium carbonei


Same

37

H2

72

Mist. desiccans

Same

37

H2

72

Mist. desiccans

'Clostridium acidi-urici Clostridium aerofoetidum

Uric acid medium 2 Cooked meat medium

XClostridium beijerinckii Clostridium bifermentans

+

+ CO2

+ C02

Remarks

Also recovered in Cooked meat medium Recovery also in Cooked meat medium and on 4% Blood agar Recovery also in Cooked meat medium and on 4% Blood agar Recovery also in Cooked meat medium and on 4% Blood agar

I

CI b 00

I . MEDIA TABLES

00

d

?

x"

CI

+ x"

b

5

d

+ x" b

m

b

m

b

m

b

m

31

List of species-continued Pre-drying culture requirements Organism

Maintenance medium

Pre-drying medium

A

f

T,"C

Gaseous

Time

,


Remarks

~

+ C02

48

7.5% Glucose serum

Ha

72

Mist. desiccans

30

H2

72

Mist. desiccans

22

H2

48

7.5% Glucose

Clostridium oedematiens

Cooked meat medium

Blood agar

37

H2

+Clostridium pasteurianum *Clostridium pectinovorum Clostridium putrefaciens

Cooked meat medium Cooked meat medium Cooked meat medium

Glucose broth

37

Same Blood agar

Addition of 1% glucose to media often improves

growth

Clostridium putrificum

Cooked meat medium

Tlostridium rubrum Cooked meat medium Wlostridium Cooked meat scatologenes medium Clostridium Cooked meat septicum medium

+ C02

serum

Blood agar

37

H2

+ C02

2448

7.5% Glucose serum

Glucose broth

30

H2

72

Mist. desiccans

Same

37

H2

72

Mist. desiccans

Blood agar

37

H2+ CO2

48-96

7.5% Glucose serum

Also recovered on4% Blood agar and in Cooked meat medium Also recovered in Cooked meat medium

Also recovered on 4% Blood agar and in Cooked meat medium

Clostridium sphenoides

Cooked meat medium

Blood agar

37

Hz + COz

Clostridium sporogenes

Cooked meat medium

Blood agar

37

Hz COz

Clostridium sticklandii medium Cooked meat medium Cooked meat medium

Same

30

Same Blood agar

Tlostridium sticklandii Tlostridium subterminale Clostridium tertium

Clostridium tetani

+Clostridium tetanomorphum

7.5”/0 Glucose serum

48

7.5% Glucose serum

Hz

5 days

Mist. desiccans

37

Hz

72

Mist. desiccans

37

Hz COz

48

7.5% Glucose

+

+

serum

Cooked meat medium

Blood agar

Clostridium tetanomorphum medium

Same


48

37

Hz + COz

48

7.5% Glucose serum

30

Hz

5 days

Also recovered on 4% Blood agar and in Cooked meat medium Also recovered on 4% Blood agar and in Cooked meat medium

Also recovered on 4% Blood agar and in Cooked meat medium Also recovered on 4% Blood agar and in Cooked meat medium

m

H

2

E

Mist. desiccans

w w

w


P

Pre-drying culture requirements Maintenance medium

Pre-drying medium

r

A

, Suspending fluid

T,"C

Gaseous

Time

Blood agar

37

Ha + COa

48

7.5% Glucose serum

Clostridium Same thennoaceticum medium 'Clostridium Clostridium Same thermosaccharo- thermosaccharolyticum lyticum medium Clostridium welchii Cooked meat Blood agar medium

55

Hz

4 days

Mist. desiccans

Organism Clostridium tetanomorphum

Cooked meat medium

'Clostridium thermoaceticum

for drying

Remarks Also recovered on 4% Blood agar and in Cooked meat medium rm

55

Hz

37

Ha COz

+

48

Mist. desiccans

24-48

7.5% Glucose serum

Eir

Also recovered in Cooked meat medium

Coca., anaerobic-see

Anaerobic cocci, Peptococcus and Veillonella

Comamonas percolans

Nutrient agar

*Corynebacterium acetoacidophilum Corynebacterium acnes 'Corynebacterium aquaticum

Same

37

Air

24

7.5% Glucose serum

Nutrient agar

Same

30

Air

72

Mist. desiccans

Cooked meat medium Nutrient agar

Blood agar

37

H z + C02

48

Same

30

Air


5 days

'Corynebacterium barkeri +Corynebacterium betae Corynebacterium bovis Corynebacterium diphtheriae Corynebacterium equi Corynebacterium fascians Corynebacterium h i 'Corynebacterium flaccumfaciens Corynebacterium flavidum Corynebacterium haemolyticum *Corynebacterium herculis Corynebacterium hofmannii Torynebacterium ilicis *Corynebacterium insidiosum 'Corynebacterium laevaniformans f

Nutrient agar

Same

30

Air

5 days

Mist. desiccans

Nutrient agar

Same

30

Air

5 days

Mist. desiccans

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

2448

Blood agar

Same

37

Air

2448

Nutrient agar

Same

25

Air

5 days

7.5% Glucose serum 7.5yo Glucose serum 7.5% Glucose serum Mist. desiccans

Blood agar

Same

30

Air

2448

Nutrient agar

Same

30

Air

5 days

Blood agar

Same

37

Air

24

Blood agar

Same

37

Air

24

Nutrient agar

Same

30

Air

72

Blood agar

Same

37

Air

24

Nutrient agar

Same

30

Air

72


Nutrient agar

Same

25

Air

7 days

Mist. desiccans

Nutrient agar

Same

30

Air

5 days

Mist. desiccans



7.5% Glucose serum 7.5% Glucose serum Mist. desiccans

w

m

List of species-cuntinued Pre-drying culture requirements Organism

Maintenance medium

Pre-drying medium

I

Time


A

T,"C

Gaseous

\

Remarks

-

Nutrient agar Same XCorynebacterium manihot Nutrient agar Same 'Corynebacterium mediolanum Nutrient agar Same 'Corynebacterium michiganense Blood agar Same Corynebacterium minutissimum Blood agar Same Corynebacterium murium Blood agar Nutrient agar Corynebacterium mycetoides Blood agar Same Corynebacterium ovis Cooked meat Blood agar Corynebacterium Pamedium Nutrient agar Same 'Corynebacterium poinsettiae Corynebacterium Blood agar Same PYogenm 'Corynebacterium Nutrient agar Same rathayi Corynebacterium Blood agar Same renale Cwynebacterium rubrum-see also Mycobacterium rhodochrous

30

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

25

Air

72

Mist. desiccans

37

Air

2448

37

Air

24

37

Air

48

37

Air

24

37

Hz COz

30

Air

72

7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum Mist. desiccans

37

Air

24

25

Air

5 days


37

Air

24

7.5% Glucose

+

24-48

serum

Growth good on Nutrient agar

U 4

?

Corynebacterium rubrum *Corynebacterium tritici

Blood agar

Nutrient agar

37

Air

48

Nutrient agar

Same

25

Air

"Cytophaga aurantiaca

Dubos' salts solution

Same with agar

25

Air

Tytophaga ferrnentans Tytophaga hutchinsonii

Cytophaga agar No. 1 . Dubos' salts solution

25

Hz

Same with agar

25

Wytophaga johnsonii Tytophaga marinoflava *Cytophaga salmonicolor Tytophaga succinicans

Cytophaga medium No. 4. Sea water agar

Same Same

Cytophaga medium No. 3. Cytophaga agar No. 2.

*Dactylosporangium Oatmeal agar aurantiacum *Dactylosporangium Oatmeal agar thailandensis

Derrnatophilus congolensis-see *Derxia gummosa

5 days

Mist. desiccans

Air

5 days

Mist. desiccans

L-dry. Agar medium for pre-drying. May be grown in air as stab culture L-dry. Agar medium for pre-drying.

25

Air

72

Mist. desiccans

20

Air

72

Mist. desiccans

25

H2

25

Air

28

Air

28

Air

30

Air

Maybegrownin airasstab culture

Polysepta pedis

Nitrogen-free agar


Growth good on Nutrient agar

5 days


Same

72

Mist. desiccans

L-dry

!-

2 E

w

List of species-continued

00


Maintenance medium

*Desulfotomacdum nigrificans *Desulfotomaculum orientis *Desulfotomaculum ruminis

Postgate's medium Postgate's medium Postgate's medium

'Desulfovibrio africanus 'Desulfovibrio desulfuricans 'Desulfovibrio desulfuricans var. aestuarii +Desulfovibrio desulfuricans var. azotovorans *Desulfovibrio gigas

Postgate's medium Postgate's medium Postgate's medium +2.5% NaCl Postgate's medium

*Desulfovibrio salexigens *Desulfovibrio vulgaris

Postgate's medium Postgate's medium + 2.5% NaCl Postgate's medium

Pre-drying medium

r

A

T,"C

Gaseous

Time

Suspendingfluid for drying

Same

55

H2

72

Mist. desiccans

Same

30

H2

5 days

Mist. desiccans

Same

37

H2

72

Mist. dericcans

Same

30

H2

5 days

Mist. desiccans

Same

30

H2

5 days

Mist. desiccans

Same

30

H2

5 days

Mist. desiccans

Same

30

H2

5 days

Mist. desiccans

Same

30

H2

14 days

Mist. desiccans

Same

30

H2

5 days

Mist. desiccans

Same

30

H2

5 days

Mist. desiccans

Remarks

+Desulfovibrio vulgaris var. oxamicus

Postgate’s medium

Same

30

Hz

5 days

37

Air

24

Mist. desiccans

Diplococcus pneumoniae-see Streptococcus pneumoniae Edwardsiella tarda

Nutrient agar

+Elytrosporangium brasiliense

Same

Yeast malt agar

25

Air Air

24

Nutrient agar

Same

37

Nutrient agar

Same

37

Air

24

Nutrient agar

Same

37

Air

24

Nutrient agar Nutrient agar Nutrient agar

Same Same Same

30 30

Erysipelothrix inridiosa

Blood agar

Escherichia alkalescens Escherichia coli Escherichia dispar

Enterobacter aerogenes Enterobacter cloacae Enterobacter liquefaciens *Erwinia amylovora *Erwinia carotovora ‘Erwinia herbicola

Ferrobacillus-see

7.5% Glucose broth

7.5% Glucose broth

7.5% Glucose broth

7.5% Glucose broth

30

Air Air Air

72 72 72

Mist. desiccans Mist. desiccans Mist. desiccans

Same

37

Air

2448

7.5”/0Glucose serum

Nutrient agar

Same

37

Air

24

Nutrient agar

Same

37

Air

24

Nutrient agar

Same

37

Air

24

7.5% Glucose broth 7.5% Glucose broth 7.5% Glucose broth

Thiobacillus ferrooxydans


w

9

List of species-Continued Pre-drying culture requirements Organism *Flavobacterium acidificum *Flavobacterium aquatile *Flavobacterium capsulatum *Flavobacterium devorans *Flavobacterium halmephilum +Flavobacterium heparinum *Flavobacterium, marine Flavobacterium meningosepticum *Flavobacterium pectinovonun *Flavobacterium proteus *Flavobacterium resinovorum *Flavobacterium rhenanum

Maintenance medium

Pre-drying medium

I

A

T,"C

Gaseous

Time

,


Same

25

Air

48

Mist. desiccans

Sodium caseinate Same agar Nutrient agar Same

25

Air

72

Mist. desiccans

30

Air

72

Mist. desiccans

Nutrient agar

Same

25

Air

72

Mist. desiccans

Halophile medium Flavobacterium heparinum medium Sea water agar

Same

25

Air

7 days

Mist. desiccans

Same

25

Air

72

Mist. desiccans

Nutrient agar

Same

20

Air

72

Mist. desiccans

Nutrient agar

Same

37

Air

24

7.5% Glucose

Nutrient agar

Same

25

Air

72

serum Mist. desiccans

Nutrient agar

Same

25

Air

3-5 days

Mist. desiccans

Nutrient agar

Same

25

Air

5 days

Mist. desiccans

Nutrient agar

Same

25

Air

72

Mist. desiccans

Remarks

a-

3 9

n

P

*Flavobacterium suaveolens Flavobacterium typhiflavum Gaffkya-see

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

37

Air

24

7.5% Glucose serum

Pediococcus

Gemella haemolysans

Blood agar

Same

37

Air

24

7.5% Glucose serum

Haemophilus aegyptius Haemophilus aphrophilus Haemophilus canis

Chocolate blood agar Chocolate blood agar Chocolate blood agar Chocolate blood agar Chocolate blood agar Chocolate blood agar

Same

37

CO2

24-48

Same

37

24-48

Same

37

coz coz

Same

37

c02

24-48

Same

37

c02

24-48

Same

37

coz

2448

7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum

Chocolate blood agar Chocolate blood agar

Same

37

COa

24-48

Same

37

c02

24-48

Haemophilus gallinarum Haemophilus haemolyticus Haemophilus influenzae

Haemophilus parainfluenzae Haemophilus suis


24-48


3W Some cultures of this organism may be difficult to maintain by freeze-drying. Some cultures of this organism may be difficult to maintain by freeze-drying.

E

R

List of species-continued


Maintenance medium

Pre-drying medium

A

I

T,"C

Gaseous

Time


Remarks -

Haemophilus VagiMliS

Hafnia alvei

Casman's blood agar

Same

37

coz

48

7.5% Glucose serum

Nutrient agar

Same

37

Air

24

7.5% Glucose broth

37

Air

37

Air

37

Air

37

Air

*Halobacterium Cutirubnun #Halobacterium halobium *Halobacterium salinarium *Halobacterium trapanicum

Halophile medium Halophile medium Halophile medium Halophile medium

#Hydrogenomonas facilis 'Hydrogenomonas thermophilus

Nutrient agar

Same

25

Air

5 days

Mist. desiccans

Nutrient agar

Same

45

Air

5 days

Mist. desiccans

*Hyphomicrobium vulgare

Hyphomicrobium medium

Same

25

Air

7 days

Mist. desiccans

37

Air

24

7.5% Glucose broth

Jensenka cattimuria- see Mycobacterium rhodochrous

Klebsiella aerogenes Nutrient agar

Same

(II

Klebsiella edwardsii Nutrient agar var. atlantae Klebsiella edwardsii Nutrient agar var. edwardsii Klebsiella ozaenae Nutrient agar

Same

37

Air

24

7.5% Glucose

Same

37

Air

24

7.5% Glucose

Same

37

Air

24

broth broth

7.5% Glucose broth

37

24

Air

Klebsiella pneumoniae Klebsiella rhinoscleromatis

Nutrient agar

Kurthia zopfii

Nutrient agar

Same

30

Air

24-72

MRS medium

Same

37

Air

48

*Lactobacillus acidophilus Lactobacillus acidophilus

Same

7.5% Glucose broth

Nutrient agar

Same

37

24

Air

7.5% Glucose broth

75% Glucose serum Mist. desiccans


J

E Tomato juice agar

37

with or without

48

7.5% Glucose serum

c02

*Lactobacillus bifidus MRS medium Lactobacillus bifidus Cooked meat medium *Lactobacillus bifidus Gyorgy and Rose medium var. pennsylVaniCUS *Lactobacillus brevis MRS medium *Lactobacillus brevis MRS medium var. rudensis MRS medium *Lactobacillus buchneri

Y

Same Glucose agar or GalactoseIagar Same

37 37

H 2 H 2

37

Ha

72

Mist. desiccans 7.5 % Glucose serum Mist. desiccans

Same Same

30 30

Air Air

72 72


Same

37

Air

48

Mist. desiccans

+ C02

72 3-4 days

Also recovered oncaseinagar andinpurple milk

E! *

+

r

!2

Also recovered on Casein agar

ew


Maintenance medium

Pre-drying medium

I

A

T,"C

Gaseous

Time


*Lactobacillus bulgaricus 'Lactobacillus casei (and varieties) Lactobacillus casei

MRS medium

Same

37

c02

48

Mist. desiccans

MRS medium

Same

37

Air

48

Mist. desiccans

Tomato juice agar

37

coz

48

7.5% Glucose serum

*Lactobacillus cellobiosus *Lactobacillus delbrueckii *Lactobacillus fermenti 'Lactobacillus fructovorans 'Lactobacillus helveticus 'Lactobacillus hilgardii 'Lactobacillus jugurti 'Lactobacillus lactis *Lactobacillus leichmannii

MRS medium

Same

30

Air

72

Mist. desiccans

MRS medium

Same

37

Air

48

Mist. desiccans

MRS medium

Same

37

Air

48

Mist. desiccans

MRS medium

Same

30

Air

72

Mist. desiccans

MRS medium

Same

30

Air

72

Mist. desiccans

MRS medium

Same

30

Air

72

Mist. desiccans

MRS medium MRS medium MRS medium

Same

37 37 37

Air Air Air

48 48 48

Mist. desiccans Mist. desiccans Mist. desiccans

Same Same

Remarks

Also recovered

on Casein agar and in Purple milk.

2-4 days

7.5 % Glucose

37 30 30

Air Air

72 72


Air

*Lactobacillus parvus *Lactobacillus plantarum *Lactobacillus salivarius 'Lactobacillus viridescens

MRS medium MRS medium

Casein agar or Tomato juice agar Same Same

MRS medium

Same

37

Air

48

Mist. desiccans

MRS medium

Same

37

Air

48

Mist. desiccans

*Lampropedia hyalina

Acetate agar

25

Air

Leptotrichia buccalis Leptotrichia dentium

Cooked meat medium Blood agar

Blood agar

37

H2

3 days

7.5% Glucose

Same

37

Air

4 days

7.5% Glucose

'Leuconostoc dextranicum *Leuconostoc mesenteroides *Leuconostoc oenos

MRS medium

Same

25

Air

72

Mist. desiccans

MRS medium

Same

25

Air

72

Mist. desiccans

Leuconostoc oenos medium

Same

22

Air

4 days

Mist. desiccans

'Leucothrix mucor

Leucothrix mucor medium

Same

20

Air

5 days

Mist. desiccans

Same

37

Air

24

7.5% Glucose serum

Lactobacillus odontolyticus

serum

+ C02

CI

serum serum

Lineola Zonga--see Bacillus macroides Listeria monocytogenes

Blood agar


Also recovered in Purple milk

List of species-Continued Pre-drying culture requirements Organism Loefflerella mallei

Maintenance medium Blood agar


r

A

T,"C 37

Gaseous

Time

Air

24-48

,


7.5% Glucose serum

Remarks Y

L cd

3

LoeJikella pseudomallei-see Pseudomonas pseudomallei

p

Laphomonas-see Comamonas

?

m

Macrospora-see Microellobosporia *Metallogenium symbioticum

Manganous acetate agar

25

Air

'Methanobacillus omelianskii

Methanobacillus medium

30

H2

'Methanococcus vannielii

Methanococcus medium

30

H2

'Methanosarcina barkeri

Methanosarcina barkeri medium

30

Hz

'Methylococcus capsulatus

Methylococcus medium

37

50% Air 50% CH4

'Microbacterium flaw 'Microbacterium lacticum

Nutrient agar

Same

30

Air

5 days

Mist. desiccans

Nutrient agar

Same

30

Air

5 days

Mist. desiccans

*Microbacterium liquefaciens *Microbacterium thermosphactum

Nutrient agar

Same

30

Air

5 days

Mist. desiccans

Nutrient agar

Same

25

Air

5 days

Mist. desiccans

*Microbispora aerata *Microbispora bispora XMicrobispora rosea

Czapek's peptone Same agar Czapek's Same peptone agar Czapek's Same peptone agar

30

Air

7 days

Mist. desiccans

55

Air

7 days

Mist. desiccans

30

Air

14 days

Mist. desiccans

72

Mist. desiccans

48

Mist. desiccans

72

Mist. desiccans

72

Mist. desiccans

24-48

7.5% Glucose serum

Micrococcussee also Staphylococcus afermentans, Staph. lactis and Staph. roseus Same 30 Air Nutrient agar *Micrococcus denitrificans 30 Air Same Nutrient agar *Micrococcus glutamicus Same 20 Air Nutrient agar *Micrococcus halodenitrificans 6% NaCl Micrococcus lysodeikticus-see Staphylococcus afermentans *Micrococcus Halophile 37 Air morrhuae medium 30 Air Same Nutrient agar *Micrococcus radiodurans Same 37 Air Nutrient agar Micrococcus violagabriellae

+

*Microcyclus aquaticus

Nutrient agar

Same

25

Air

72

Mist. desiccans

*Microechinospora grisea

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans


c

t

&


Maintenance medium

Pre-drying medium

r

A

T,"C

Gaseous

Time


'Microellobosporia cinerea

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

'Microellobosporia flavea

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

'Micromonospora chalcea 'Micromonospora vulgaris *Micropolyspora faeni Moraxella lacunata

Glycerol asparagine agar Yeast malt agar

Same

30

Air

14 days

Mist. desiccans

Same

45

Air

7 days

Mist. desiccans

Yeast malt agar

Same

45

Air

5 days

Mist. desiccans

Blood agar

Same

37

Air

24

7.5% Glucose serum

Blood agar

Same

37

Air

24

Blood agar

Same

37

Air

24


Same

37

Air

4-5 days

7.5% Glucose serum

Same

37

Air

6-7 days

7.5% Glucose serum

Moraxella liquefaciens Moraxella nonliquefaciens

Morganella--see Proteus m o r g d Mycobacterium Dorset's egg acapulcensis medium Mycobacterium americae

Dorset's egg medium

Remarks

Also recovered on Blood agar and Glucoseagar Also recovered on Glucose agar

30 or 37

Air

2-7 days

30

Air

5-10 days

7.5% Glucose serum 7.5% Glucose

Dorset’s egg medium Dorset’s egg medium

Same or Glucose agar Same or Blood agar, or Nutrient agar

Dorset’s egg medium Nutrient agar

Glucose agar

30

Air

5-7 days 7.5% Glucose

Same

30

Air

7 days


Dorset’s egg medium

Glucose agar

30

Air

2-5 days

7.5% Glucose

Mycobacterium gordonae Mycobacterium johnei Mycobacterium kanSaSii Mycobacterium marinum

Dorset’s egg medium Mycobacterium johnei medium Dorset’s egg medium Dorset’s egg medium

Same

37

Air

10 days

Same

37

Air

5 weeks

Same

37

Air

10 days

Glucose agar

30

Air

5-7 days

Mycobacterium parafortuitum


Nutrient agar

37

Air

48

Mycobacterium aurum Mycobacterium balnei

Mycobacterium flavescens *Mycobacterium f l a w var. methanicum Mycobacterium fortuitum


serum

serum

7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum

Also recovered on Glucose agar. Growth usually best on Dorset egg and Blood agar

Growth on Nutrient agar as good as on Glucose agar

Also recovered on Blood agar and Nutrient agar Also recovered on Glucose agar


Maintenance medium

Mycobacterium peregrinum Mycobacterium phlei Mycobacterium rhodochrous Mycobacterium runyonii Mycobacterium smegmatis Mycobacterium termoresistible

Dorset’s egg medium Dorset’s egg medium Dorset’s egg medium Dorset’s egg medium Dorset’s egg medium Dorset’s egg medium

Mycobacterium terrae


Mycobacterium

Dorset’s egg medium Dorset’s egg medium Dorset’s egg medium

tomidae Mycobacterium tubercu1osis Mycobacterium UlCeranS

Pre-drying medium

A

I

T,”C

Gaseous

Time

,

37

Air

4 days

Glucose agar or Nutrient agar Glucose agar or Nutrient agar Glucose agar

30 or 37

Air

3-4 days

30

Air

4-5 days

37

Air

4 days

Glucose agar or Nutrient agar Glucose agar

30 or 37

Air

2-4 days

37

Air

4 days

Same

37

Air

7 days

Same

37

Air

7 days

Same

37

Air

Same

30

Air

Glucose agar


7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum

7.5% Glucose serum 5-7 weeks 7.5% Glucose serum 8 weeks 7-57!, Glucose serum

Remarks



Also recovered on Blood agar and Nutrient agar Also recovered on Glucose agar and Soil extract agar Also recovered on Glucose agar

Mycobacterium xenopei Mycococcus luteus

Dorset's egg medium Nutrient agar

Same

37

Air

4 weeks

Same

30

Air

48-72

Mycococcus ruber

hhtrient agar

Same

30

Air

48-72

'Mycoplana bullata *Mycoplana dimorpha Mycoplasma bovirhinis


Same Same

25 25

Air

72 72

Horse serum broth

Same

37

Air

Air

About 2-7days

7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum Mist. desiccans Mist. desiccans 7.5% Glucose serum

Mycoplasma fermentans

Horse serum broth

Same

37

Air

About 2-7 days

7.5% Glucose serum

Mycoplasma gallisepticum

Horseserum broth

Same

37

Air

About 2-7days

7.5% Glucose serum


Adequate growth in predrying medium checked by plating-out counts on Horse serum agar Adequate growth in predryingmedium checked by plating-out counts on Horse serum agar Adequate growth in predrying medium checked by plating-out counts on Horse serum agar

7

5

* 5m

52

rn r l h d

A

aa,

r l d

P

CA

A

ga,

n

44

g:

CA

m

P

r l d

S. P. LAPAGE, J. E. SHELTON AND T. G . MITCHELL

rn

x

rld

1.0

Bt-

4A

tm

b

tm

Q

9

rn

Mycoplasma pneumoniae

Mycoplasma salivarium

Mycoplasma suipneumoniae

*M~XOCOCCUS fulvus *M yxococcus

Horse serum broth

Same

37

Air

About 7.5 % Glucose 3-10 days serum

Horse serum broth

Same

37

Air

About 2-7 days

M. suipneumoniae broth

Same

Klebsiella agar Klebsiella agar

virescens *Myxococcus xanthus Casitone agar Neisseria animalis

Blood agar


Same

37

Air

About 2-7 days

7-5% Glucose serum

7.5% Glucose serum

Adequate growth in predrying medium checked by plating-out counts on Horse serum agar Adequate growth in predrying medium checked by plating-out counts onHorse serum agar Adequate growth in predryingmedium checked by plating-out ~~unts,which are incubated in 5% COa in Na on M. suipneumoniae agar

-_

I

s

*

2m

?i

Air

25 25

Air

25

Air

37

Air

24

7.5% Glucose serum wl

w

wl


P


Maintenance medium

Pre-drying medium

A

I

T,"C

Gaseous

Time


Neisseria canis

Blood agar

Same

37

Air

24

7.5% Glucose

Neisseria catarrhalis Neisseria caviae

Chocolate blood agar Blood agar

Same

37

Air

2448

7.5% Glucose

Same

37

Air

24

Neisseria cinerea

Blood agar

Same

37

Air

24

Neisseria cuniculi

Blood agar

Same

37

Air

24

Neisseria Blood agar denitriiicans Neisseria flavescens Chocolate blood agar Neisseria Chocolate blood gonorrhoeae agar

Same

37

Air

24

Same

37

coz

24

Same

37

c02

2448

Neisseria meningitidis Neisseria pharyngis

Same

37

c02

2448

Same

37

c02

24

Remarks

serum

Chocolate blood agar Chocolate blood agar

serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 75% Glucose serum 75% Glucose serum



Also recovered on Blood agar Some cultures of this organism may be difiicult to maintain by freeze-drying


rn

.Nitrobacter agilis

Nitrobacter agilis medium 'Nitrocystis oceanus Nitrosomonas europaea medium in sea water *Nitrosomonas Nitrosomonas europaea europaea medium 'Nocardia apis Glycerol asparagine agar Nocardia asteroides Dorset's egg medium Nocardia Dorset's egg blackwellii medium Nocardia brasiliensis Dorset's egg medium

'Nocardia calcarea Nocardia caviae 'Nocardia cellulans 'Nocardia coeliaca Nocardia congolensis Nocardia cuniculi

Nutrient agar Dorset's egg medium Nutrient agar Glycerol asparagine agar Dorset's egg medium Dorset's egg medium


Same Glucose agar or Nutrient agar Glucose agar Glucose agar

Same Glucose agar

25

Air

25

Air

25

Air

25

Air

7 days

Mist. desiccans

30 or 37

Air

24-48

30

Air

5 days

37

Air

5 days

7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum

25 30 or 37

Air Air

5 days 24-72

Same Same

25

25

Air Air

5 days 5 days

Glucose agar

30

Air

10 days

Glucose agar

30

Air

72

Mist. desiccans 7.5% Glucose serum Mist. desiccans Mist. desiccans



r

E Also recovered onBloodagar and Nutrient agar; Blood agar invariably best

E 4

r



cn cn

vl

List of species-Cmtinued

m

Pre-drying culture requirements Organism *Nocardia erythropolis Nocardia farcinica 'Nocardia globerula *Nocardia hydrocarbonoxydans 'Nocardia italica 'Nocardia lurida Nocardia madurae

Maintenance medium

Pre-drying medium

A

I

T,"C

Gaseous

Time


Nutrient agar

Same

25

Air

5 days

Mist. desiccans

Dorset's egg medium Nutrient agar Glycerol asparagine agar Glycerol asparagine agar Glycerol asparagine agar Dorset's egg medium

Glucose agar

30

Air

4 days

Same Same

25 25

Air

Air

5 days 5 days

7.5% Glucose serum Mist. desiccans Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

7 days

Mist. desiccans

Glucose agar

30

Air

10 days

7.5% Glucose serum

'Nocardia opaca Nocardia pelletieri

Nutrient agar Dorset's egg medium

Same Glucose agar

25 30

Air Air

5 days 10 days


*Nocardia petroleophila 'Nocardia restrictus 'Nocardia rubropertincta

Glycerol asparagine agar Nutrient agar Nutrient agar

Same

25

Air

7 days

Mist. desiccans

Same Same

25 25

Air Air

5 days 5 days


Remarks

cn cd I-

$ "2 ?

m

Also recovered on Blood agar and Nutrient agar; Blood agar often better Recovery on Blood agar often better

Glycerol asparagine agar Dorset's egg medium

Same

25

Air

5 days

Mist. desiccans

Glucose agar

37

Air

48

7.5% Glucose serum

Nutrient agar

Same

25

Air

5 days

Mist. desiccans

Same Glycerol asparagine agar Same *Nocardia uniformis Glycerol asparagine agar Obesumbacterium--see Flavobacterium proteus Pasteurella Blood agar Same haemolytica Pasteurella haemo- Blood agar Same lytica var. ureae

25

Air

7 days

Mist. desiccans

25

Air

5 days

Mist. desiccans

37

Air

24

37

Air

24


'Nocardia rugosa Nocardia salivae

'Nocardia salmonicolor +Nocardia satumea

CI

Pasteurella pestis

Blood agar

Same

37

Air

24

Pasteurella pseudotuberculosis Pasteurella septica

Blood agar

Same

37

Air

24

Blood agar

Same

37

Air

24

Pasteurella ureae-see Pasteurella haemolytica var. ureae Pasteurella X - s e e Yersinia enterocolitica Pediococcus-see also Aerococcus For explanation, see pp. 6-9.

Also recovered on Blood agar and Nutrient agar

7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum

Viability on maintenance media shortlived ; requires frequent subculturing

3

E 4

G

E

wl

00

List of species-Continued Pre-drying culture requirements Maintenance medium

Organism

Pre-drying medium

f

4

T,"C

Gaseous

Time

,


*Pediococcus acidilactici *Pediococcus cerevisiae *Pediococcus halophilus *Pediowccus parvulus *Pediococcus pentosaceus *Pediococcus soyae

MRSmedium

Same

25

Air

72

Mist. desiccans

MRSmedium

Same

25

Air

72

Mist. desiccans

MRSmedium 18% NaCl MRSmedium

Same

25

Air

72

Mist. desiccans

Same

25

Air

72

Mist. desiccans

MRS medium

Same

25

Air

72

Mist. desiccans

MRS medium

Same

25

Air

72

Mist. desiccans

*Pelodictyon clathratiforme

Pfennig's medium

20

H2 COz

*Peptococcus aerogenes *Peptococcus glycinophilus

Peptone yeast Same glutamate Tryptone glycine Same medium

37

H2

48

Mist. desiccans

37

H2

72

Mist. desiccans

20

Air

72

Mist. desiccans

+

+

Peptostreptococcus-see Anaerobic cocci Pfeiyerelh-see *Photobacterium fischeri

Loefflerella Sea water agar

Same

Remarks

VI

N

I. MEDIA TABLES

0

m

M

.S

M

e

'4

59

6 I \o

ti u

a

P I . '

.-6

9

a

e

5

M

45

N b

M

.?5

M


N b

N b

XPropionibacterium pituitosum

Krebs’ yeast lactate medium

Same

30

Air

72

Mist. desiccans

+Propionibacterium raffinosaceum


Same

30

Air

72

Mist. desiccans

XPropionibacterium rubrum


Same

30

Air

72

Mist. desiccans

30

Air

+Propionibacterium sanguineum


Same

XPropionibacterium shermanii


Same

30

Air

72

Mist. desiccans

+Propionibacterium technicum


Same

30

Air

72

Mist. desiccans


72

Mist. desiccans

0 2 tension should be reduced by using well-filledbottles of medium 0 2 tension should be reduced by using well-filledbottles of medium 0 2 tension should be reduced by using well-filledbottles of medium 0 2 tension should be reduced by using well-filledbottles of medium 0 2 tension should be reduced by using well-filled bottles of medium 0 2 tension should be reduced by using well-filledbottles of medium

. 8U c-(

I+

k.

2 cn

QI

List of s p e c i e s 2 o n t i n u e d

N


Maintenance medium

Pre-drying medium

A

I

T,"C

Gaseous

Time

,


*Propionibacterium thoenii

Krebs' yeast lactate medium

Same

30

Air

72

Mist. desiccans

'Propionibactenum wentii


Same

30

Air

72

Mist. desiccans

*Propionibacterium zeae


Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Nutrient agar

Same

30

Air

72

Mist. desiccans

Proteus inconstans

Nutrient agar

Same

37

Air

24

Proteus mirabilis

Nutrient agar

Same

37

Air

24

7.5% Glucose broth 7-57! Glucose broth

'Protaminobacter alboflavus *Protaminobacter ruber

Remarks 0 2 tension should be reduced by using well-fdledbottles of medium 0 2 tension should be reduced by using well-fdledbottles of medium 0 2 tension should be reduced by using well-filled bottles of medium

r-1 0

Proteus morganii

Nutrient agar

Same

37

Air

24

Air

24

7.5% Glucose broth

7.5% Glucose

Proteus rettgeri

Nutrient agar

Same

37

Proteus vulgaris

Nutrient agar

Same

37

Same

30

Air

48

Mist. desiccans

Same

37

Air

24

Mist. desiccans

Same

37

Air

24

Mist. desiccans

Providenciu-see Proteus inconstans 'Pseudomonas Nutrient agar acidovorans 'Pseudomonas Nutrient agar aeruginosa (and varieties) *Pseudomonas alcaligenes 'Pseudomonas aminovorans *Pseudomonas antimyceticum 'Pseudomonas arOtnatica *Pseudomonas arsenoxydans *Pseudomonas atlantica 'Pseudomonas aureofaciens +Pseudomonas azotocolligans

Nutrient agar

broth Air

24

7.5% Glucose broth

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Sea water agar

Same

20

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans


NCTC: SUSpending fluid 7.5% Glucose serum NCTC: SUSpending fluid 7.5% Glucose serum

c (

3 2-

Cl

%

E

List of species--cOntinued Pre-drying culture requirements Organism

Maintenance medium

Pre-drying medium

f

A

T,"C

Gaseous

Time

,


'Pseudomonas azotogensis 'Pseudomonas beijerinckii 'Pseudomonas boreopolis 'Pseudomonas calcoacetica 'Pseudomonas caryocyaneus *Pseudomonas chlororaphis

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar + 3% NaCl Nutrient agar

Same

30

Air

48

Mist. desiccans

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

25

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

*Pseudomonas cocovenenans *Pseudomonas convexa XPseudornonas cruciviae *Pseudomonas dehalogenans *Pseudomonas denitrificans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Remarks

NCTC: suspending fluid 7.5% Glucose serum

P

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

37

Air

24

7.5% Glucose

Nutrient agar

Same

30

Air

48


Nutrient agar

Same

30

Air

48

Mist. desiccans

Sea water agar

Same

20

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

m

Nutrient agar

Same

30

Air

48

Mist. desiccans

5

Nutrient agar

Same

30

Air

24

7-5 % Glucose serum

'Pseudomonas fragi 'Pseudomonas geniculata Pseudomonas graveolens


Same Same

30 25

Air Air

48 48


Nutrient agar

Same

30

Air

24

7.5 % Glucose serum

'Pseudomonas huttiensis 'Pseudomonas indigofera 'Pseudomonas indoloxidans

Nutrient agar

Same

25

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

'Pseudomonas desmolyticum 'Pseudomonas diminuta Pseudomonas diminuta 'Pseudomonas echinoides 'Pseudomonas eisenbergii 'Pseudomonas elongata *Pseudomonas extorquens 'Pseudomonas fluorescens Pseudomonas fluorescens


Recovery on Blood agar often better


2

2

i

List of Species-Contirmed Pie-drying culture requirements Organism

Maintenance medium

Pre-drying medium

Pseudomonas Nutrient agar Same iodinum 'Pseudomonas Nutrient agar Same lanceolata 'Pseudomonas Nutrientagar Same lemoignei 'Pseudomonas Nutrientagar Same lemonnieri PscudomoMs mahi-aee Loderella mallei Same Nutrient agar CPseudomonas maltophilia Same Sea water agar 'Pseudomonas, marine Same Nutrient agar 'Pseudomonas mephitics Pseudomonas 8Pseudomonas methanica methanica medium Same Methanol d m *Pseudomonas, methanol oxidisers medium Same Nutrient agar *Pseudomonas mucidolens Same Nutrient agar Pseudomonas mucidolens

A

I

T,"C

Gaseous

Time


37

Air

24

25

Air

48

75% Glucose serum Mist. desiccans

30

Air

48

Mist. desiccans

30

Air

48

Mist. desiccans

30

Air

48

Mist. desiccans

20

Air

48

Mist. desiccans

30

Air

48

Mist. desiccans

30

50% Air 50% CHI

30

Air

5 days

Mist. desiccans

30

Air

48

Mist. desiccans

30

Air

24-48

7.5% Glucose serum

Remarks


*Pseudomonas multivorans +Pseudomonas myxogenes 'Pseudomonas nigrifaciens 'Pseudomonas oleovorans 'Pseudomonas ovalis Pseudomonas ovalis +Pseudomonas oxaliticus *Pseudomonas pavonacea +Pseudomonas perolens +Pseudomonas pictorum +Pseudomonas pseudoalcaligenes 'Pseudomonas pseudomallei Pseudomonas pseudomallei +Pseudomonas punctatum 'Pseudomonas putida

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans


Same Same

30 30or37

Air Air

48 24


Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

37

Air

24

Mist. desiccans

Blood agar

Same

37

Air

24

Nutrient agar

Same

37

Air

48


Nutrient agar

Same

30

Air

48

Mist. desiccans


Recovery on Blood agar often better F


Pre-drying culture requirements Organism *Pseudomonas putrefaciens 'Pseudomonas rathonis 'Pseudomonas reptilivora 'Pseudomonas resinovorans 'Pseudomonas rhodos 'Pseudomonas riboflavina 'Pseudomonas saccharophila *Pseudomonas septica 'Pseudomonas striata *Pseudomonas stutzeri *Pseudomonas syncyanea Pseudomonas syncyanea

Maintenance medium

Pre-drying medium

f

A

T,"C

Gaseous

Time

,


Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

48

Mist. desiccans

Pseudomonas saccharophila medium Nutrient agar

Same

30

Air

48

Mist. desiccans

Same

30

Air

48

Mist. desiccans


Same Same

30 30

Air Air

48 48


Nutrient agar

Same

30

Air

48

Mist. desiccans

Nutrient agar

Same

30

Air

2448

7.5% Glucose serum

Remarks

? m


+Pseudomonas Nutrient agar synxantha +Pseudomoms Nutrient agar taetrolens +Pseudomonas Nutrient agar testosteroni 'Pseudomonas trifolii Nutrient agar +Pseudomoms Nutrient agar tumefaciens +Pseudonocardia Yeast malt agar thermophila Ramibacterium Cooked meat pseudoramosum medium Rettgerella-see Proteus rettgeri +Rhizobium Rhizobium japonicum medium 2 +Rhizobium Rhizobium leguminosarum medium 1 +Rhhbium meliloti Rhizobium medium 1 +Rhodomicrobium Nutrient agar vannielii +Rhodopseudomoms Yeast peptone capsulata broth +Rhodopseudomonas Yeast peptone gelatinosa broth +Rhodopseudomonas Yeast peptone PalUStriS broth


Same

30

Air

48

Mist. desiccans

Same

30

Air

48

Mist. desiccans

Same

30

Air

48

Mist. desiccans

Same Same

30 30

Air Air

48 48


Same

45

Air

5 days

Mist. desiccans

Blood agar

37

H2

25

Air

25

Air

25

Air

Same

30

Ha

10 days

Mist. desiccans

Incubate in light

Same

30

H 2

5 days

Mist. desiccans

Incubate in light

Same

30

Ha

5 days

Mist. desiccans

Incubate in light

Same

30

Hz

5 days

Mist. desiccans

Incubate in light

+ C02

48

7.5% Glucose serum


0


Maintenance medium

Pre-drying medium

L

r

T,"C

Gaseous

3

Time


Remarks

*Rhodopseudomonas spheroides *Rhodopseudomonas viridis

Yeast peptone broth Yeast malate medium

Same

30

H2

5 days

Mist. desiccans

Incubate in light

Same

30

€32

7 days

Mist. desiccans

Incubate in light

Whodospirillum molischianum %hodospirillum rubrum

Yeast peptone broth Yeast peptone broth

Same

30

H2

5 days

Mist. desiccans

Incubate in light

Same

30

Ha

7 days

Mist. desiccans

Incubate in light

37

Air

24

75% Glucose

Salmonella mizona-see Arizona arizonae Salmonella spp. Nutrient agar Same (Over 350 serotypes including Salmonella abortusovis, Salmonella paratyphi A, Salmonella pullorum, Salmonella sendai, Salmonella typhi, Salmonella typhisuis)

broth

*Saprospira grandis

Marine flexibacteria medium fsaprospira thermalis Freshwater flexi- Same bacteria medium fsarcina littoralis Halophile medium Sarcina lutea-see Staphylococcus afermentans *Sarcina marina Halophile medium fsarcina maxima Sarcina maxima medium Sarcina morrhuae Halophile medium fSarcina Halophile sreenivasani medium fsarcina ventriculi Sarcina ventriculi medium Serratia marcescens Nutrient agar Same

25

Air

37

Air

37

Air

37

Air

37

H2

37

Air

37

Air

37

Air

22,30or37

Air

72

Mist. desiccans

L-dry

24

7.5% Glucose

Growth temperature preferred may vary among different strains

4

serum

Nutrient agar

Same

37

Air

24

Shigella dysenteriae Nutrient agar

Same

37

Air

24

Shigella flexneri

Nutrient agar

Same

37

Air

24

Shigella sonnei

Nutrient agar

Same

37

Air

24

Shigella boydii


7.5% Glucose broth 7.5% Glucose broth 7 5 % Glucose broth 7.5% Glucose broth

G

bi

4 t3

Lit of species-Continued

Organism Simonsiellacrassa *sorangium sp.

Maintenance medium Blood agar

Pre-drying medium

Pre-drying culture requirements A

I

T,"C

Same

37

Gaseous Air

Time

48


Remarks

cd

7-57!, Glucose

E*

serum

Casitone agar

25

"g

Air

? m

Sphocrophorus-see Bacteroides *Sphaerotilus Peptone ferric discophorus citrate agar *Sphaerotilus natans Glycerol casitone agar *Spirillospora albida

Same


Spirill~m medium *Spirillum Marine atlanticum Spirillum medium *Spirillum Marine beijerinckii Spirillum medium *Spirillum delicaturn Spirillum medium 'Spirillum andus

rn

30

Air

25

Air

25

Air

25 25

5 days

Mist. desiccans

Air

5 days

Mist. desiccans

L-dry

Air

5 days

Mist. desiccans

L-dry

P Same Same

.

z

=i

r Same

25

Air

5 days

Mist. desiccans

L-dry

Same

25

Air

5 days

Mist. desiccans

L-dry

*spirillum giesbergeri *Spirillum graniferum *Spirillum itersonii var. itersonii *Spirillum itersonii var. vulgatum *Spirillum linum

spirillum medium spirillum medium spirillum medium spirillum medium Marine spirillum medium *Spirillum lunatum Marine Spirillum medium spirillum *SpirUum metamorphum medium spirillum *Spirillum peregrinum medium spirillum *spirillum medium polymorphum Spirillum 'Spirillum putridiconchylium medium spirillum *Spirillum serpens medium var. azotum Spirillum *Spirillum serpens medium var. serpens Spirillum *Spirillum medium sinuosum spirillum *Spirillum medium virginianum


Same

25

Air

5days

Mist.desiccans

L-dry

Same

25

Air

5 days

Mist. desiccans

L-dry

Same

25

Air

5 days

Mist. desiccans

L-dry

Same

25

Air

5 days

Mist. desiccans

L-dry

Same

25

Air

5 days

Mist. desiccans

L-dry

Same

25

Air

5 days

Mist. desiccans

L-dry

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans -1

w

List of species-Cmtinued Pre-drying culture requirements Organism 'Spirillum volutans .Sporocytophaga caulifonnis

+Sporocytophaga myxococcoides *Sporolactobacillus inulinus *Sporosarcina ureae Staphylococcus afermentans

Maintenance medium

Pre-drying medium

Spirillum volutans medium Same CYtOPhV medium No. 4 Same with agar Dubos' salts solution Same MRS medium

A

T,O

C

Gaseous


25

Air

25

Air

72

Mist. desiccans

25

Air

5 days

Mist. desiccans

37

Air

48

Mist. desiccans

30

Air

72

Mist. desiccans

Urea nutrient agar Nutrient agar

Same

Nutrient agar

Same

37

Nutrient agar

Same

30or37

Nutrient agar

Same

Same

>

Time

I-

48-72

7.5% Glucose serum

Air

24

7.5% Glucose

Air

2448

22,30or37 Air

24-72

22,30 or 37 Air

Remarks

Agar medium for pre-drying

Growth temperature preferred may vary among different Strains

Staphylococcus aureus Staphylococcus Iactis Staphylococcus roseus

serum 7.5% Glucose serum 7.5% Glucose serum

Growth temperature preferred m a y vary among different Strains

Air

24-48

7.5% Glucose serum

3 4 days

7.5 % Glucose

Staphylococcus saprophyticus

Nutrient agar

Same

30or37

Streptobacillus moniliformis

Chocolate blood agar

Same

37

Streptococcus Blood agar agalactiae Streptococcus bovis Blood agar

Same

37

Air

24-48

Same

37

Air

2448

Streptococcus durans Streptococcus dysgalactiae Streptococcus equi

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

2448

Blood agar

Same

37

Air

24-48

Streptococcus equinus Streptococcus equisimilis

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

2448


+

H2 CO2

serum

7 5 % Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum

Requires regular sub-cultuting to maintain viability on laboratory media. Some culhues of this organism may be difficult to maintain by

List of species--Contimced Pre-drying culture requirements

Organism

Maintenance medium

Pre-drying medium

A

f

T,"C

Gaseous

>

Time

Streptococcus faecalis streptococcus faecium Streptococcus haemolyticus Streptococcus hominis Streptococcus infrequens Streptococcus lactis

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

2448

Streptococcus liquefaciens

Blood agar

Same

37

Air

2448

StreptOCOCCUS

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

24-48

Blood agar

Same

37

Air

2U8

mastitidis streptococcus mutans streptocM.rxls pneurnoniae Streptccoccus PYOgeneS streptococcus salivarius


7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7-57!, Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum

Remarks

rn

Streptococcus Blood agar sanguis Streptococcus Blood agar thermophilus Streptococcus Blood agar uberis Streptococcus Blood agar zooepidemicus Streptococcus Blood agar zymogenes Streptococcus: un- Blood agar named members of groups A-H and K-T (inclusive)

Same

37

Air

24-48

Same

37

Air

24-48

Same

37

Air

2448

Same

37

Air

2448

Same

37

Air

2448

Same

37

Air

2448

7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum m

Same

25

Air

10 days

Mist. desiccans

Same

25

Air

14 days

Mist. desiccans

Same

25

Air

14 days

Mist. desiccans

Same

25

Air

10 days

Mist. desiccans

f

Same

25

Air

14 days

Mist. desiccans

f

Same

25 25

Air

10 days

Air

10 days


Yeast malt agar Streptomyces achromogenes (and varieties) Yeast malt agar Y3reptomyces albidoflavus Starch salts agar #Streptomyces albireticuli Yeast malt agar *Streptomyas albogriseolus Oatmeal agar Streptomyces albolongus Streptomyces albus Yeast malt agar Yeast malt agar Streptomyces ambofaciens


Same

4

List of species4mtimred

00

Pre-drying culture requirements Organism +Streptomyces antibioticus *Streptomyces argenteolus +Streptomyes armentosus 'Strep tomyces atroolivaceus +Streptomyces aurantiogriseus +Streptomyces aureofaciens +Streptomyes aureus +Streptomyces autotrophicus +Streptomycesbellus *Streptomycesbellus var. cirolerosus Wreptomyces blastmyceticus +Streptomyces bluensis Streptomyces buccalis

Maintenance medium

Pre-drying medium

L

I

T,"C

Gaseous

Time

,


Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Starch salts agar Same

25

Air

21 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar Glycerol asparagine agar Yeast malt agar Yeast malt agar

Same Same

25 25

Air Air

10 days 21 days


Same Same

25 25

Air Air

10 days 10 days


Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Glucose agar

Same

30

Air

2-7 days

7.5% Glucose serum

Remarks

Also recovered on Blood agar and Nutrient agar; Blood agar often better

v1

*Streptomyces cacaoi 'Streptomyces caelestis 'Streptomyes canus 'Strep tomyces capreolus *Streptomyces capuensis %reptomyes cavourensis 'Streptomyces cinereoruber *Streptomyces cinnamomeus 'Streptomyces coelicolor +Streptomyces COeruleOfUSCUS *Streptomyces coeruleorubidus *Streptomyces

Same Same

25 25

Air Air

14 days 10 days


Starch salts agar Same Starch salts agar Same

25 25

Air Air

14 days 14 days


Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Starch salts agar

Same

25

Air

10 days

Mist. desiccans

Starch salts agar Yeast malt a&

coerulescens

*Streptomyces WdUS

+Streptomyces corchorusii 'Streptomyces cremeus


00

0


Pre-drying culture requirements Organism +Streptomyces

Maintenance medium

Pre-drying medium

f

A

T,"C

Gaseous

Time

,



25

Air

14 days

Mist. desiccans


25

Air

14 days

Mist. desiccans

Same

25

Air

14 days

Mist. desiccans

Starch salts agar Same Starch salts agar Same

25 25

Air Air

14 days 14 days


Yeast malt agar Yeast malt agar

Same Same

25 25

Air Air

10 days 14 days


Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans


Same

25

Air

21 days

Mist. desiccans

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

diastaticus

%reptomyces distallicus *Streptomyces echinatus *Streptomyces endus %reptornyces erythraeus eStreptomycesfinlayi %treptomyces fluorescens 'Streptom yces fradiae +Streptomyces fragills +Streptomyces fulvissimus 'Strep tomyces fulvoviridis +Streptomyces furlongus +Streptomyces galilaeus

Yeast malt agar

Remarks

'Streptomyces gardneri * Streptomyces gelaticus 'Streptomyces glaucescens 5Streptomyces globisporus Streptomyces graminis

*Streptomyces griseocarneus +Streptomyces griseolus 'Streptomyces griseomycini 'Streptomyces griseoplanus *Streptomyces griseorubens +Streptomyces griseoviridis Streptomyces griseus

Same

25

Air

14 days

Mist. desiccans


25

Air

21 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Glucose agar

Same

30

Air

2-7 days

7.5% Glucose serum

Oatmeal agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Oatmeal agar

Same

25

Air

14 days

Mist. desiccans

Glucose agar

Same

30

Air

2-7 days

7.5% Glucose serum

Oatmeal agar


Also recovered on Blood agar and Nutrient agar; Blood agar often best


c (

List of species-Continued Pre-drying culture requirements Organism 'Streptomyces griseus (and varieties) *Streptomyas halstedii 'Streptomyces hawaiiensis CStreptomyces hiroshimensis Streptomyces hominis

Streptomyces hortonensis

'Streptomyces hygroscopicus 'Strep tomyces hygroscopicus var. angustomyceticus

Maintenance medium

Pre-drying medium

r

A

T,"C

Gaseous

Time


>

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Oatmeal agar

Same

25

Air

14 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Glucose agar

Same

30

Air

2-7 days

7-57!, Glucose serum

Glucose agar

Same

30

Air

2-7 days

7-57!, Glucose serum

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Remarks

Also recovered on Blood agar and Nutrient agar; Blood agar often best Also recovered on Blood agar and Nutrient agar; Blood agar often best

f S treptomyces hygroscopicus var. decoyicus +Streptomyces indigoferus *Streptomyces kanamyceticus 'Streptomyces kentuckensis 'Streptomyces krestomyceticus 'Streptomyces lavendulae *Streptomyces limpmannii *Streptomyces lincolnensis Streptomyces listeri

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Glucose agar

Same

30

Air

2-7 days

7.5% Glucose serum

CStreptomyces longispororuber CStreptomyces longisporus +Streptomyces lusitanus

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans



*

4 W

li

00

w

List of species-continued Pre-drying culture requirements Organism 'Streptomyces lusitanus var. tetracyclini 'Streptomyces luteoverticillatus 'Streptomyces macrosporeus 'Streptomyces matensis 'Streptomyces mediocidicus 'Streptomyces mediterranei 'Streptomyces melanogenus 'Streptomyces misakiensis 'Streptomyces naraensis 'Streptomyces netropsis 'Streptomyces nigrescens 'Streptomyces nitrosporeus

Maintenance medium

Pre-drying medium

r

A

T,"C

Gaseous

Time

,


Same

25

Air

10 days

Mist. desiccans

Glycerol Same asparagine agar Starch salts agar Same

25

Air

21 days

Mist. desiccans

25

Air

14 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans


Same

25

Air

14 days

Mist. desiccans

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Oatmeal agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Starch salts agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Remarks

'Streptomyces niveus 'Streptomyces nogalater 'Streptomyces noursei 'Streptomyces olivaceus 'Streptomyces olivovertici11atus 'Streptomyces Pa'Streptomyces parvus 'Streptomyces paucisporogenes Streptomyces pelletieri

*Streptomyces peUcetius *Streptomyces phaeochromogenes *Streptomyces phaeoverticillatus *Streptomyas phaeoviridis *Streptomyces pilosus *Streptomycee platensis


Same Same

25 25

Air Air

14 days 14 days


Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Oatmeal agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans


Same Same

25 25

Air

Air

10 days 10 days


Glucose agar

Same

30

Air

2-7 days

7.5% Glucose serum

Starch salts agar

Same

25

Air

14 days

Mist. desiccans


25

Air

14 days

Mist. desiccans


25

Air

14 days

Mist. desiccans

Yeastmaltagar

Same

25

Air

14 days

Mist. desiccans

Starch salts agar Yeastmaltagar

Same Same

25 25

Air Air

14 days 14 days

Mist. deaiccans Mist. desiccans



E 5

00

List of species-Cmtirmed

0.

Pre-drying culture requirements Organism *Streptomyces pluricolorescens +Streptomyces polychromogenes *Streptomyces prasinopilosus Streptomyces pseudogriseolus +Streptomyces Purpl===? 'Streptomyces rectus +Streptomyces resistomycificus Y3treptomyces rimosus Y3treptomyces roseochromogenes *Streptomyces roseoluteus 'Streptomyces roseoverticillatus

Maintenance medium

Pre-drying medium

r

A

T,"C

Gaseous

Time

, Suspendingfluid for drying

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Oatmeal agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Starch salts agar

Same

50

Air

5 days

Mist. desiccans

Oatmeal agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Remarks

P

Streptomyces somaliensis

'Streptomyces sparsogenes 'Streptomyces spectabilis 'Streptomyces spheroides 'Strep tomyces spiroverticillatus 'Streptomyces tendae 'Strep tomyces therrnonitrificans +Streptomyces thermoviolaceus subsp. apingens Yheptomyces thermoviolaceus subsp. thermoviolaceus 'Streptomyces thermovulgaris +Streptomyces toxytricini Streptomyces upcottii

Glucose agar

Same

30

Air

2-7 days

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans

Yeast malt agar

Same

25

Air

10 days

Mist. desiccans


Same Same

25 50

Air Air

10 days 5 days


Yeast malt agar

Same

50

Air

5 days

Mist. desiccans

Yeast malt agar

Same

50

Air

5 days

Mist. desiccans

Yeast malt agar

Same

50

Air

5 days

Mist. desiccans

Yeast malt agar

Same

25

Air

14 days

Mist. desiccans

Blood agar

Same

30

Air

3-5 days

75% Glucose serum


7.5% Glucose serum


3m

Fl

Also recovered on Glucose agar and Nutrient agar

00

List of species-Cmtinued

oc


Maintenance medium

+Streptomyces Yeast malt agar varsoviensis +Streptomyces Yeast malt agar venemelae +Streptomyces Starch salts agar violaceoruber +Streptomyces Yeast malt agar Viridans *Streptomyces Glycerol viridifaciens asparaghe agar *Streptomyces Yeast malt agar viridochtomogenes +Streptomyces Starch salts agar xantholiticus *Streptomyces Glycerol zaomyceticus asparagine agar *Streptosporangium indianesis *Streptosporangium rubrum

Czapek's peptone agar Czapek's peptone agar

mennomonospora awata


Pre-drying medium

Suspendingfluid 7-

T,"C

Gaseous

Time

for drying

Same

25

Air

14 days

Mist. desiccans

Same

25

Air

10 days

Mist. desiccans

Same

25

Air

10 days

Mist. desiccans

Same

25

Air

10 days

Mist. desiccans

Same

25

Air

14 days

Mist. desiccans

Same

25

Air

14 days

Mist. desiccans

Same

25

Air

7 days

Mist. desiccans

Same

25

Air

21 days

Mist. desiccans

Same

25

Air

7 days

Mist. desiccans

Same

25

Air

14 days

Mist. desiccans

Same

50

Air

5 days

Mist. desiccans

Remarks

1

P

+

Vhermomonospora viridis *Thiobacillus concretivorus

Nutrient agar

Same

45

Air

7 days

Mist. desiccans

Sulphur medium

Same

25

Air

7 days

Mist. desiccans

*Thiobacillus denitrificans

Thiobacillus denitrificans medium Ferrooxydans medium Sulphur medium

Same

30

H2

5 days

Mist. desiccans

Sulphur medium Same

25

Air

7 days

Mist. desiccans

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

30

Air

48

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

Same

25

Air

7 days

Mist. desiccans

Same

25

Air

5 days

Mist. desiccans

25

H2 C02

Thiobacillus ferrooxydans Thiobacillus intermedius Thiobacillus neapolitanus Thiobacillus novellus *Thiobacillus perometabolis Thiobacillus thioCyanOXidanS *Thiobacillus thiooxidans

*Thiobacillus thioparus Thiococcus

Thiosulphate agar No. 1. Thiosulphate agar No. 1. Thiobacillus perometabolis medium Thiosulphate agar No. 1. Sulphur medium Thiosulphate agar No. 1. Pfennig's medium

For explanation,see pp. 6-9.

+

Resuscitate on Thiosulphate agar No. 2.

Resuscitate on Thiosulphate agar No. 2.

Resuscitate on Thiosulp hate agar No. 2.

Incubate in light

-

List of species-Continued Pre-drying culture requirements Organism Ybiopedia rosea 'Thiospirillum jenense 'ThiOthriX 'Treponema zuelzerae 'Veillonella

Maintenance medium

Pre-drying medium

Same

I

A

T,"C

Gaseous

30 20

€32

10

Air

30

H2

37

H2

V 17 medium

37

H2

V 17 medium

37

€32

V 17 medium

37

H2

V 17 medium

37

Ha

V 17 medium

37

H2

CCTmedium Pfennig's medium ThiOthriX medium Treponema zuelzerae medium V 17 medium

7 days

+

Ha C02

alcalescens subsp. alcalescens

*Veillonella alcaleOCenS

subsp. criceti Veillonella alcalescens subsp. dispar *Veillonella alcalescens subsp. ratti Veillonella parvula subsp. atypica *Veillonella parvula subsp. parvula

Time

,


Mist. desiccans

Remarks Incubate in light Incubate in light

*Veillonella parvula V 17 medium subsp. rodentium Vibrio alcaligenes Vibrio anguillarum Vibrio bubulus Vibrio choleraeasiaticae Vibro eltor Vibrio fetus

37

Ha

Blood agar

Same

37

Air

Sea water agar Cooked meat medium Blood agar

Same Blood agar

20 37

Air H2+ CO2

Same

37

Air

24

Blood agar

Same

37

Air

24

Cooked meat medium

Blood agar

37

Ha+ COz

Vibrio ichthyodermis Sea water agar Vibrio marinus Yeast peptone broth in sea water Vibrio Blood agar metchnikovii Vibrio paraSea water agar haemolyticua Vibrio paraMarine agar haemolyticus


24 72 2 4 days

2-4 days

Same Same

20 15

Air Air

72

Same

37

Air

24

Same

20

Air

72

Same

22

Air

24

72

7.5% Glucose serum Mist. desiccans 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum 7.5% Glucose serum

Also recovered on Chocolate blood agar. Some cultures of thisorganism may be difEcult to maintain by freeze-drying.

Mist. desiccans Mist.desiccans 7.5% Glucose serum Mist. desiccans

7.5% Glucose serum

Recovery on Blood agar as good

F

E

F

2


Pre-drying culture requirements Maintenance medium

Organism ~

~~

Pre-drying medium

A

f

T,"C

Gaseous


Remarks

~

~

24

7.5 % Glucose serum

Air

24

7.5% Glucose serum

Air

24

7.5% Glucose serum

37

Air

LVitreoscilla 25 Acetate agar -aW Micmbispora Yersinia Blood agar Blood agar or 37 Nutrient agar entermlitica Yersiniiapcstis-see Pasteurella peatis Yersiniop s d t u b e r d h - e e Pasteurella pseudotuberculosis Zoogloea ramigera Nutrient agar Same 30

Air

Vibrio proteus

Time

Blood agar

ZopfiuS--see Kurthia zopfi 'Zymobacterium Zymobacterium oroticum medium *Zymomonas Zymomonas anaerobia medium *Zymomonas Zymomonas anaerobia vat. medium pomaceae +Zymomonas Zymomonas mobilis medium


Nutrient agar

Same

37

H2

5 days

Mist. desiccans

Same

30

H2

72

Mist. desiccans

Same

30

H2

72

Mist. desiccans

Same

30

72

Mist. desiccans

Recoveryon Blood agar better than on Nutrient agar

93

I. MEDIA TABLES

V. FORMULAE OF MEDIAt A. Growth media 1. Acetate agar (Pringsheim, 1950) Yeast extract Peptone Sodium acetate Agar powder Distilled water

5g 5g

0.1 g 15 g 1 litre

Dissolve all the ingredients except the agar in the water and adjust the p H to

74-7.6. Add the agar to the medium, steam the medium and distribute the complete medium in bottles or tubes and autoclave at 15 psi for 15 min.

2. Alcaligenes tolerans agar (Abd-el-Malek and Gibson, 1952) Peptone Meat extract Ammonium lactate Ferric citrate Agar powder Distilled water

5g 3g 3g 0.2 g 10 €! 1 litre

Dissolve the ferric citrate in a small part of the water and add to complete the medium after both it and the remainder have been sterilized. Dissolve the peptone, meat extract and lactate in the remainder of the water and adjust the pH to 7.0.Add the agar and steam the medium. Sterilize the two parts of the medium separately by steaming for 20 min on three successive days. Mix the parts and distribute aseptically. 3. Bdellovibrio medium (Stolp and Starr, 1963) Basal medium Yeast extract 3g Peptone 0.6 g 19 g Agar powder Distilled water 1 litre Overlay medium As basal medium but only 6 g of agar. For both media, dissolve the yeast extract and peptone in the water and adjust the pH to 7.2. Add the agar and steam the medium. Dispense into bottles and sterilize at 15 psi for 15 min in the autoclave. To use, melt a suitable quantity of the basal medium and pour plates. Grow cultures of the appropriate host organism on suitable nutrient media and harvest the cells. Melt a bottle of Overlay medium and after cooling add the inoculum of host cells to it. Use this to prepare a top layer on the plates which are then ready for inoculation with the Bdellm'brio.

t

For index of manufacturers, see Section VI, p. 131.

94


4. BeggMtoa medium (Burton and Morita, 1964)

Yeast extract CaCla Sodium acetate Agar powder Tap water Catalase

28 0.1 g 0.5 g

28 1 litre 10 Sigma unitslml medium (1 Sigma unit decomposes 1 pmole of HaOa/min at pH 7.0, 25°C) Dissolve the yeast extract, CaCla and sodium acetate in the water and adjust the pH to approximately 7.0.Add the agar and steam the medium. After distribution into bottles, tubes or flasks, autoclave at 15 psi for 15 min. Add the catalase aseptically to the medium when it is required.

5 . Bloodagar 250 ml Nutrient agar (medium No. 68) Sterile horse blood (Oxoid, Code No. SRSO.), defibrinated, no preservative 12.5 ml Autoclave nutrient agar at 10 psi for 10 min to melt and allow to cool. When approximately 50°-55"C, add the blood aseptically and mix well before distributing in sterile tubes or plates. Note Layered blood agar plates are routinely used. These are prepared by pouring a thin layer of peptone saline agar (medium No. 74) (approximately 10-15 mi) into the Petri dish and allowing this to set before pouring the blood agar (approx. 15 ml) on top. When indicated, glucose is added to the above medium at a concentration of 1%.

6 . 4% Blood agar The constituents and method of preparation follow the same formula as blood agar (medium No. 5), but the agar is incorporated at 4 times the concentration there stated. 7 . Bordet-Gengou agar (modijiedf r m Bordet and Gengou, 1906) Peptone (Evans) 10 8 NaCl 5g 10 ml Glycerol (BDH) Soluble starch (BDH) 2.5 g Tapwater 1 litre Standard Davis agar (NewZealand) 11 8 500 ml Sterile horse blood (Oxoid) Mix the starch with a few millilitres of water to produce a smooth paste. The remaining water is heated and the peptone, NaCl and glycerol are dissolved in it; the starch suspension is then added. Adjust the pH to 7.5. Add the agar and

I. MEDIA TABLES

95

dissolve by autoclaving at 10 psi for 10 min, check the pH and adjust to pH 7.5 if necessary. While still hot, filter through 2-3 thicknesses of absorbent surgical gauze and distribute in 200 ml amounts in screw-capped bottles. Sterilize by autoclaving at 10 psi for 20 min. Warm 100 ml of sterile horse blood to about 45°C and add aseptically to 200 ml of previously melted base, cooled to between 50"-55"C. Mix thoroughly and distribute aseptically into sterile test-tubes or Petri dishes. 8 . Butyribacterium madium (King and Rettger, 1942) Tryptone 20 g Beef extract 10 g Glucose 5g NaaHPOa. 12Ha0 48 Cysteine HCl 0.5 g Beef infusion (or equivalent of dehydrated medium) 500 ml 15 g Agar powder ?Distilled water 500 ml ? If dehydrated meat infusion is used rather than 5 0 0 ml of beef infusion, then 1 litre of distilled water will be required. Mix all the ingredients except the agar with, and dissolve in, the water. Add the agar powder and steam the medium. Dispense into tubes or bottles and autoclave at 15 psi for 15 min. It is preferable to use the medium freshly prepared.

(NCTCRecords) Skim milk powder (Oxoid, Code No. L31) 50 g 9. Casein agar

Standard Davis agar (New Zealand) 25 g Distilled water 1- 5 litres Prepare 10% Skim milk solution by dissolving the Skim milk powder carefully in 500 ml of distilled water. Distribute the solution in 50 ml amounts in screw-capped bottles, and sterilize by autoclaving at 15 psi for 5 min. Prepare 2.5% distilled water agar by dissolving the agar in 1 litre of distilled water by autoclaving at 10 psi for 10 min. After distribution in 100 ml amounts in 150 ml screw-capped bottles, sterilize by autoclaving at 20 psi for 20 min. For use, 100 ml of distilled water agar is melted by autoclaving at 10 psi for lOmin, and to this is added aseptically 5 O m l of the 10% Skim milk solution. After thorough mixing, the medium is poured aseptically into sterile Petri dishes or test tubes. 10. Casitone agar (H. Reichenbach, personal commumcation)

Casitone (Difco) 1 g Meat extract I g Glucose I g Agar powder 12 g Distilled water 1 litre Dissolve the glucose in a little of the water and autoclave separately.

96


Dissolve the meat extract and casitone in the remaining water and adjust the pH to 7.2. Add the agar and steam to dissolve. Autoclave the medium at 15 psi for 15 min and add the sterile glucose. Pour the medium into Petri dishes or into sterile bottles for slopes. 11. Casman's blood agar (modifiedfrom Casman, 1947) Difco proteose peptone 200 g Bacto tryptose 200 g Bacto beef extract 60 g 100 g NaCl Nicotinamide I g p-Aminobenzoic acid I g Brown and Polson cornflour 20 g . Soluble starch 20 g "Ionagar" No. 2 (Oxoid) 280 g 20 litres Distilled water Defibrinated human blood 1 litre Blend the cornflour and starch to a paste with a little of the water. Heat the remainder of the water to boiling and add it to the paste, stirring continually until the mixture clarifies. Return this suspension to the vessel containing the rest of the boiling water, mix well, and add the remainder of the constituents with the exception of the blood. Dissolve the agar by boiling or by autoclaving. After the addition of 20 ml of ~ O M N ~ O check H , the pH to 7.5. Filter the medium through gauze and distribute it. Sterilization is by autoclaving at 10 psi for 20 min. When the medium has cooled to between 5Oo-55"C, add the blood aseptically. 12. Caulobacter medium (Poindexter, 1964) Peptone 2g Yeast extract I g 0.2 g MgSOi. 7He0 Riboflavin PI3 10 g Agar powder Distilled water 1 litre Dissolve all the ingredients except the agar in the water and adjust the pH to 7.0. Add the agar and dissolve by steaming. Distribute as required and sterilize by autoclaving at 15 psi for 15 min. Note This medium is also prepared, where indicated, with 2.5% NaC1. 13. CCTmedium Trace elements solution FeCls. 6HzO 2.7 g HsBOs 0.1 g ZnSO4.7HaO 0.1 g 0.05 g Co(N0s)a.6Ha0 Cusos. 5Ha0 0-005 g MnCla. 6Ha0 0.005 g 1 litre Distilled water N.B. A precipitate of iron oxide forms, but does not appear to interfere with the success of the medium.

I. MEDIA TABLES

97

Basal medium KHzPOi NHiC1 MgCl2.6HzO NaCl Distilled water Trace elements solution

Sodium bicarbonate 10% w/v in distilled water.

Sodium sulphide (Na2S.9HzO) 10% w/v in distilled water.

Sodium thiosulphate (NazS203.5HaO) 10% w/v in distilled water

Sodium malate 10% w/v in distilled water. Autoclave all solutions separately at 15 psi for 15 min. Chromatium sp. (small-celled, such as strain D) For every 10 ml of freshly boiled basal medium add aseptically 0.2 ml of bicarbonate, 0.02 ml of sulphide, 0.1 ml of thiosulphate and 0.1 ml of malate. Thiopedia Follow same procedure as for Chromatiurn. Chlorobium limicola For every 10 ml of freshly boiled basal medium add aseptically: 0.2 ml of bicarbonate and 0.1 ml of sulphide. Adjust the pH of the complete medium to 7.0-7.2 with sterile phosphoric acid. Chlorobium thiosulfatophilum For every 10 ml of freshly boiled basal medium add aseptically: 0.2 ml of bicarbonate, 0.02 ml of sulphide and 0.1 ml of thiosulphate. Adjust the pH of the complete medium to 7.0-7.2 with sterile phosphoric acid. Certain strains of Chlorobium are known to require vitamin B12 or related compounds; for these it is necessary to add the vitamin to the growth medium. If an agar medium is required, e.g., for stab cultures or purification procedures, 1% w/v “Ionagar” No. 2 may be added to the basal medium. 14. Charcoal blood agar Charcoal agar (Oxoid) 13.8 g Distilled water 270 ml Sterile horse blood (Oxoid) 30 ml Soak the charcoal agar in the water for 15 min, and then autoclave at 15 psi for 15 min. Add the blood aseptically to the medium when the latter has cooled to approximately 50°C, and mix the medium gently before dispensing aseptically into sterile test tubes or Petri dishes. 5

98


15. Chloropseudomonas medium (Bose, 1963) Versenol iron solution 59 g in 500 ml water EDTA FeS04.7HaO 24.9 g Dilute to 1 litre with distilled water, and leave to aerate overnight. Trace elements

FeCls. 6HaO NazB407. lOHaO ZnSO4.7Hao COSoi. 7Ha0 CuCln. 2Ha0 MnSO4.4HzO Versenol iron solution Distilled water

1.6 g 0.88 g 0.44 g

0.24 g 0.0135 g 0.0165 g 110 ml to 1 litre

Double strength salts solution

WBPOI NHiCl MgCla. 6HaO NaCl CaCln Trace elements solution Distilled water

2g 2g

10 8 4og

0.08 g

2nd 998 ml

Final medium Double-strength salts solution 500 ml Distilled water 450 ml NaaS. 9He0, 10% w/v in distilled water 2 ml NaHCOs, 10% w/v in distilled water 40 ml FeS04.7Ha0,0*05% w/v in 0.3 M HCl 5 ml 3ml Ethanol, 70% v/v in distilled water Mix together the double-salts solution and the distilled water of the final medium and autoclave them at 15 psi for 15 min. Autoclave the NaaS, NaHCOs and FeS04 solutions in separate containers at 15 psi for 15 min. Filter-sterilize the ethanol. After sterilization, mix the ingredients aseptically and adjust the pH to 7.3 with concentrated HC1. 16. Chocolate blood agar Place tubes or plates containing the blood agar medium (medium No. 5) in a 60°C anhydric incubator for 14-2 h, until the medium has attained a uniform characteristic “chocolate” colour. 17. Clostridium aceticum medium (El-Ghazzawi, 1967) A. Fructose 10 g Distilled water 200 ml B. NaHCOs 20 i3 Distilled water 200 ml 1 ml C. Vitamin BIZ(2 mg/lOO ml distilled water)

99

I. MEDIA TABLES

D. Peptone KzHPOi Co(N0a)a Distilled water E. Vitamin solution F. Heavy metal solution

10 l3 10 g

19.7 mg 600 ml 10 ml 25 ml

Vitamin solution 0.1 mg 1 mg 0.5 mg 0.5 mg 0.25 mg 2.5 mg 100 ml

Biotin Nicotinic acid Thiamin p-Aminobenzoic acid Pantothenic acid Pyridoxin Distilled water

Heavy metal solution EDTA FeS04.7HzO ZnSO4. 7H20 MnCla .4Hz0 HaBOa CuCla. 2He0 NiCla .6HaO NaaMo04.2H20 Distilled water

1.5 g 0.2 g 0.1 g 0.02 g 0.03 g 0.001 g 0.002 g 0.003 g 1 litre

Dissolve the EDTA in the water first. Prepare the vitamin Bla solution (C), vitamin solution (E) and heavy metal solution (F) separately and sterilize by filtration. Dissolve the fructose in the appropriate amount of water and sterilize by autoclaving at 15 psi for 15 min (A). Dissolvethe bicarbonate (B) in the water and sterilizethe solution by filtration under positive pressure of COa. Prepare the medium (D) and sterilize by autoclaving at 15 psi for 15 min. Mix the sterile solutions A, B, D, E and F, add 1 ml of solution C and mix again. Adjust the pH aseptically to 8.0and distribute the medium into sterile tubes or bottles. 18. Clostridium chauvoei medium (Batty and Walker, 1965) Nutrient agar (medium No. 68b with 1.8% Standard Davis agar) 75 ml Glucose, Anhydrous D (+), 50% aqueous solution 2ml 3 ml Liver extract (Bacto) Sterile sheep blood (“Wellcome” Brand), defibrinated, no preservative 5 ml Prepare stock nutrient agar, but with the agar incorporated t o give a final concentration of 1.8%) and with the inclusion of the liver extract. Distribute

100


the medium in 75 ml amounts in screw-capped bottles, and sterilize by autoclaving at 15 psi for 15 min. Sterilize the solution of glucose, 50% w/v in distilled water by sintered-glass filtration, and add 2 ml aseptically to the molten cooled nutrient agar. Also add 5 ml of sterile sheep blood to this molten medium when the temperature is at approximately 50°C.After thorough mixing, dispense the medium aseptically into sterile tubes or Petri dishes. 19. Clostridium kluyveri medium (Stadtman and Barker, 1949) Part 1 Ethanol 20 ml Potassium acetate 4.3 g 3.5 ml M Phosphate buffer, pH 7.1 Acetic acid (glacial) 2.5 ml 0.5 g (NH4)zS04 MgS04.7HzO 0-2g CaS04.2HzO 10 mg MnS04.4HzO 2.5 mg NazMoOe .2H20 2.5 mg Biotin 10 Pg 1 mg p-Aminobenzoic acid Distilled water 900 ml

Part 2 5.9 g

KzCOs

100 ml

Distilled water

Part 3 NazS .9H& Distilled water

1 g

10 ml

Part 4 0.1 g FeS04.7HzO HzS04 0.1N 10 ml Dissolve the solid ingredients of each part of the medium in the respective liquids. Sterilize each part in separate containers by autoclaving at 15 psi for 15 min. Add Part 2 to Part 1 aseptically, then add 2 ml of Part 3 and 0.5 ml of Part 4. Mix well and check the pH; adjust if necessary to approximately 7.0. Distribute aseptically.

20. Clostridium sticklandii medium (Stadtman and White, 1954) L-Arginine HC1 1.5 g L-Lysine HC1 1.5 g Yeast extract 2 g NHG1 0.5 g CaClz .2H20 0.01 g MgS04.7Hz0 0.2 g 0.01 g FeS04.7HzO

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I. MEDIA TABLES

MnS04.4Hz0 NazMoOa. 2H20 K2HPO4-KH2P04,0.04

M,

pH 7.5

0.001 g 0.001 g 1 litre

Dissolve the ingredients in the phosphate buffer, distribute the medium in bottles or tubes and sterilize by autoclaving at 15 psi for 15 min.

21. Clostridium tetanomorphum medium (modifiedfrom Barker et al., 1959) Yeast extract Monosodium glutamate MgS04.7HzO FeS04.7HzO MnS04.4H20 NazMo04.2H20 CaCla .6H2O KH2P04-Na2HPO4,0.04

10 g

M,

pH 7.0

15 g 0.25 g 0.01 g 0.002 g 0.0025 g 0.022 g 1 litre

Dissolve the ingredients in the buffer solution and distribute the medium in bottles. Autoclave the medium at 15 psi for 15 min. T o avoid excessive precipitation, FeS04 may be omitted from the bulk of the medium before autoclaving, and added afterwards as 0.01 ml of a 10% w/v solution of FeS04 in 0.1 M sulphuric acid that has been autoclaved at 15 psi for 15 min.

22. Clostridium thermoaceticum medium (Lentz and Wood, 1955) Part 1 K2HP04 7 g KHaP04 5-5 g MgS04.7HzO 0.1 g 0.5 g (NH4M04 Tryptone 5 g Yeast extract 5 g Glucose 12.6 g Sodium thioglycollate 0.5 g Distilled water 900 ml Part 2

NaHC03 Distilled water

10.5 g 100 ml

Dissolve the ingredients of the two parts in the respective quantities of water and sterilize separately by autoclaving at 15 psi for 15 min. Mix the two parts aseptically and distribute as required. If the medium is to be stored, keep the parts separately and boil immediately before use ;it is also advisable to keep the thioglycollate separately.

102


23. Clostridium thermosaccharolytimm medium (Phkl and Ordal, 1967) Part 1 Peptone 5 g Yeast extract 5 g 0.1 g CaCla. 2Ha0 (NHdaSOr 1 g MgSOlr 0.1 g 0.1 g Mnso4.4Ha0 ZnSO4. 7Ha0 0.005 g CuS04.5HaO 0.005 g NaaMoOlr .2Hz0 0.001 g o.oO05 g FeS04.7Hzo Distilled water 900 ml Part 2 L-Arabinose 5% wlv in distilled water Dissolve the ingredients of Part 1 in the water and adjust the pH to 6.8. Autoclave the medium at 15 psi for 15 min. Filter-sterilize 100ml of 5% L-arabinose, Part 2, and add it aseptically to the autoclaved medium, Part 1; distribute the medium aseptically into sterile bottles. 24. Cooked meat medium (NCTC Records) Horse meat (fat-free, minced) 2 lb (900 g) NaCl 10 g Peptone (Evans) 20 g 2 ml Sodium thioglycollate (45% aqueous solution) Distilled water 2 litres Boil the meat in distilled water for 1 h, and then filter through 2-3 thicknesses of absorbent surgical gauze, pressing the meat dry. Add the remainder of the ingredients to the filtrate and mix them well, before adjusting the pH value of the solution to pH 8.4 with 10 M NaOH. Then bring the solution to the boil and filter it. The dry meat is distributed into screw-capped (1 oz McCartney) bottles to a depth of approximately 2.5 cm, and the broth is dispensed on top to a depth of approximately 5 cm. Sterilization is by autoclaving at 20 psi for 20 min, allowing slow reduction in pressure after sterilization. Note When indicated, glucose is added to the above medium at a concentration of 1%. 25. Cytophaga agar No. 1 (Bachmann, 1955) Yeast extract 10 6 NaCl 20 g KaHP04 0.2 g MgSOr 0.5 g NHiCl 1 g FeC13.6HzO 0.25 mg Distilled water 1 litre Dissolve the ingredients in the water and adjust the pH to 7.0-7.4. After distribution in bottles or tubes, autoclave the medium at 15 psi for 15 min.

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26. Cytophaga agar No. 2 (Anacker and Ordal, 1959) Tryptone 0.5 g Yeast extract 0.5 g Sodium acetate 0.2 g Beef extract 0.2 g Agar 9 g 1 litre Distilled water Dissolve all the ingredients except the agar in the water and adjust the pH to 7.2-7.4. Add the agar, steam the medium to dissolve, then distribute as required. Sterilized by autoclaving at 15 psi for 15 min. 27. Cytophaga medium No. 3 (Veldkamp, 1961) Nutrient broth, dehydrated (Difco) I g Yeast extract 1 g 1 ml Corn steep liquor 0.5 g NaHCOs NaCl 30 g Agar powder 10 g Distilled water 1 litre Mix all the ingredients except the agar with the water and adjust the pH to 7.2. The agar is added and the complete medium is steamed. Sterilization is by autoclaving at 15 psi for 15 min. 28. Cytophuga medium No. 4 ( T . Gibson, unpublished work) Yeast extract 2.5 g 10 8 Agar Distilled water 1 litre Dissolve the yeast extract in the water and adjust the pH to 7.2-7-4. Add the agar, steam to dissolve, distribute in bottles and autoclave at 15 psi for 15 min. 29. Czapek's peptone agar (Kane, 1966) Sucrose 30 g KzHPOa I g MgS04.7HzO 0.5 g 0.5 g KCI FeS04.7HaO 0.01 g 5 g Peptone Agar powder 15 g Distilled water 1 litre Dissolve all the ingredients except the agar in the water and adjust the pH to 7.0-7.3. Add the agar and steam the medium. After dispensing into bottles, autoclave the medium at 15 psi for 15 min. 30. Dorset's egg medium Hen eggs NaCI, 0.85 yo sterile aqueous solution

8 100 ml

104


Wash the eggs in 70% alcohol and place them on a sterile cloth. Break the shells with a sterile knife so that the contents fall into a sterile flask containing a few pieces of sterile broken glass. After shaking to break up the eggs, add the saline aseptically, and thoroughly shake the mixture to produce a homogenous suspension. Distribute the mixture aseptically in either 2 ml volumes in sterile 5 ml screw-capped bijou bottles, or in 5 ml volumes in sterile screwcapped 1 oz McCartney bottles. Slope the containers in an inspissator, heat them slowly to 75°C and then hold them at this temperature for 1 h. Repeat this inspissation treatment on the following two successive days.

31. Dubos’ salts solution NaN03 0.5 g KaHP04 1 g MgSO4.7HzO 0.5 g KC1 0.5 g FeS04.7HzO 0.01 g Distilled water 1 litre Dissolve the salts in the water and adjust the pH to 7.2. Distribute the medium in 10 ml quantities in 1 oz bottles and add a strip of Whatman No. 1 filter paper to each. Autoclave the bottles at 15 psi for 15 min. When used as an agar medium, sterilize the filter paper strips separately and place them aseptically on the surface of the solidified medium. For Cellulomonas and Cellvibrio, the addition of 0.5 g yeast extract to the basal medium is recommended. 32. Ethanol malt agar (NCIB Records) Malt extract 15 g Yeast extract 5 g Ethanol 30 ml 25 g Agar powder Distilled water 1 litre Dissolve the malt and yeast extracts in the water, add the agar and steam the medium. The ethanol can be added immediately before the medium is autoclaved at 10 psi for 20 min, or, preferably, added after sterilization of the basal medium. In the latter case, sterilization of the ethanol is carried out separately by filtration.

33. Ferrooxydans medium (Hutchinson et al., Part 1 KHzPO4 MgS04.7HzO (NH3aSOa CaClz MnS04.4HzO NaCl Ah(so4)s. 12Hz0 Distilled water

1966)

0-4 g 0.1 g 0.1 g 0.03 g 0.02 g 1 g 1.4 g 1 litre

I. MEDIA TABLES

105

Part 2 FeS04.7HaO 10 lz HzS04 concentrated 0.09 ml Distilled water 100 ml Prepare the two parts separately by dissolving the respective constituents in the liquids. Distribute the basal medium (Part 1) in 90 ml amounts in 250 ml capacity conical flasks. Place the FeS04 solution in a bottle and sterilize it with the flasks of basal medium by autoclaving at 15 psi for 15 min. Before use, add 10 ml of FeS04 solution to each flask aseptically. 34. Flavobacteriurn heparinurn medium (Payza and Korn, 1956) Trypticase (BBL) 3.5 g Phytone (BBL) 0.6 g Glucose 0.5 g NaCl 1 g KzHPOi 0.5 g Agar powder 15 g 1 litre Distilled water Dissolve all the ingredients except the agar in the water and adjust the pH to 6.5. Add the agar and steam the medium, then dispense into tubes or bottles as required and autoclave at 15 psi for 15 min to sterilize. For studies on heparin breakdown, a concentration of 0.002% sterile sodium heparin should be added to the medium. 35. Freshwater jlexibacteria medium (Fox and Lewin, 1963) KNOB 0.1 g MgS04.7Hz0 0.1 g CaClz .2H20 0.1 g Sodium glycerophosphate 0.1 g Tris buffer I g Casamino acids 1 g Thiamine 1 mi% 1 Pg Vitamin Biz Trace elements solution (formula as in CCT medium, No. 13) 1 ml NazMo04.2HaO 0.0025 g Glucose 1 g Agar powder 10 g Distilled water 1 litre Dissolve the glucose in a little of the water and sterilize separately by filtration. Dissolve the other ingredients, apart from the agar, in the remaining water and adjust the pH to 7.0. Add the agar and steam the medium. Autoclave the medium in bulk at 15 psi for 15 min and add the glucose solution. Distribute aseptically into sterile bottles. 36. Galactose agar (NCTC Records) 200 ml Nutrient agar (medium No. 68b) Galactose 50% aqueous solution 4 ml

106


Sterilize the galactose solution by sintered-glass filtration, and add 4 ml aseptically to the molten nutrient agar that has been cooled to approximately 50°C. Thoroughly mix the medium before distributing aseptically into sterile tubes or Petri dishes. 37. Glucose agar (NCTCRecord) Glucose, anhydrous D (+) 10 g Nutrient agar (medium No. 68b) 1 litre Add the glucose to the nutrient agar and autoclave the mixture at 10 psi for 15 min. After thorough mixing, dispense the medium aseptically into test tubes or Petri dishes.

38. Glucose broth To 1 litre of nutrient broth (medium No. 71) add 10 g of glucose. When dissolved, distribute the medium in bottles or tubes and autoclave at 15 psi for 15 min. 39. Glucose yeast extract agar (NCIB Records) Glucose 100 E! Yeast extract 10 B CaCOs 20 8 Agar powder 25 8 Distilled water 1 litre Dissolve all the ingredients except the agar in the distilled water. Add the agar and steam the medium. After dispensing into bottles, autoclavethe medium at 15 psi for 15 min.

40. Glycerol asparagine agar (Pridhum and Lyons, 1961) L-Asparagine 1 g Glycerol 10 g KaHP04 1 g Trace salts (formula as for Oatmeal agar, medium No. 72) 1 ml Agar powder 20 8 Distilled water 1 litre Dissolve all the ingredients except the agar in the water and adjust the pH to 7.0-7.4. Add the agar and steam the medium. After distribution into bottles, autoclave the medium at 15 psi for 15 min. 41. Glycerol casitone agar (Dmdero et al., 1961) Glycerol 10 g Casitone 5 g Yeast extract 1 g Agar powder 15 8 Distilled water 1 litre Dissolve all the ingredients except the agar in the water and adjust the pH to 7.0. Add the agar and steam the medium. Sterilization is by autoclaving at 15 psi for 15 min.

107

I. MEDIA TABLES

42. Gylirgy and Rose medium (1955) KzHPOi 2.5 g Lactose 35 g 25 g Sodium acetate N-Z Case (enzymatic digest of casein; 25 8 Sheffield Chemical Division) 0.01 g ofeach Adenine, guanine, uracil and xanthine 0.2 g of each Alanine, cystine, and tryptophan Asparagine 0.1 g Thiamin HCl 0.2 mg Riboflavin 0.2 mg 1.2 mg Pyridoxine HCl Nicotinic acid 0 - 6 mg p-Aminobenzoic acid 0.01 mg 0.01 mg Folic acid Biotin 0.004 mg 5 ml Salts solution (see below) Ascorbic acid 5 mg 1 litre Distilled water

Salts solution MgS04.7HaO FeS04.7Hzo NaCl MnSOr. 4Hz0 Distilled water

10 g 0.5 g 0.5 g 0.337 g 250 ml

Mix all the ingredients except the ascorbic acid with the water and adjust the pH to 6.8 with NaOH. Prepare the ascorbic acid as a 1% w/v solution in distilled water and sterilize in a well-filled screw-capped bottle by autoclaving at 15 psi for 15 min. Autoclave the bulk of the medium under the same conditions and add the ascorbic acid (0.5ml) afterwards. Distribute the complete medium aseptically in bottles or tubes.

43. Halophile medium (Payne et al., 1960) Casamino acids Yeast extract Trisodium citrate KCl MgS04.7Hzo FeS04.7Hzo Mns04.4HzO Agar powder NaCl Distilled water Dissolve all ingredients except the agar in the water and adjust the pH to 7.4. Add the agar, steam the medium and distribute as required. Autoclave at 15 psi for 15 min.

108


44. Horse serum agar (modifiedfrom Chanock et al., 1962) Make as horse serum broth (medium No. 4 3 , except 70 ml of Bacto PPLO agar should be used instead of 70 ml of Bacto PPLO enrichment broth. Plates should be prepared on the day of inoculation. Melt the agar and cool to 5O"C,mix with the other ingredients (warmed to SOOC) and pour plates-8 ml of medium per 5 cm diameter plastic Petri dish. 45. Horse serum broth (modifiedfrom Chanock et al., 1962) Bacto PPLO broth (without crystal violet) 70 ml Horse serum 20 ml Yeast extract 10 ml 2.5 ml Thallous acetate Penicillin 0.2 ml

Bacto PPLO broth (without crystal violet) (Difco code 0554) Suspend 21 g of powder in 1 litre of de-ionized water. Distribute in 70 ml amounts in4 oz medical flats (or 350 ml amounts in 20 oz bottles). Autoclave at 15 psi for 15 min. Store at room temperature. (Bacto PPLO broth with crystal violet is not recommended.) Horse serum "Wellcome" Brand, Horse serum No. 3 is satisfactory; do not inactivate. Store at - 30°C. Yeast extract Suspend 250 g of baker's yeast in 1 litre of de-ionized water. Heat at 100°C for 30 min, cool rapidly and clarify by centrifugation. (M.S.E. Major, 2000 rev/min for 30 min.) Discard the sediment and re-centrifuge the supernatant if necessary. Dispense in 10 ml amounts, autoclave at 10 psi for 10 min and store at -30°C. Thallousacetate: SCHEDULE 1 POISON (U.K.) Make a 1% w/v solution in de-ionized water. Seitz-filter and store in 2.5 ml amounts at room temperature. Penicillin Use benzylpenicillin sodium B.P., 100,000 units/ml in sterile de-ionized water. Store at f 4 " C (discard after 2-3 weeks). 46. Hyphomicrobium medium (adapted from Hirsch and Conti, 1964) KHaP04 1-36 g NaaHPO4 2-13 g MgS04.7HzO 0.2 g 9-95 mg CaCla. 2Ha0 5 mg FeS04.7Hao 2 - 5 mg MnS04.4HaO NaeMoOr .2Hz0 2 - 5 mg 15 g Agar powder Distilled water 970 ml Methanol 4 ml Urea (20% w/v solution in distilled water) 30 ml

109

I. MEDIA TABLES

Sterilize the methanol and urea separately by filtration. Dissolve the salts in the water and add the agar. Steam the medium and then autoclave it in bulk at 15 psi for 15 min. Add the methanol and 30 ml of the urea solution to the molten agar and dispense the complete medium aseptically into tubes or bottles. 47. Klebsiella agar Prepare plates of water agar by dissolving 1% w/v agar powder in distilled water and autoclaving it at 15 psi for 15 min. Pour 15-20 ml quantities of the agar into 3+ in. Petri dishes and allow the medium to set. Grow a culture of Klebsiella aerogenes on nutrient agar at 37°C for 24 h and harvest the cells by washing off with sterile distilled water. Centrifuge the cells once and re-suspend them in distilled water. Spread the cell suspension over the surface of the water agar plates and drain off the excess liquid after 20-30 min. Dry the surface of the plates byplacing them inan incubatorat 45°C. Theplates should be used the same day as they are prepared, but the water agar base may be stored. The growth from one 10 mlnutrient agar culture will provide 1-3 ml of cell suspension. The method can be employed using other eubacteria if desired. A useful alternative medium for the cultivation of fruiting myxobacteria is yeast agar (medium No. 106). 48. Krebs’ yeast lactate medium Yeast extract

10 g

mzpoi NazHPO4.2HzO 3 g Sodium lactate (70%) 40 ml 960 ml Distilled water Dissolve the solid ingredients in the water and add the lactate. Adjust the pH to 7.0 and distribute the medium into 1 oz screw-capped bottles. The bottles should be well filled, but to save undue loss of medium during sterilizing, the bottle can be “topped-up’’ to the desired level of at least 2 full immediately after inoculation. Sterilization is by autoclaving at 15 psi for 15 min. 49. Leuconostoc oenos medium (Garwie, 1967)

A. Peptone Yeastrel Glucose MgS04.7HzO MnS04.4HzO Tomato juice Distilled water B. Cysteine HCl, 1% w/v in distilled water.

10 g 5 g

10 g 0.2 g 0.05 g 250 ml 750 ml

Dissolve the solid ingredients of medium A in the water and add the tomato juice. Adjust the pH if necessary to 4.8. Sterilize the medium by autoclaving at 15 psi for 15 min.

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Sterilize the cysteine HCl solution separately by Seitz-filtration or a similar method, and add 0-5 ml to 10 rnl of medium A before use.

50. Leucothrix mucor medium (Brock, 1964) NaCl 11.75 g MgCla 2.5 g NanSOi 2 g CaCla. 2H20 0.75 g 0.35 g KC1 NaHCOs 0.1 g Yeast extract 1 g If3 Tryptone Distilled water 1 litre Dissolve the salts and other ingredients in the water and adjust the pH to approximately 7.0.Dispense into bottles or tubes and autoclave at 15 psi for 15 min. As an alternative to the salts used in this medium, 2 strength aged filtered sea water may be used (see medium No. 85). 51. Maixe mash Mix 50 g of freshly ground maize meal with 1litre of distilled water and boil the mixture until a sample will gelatinize on cooling. The medium is then dispensed into bottles or tubes, keeping it well agitated during the process. Autoclave the medium for 1 h at 15 psi.

52. Manganouc acetate agar (Zawarzt’n, 1964) 0.1 g Manganous acetate “Ionagar” No. 2 (Oxoid) 10 g 1 litre Distilled water Dissolve the manganous acetate in the water and check the pH to approximately 7.0. Add the agar and steam the medium. Dispense the medium into screw-capped bottles, and autoclave at 15 psi for 15 min. Although there is no apparent source of N, P, K or other nutrients in the medium, it proves to be successful in practice. The organisms may actually be using traces of volatile compounds from the atmosphere.

53. Marine agar Beef-extract (Oxoid, “Lab-Lemco”) I g Peptone (Evans) NaCl 2.25 g Standard Davis agar (New Zealand) 1.5 g loo ml Distilled water Dissolve all the ingredients except the agar, heating if necessary, and adjust the pH of the solution to 7.8. After boiling the solution for 3-5 min, filter it through Green’s “Hyduro” 9 0 4 ) filter paper, before re-adjusting the p H value of the medium to 7.3.Add the agar and dissolve by autoclaving at 10 psi for 10 min. After distribution in 200 ml amounts in screw-capped bottles, sterilize the medium by autoclaving at 15 psi for 15 min.

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54. Marine Jlexibacteria medium (Fox and Lewin, 1963) KN03 0.5 g 0.1 g Sodium glycerophosphate Tris buffer 5 g Tryptone Yeast extract 5 g Trace elements solution (formula as in CCT medium, No. 13) 1 ml NaaMoOi. 2HaO 0.0025 g Agar powder 10 g Aged filtered sea water 1 litre Mix all the ingredients except the agar with the sea water and adjust the pH to 7.0. Add the agar and steam the medium. After distribution into bottles, autoclave the medium at 15 psi for 15 min. 55. Marine Spivillum medium (modifiedfrom Williamsand Rittenberg, 1957) Prepare the medium in the same way as Spirillum medium (medium No. 89), except that 750 ml of the distilled water should be replaced by an equal volume of aged sea water.

56. Methanobacillus medium (Johns and Barker, 1960) Ethanol 10 g KaHP04 6 g KHaP04 9 g NHcCl 5 g MgCla FeS04.7Ha0 0.01 g CaCla 0.01 g 1 litre Tap water Mix and dissolve the ingredients in the water and adjust the pH to 7.4. Dispense the medium in tubes or bottles and autoclave at 10 psi for 20 A n . N.B. It has been shown that the cultures usually grown on this medium as Methanobacillus omelianskii are mixtures (Bryant et al., 1967), and the above medium would not be suitable for pure cultures of the methane-forming organism. 57. Methanococcus medium (Stadtman and Barker, 1951) Sodium formate 15 g (NH4)aS04 1 g CaCla. 2Ha0 0.01 g 0.01 g MgClz. 2 H 2 0 FeCl3.6HaO 0.02 g MnS04.4HaO 0.01 g 0.001 g NaaMoOc .2Ha0 KaHP04 2 g Sodium thioglycollate 0.5 g Distilled water 1 litre Dissolve the solid ingredientsin the water and adjust the pH to approximately 8.0. Sterilize by autoclaving at 15 psi for 15 min. If the medium is not to be used immediately, it is advisable to store it in well-filled bottles, and keep the

112


sodium thioglycollate as a separate solution to be added to the medium immediately before use.

58. Methanol salts medium (Peel and Quayle, 1961) WzPOa 1.36 g NaaHPO4 2.13 g (NH3aSOa 0.5 g MgS04.7HzO 0.2 g CaCl2.2H20 0.01 g FeSO4.7H20 5 mg MnSO4.4HaO 2.5 mg NaaMoO4.2HaO 2.5 mg Distilled water 1 litre Methanol 5 ml Add all the ingredients except the methanol to the water and autoclave the mixture at 15 psi for 15 min. Seitz-filter the methanol to sterilize it and add aseptically to the previously sterilized salts solution. 59. Methanosarcina barkeri medium (Blaylock and Stadtman, 1966) A. NHiCl 0.5 g 0.01 g CaClz .2Hz0 0.01 g MgCla .2H20 2 mg FeCls .6H2O 1 mg MnS04.4Ha0 1 mg NaaMoO1.2H20 KaHP04 3-48 g KHaP04 2-72 g 1 litre Distilled water B. NaaS. 9Hz0 10% w/v in distilled water C. NaaCOs 10% w/v in distilled water D. Methanol Prepare the salts solution A by dissolving the constituents in the water and sterilize by autocalving at 15 psi for 15 min. Also sterilize solutions B and C by autoclaving at 15 psi for 15 min. Seitz-filter the methanol. T o 1 litre of salts medium A, add aseptically2 ml of B, 20 ml of C and 10 ml of D. After mixing, adjust the pH with 10% HCI to 6.8.

60. Methylococcus medium (Foster and Davis, 1966) NaNO3 2 g MgS04.7Hz0 0.2 g 0.04 KC1 CaCl2 0.015 g NazHPOo 0.21 g NaHaPO4 0.09 g FeS04.7HzO 1 mg CuSOe. 5Hz0 5 Pg H3B03 10 P€! MnS04.5HzO 10 Pg

I. MEDIA TABLES

113

ZnSO4.7Hz0 70 CLg NazMoO4.2HzO 10 Pg 1 litre De-ionized water Dissolve the constituents in the water and dispense the medium in bottles or flasks. Autoclave the medium at 15 psi for 15 min.

61. MRS medium (de Man et al., 1960) Peptone 10 g 10 g Beef extract Yeast extract 5 g Glucose 20 g Tween 80 1 ml 2 g KzHP04 Sodium acetate 5 g Tri-ammonium citrate 2 g MgS04.7HaO 0.2 g MnS04.4HzO 0.2 g Distilled water 1 litre Dissolve the ingredients in the water and check the pH to within 6.2-6.6. Distribute the medium as required in bottles and tubes, and sterilize by autoclaving at 15 psi for 15 min. When used for culture maintenance, a little chalk should be placed in each tube and 1 % agar included in the medium. Note This medium is prepared, when indicated, with 18% NaCl. 62. Mycobacteriumjohnei medium (Stuart, 1965) Basal medium Casamino acids (Difco) 2.5 g L- Asparagine 0.3 g NazHP04, anhydrous 2.5 g KHzPO4 1.0 g Sodium citrate 1.5 g 0.6 g MgS04.7Hz0 Glycerol 25 mt 50 ml Tween 80 (1 % solution) 15 g Standard Davis agar (New Zealand) Distilled water to800 ml Dissolve the constituents with minimum heat to give a total volume of 800 ml. Sterilize this basal medium at 10 psi for 15 min, and store at 4"-6"C until required. Crude mycobactin Grow Mycobacteriumphlei for 3-4 weeks in a beef infusion broth containing 10% glycerol and 4% Difco-Bacto peptone, p H 7.2. Kill the growth by autoclaving, then collect by filtration and wash with large amounts of distilled water, drain and dry over CaC12. Extract 100 g of dried culture, for 30 min with 3 successive 500 ml amounts of acetone in a 1 litre flask fitted with a reflux condenser.

114

S. P . LAPAGE, J. E. SHELTON AND T. G . MITCHELL

Bulk the acetone extracts, evaporate them to dryness, and extract the resulting waxy residue in a Soxhlet for 18-20 h with petroleum ether (40”6OOC). Dissolve the hard brick-red deposit (approx. 3 g) that is left in warm absolute alcohol and centrifuge the solution at 710g for 30 min, thereby removing any bacilli that may have been carried over. Evaporate the supernatant to dryness, and grind the product to a fine powder of crude mycobactin free from acid-fast bacilli and debris.

Final solid medium Basal medium 800 ml Crude mycobactin 0.16 g 100,000 units Penicillin Chloramphenicol 0.05 g Pimaricine (Royal Netherlands Fermentation Industries Ltd., Delft, Holland) 0.05 g Sterile bovine serum 200 ml Melt the basal medium, cool to 56°C in a water bath and add the remaining constituents, the bovine serum having been inactivated by heat at 56°C for 1 h. Thoroughly mix the medium and check the final pH value 7.2, before distributing aseptically in 5 ml amounts into sterile screw-capped bottles, which are sloped. 63. Mycoplasma suipneumoniae agar (modijied from Goodwin et al., 1967) Prepare Mycoplasma suipneumoniae broth (medium No. 64) with the addition of 1% Oxoid “Ionagar” No.2. 64. M y c o p l a m suipneumoniae broth (modijiedfrom Goodwin et al., 1967) Hanks’ balanced salt solution 40 ml 30 ml Hartley digest broth 20 ml Pig serum 10 ml (autoclaved at 5% Lactalbumin hydrolysate 10 psi for 10 min) Penicillin 0.2 ml 1.25 ml Thallous acetate Yeast extract 0.5 ml The complete, mixed medium is stored at about - 30°C.

Hanks’balanced salt solution Prepare from Hanks’ stock solution without bicarbonate (Oxoid, BR 19a). Hartley digest broth 16 lb (7.25 kg) Meat (ox heart) NaaCOa 96 8 Pancreatic extract 240 ml 240 ml Chloroform 192 ml Technical HCl T a p water 12 litres Mince the meat finely and add to the water. Heat to 80”C, add Na2COs, stir well and infuse in refrigerator overnight. Next day, heat to 45°C and add

115

I. MEDIA TABLES

pancreatic extract and chloroform. Stir well and maintain at 45°C for 3 h. Add HC1 and boil for 30 min, then filter through Green’s “Hyduro” 904) filter paper. Add 10 M NaOH to give pH 8.4.Boil for 30 min, and then filter through Green’s “Hyduro” 904f filter paper into 2 litre bottles. Autoclave at 10 psi for 20 min.

Pig serum Seitz-filter and then inactivate (in 500 ml amounts) in a 56°C water bath for 1 h.

Penicillin, Thullous acetate, and Yeast extract For method of preparation see Horse serum broth (medium No. 45).

65. Nitrobacter agilis medium (Krulurich and Funk, 1965) KNOa 0.17 g CaC03 10 g MgSOj. 7Hz0 0.14 g FeS04.7HaO 0.03 g MnS04.4HaO 0.01 g NaCl 0.3 g KaHP04 0.14 g NaaCOs 0.25 g 1 litre Distilled water Biotin 150 mg Prepare the biotin separately as a filter-sterilized solution. Dissolve the NaaCOs in a little of the water and sterilize by autoclaving at 15 psi for 15 min. Dissolve the remaining salts in the rest of the water and sterilize by autoclaving at 15 psi for 15 min. Mix the carbonate and salts solutions and add the biotin. Distribute the complete medium into tubes or flasks aseptically. 66. Nitrogen-free agar (Norris, 1959) Part 1 KaHPOlr MgSO4.7HaO CaCOs NaCl FeSOj.7HzO NaaMoO4.2HaO Agar powder Distilled water

0.2 g I g 0.2 g 0.1 g 0.005 g 15 g 1litre

Part 2 D-Glucose, anhydrous 10 g Distilled water 50 ml Dissolve the salts of Part 1 in the water and adjust the pH to approximately 7.0. Add the agar, steam the basal medium and sterilize by autoclaving at 15 psi for 15 min.

116


Dissolve the glucose (Part 2) in the separate quantity of water and sterilize by Seitz-filtration. Add the glucose solution aseptically to the basal medium and dispense the complete medium as required into tubes, bottles or Petri dishes. 67. Nitrosomonas europaea medium (adaptedfrom Hooper et al., 1967) A. NaaHPOi 13.4 g KHaP04 0.773 g 0.5 g NaHCOs (NH4)2S04 2.5 g Distilled water 1 litre B. MgS04.7H20 8.5 mg CaC12.2H20 310 mg Sodium ferric ethylenediamine di-o-hydroxyphenylacetate (Sequestrene 138-Fe, Geigy Chemical Co.) 3.0 mg Distilled water 100 ml Dissolve the components of medium A in the distilled water and check the pH (approximately8.0). Sterilize the medium by filtration through a Millipore filter. If it is desired to heat-sterilize medium A, the bicarbonate should be prepared separately and the two parts autoclaved. Autoclave medium B a t 15 psi for 15 min and add 6 ml aseptically to 1 litre of medium A.

Nitrocystis oceanus Prepare the medium in the same way as for Nitrosomonas europaea, except that aged filtered sea water is substituted for the distilled water. 68a. Nutrient agar (The Oxoid Manual, 1965) For species marked with an asterisk (NCIB) in the list, this formula has been used. For species not marked with an asterisk, the NCTC formula (medium No. 68b) has been used. It is recommended that the correct formula should be used, especiallywith some species covered by the NCIB, e.g., flavobacteria. In many cases it would appear that it would make little difference which formula is used, but growth on the NCIB formula may be slower. The medium is conveniently prepared from "Oxoid" nutrient agar granulesNutrient agar granules 28 g 1 litre Distilled water "Oxoid" Nutrient agar granules containBeef extract 1 g Yeast extract 2 g Peptone 5 g NaCl 5 g 15 g Agar powder in 28 g of granules. Dissolve the granules in the water by steaming and distribute as required in bottles or tubes. Sterilization is by autoclaving at 15 psi for 15 min. Note This medium is prepared when indicated with 3% or 6% NaCl.

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68b. Nutrient agar For species not marked with an asterisk (NCTC) in the list, this formula has been used. For species marked with an asterisk the NCIB formula (medium No. 68a) has been used. It is recommended that the correct formula should be used especiallywith some species covered by the NCIB, e.g., flavobacteria. In many cases it would appear that it would make little difference which formula is used, but growth on the NCIB formula may be slower. Oxoid Nutrient Broth No. 2 (Granules, CM67) 25 g Standard Davis agar (New Zealand) 10 g 1 litre Distilled water The manufacturers state that Oxoid Nutrient Broth No. 2 when prepared at this concentration contains“Lab-Lemco” Beef Extract Peptone (Oxoid, L37) NaCl pH = 7.5 (approx.)

10 g

10 g 5 g

Thoroughly mix the solids and water before adjusting the pH to 8.4. Autoclave the medium at 10 psi for 20 min to dissolve the agar, and filter it hot through Green’s “Hyduro” 904+ filter paper. The latter removes phosphates precipitated by the higher pH of the medium at this stage. Adjust the pH to 7-6, and distribute the medium in 250 ml amounts in screw-capped bottles and sterilize at 15 psi for 15 min. 69. 4% Nutrient agar The constituents and method of preparation follow the same formula as Nutrient agar (medium No. 68b), but the agar is incorporated at 4 times the concentration there stated. 70. Nutrient agar, p H 8.0 The final pH adjustment during the preparation of nutrient agar (medium No. 68b) is to pH 8.0instead of pH 7.6.

71. Nutrient broth (The Oxoid Manual, 1965) This medium is prepared from commercially available Oxoid Nutrient Broth No. 2. Oxoid Nutrient Broth No. 2 (Granules, CM67) 25 g 1 litre Distilled water The manufacturers state that Oxoid Nutrient Broth No. 2 when prepared at this concentration mntains“Lab-Lemco” Beef Extract Peptone (Oxoid, L 37) NaCl pH = 7.5 (approx.)

10 g 10 g 5 g

118


Dissolve the Oxoid nutrient broth in the water and mix well, before distributing into final containers. Sterilization is by autoclaving at 15 psi for 15 min. 72. Oatmeal agar (Kiister, 1959) Oatmeal Agar powder Distilled water Trace salts solution (see below)

20 g 18 g 1 litre

1 ml

Trace salts solution FeS04.7HaO 0.1 g MnC12.4Hzo 0.1 g 0.1 g ZnSO4.7HzO Distilled water 100 ml Steam the oatmeal in the distilled water for 20 min and filter the mixture through cheese-cloth. Restore the volume of atrate to 1 litre with distilled water and add the trace salts solution and agar. Adjust the pH to 7-2, steam the medium and autoclave in bottles at 15 psi for 15 min. The bulk of the medium should be well mixed before distribution or pouring of plates.

73. Peptone ferric citrate agar (Rouf and Stokes, 1964) Peptone 5 g Ferric ammonium citrate 0.5 g 0-2 g MgS04.7HaO CaCla 0.05 g 0.05 g MnS04. H a 0 FeCls .6HaO 0.01 g Agar powder 12 g Tap water 1 litre Dissolve the peptone and salts in the water and adjust the p H to 7.0. Add the agar and steam the medium. After distribution into bottles autoclave the medium at 15 psi for 15 min. T o avoid excessive precipitation during sterilization, it is desirable to sterilize the iron compounds separately and add them aseptically at the end. 74. Peptune saline agar (Peptone agar base for blood agar plates) Peptone (Evans) 10 B Standard Davis agar (New Zealand) 10 g NaCl 5 g Tap water 1 litre Dissolve the ingredients in the water by autoclaving at 10 psi for 10 min. wfilter paper, and adjust Filter the hot solution through Green’s “Hyduro” 9 the pH to 7.6. Distribute in 200 ml amounts in screw-capped bottles, and sterilize by autoclaving at 10 psi for 10 min.

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75. Peptone yeast glutamate (Horler et al., 1966) Peptone 20 8 Yeast extract 10 g Monosodium glutamate 4 g Sodium thioglycollate 1 g Distilled water 1 litre Dissolve the ingredients in the water and adjust the pH to 7-0-7.2. Dispense the medium in tubes or bottles and autoclave at 15 psi for 15 min. 76. Pfennig’s medium (Postgate, 1966) Heavy metal solution EDTA FeS04.7H20 ZnSO4.7H20 MnClz .4Hz0 HsBOs CoCl2.6HzO CuCla. 2H20 NiCla. 6H2O NaaMoOi. 2H20 Distilled water The EDTA is dissolved in the water first.

0.5 g

0.2 g 10 mg 3 mg 30 mg 20 mg 1 mg 2 mi3 3 mg 1 litre

CaCla 0.04% w/v in distilled water.

Vitamin Biz 0.002% w/v in distilled water. Salts solution KH2P04 KCl NHeCl MgCl2. 6H2O Heavy metal solution (see above) Vitamin Biz solution (see above) Distilled water

1 g 30 ml 3ml 70 ml

NaHCOs 4.5 g in 900 ml of distilled water.

NaaS .9Hao 1.5% w/v in distilled water.

Sterilize the CaCl2 and NazS by autoclaving at 15 psi for 15 min. Saturate the bicarbonate with CO2 by bubbling through C02 gas and mix with the salts solution. Sterilize the mixture by filtration under positive pressure of C02. For every litre of this sterile solution add aseptically 500 ml of CaClz. Adjust the pH to between 6.7 and 7.2 after the addition of the quantity

120


of sulphide appropriate to the specificorganism. Some specificorganisms areChromatium okmii, final concentration of 0*06-0.09% NazS .9Hz0.

Chromatium vinosum, final concentration of 0.01-0-03% NazS .9Ha0. Chromatium warmingii, final concentration of 0-06-0.09% NazS. 9Ha0. Pelodictym clathratiforme, final concentration of 0.03 % NazS .9Hz0 and 0.04% ammonium acetate replacing NHlCl in salts solution. Thiococcus sp., final concentration of 0.075% NazS.9HzO and 0.02% ammonium acetate replacing NH4Cl in salts solution. Thiospirillumjmmse, final concentration of 0.045 % NazS .9H& and 0.02% ammonium acetate replacing NH4Cl in salts solution. 77. Postgate’s medium (Postgate, 1963)

Part 1 KaHP04 NHiCl NazSO4 CaCl2.2HzO MgS04.7HaO Sodium lactate (70%) Yeast extract Distilled water

0.5 g 1 g

1 g 0.1 g 2 g 3-5g 1 g 980 ml

Part 2 FeS04.7H20 Distilled water

0.5 g

10 ml

Part 3 0.1 g Sodium thioglycollate Ascorbic acid 0.1 g 10 ml Distilled water Dissolve the ingredients of each part in the appropriate quantities of water. Adjust the pH of Parts 1 and 3 to 7.4. Sterilize the three parts separately by autoclaving at 15 psi for 15 min. Combine the three parts, mix and distribute the medium aseptically as required. If the medium is to be stored, Part 1 should be boiled immediately before use. When the whole batch of medium is to be employed immediately following preparation, all of the constituents may be mixed before sterilization. Note This medium is also prepared, where indicated, with 2.5% NaCl.

78. Pseudomonas methanica medium (Leadbetter and Foster, 1958) NaNOa 2 g MgSOi. 7Hz0 0.2 g FeS04.7Hzo 0.001 g NaaHPO4 0.2 g NaHzPO4 0.09 g CuSo4.5Ha0 50 I% HaBOs 10 Mi!

I. MEDIA TABLES

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MnS04.5HzO 10 Pg ZnSOi. 7Hz0 70 Pg Moo3 10 Pg KCl 0.04 g CaClz 0.015 g De-ionized water to 1 litre Mix the ingredients with the water and adjust the pH to approximately 7.0. Distribute the medium into tubes and sterilize by autoclaving at 15 psi for 15 min. 79. Pseudomonas saccharophila medium (Doitdoroff et al., 1943) 1 litre KHzP04-NazHP04, ~/30, pH 6.64 1 g NHiCl MgS04.7HzO 0.5 g FeC13.6HzO 0.05 g CaClz .2Hz0 0.01 g Sucrose 2 g Dissolve the salts and sucrose in the buffer solution and distribute in bottles or tubes. Sterilize by autoclaving at 15 psi for 15 min. 80. Purple milk (NCTC Records) 1 pint (473 ml) Fresh milk 0 4-0 * 5 ml Bromocresol purple (1% alcoholic solution) Store the milk in the refrigerator overnight, and pipette or siphon off the cream from the top. To the remainder add 04-0.5 ml of 1%alcoholic bromocresol purple, so that the milk is just noticeably coloured. Dispense the medium into test tubes and sterilize by autoclaving at 10 psi for 10 min. 81. Rhizobium medium 1 (modified from Heberlein et al., 1967) Yeast extract 10 g KzHP04 0.5 g MgS04.7Hz0 0.2 g NaCl 0.2 g FeC13.6HzO 0.002 g Agar powder 15 g Distilled water 1 litre Dissolve all the ingredients except the agar in the water and adjust the pH to 7.2. Add the agar and steam the medium. After distribution into bottles or tubes, autoclave the medium at 15 psi for 15 min. 82. Rhizobium medium 2 (modified from Heberlkn et al., 1967) Glycerol 4.6 g L-Arabinose I&! Yeast extract 1 g KzHPOi 1g CaS04 1.3 g KN03 0 - 7g FeC13.6HzO 0.004 g MgS04.7HzO 0.36 g Agar powder 15 g Distilled water 1 litre

122

S. P. LAPACE, J. E. SHELTON AND T. C. MITCHELL

Mix all the ingredients except the agar with the water and adjust the pH to 7.2. Add the agar and steam the medium. After distribution into bottles or tubes autoclave the medium at 15 psi for 15 min. 83. Sarcina maxima medium (Holt and Canale-Parola, 1967) Glucose 10 g Peptone 10 g Yeast extract 5 g L-Cysteine HC1 0.5 g Distilled water 1 litre Dissolve the constituents in the water and adjust the pH to 6.0. Autoclave the medium at 15 psi for 15 min. If the medium is not to be used immediatelyafter preparation, it is desirable to sterilize the cysteine separately by filtration and to add it to the freshly boiled basal medium immediately before use. 84. Sarcina ventriculi medium (Holt and Canale-Parola, 1967) Glucose 20 g Yeast extract 20 g Distilled water 1 litre Dissolve the glucose and yeast extract in the water and adjust the pH to 6.0. Dispense the medium into bottles and autoclave at 15 psi for 15 min. If the medium is not used immediately, it should be boiled and cooled before inoculation. 85. Sea water agar (NCMB Records)

Meat extract Peptone Aged sea water Distilled water Agar powder

Preparation of aged sea water (ZoBell, 1946) Collect and store sea water in the dark for at least 3 weeks before use, at about 5°C. When required, filter through Green’s “Hyduro” 9049 filter paper to remove particulate matter. Sea water agar Dissolve the peptone and meat extract in the sea water plus distilled water. Adjust the pH to 7.8 and boil the medium for 3-5 min. Filter the medium through Green’s “Hyduro” 9049 filter paper and re-adjust the pH to 7.3. Add the agar and steam the medium. After distribution into bottles, autoclave the medium at 15 psi for 15 . min. As an alternative to the use of sea water, a synthetic salts mix can be used at 3 of nominal aquarium or sea water strength. Commercialproducts are available for this purpose, e.g., that of Rila Products, Teaneck, N. J., U S A .

I. MEDIATABLES

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86. Sodium caseinate agar (Weeks and Beck, 1960) Sodium caseinate 2 g 0.5 g Yeast extract 0.5 g Proteose peptone 0.5 g KaHPOlr Agar powder 15 g 1 litre Distilled water Dissolve all constituents except the agar in the water and adjust the pH to 7.4-7-6. Add the agar and steam the medium. Dispense the complete medium in bottles or tubes and sterilize by autoclaving at 15 psi for 15 min. 87. Soil extract agar (Gordon and Smith, 1953) Soil extract Sift a suitable amount of air-dried garden soil through 2-3 thicknesses of absorbent surgical gauze, and add 1000 g of this sifted soil to 2 . 4 litres of tap water. If the soil is rich in organic matter, 500 g of soil is sufficientper 2 - 4litres of water. Autoclave the suspension at either 15 psi for 1 h or at 20 psi for 20 min; then stir well and filter immediately through Green’s “Hyduro” 9043 filter paper. Use the filtrate as the soil extract, which if the soil was not sufficiently dried, may be clarified by the addition of talc and refiltration. Soil extract agar Peptone (Evans) 5 g Lab Lemco (Oxoid) 3 g Standard Davis agar (New Zealand) 15 8 Soil extract 1 litre Thoroughly mix the constituents, and autoclave the mixture at 10 psi for 10 min in order to dissolve the agar. Adjust the pH value of the medium to 7.0 before distribution. Sterilizationis by autoclaving at 10 psi for 10 min.

88. Soil extract agar, p H 8.0 The final pH adjustment during the preparation of soil extract agar (medium

No.87) is to pH 8.0 instead of pH 7.0. 89. Spirillum medium (modi’ed from Williams and Rittenberg, 1957) Peptone Beef extract Yeast extract Calcium lactate Distilled water Dissolve the constituents in the water and adjust the pH to 7.0.Dispense the medium as required into bottles or tubes and sterilize by autoclaving at 10 psi for 20 min. The precipitate that forms is not removed. 90. Spirillum volutans medium Place a grain of wheat in a 20 x 180 mm test tube and cover it with 1 in. of garden soil. Add water to a total height of 2 in. in the tube. After plugging the

124


mouth of the tube, autoclave at 15 psi for 15 min. Check the pH and adjust if necessary to 6.5-7-5. 91. Starch salts agar (Kiister, 1959) KzHPO4 I?z MgS04.7Hzo 1 g NaCl 1 g (NHS~SOI 2 g CaC03 2 g Trace salts solution (composition as in Oatmeal agar, medium No. 72) 1 ml Agar powder 20 g 1 litre Distilled water Starch, soluble 10 g Make a paste of the starch with a little of the water and add further water gradually to a volume of 500 ml. Add all the other ingredients except the agar to the remaining water, and mix with the starch suspension. Adjust the pH to 7.2, add the agar and steam the medium. Sterilization is by autoclaving at 15 psi for 15 min. Before pouring into Petri dishes or allowing slopes of the medium to set, thoroughly mix the medium to ensure reasonable distribution of the chalk. 92. Sulphur medium (Parker and Prisk, 1953) NH4C1 0.1 g 3.0 g KHzPOi MgCla. 6HzO 0.1 g CaClz 0.1 g Sulphur 10 g Distilled water 1 litre Dissolve all the ingredients except the sulphur in the distilled water and check the pH to 4.2. Distribute in 100 ml amounts into 250 ml conical flasks. Distribute the sulphur into screw-capped bottles, each to contain approximately 1 g. Sterilize both the basal medium and the sulphur by autoclaving a t 15 psi for 15 min. Before use, aseptically layer approximately 1 g of sulphur onto the surface of each flask of liquid basal medium.

93. Thiobacillusdenitrificans medium (Baalsrud and Baalsrud, 1954) Part 1 NazSzOs. 5Hz0 KNOS NH4C1 MgClz .6HzO Distilled water

5 g 2 g

0.5 g 0-5 g 800 ml

Part 2 KHzP04 Distilled water

2 g 100 ml

Part 3 NaHC03 Distilled water

I g 100 ml

1. MEDIA TABLES

125

Part 4 FeS04.7HzO 1 g HCl, 0.1 M 100 ml Dissolve the solid ingredients of each part in the appropriate quantities of liquid. If a solid medium is required, add 10 g of “Ionagar ” No. 2 (Oxoid) to Part 1. Sterilize the four parts separately by autoclaving at 15 psi for 15 min. Add Part 2 and 1 ml of Part 4 to Part 1 and adjust pH to approximately6.5 with sterile 0.1 M NaOH. Add Part 3 to the bulk and check the pH, which should be about 6.9. Distribute as required into bottles or tubes. 94. Thiobacillw perometabolis medium (London and Rittenberg, 1967) NHiCl 1 g KHzPO4 1 g 0.5 g MgCh Trace elements solution (composition as in Pfennig’s medium, medium No. 76, as Heavy 20 ml metal solution) Na~S~03.sHzo 5 g Yeast extract 5 g 15 g Agar powder Distilled water 980 ml Dissolve the yeast extract in 50 ml of the water, adjust the pH to 7.0 and sterilize by filtration. Dissolve all ingredients except the yeast extract and agar in the remaining water and adjust the pH to 6.9. Add the agar and steam the medium. Autoclave the medium at 15 psi for 15 min and add the sterile yeast extract. Dispense the complete medium in sterile tubes or bottles. 95. Thiosulphate agar No. 1 (Parker and Prisk, 1953) (NH4)zSOd 0.1 g KzHP04 4-0 g KHzPO4 4.0 g MgS04.7HzO 0.1 g 0.1 g CaClz FeC13.6HzO 0-02 g MnS04.4HzO 0.02 g NazSz03.5HzO 10 g “Ionagar” No. 2 (Oxoid) 12 g Distilled water 1 litre Dissolve all the ingredients except the agar in the water and adjust the pH to 6.6. Add the agar and sterilize by autoclaving at 10 psi for 20 min. 96. Thiosulphate agar No. 2 (Parker and Prisk, 1953) NH4Cl 0.1 g KHzPO4 3 g MgClz. 6HzO 0.1 g CaClz 0.1 g NazSz03.5HzO 5 g “Ionagar” No. 2 (Oxoid) 20 g 1 litre Distilled water

126


Dissolve all the ingredients except the agar in the water and check the pH to 4.2. Add the agar and steam the medium. Distribute in bottles or tubes and sterilize by autoclaving at 15 psi for 15 min. 97. Thiothrb medium (Morita and Burton, 1965) Yeast extract CaCla Sodium acetate Agar powder Sulphur spring water

2 g 0.1 g 0.5 g 15 8 1 litre

Dissolve the yeast extract, CaCla and sodium acetate in the water and adjust the pH to approximately 7.0. Add the agar, steam the medium and sterilize by autoclaving at 15 psi for 15 min. Pour the sterile medium into Petri dishes which, after inoculation, are incubated in desiccators containing 5 g of solid NazS .9Hz0. Cultures of Thiothrix are probably only obtainable as enrichments obtained from a suitable natural source,such as sulphur springs. Since exact detailsof the growth requirements of this organism are not known,the medium should be prepared with a sample of the spring water from which enrichment is being attempted. 98. Tomato juice agar (TheOxoid Manual, 1965) Tomato juice agar (Oxoid, Tablets, CM 14) 200 tablets Distilled water 1 litre The manufacturers state that Oxoid tomato juice agar when prepared at this concentration contains per litreTomato juice (solids from 400 ml) 20 g Peptone (Oxoid, L 37) 10 8 Peptonized milk (Oxoid, L 32) 10 g Agar 12 8 pH = 6.1 (approx) Add the tablets to the water and soak for 15 min. Sterilize the medium by autoclaving at 15 psi for 15 min; mix well before pouring aseptically into sterile Petri dishes or test tubes. 99. Treponema zuelzerae medium (modifiedfrom Veldkam$, 1960) Basal medium KHzP04 1 g NH4Cl I g MgClz. 6HzO 0.5 g CaClz 0.04 g FeC13.6HzO 0-0025 g 2 ml Trace element solution (see below) Agar powder 15 8 Distilled water 1 litre

127

I. MEDIA TABLES

Trace element solution 56 mg

HsB03 ZnSO4.7HzO CaCla.6H2O cus04.5H20 MnCls NazMoO4.2HsO Distilled water

44 mg

20 mg 0.2 mg 2 mg 75 mg 100 ml NaHCO3

10% w/v in distilled water.

Nu&. 9HzO 10% w/v in distilled water. Glucose 10% w/v in distilled water.

Yeast extract 10% w/v in distilled water. Prepare the basal medium and the additives separatelyand autoclave them at 15 psi for 15 min in separate containers. For use, to 1 litre of basal medium add aseptically, 10 ml of bicarbonate, 5 ml of sulphide, 10 ml of glucose and 10 ml of yeast extract as detailed above.

100. Tryptone glycine medium (Klein and Sagers, 1966)

Tryptone 5 g Yeast extract 5 g Glycine 3 g 5 ml KHsP04-NagHP04 buffer, 0.1 M, pH 7.0 MgS04.7Hso 0.2 g 0.01 g FeS04.7Hzo MnS04.4HzO 0.005 g Distilled water 1 litre Dissolve the ingredients in the water and check the pH (approx. 7.0). Dispense the medium into bottles and autoclave at 15 psi for 15 min.

101. Urea nutrient agar Filter-sterilize a 20% w/v solution of urea in distilled water and add 5 ml aseptically to 100 ml of molten cooled sterile nutrient agar (medium No. 68a). After mixing, dispense the medium aseptically.

102. Urea soil extract agar Sterilize a 20% w/v solution of urea in distilled water by Seitz-filtration, and add 5 ml aseptically to 100 ml of molten cooled sterile Soil extract agar (medium No. 87). After mixing, dispense the medium aseptically.

128


103. Uric acid medium 2 (Schefferle,1965) Uric acid 1 g NazHP04.12 H a 0 6 g Yeast extract 2.5 g 100 ml Mineral solution (see below) Agar powder 15 8 Distilled water 900 ml Mineral solution KHaP04 1 g CaClz 0.1 g MgS04.7HzO 0.3 g NaCl 0.1 g FeCls. 6H2O 0.01 g Distilled water 1 litre Add all the ingredients except the agar to the water and boil. Adjust the pH of the medium to 7.0 and add the agar. Steam the medium, distribute in bottles and autoclave at 15 psi for 15 min.

104. Uric acid medium 2 (Rabinouitz, 1963)

Uric acid 2 g KOH, 10 M 1 - 2 ml KzHPO4.3HzO 0-91 g MgS04.7HzO 0.035 g FeS04.7HzO 1-75 mg CaCla. 2Hz0 4.2 mg Yeast extract 1 g Mercaptoacetic acid 2 ml Distilled water to 1 litre Mix the uric acid with the bulk of the water and the KOH and boil until the uric acid is dissolved.Add the Mg, Fe and Ca salts, followed by the yeast extract, which should be first dissolvedin a little water. Add the mercaptoacetic acid and adjust the pH to 7-0-7.2. Distribute the medium in bottles and autoclave at 15 psi for 15 min. Store the medium at 37°C until required to prevent precipitation of the uric acid.

10.5. V 17 medium (Rogosu, 1964)

Trypticase (BBL) 5 g Yeast extract 3g Sodium thioglycollate 0.75 g Tween 80 1 g Sodium lactate (50%) 25 ml Distilled water 1 litre Mix the ingredients with the water and adjust the pH to 7.5. Dispense the medium into bottles and sterilize by autoclaving at 15 psi for 15 min.

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I. MEDIA TABLES

106. Yeast agar ( H . Reichenbach,personal communication)

Supplementary to Klebisella agar (medium No. 47) 10 g 10 g freshweight 12 g 1 litre

Potatoes, peeled and diced Baker’s yeast Agar powder Distilled water

Add the diced potato to the water and steam for 30 min. Remove the particulate matter by passing through cheese cloth and restore the volume to 1 litre with water. Add the yeast and agar and steam the medium after adjusting the pH to 7.2. Autoclave the medium at 15 psi for 15 min.

107. Yeast malate medium Yeast extract Sodium malate Distilled water

5 g 1 g 1 litre

Dissolve the yeast and malate in the water and adjust the pH to 7.0. Dispense the medium in 1 oz screw-capped bottles and autoclave at 15 psi for 15 min. The medium should be freshly boiled before use.

108. Yeast malt agar (Pridham et al., 1957) Yeast extract 4 g Malt extract 10 g Glucose Agar powder Distilled water

4 g

20 g 1 litre

Dissolve the yeast, malt and giucose in the water and adjust the pH to 7.2. Add the agar, steam the medium and dispense into bottles. Autoclave the bottles at 15 psi for 15 min and use the medium as slopes or plates.

109. Yeast-peptone broth Yeast extract Peptone Distilled water

2.5 g 2-5 g 1 litre

Dissolve the ingredients in the water and adjust the pH to 7.0-7.4. Dispense the medium into screw-capped bottles (1 oz) in 20 ml amounts and sterilize by autoclaving at 15 psi for 15 min.

Note Where stated, this medium is prepared with sea water.

6

130

S . P. LAPAGE, f. 8. SHELTON AND T. 6.MITCHELL 110. Zymobacterium medium (adaptedfrom Wachsman and Barker, 1954)

Tryptone 20 g Glucose 5 g Yeast extract 0.5 g Orotic acid 2 g 0.5 g Sodium thioglycollate MgS04.7HaO 0.2 g FeS04.7Hz0 0.005 g MnS04.4HaO 0.005 g NazMoO4.2HaO 0.005 g 15 mg Riboflavin KHaP04-Na~HP04,0.05 M, pH 7.4 1 litre 15 g Agar powder Add the ingredients except the agar, to half the phosphate buffer. Check the pH and adjust it to 7.4 with NaOH. Add the remainder of the phosphate then the agar and steam the medium. After dispensing into bottles, autoclave the medium at 15 psi for 15 min. It is advisable to use the medium fresh. 111. Zymomonas medium (NCIB Records-originally from Dr. J. G. Carr)

Yeast extract 10 g 10 g Glucose Tap water 1 litre Dissolve the glucose and yeast extract in the water and adjust the p H to 4.8. After distribution in bottles, autoclave the medium at 10 psi for 20 min. It is advisable to boil the medium immediately before use.

B. Suspending fluids 112. 7.5% Glucose broth (NCTC Records) 400 ml Nutrient broth (medium No.67) Glucose (Dextrose, anhydrous D( +)) 30 g Add the glucose to the nutrient broth and mix well until dissolved. Check the pH of the glucose broth to 7.6. Distribute the medium in approximately 5 ml amounts into sterile screwcapped bijou bottles, and sterilize by autoclaving at 10 psi for 10 min. 113. 7.5% Glucose serum (NCTC Records) Sterile Horse serum (“Wellcome” Brand No. 3; 400 ml natural clot, no preservative, filtered) Glucose (Dextrose, anhydrous D( +)) 30 g When the serum is at approximately room temperature, add the glucose and gently shake the suspension (to avoid frothing) until the glucose has dissolved. Seitz-filter (14 cm &a. Seitz-pad) using gentle suction, into a sterile container. Duration of filtration using this volume is approximately 2 h. Distribute the glucose serum aseptically in approximately 5 ml amounts into sterile screw-capped bijou bottles.

I. MEDIA TABLES

131

114. Mist.desiccans (Fry and Greaves, 1951) Sterile Horse serum (“Wellcome” Brand No. 3; 300 ml natural clot, no preservative, filtered) Glucose 30 g 1-3 g Nutrient broth (Difco, dehydrated) Distilled water 100 ml

Dissolve the glucose and nutrient broth in the distilled water and add to the serum. After careful mixing, sterilize by Seitz-filtration using positive pressure. Dispense the sterile medium aseptically into sterile bottles or tubes.

BBL

VI. INDEX OF MANUFACTURERS Baltimore Biological Laboratory Inc. , 2201, Aisquith Street, Baltimore 18, Maryland, U.S.A. Agents in the British IslesBecton, Dickinson and Co. Ltd, Irish Plastic Packaging Buildings, Ring Road, Ballyfermot, Dublin 10, Eire.

BDH British Drug Houses Ltd, BDH Laboratory Chemicals Division, Poole, Dorset. Davis Davis Gelatine (N.Z.) Ltd, Christchurch, New Zealand. Difco Difco Laboratories Inc., (Bacto) 920, Henry Street, Detroit 1, Michigan, U.S.A. U.K. agentsBaird and Tatlock (London) Ltd, Freshwater Road, Chadwell Heath, Essex. Evans Evans Medical Ltd, Speke, Liverpool. Oxoid Oxoid Division of 0 x 0 Ltd, Southwark Bridge Road, London S.E.l. Sheffield Sheffield Chemical Division, Chemical National Dairy Products Corporation, Division Nonvich, New York, U.S.A. Wellcome Wcllcome Research Laboratories, Beckenham, Kent.

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J. E. SHELTON AND T. G . MITCHELL ACKNOWLEDGMENTS

The NCTC authors wish to pay tribute to Dr. Cowan under whose Curatorship freeze-drying was initiated in the NCTC and who devised many of the media and methods described in this Chapter. We also wish to thank Dr. B. E. Andrews for his help with the preparation of the Mycoplasma section. The media formulae marked “NCIB records” and “NCMB records”, have been drawn from the official records of these Collections and are Crown Copyright. The permission of the Director of Torry Research Station to quote from these records is gratefully acknowledged. We also thank Mrs. Barbara Buckton for her patience and care in typing the manuscript and without whose help the Chapter would never have been finished. REFERENCES Abd-el-Malek, Y., and Gibson, T. (1952).J. Dairy Res., 19, 294-301. hacker, R. L., and Ordal, E. J. (1959).J. Bact., 78,25-32. Annear, D. I. (1962). Aust. J. exp. Biol. med. Sci., 40, 1-8. Baalsrud, K., and Baalsrud, K. S. (1954). Arch. Mikrobiol., 20, 34-62. Bachmann, B. J. (1955).J. gen. Microbiol., 13, 541-551. Barker, H. A., Smyth, R. D., Wilson, R. M., and Weissbach, H. (1959).J. biol. Chem., 234,320-328. Batty, I., and Walker, P. D. (1965). J. appl. Bact.,28, 112-1 18. Blaylock, B. A., and Stadtman, T. C. (1966). Arch. Biochem. Biophys., 116,138-152. Bordet, J., and Gengou, 0. (1906). Ann. Inst. Pasteur, Paris, 20, 731-741. Bose, S. K. (1963). In “Bacterial Photosynthesis” (Ed. H. Gest, A. San Pietro and L. P. Vernon), pp. 501-510. Antioch Press, Yellow Springs, Ohio. Brock, T. D. (1964). Science, N.Y., 144, 870-871. Bryant, M. P., Wolin, E. A., Wolin, M. J., and Wolfe, R. S. (1967). Arch. Mikrobiol., 59,20-36. Burton, S. D., and Morita, R. Y. (1964).J. Bact., 88, 1755-1761. Casman, E. P. (1947) Am. J. clin. Path., 17,281-289. Chanock, R. M., James, W. D., Fox, H. H., Turner, H. C., Mufson, M. A., and Hayflick, L. (1962). Proc. SOC. exp. Biol. Med., 110, 884-889. de Man, J. C., Rogosa, M., and Sharpe, M. E. (1960). J. appl. Bact., 23, 130-135. Dondero, N. C., Phillips, R. A., and Heukelekian, H. (1961). Appl. Microbiol., 9, 219-227. Doudoroff, M., Kaplan, N., and Hassid, W. 2.(1943). J. biol. Chem., 148, 67-75. El-Ghazzawi, E. (1967). Arch. Microbiol., 57,l-19. Foster, J. W., and Davis, R. H. (1966).J. Bact., 91, 1924-1931. Fox, D. L., and Lewin, R. A. (1963). Can.J. Microbiol., 9, 753-768. Fry, R. M., and Greaves, R. I. N. (1951). J. Hyg., Camb., 49, 220-246. Garvie, E. I. (1967). J. gen. Microbiol., 48,431-438. Goodwin, R. F. W., Pomeroy, A. P., and Whittlestone, P. (1967). J. Hyg., Camb., 65, 85-96. Gordon, R. E., and Smith, M. M. (1953).J. Bact., 66,4148. Gyorgy, P., and Rose, C. S. (1955).J. Bact., 69,483-490. Heberlein, G. T., De Ley, J., and Tijtgat, R. (1967). J. Bact., 94, 116-1 24. Hirsch, P., and Conti, S. F. (1964). Arch. Mikrobiol., 48, 339-357. Holt, S. C., and Canale-Parola, E. (1967).,J. Bact., 93, 399410.

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Hooper, A. B., Hansen, J., and Bell, R. (1967). J. biol. Chem., 242,288-296. Horler, D. F. ,Westlake, D. W. S., and McConnell, W. B. (1966). Can.J. Microbiol., 12,47-53. Hutchinson, M., Johnstone, K. I., and White, D. (1966). J. gen. Microbiol., 44, 373-381. Johns, A. T., and Barker, H. A. (1960). J. Bact., 80, 837-841. Kane, W. D. (1966). J. Elisha Mitchell Scient. Soc., 82, 220-230. King, J. W., and Rettger, L. F. (1942).J. Bact., 44, 301-316. Klein, S. M., and Sagers, R. D. (1966). J. biol. Chem., 241, 197-205. Krulwich, T. A., and Funk, H. B. (1965).J. Bact., 90,729-733 Kiister, E. (1959). Int. Bull. bact. Nomencl. Taxon., 9,97-104. Leadbetter, E. R., and Foster, J. W. (1958). Arch. Mikrobiol., 30, 91-118. Lentz, K., and Wood, H. G. (1955).J. biol. Chem., 215,645-654. London, J., and Rittenberg, S. C. (1967). Arch. Mikrobiol., 59, 218-225. Morita, R. Y., and Burton, S. D. (1965). 2. allg. Mikrobiol., 5, 117-179. Norris, J. R. (1959). Lab. Pract., 8, 239-243. Parker, C. D., and Prisk, J. (1953). J. gen. Microbiol., 8, 344-364. Payne, J. I., Sehgal, S. N., and Gibbons, N. E. (1960). Can.J. Microbiol., 6,9-15. Payza, A. N., and Kom, E. D. (1956).J. biol. Chem., 223,853-858. Peel, D., and Quayle, J. R. (1961). Biochem.J., 81,465469. Pheil, C. G., and Ordal, 2. J. (1967). Appl. Microbiol., 15, 893-898. Poindexter, J. S. (1964). Bact. Rev., 28, 231-295. Postgate, J. R. (1963). Appl. Microbiol., 11, 265-267. Postgate, J. R. (1966). Lab. Pract., 15, 1239-1244. Pridham, T. G., and Lyons, A. J. (1961).J. Bact., 81,431-441. Pridham, T. G., Anderson, P., Foley, C.,Lindenfelser, L. A., Hesseltine, C. W., and Benedict, R. G. (1957). In “Antibiotics Annual 1956-7” (Eds. H. Welch and F. Marti-Ibanez), p. 947. Medical Encylopedia Inc., New York. Pringsheim, E. G. (1950).J. gen. Microbiol., 4, 198-209. Rabinowitz, J. C. (1963). Meth. Enzym., 6, 703-713. Rogosa, M. (1964). J. Bact., 87, 162-170. Rouf, M. A., and Stokes, J. L. (1964). Arch. Mikrobiol., 49, 132-149. Schefferle, H. E. (1965).J. appl. Bact., 28,412-420. Stadtman, E. R., and Barker, H. A. (1949).J. biol. Chem., 180,1085-1093. Stadtman, T. C., and Barker, H. A. (1951).J. Bact., 62, 269-280. Stadtman, T. C., and White, F. H. (1954).J. Bact., 67, 651-657. Stolp, H., and Starr, M. P. (1963). Antonie van Leeuwenhoek, 29, 217-248. Stuart, P. (1965). Br. vet.J., 121, 289-318. “The Oxoid Manual”, 3rd Edn, 1965. Oxoid Ltd., London. Veldkamp, H. (1960). Antonie van Leeuwenhoek, 26, 103-125. Veldkamp, H. (1961). J.gen. Microbiol., 26, 331-342. Wachsman, J. T., and Barker, H. A. (1954). J. Bact., 68, 400-404. Weeks, 0. B., and Beck, S. M. (1960).J. gen. Microbiol., 23, 217-229. Williams, M. A., and Rittenberg, S. C. (1957). Int. Bull. bact. Nomencl. Taxon., 7, 49-103. Zavarzin, G. A. (1964). 2. allg. Mikrobiol., 4,390-395. ZoBell, C. E. (1946). “Marine Microbiology”. Chronica Botanica Co., Waltham, Mass.


CHAPTER I1

Culture Collections and the Preservation of Bacteria S. P. LAPAGE AND JEAN E. SHELTON National Collection of Type Cultures, Central Public Health Laboratory, London, England AND

T. G. MITCHELL* AND A . R. MACKENZIE National Collectaon of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland

.

I. Introduction . 136 11. Culture Collections, General . . 136 A. Functions and purpose . . 136 B. Kinds of culture collection . . 137 C. Type cultures and nomenclature . . 138 D. Patent cultures . . 141 111. Operation and Management of a Culture Collection . 143 A. Summary of operation . . 143 B. Recordkeeping . . 145 C. Accessions . . 152 D. Batches . . 154 E. Supplyanddemand . . 155 F. Safety precautions with pathogens . . 159 G. Allotment of cultures . 162 H. Weeklymeeting . . 162 IV. Preservation . . 163 A. Subculture . . 163 B. Reduced metabolism and periodic transfer . . 166 C. Drying . . 167 D. Freezing . . 169 V. Freeze-drying . . 170 A. Principles of freeze-drying . . 171 B. Practice of freeze-drying . . . 171 C. The stages in freeze-drying . . 173 VI. OtherMethods . . 194 A. Separate freezing and drying for pathogens . . 194 B. Sordelli’s method . . 195 C. L-drying . . 197 VII. Methods for Bacteria Unsuited to Freeze-drying . . 200 * Present address : Research and Development Establishment, British-American Tobacco Co. Ltd., Southampton, Hants, England.

.

.

136 s.

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VIII. Stabilityof characters . IX. Viability . A. General . B. Viable count technique . C. Estimation of shelf life . D. Survival of bacteria by freeze-drying . X. List of Some Culture Collections . References

.

. .

. . . . . .

202 202 202 203 204 204 210 227

I. INTRODUCTION This chapter is divided into five sections. Firstly a general account of culture collections of bacteria is given (Section II), then an account of the day to day management and operation of a culture collection (Section 111). Methods of maintenance and preservation are next discussed (Section IV-VIII), followed by our experience of the viability of many bacterial species, maintained largely in the freeze-dried state (Section IX). Finally, a list of some culture collections in the world is given for reference (Section X). Many points concerning culture collections and a general discussion of their problems were reviewed by Clark and Loegering (1967), Martin (1963) and Martin (1964). This account is largely derived from the practice of the National Collection of Type Cultures (NCTC), and of the National Collection of Industrial Bacteria (NCIB), the management and operation of which are very similar. The authors have worked only in these two Collections and do not feel competent to discuss the preservation of micro-organisms other than bacteria. We have attempted to write a general account of our own procedures and problems rather than an additional review article. 11. CULTURE COLLECTIONS, GENERAL

A. Functions and purpose The prime function of a culture collection is the preservation of organisms, and related to this is the selection of the holding within the resources of the collection. Preservation of reference individuals is the pivot on which adequate classification and nomenclature are based. Without reference individuals, the edges of groups would blur as new tests were introduced and reference to past work would become difficult to apply. In the case of bacteria, preservation of living cultures is essential. In the case of other biological specimens, e.g., plants or animals, preservation of dead individuals may be satisfactory. Supply on demand naturally follows, so that reference cultures are available to other workers.

11. CULTURE COLLECTIONS

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Storage of a duplicate collection of cultures and key records is also advisable. Among the ancillary activities necessary to maintain a culture collection in an active, growing state and with a progressive approach are: the study of nomenclature, classification and taxonomy, and the production of catalogues; scanning of the literature and the provision of information services ; identification services; teaching and provision for visitors; membership of committees and attendance at meetings; co-operation with other culture collections; research into culture collection problems and the presentation and publication of scientific papers. All these play an important part and duty of a culture collection and help to attract high quality staff and to maintain their interest.

B. Kinds of culture collection 1. Service and other collections Culture collections range from large service collections, such as the NCTC, whose prime function is the preservation and supply of cultures on demand, to small collections kept in individual laboratories for their own use. Service collections keep organisms from as wide a range of genera and species as are within the scope of the collection, e.g., medical or plant pathogenic bacteria. These collections send cultures all over the world, are internationally orientated, and cultures are deposited in them by workers for maintenance and reference. Between the large service collections and the collection for internal use in the laboratory lie a host of intermediates. World Health Organization (WHO) reference laboratories can be cited whose terms of reference include the supply of cultures. Specialist collections, usually held by individual workers or groups, may be prepared to supply cultures as a personal favour. These specialist collections frequently maintain only one species or group of organisms and are often disbanded when the worker responsible retires or dies; the cultures may be lost unless a service collection can be found prepared to accept them. This may be a difficult problem. A culture collection may act as a bureau or register providing information on sources of supply for organisms which it does not maintain. Cultures may be prepared by other workers and distribution be undertaken by a collection; or a closer liason may exist as, for example, in the NCTC, where the suspensions of Mycoplasma are prepared in the Mycoplasma Reference Laboratory but freeze-dried and distributed by the NCTC. This avoids duplication of workers and ensures that the organisms are handled by experts in that group. It does however, in general, require location in the same building.

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MACKENZIE

2. Specialization Culture collections are usually specialized to a certain degree and do not keep organisms of every type, e.g. ,the Commonwealth Mycological Institute Collection of Fungus Cultures or the National Collection of Marine Bacteria. Specialization may be by subject, e.g., the National Collection of Dairy Organisms or by the type of organism, e.g., the National Collection of Yeast Cultures. The American Type Culture Collection (ATCC) is, however, the prime example of a Collection maintaining a wide variety of types of organism: bacteria, bacteriophages, phycoviruses, fungi, yeasts, plant rusts and protozoa. Types of larger culture collections specialized by subject commonly found are : medical, veterinary, plant pathological, fermentation, marine, industrial and dairy. Culture collectionsspecialized by micro-organisms may contain one or more of: yeasts; fungi; protozoa; bacteria; algae or viruses. C. Type cultures and nomenclature 1. Notes on type cultures For the reader’s convenience the various kinds of type culture are summarized with some relevant quotations from the Bacteriological Code (1966). Reference should also be made to Sneath and Skerman (1966) and to Cowan (1968) for a discussion of type strains. Recommendation 8a (3) states“Type is a term which has frequently been used incorrectly in order to designate a sub-division of a species, particularly in cases where the distinguishable characters are regarded as insufficient to justify the recognition of a subspecies. The term ‘type’in bacteriology (as throughout biology) should be used strictly in the sense defined in Principle 11 and Rule 9. It should not be used to designate infrasubspecific forms based on antigenic characters.” Principle 11 states“The application of the names of taxa is determined by means of nomenclatural types (nominifers). A nomenclatural type is that constituent element of a taxon to which the name of the taxon is permanently attached, whether as the accepted name or as a synonym. Note. The phrase ‘constituent element of a taxon’ for a species or a subspecies of bacteria is the type specimen, usually a type strain or culture.” The kinds of type strains areHolotype. The specimen designated as the type culture in the original description, or if the original description was based on only one strain, see Rule 9 d (1) Note 2 and Rule 9 d (2).


139

Paratype. A strain, not designated as the type strain, which was used by the author to compile his original description. Cotype (syntype). Any strain of the original author’s collection if none was designated as a type strain. Lectotype. Strain selected from the original material of the description designated later as a type strain by a subsequent author. Neotype. A strain chosen to replace a type strain which has been lost or for a group for which a type was not designated, The definition in the Code Rule 9 d (1) Note 2 (c) is of importance to authors intending to propose a neotype : “A neotype strain is one which has been accepted by international agreement to replace a type strain which is no longer in existence. It should agree with the diagnosis given by the original describer. A neotype strain must be proposed by an author in the International Journal of Systematic Bacteriology, together with a full citation, a description or reference to a validly published description and a record of the permanently established culture collection where the strain is deposited. The neotype strain becomes established from the date of publication in the Journal. Any objection should be referred to the Judicial Commission. A neotype strain shall be proposed only after a careful search for the original culture. If the original culture is subsequently rediscovered the matter shall be referred immediately to the Judicial Commission.” The code also recommends deposition of a type strain in one of the existing culture collections, see Rule 9 d (1). Reference strain. This is defined in the code as: “a culture (strain) which is neither a type strain nor a neotype strain, but which is used by an author as a standard in a study of a related group of organisms. A reference strain has no nomenclatural status.” Rule 9 d (1) Note 2 (d). Note that a type strain is not necessarily “typical” in its reactions. It is one of the strains on which the original description was based. I n the case of neotypes many authors take trouble to choose a “typical” culture provided it conforms to the original description.

2. Notes on nomenclature It was considered useful to include a short account of the categories of taxa used in bacteriology (see Bacteriological Code, 1966, for further details). The Note to Principle 7 states“A summary of the names of the categories of taxa that may be recognized

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s. P. LAPAGE, J.

E. SHELTON, T. G. MITCHELL AND A. R. MACKENZIE

in the Bacteriological Code is given below. Those names that should be recognized where pertinent in taxonomy of bacteria are printed in boldfaced type. Those which are optional are printed in Roman. The equivalent Latin is given in parentheses.

1. Division (divisio) 2. Subdivision (subdiwisio) 3. Class (CZuss;S) 4. Subclass(Subclassis)

5. Order (Ordo) 6. Suborder (Subordo) 7. Family (FurniZiu) 8. Subfamily (SubfumiZiu) 9. Tribe (Tribus) 10. Subtribe (Subtribus)

1 1. Genus (Genus) 12. Subgenus (Subgenus) 13. Section(Sectio) 14. Subsection (Subsectio)

15. Series (Series) 16. Subseries(Subseries) 17. Species (Species) 18. Subspecies (Subspecies) Variety (Vurietus)

19. Individual (Indiuiduum)” Recommendation 8a (l), (2), part of (3), and (7) state(8a) “Authors of names of infrasubspecific subdivisions of species of bacteria should attend to the following recommendations and definitions: (1) A strain is made up of the descendants of a single isolation in pure culture. It may be designated in any manner, as by the name of the individual responsible for its isolation (as Corynebucterium diphtheriue strain Park-Williams); by the name of a locality, by a number, or by some similar laboratory distinguishing mark, ‘Strain’ may also be used to designate cultures of bacteria which correspond to cultivated ‘varieties’


141

(cultivars) of higher plants, and which have some special economic significance. (2) A special form (forma specialis) is an infrasubspecific form included in an infrasubspecific subdivision of a species of a parasitic, symbiotic or commensal micro-organism distinguished primarily by adaptation to a particular host or habitat. It is named, preferably, by use of the scientific name of the host written in the genitive. (3). . ..It is recommended that wherever appropriate the following terms be used to designate infrasubspecific subdivisions of a species : Forma specialis, biotype, serotype, morphotype, lysotype (phagotype). Less commonly used are the name (terms) state, stage, chemovar, chemotype and cultivar. It is recommended that the term serotype (or serological type) be used for infrasubspecific forms based upon antigenic characters. . . . (7) The term ‘group’ in bacteriology should be used with great care and be well-defined if ambiguity is to be avoided. It has been used in somewhat different senses by those in various fields of bacteriology. ‘Group’ may be appropriately used to designate congeries of organisms having common characteristics. I n many cases the groups are based upon antigenic analysis. They are assemblages of related serotypes. The term ‘group’ has in some cases been employed to avoid the use of the correct nomenclatural name of a taxon such as genus or species. This use leads to confusion and should be avoided.”

D. Patent cultures When a novel process utilizing micro-organisms has been developed, it is now an accepted practice to obtain patent protection. A common condition for acceptance of a patent application in many countries is that the method for performing the invention must be made available to the public. The usual methods of fulfilling this condition in respect of patents citing the use of micro-organisms is to deposit appropriate cultures in an acceptable culture collection. Culture collections which act in this manner include : (a) in the United Kingdom-

(i) for cultures of fungi : Commonwealth Mycological Institute. (ii) for cultures of bacteria and actinomycetes: National Collection of Industrial Bacteria. (b) in the U,S.A.(i) American Type Culture Collection. (ii) Institute of Microbiology, Rutgers University,

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P. LAPAGE, J. E. SHELTON, T. G. MITCHELLAND A. R. MACKENZIE

(iii) Northern Regional Research Laboratories (NRRL). The addresses of these collections are given in Table VI. Details of requirements for the deposition of cultures for patent purposes in one of the above or in any other culture collections, should be obtained from the appropriate curator. In some cases charges may be made to cover the cost of maintenance of the culture or cultures. Normally the procedure consists of the following(i) The culture is deposited in the appropriate culture collection before submission of the complete patent specification. The collection must be informed of the reason for the deposition, and details must accompany the culture on methods of cultivation and maintenance. Wherever possible it is helpful if three or more lyophilized cultures can be submitted, allowing for the additional ones to be stored as original reference material. (ii) The collection allots the culture an accession number and informs the depositor, in order that this may be quoted in the patent specification. (iii) When suitable cultures have been prepared by the collection, a sample is submitted to the depositor for checking, especially in respect of the special properties claimed by him. (iv) The culture is not listed by, nor distributed from the collection until it has been informed by the depositor of the publication of the patent specification. Intending patentees should recognize that the whole onus of ensuring the continued availability of the culture is theirs and is not transferred to the collection, which merely acts as an independent distribution centre. Likewise, the collection accepts no responsibility for the ability of the deposited culture to act in the manner claimed in the patent specification. Since the rules and suggested procedures regarding patents involving micro-organisms vary from one country to another and also with time, it is essential that intending patentees obtain professional advice on the requirements for deposition of cultures relating to the countries for which they wish to apply for grant of a patent. The maintenance of patent cultures involves some method of identifying cultures which are under patent application, and those for which patents have been granted. This may be done in any suitable manner, e.g., colouring of documents and labelled tags.

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111. OPERATION AND MANAGEMENT OF A CULTURE COLLECTION

A. Summary of operation This description is a summary of the operation of the NCTC, and to a great extent of the NCIB. A flow chart giving a summary of the operation of the NCTC is given in Fig. 1. Bacterial strains deposited in a collection are referred to as accessions and are accompanied by data on the strain from the depositor. An accession is checked for purity, characteristics and, if doubt exists as to its nomenclatural status, may be referred to a specialist. Not all culture collections have adequate staff to carry out these checks, or they may be considered unnecessary and accessions may be preserved in the state in which they are received. If the checks are satisfactory, then the accession is allotted a collection number and records are made. I t is then preserved, if by freeze-drying, in a batch of ampoules which is referred to as the first batch. An ampoule is checked in the collection and if satisfactory results are obtained, an ampoule of the batch is sent to the depositor for checking. The batch is not available for issue until the depositor reports that the sample ampoule is satisfactory. When the stock of ampoules is nearlyexhausted a new batch is prepared, either from seed ampoules of the first batch or more usually from the current batch. Checks for the maintenance of viability are made on the first and succeeding batches, i.e., the stock of the culture, at pre-determined intervals. If an unusual decline in viability is found, then a new batch is prepared at whatever time is considered necessary. Tests for characteristics are carried out immediately after drying each

- E

NEW CULTURE --+

NE;,TH

___*

STOCK CARDS

DRj D

if new

MASTER CARD -Depositors check

AD check

-

STORAGE

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B D check

if special group

Reterencc laboratory check

ORDERS

if falling -REGULAR

COUNTS

FIG.1. Flow chart of the NCTC. This is a simplified flow chart to illustrate the principles of the Collection. BD, before freeze-drying, AD after freeze-drying.

BD WORKINGSHEET

NEW CULTURE I CARD

2 if low +STOCK

+

CARD

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NCTC No.allocated

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ACCESSION

FORM

-

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I

decision when next to count

if fulling-

p

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FIG.2. Flow chart of the NCTC and system of records. This chart includes full details of the system used in the NCTC. BD, before freeze-drying; AD, after freeze-drying.

1 2-4

E m


145

successive batch, to ensure that the culture has maintained its characteristics. Purity checks are carried out at all stages of the process. On receipt of requests, ampoules are despatched, packed in conformity with postal regulations. A packing note, giving the collection numbers and other data, is sent with each order, and an invoice for charges if these are levied.

B. Record keeping Various records are kept in the NCTC which are described below. Fig. 2 is a more comprehensive flow chart of the functioning and system of records used in the NCTC. 1. Master cards

Permanent records on the cultures in the collection are kept on master cards of thin cardboard (see Fig. 3), filed in Shannon Visible Record Drawer Cabinets. The results of laboratory examinations of cultures are entered first on paper working sheets, which are replicas of the master cards, and then when complete, are transferred to the master cards. The master card thus prepared contains all the information on the culture, and further details are entered on it, such as data on further batches, serological checks, checks by the depositor and so on. The master cards have been in use in the Collection for many years; the layout is useful but they do not contain all the tests used in modern bacteriology. Many additional tests are carried out, and for some groups of organisms further tests are routine practice which are not used for other groups, as for example, the special tests for streptococci or mycobacteria. A list of the tests used routinely for accessions in the NCTC is given in Table I.

2. Numerical cards A numerical card index containing all but the laboratory data on the strain is prepared for convenient reference to the cultures in the collection. Numerical cards of discarded cultures are retained. One of the primary uses of this index is for checking the numbers quoted in orders, and ensuring that the strain belongs to the species quoted on the order. It also reveals at once if a culture is not present in the Collection. If this proves to be the case, reference to the cards of discarded cultures will reveal what culture was held under that number, whether it was transferred to another collection, and also whether a strain of a given species ever possessed that number.

146

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Purple milk Egg yolk plate Citrate Gluconate Malonate Phenylalanine Starch AescuIin Urea Casein Serum liquefaction Indole

HzS Decarboxylases arginine lysine omithine

KCN

Antibiotic sensitivity Pencillin Streptomycin Cldoramphenicol Tetracycline Erythromycin Ampicillin Glucose peptone water Glucose serum water Glucose mineral salt medium Adonitol Arabinose Cellobiose Dextrin Dulcitol Galactose Glycerol Glycogen

Antigenic structure-usually determined by reference laboratory Bacteriophage type-usually determined by reference laboratory Pathogenicity-rarely tested except for the production of toxins

Inositol Inulin Lactose ONPG Laevulose Maltose Mannitol Mannose Rafthose Rhamnose salicin Sorbitol Starch Sucrose Trehalose Xylose

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T. G. MITCHELL AND A. R. MACKENZIE

3. Alphabetical index A set of alphabetical cards is kept with one or more cards for each species, on which the holding of that species is listed. The species’ cards are arranged alphabetically under each genus, and the genera are then arranged alphabetically as well. Strain names and other salient details are recorded on the cards, which are of value in fulfilling orders and for other purposes.

4. Customer cards An alphabetical index of customers is kept primarily by place name, e.g., Manchester or Sheffield, and then alphabetically under the name of the Institute. This applies to the U.K., and a similar index is kept for all other countries, which are also arranged alphabetically. Alphabetical arrangement by the names of individual customers has not proved satisfactory as they change institutes, resign or retire. Order forms from a department may be signed by more than one individual at different times, and this may be an administrative not a scientific worker. A record is kept on this card of the date, customer’s order number, collection order number, and other details.

5 . Stock cards These cards provide a record of the stock of ampoulesand are filed numerically by the NCTC numbers. A set of numbers up to 60 is conveniently printed on each card, and this provides for the majority of cultures which are dried in batches of 15 or 50 ampoules. Larger batches will require more than one card. When a batch is dried, the number of ampoules prepared is marked on the card, and the appropriate number then crossed off whenever an ampoule is removed from the stock. When the stock is depleted to a pre-set figure marked on each card, the culture is recorded as requiring a new batch by an entry in the drying book (see Section IIIB. 7 below). Details of the date and NCTC order number are entered on the relevant stock card when a culture is issued. Information is, therefore, immediately available on the number of ampoules issued and the annual demand for each culture.

6. Count cards A card index arranged numerically by the NCTC numbers is also kept, on which are recorded the results of the viable counts on the cultures. Each batch is allotted a separate card and records are kept of the media and conditions of growth before drying, suspending fluid, media for revival and other details. I n addition, culture and batch numbers of other organisms dried on the same day are noted, in case of suspected cross contamination.


15 1

7. Drying book Cultures to be freeze-dried are entered in the drying book when, for example, routine counts indicate a significant loss of viability or depletion of ampoule stock falls to the pre-set figure on the stock card. Details of the number and size of the batch to be dried are also entered, so that the drying programme may be planned by reference to this book.

8. Count book When an accession is dried it is entered in the count book under the date of drying, as are further batches when they are dried. When biochemical tests, purity checks and viable counts have been satisfactorily checked for a batch, this is entered alongside the number of the culture, and a decision made as to when the batch needs the next check to ensure maintenance of viability. In the NCTC, all batches are routinely counted at 1year, 5 years, 10 years, 15 years and so on after the date of drying. However, in certain cases, such as when a significant drop was obtained between the before and after drying count of the organism, then an earlier check is necessary, e.g., after 3 months or 6 months. Results from viable counts at various time intervals similarly determine the time for subsequent checks. Reference to this count book will indicate which cultures require examining for viability at any period, e.g., after 5 years, by reference to the same date in the book 5 years before. Reference to the count cards at the same time will enable a rota for viable counts to be compiled in a work book with details of the media, temperature of incubation, etc. 9. Biochemical book Whenever a batch is dried, and providing that results at this stage appear to be satisfactory, an entry is made in the book. This is usually done on a weekly basis providing a rota for biochemical tests to be carried out on the cultures after drying. Reference to Table I will indicate the tests routinely examined. Results from 7 day old purity plates are later recorded in this book, and also that the biochemical tests examined have proved satisfactory and details of the batch recorded on the master card.

10. Tickets In the various stages of accession, of drying and viability checks, and of preparation of further batches, cultures are accompanied around the collection by tickets on which the various checks are marked as they are performed, and the initials of the worker added. These provide control of the processing of each culture. r

152

s.


11. Discarded records When a culture is discarded, the records and cards are kept, the latter in separate filing drawers provided for that purpose, so that reference to old records is easy.

12. Packing note A packing note is typed to enclose with the ampoules which are sent to the customer. This note contains the culture collection order reference number, the sender’s reference number, the NCTC numbers of the cultures, the names of the cultures and other data required as well as the batch number of each culture supplied. If for any reason a letter is sent to the customer, a copy of the letter is also enclosed with the ampoules, as the recipient of the original letter may not be the worker actually dealing with the ampoules.

13. Invoice Normal invoice forms are made out to enclose with the cultures when a charge is levied. These are sent under separate cover. C . Accessions 1. Accession A form is sent out to be completed and returned with a culture for deposition. The following information is requested(i) Name of organism. (ii) Authority for name. (iii) Strain name or number. (iv) Name and address of sender. (v) Source of culture. (vi) Date of isolation of culture. (vii) Who isolated the culture. (viii) History of the culture, e.g., has it been received from another culture collection, who has maintained it in intervening years and so on. (ix) The reason for depositing the culture in the collection, e.g., type culture, antibiotic assay, etc. (x) Morphological, cultural and other details available. These include media and methods for maintenance and preservation, if the latter are known, and special requirements for growth such as anaerobiosis or increased carbon dioxide tension. In addition, details of other characteristics of the organism found by the sender are valuable. (xi) Copies of any reprints pertinent to the organism should be enclosed, or references given if reprints are unavailable.


153

After reception of the culture and the completed form, the culture is then checked for purity and for biochemical and other characteristics. These results are assessed in the light of the sender’s findings and if all is satisfactory the culture is allotted a number and taken into the collection. I n practice, since depositors frequently send cultures when their manuscript is about to go to press, the culture is often allotted a number at once so that the collection number may appear in the publication.

2. Checks on accessions A summary of the bacteriological checks in the N C T C is given in Fig. 4. ACCESS ION

purity, characterisation, specialist it iequired

1

viable count, purity

SUSPE N S ION

I

FREEZE-DRIED

FIRST BATCH

Y’

VIABLE COUNTS 1,5.10 and succeeding years or as required

AMPOULE

viable count, purity, charocterisat ion, specialist if required, depositor’s check

BATCH DEPLETED ampoule of first batch 4 suspension for second batch, checks as above except not usually to depositor

FIG.4. Bacteriological checks in t h e NCTC.

Viable counts are made on the suspension before drying and of a reconstituted ampoule chosen at random from the batch after drying. Biochemical tests are carried out on the culture for deposition and again on a sample ampoule after the batch has been dried; these two sets of biochemical tests are compared and scored on the master cards. Each ampoule is checked for maintenance of vacuum with a high frequency spark discharge leak detector (referred to throughout as a high frequency tester) ; and put into the storage cabinets in the collectionprovided

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P. LAPAGE, J. E. SHELTON,T. G. MITCHELL AND A. R. MACKENZIE

that no undue drop in viability is shown, the count is satisfactory, the culture pure and biochemical checks are correct. An ampoule is then sent to the depositor for checking, and ampoules are not issued until it has been reported that the sample ampoule is satisfactory.

3. Cultures not accepted Ampoules which are restricted in supply and not available to all workers are not held. The collection considers that if others are to decide to whom cultures can be sent, it is better that the individuals concerned should deal directly with each other, and the collection not be involved in correspondence as an intermediary. The NCTC does not keep patent cultures, except for the case of vaccine strains of pathogens. Patent cultures are also discussed in Section IID.

D. Batches 1. Size of batches This is determined by the expected or known demand for ampoules. Ampoules are filled in batches of 18, 54, 110 or 210 which are suitable for the capacity of our machines. We obtain final batches corresponding to approximately 15, 50, 100 or 200 ampoules, since after drying, a few may be faulty for various reasons usually indicated by failure to maintain a vacuum. Batches of this size are suitable for storage in our cabinets, whose capacity and layout were designed to hold these amounts. 2. Labelling of batches NCTC ampoules are labelled by enclosing a piece of blotting paper with the number of the culture on it. The serial number of the batch is coded using different coloured blotting paper as followsBatch 1 = white Batch 6 = orange Batch 7 = brown Batch 2 = pink Batch 8 = red Batch 3 =blue Batch 9 = yellow Batch 4 = green Batch 10 = mauve Batch 5 = grey If required, the date or name of the organism or any other detail can also be typed on the paper inside the ampoule. It is also easy to write on the outside of ampoules with indian ink.

3. Conserved ampoules Two ampoules of each batch are ringed with coloured Sellotape to indicate that they are not available for issue or to be used for any routine purpose, and these two ampoules are held in reserve.


155

4. Tests on batches Viable counts are carried out on the suspension before drying, and on a sample ampoule from the batch when each new batch is prepared. Biochemical tests are also carried out on a sample ampoule which, in addition, is checked for purity. As the batch is made directly from the previous batch, and this has been checked before by biochemical tests, it is considered unnecessary to duplicate these tests before drying the culture. The results of the tests are recorded on the working sheets, and compared with the results entered on the master cards from previous times of testing. The results of the viable counts are recorded on the count cards. Batches are referred for appropriate specialist or reference tests as required.

E. Supply and demand 1. Supply of cultures It is essential that orders be sent promptly from a culture collection. We try to attain postage on the same day as the order is received. Very rarely is supply of a culture really urgent, as it takes time to open the ampoule, revive the bacteria and subculture it one or two times to get the strain into an optimal state. Orders are not taken by telephone, as this can lead to great confusion, and places an unfair strain on secretarial staff. The wrong culture may be sent from confusion of numbers and the wrong species if this is misinterpreted from pronunciation. Fig. 5 illustrates the system of dealing with orders in the NCTC. All orders are inspected by a graduate who decides which strains shall be sent. Orders for cultures not held in the collection are referred elsewhere, either directly or by referring the customer to a collection from which they can be obtained. The majority of our orders do not contain any information as to the purpose for which they are required. This can be a great disadvantage to us as the culture which we would supply may vary under different circumstances, e.g., for vaccine production, for teaching purposes, for some property or character, or a particular strain mentioned in a publication. However in some cases it would appear unnecessary, e.g., large university orders for a wide range of species, when it is assumed that these cultures are required for teaching. However, it would be a considerable advantage if customers would give a culture collection as much information as possible on their orders. Issue is limited to one ampoule of a given strain except in special cases.

156

s. P. LAPAGE, J.

E. SHELTON, T. G . MITCHELL AND A. R. MACKENZIE

It is desirable for a service culture collection to supply cultures in the easiest and cheapest form in terms of postage. Ampoules for internal post in the U.K. are placed in cotton spools 42'- x 0.305 in. internal dia., and 0.4375 in. external dia., which are obtainable from Sidney Clifford Ltd.t A cotton wool plug is placed in one end of the cotton spool, then the ampoule is inserted and held in place by a further cotton wool plug, and finally small corks are placed in each end. These spools are very strong and breakage of our ampoules has been very rare. The ORDER EDITED BY GRADUATE

I

-

.c

check numbers NUMERICAL CARDS check species ALPHABETICAL CARDS select strains NCTC RECORDS, CATALOGUE refer elsewhere OTHER CATALOGUES

TYPE PACKING NOTE

-

ENTER ON CUSTOMER CARD

SELECT AMPOULE(S1

-

ENTER ON STOCK CARD

1

1 1

VACUUM TESTED PACKED

1

DESPATCH

FIG.5. Order system in the NCTC.

cotton spools are then placed in padded envelopes which are obtainable in a variety of sizes from the Jiffy Packaging Company Ltd.$; the NCTC has found the envelope size, 5 x 10 in., convenient for 3-15 ampoules, Larger numbers can be packed in cardboard boxes, although these are not suitable for overseas mail (see Section IIIE.3). Small hollowed-out wooden boxes may be excellent but are expensive. A slip describing the method of opening ampoules is enclosed with every order (see Section VC.13). A note giving particulars for certain organisms may also be included, e.g., virulent strains of Mycobactmiurn tuberculosis.

t Sidney Clifford Ltd, Fortown Green, Huddersfield, Yorkshire. 1Jiffy Packaging Company Ltd, Winsford, Cheshire.


157

2. Restrictions in supply Apart from the postal regulations and quarantine restrictions which are discussed in the next two sections, there are certain conditions of supply of pathogens by the NCTC. (i) Cultures are supplied at the discretion of the Curator. (ii) Pathogens are not sent to countries in which they do not naturally occur unless there is a special reason. (iii) Pathogenic cultures are only sent to responsible workers, experienced in the handling of pathogens. (iv) Ampoules are not supplied to private addresses unless there is a special reason. If we receive an application from a private address we usually ask the customer to write on official headed paper with reference to the institute or firm in which the work will be carried out. Many of these requests are from schoolchildren who are asked to refer the matter to their teacher. This restriction has to be placed on cultures which are pathogenic for obvious reasons. I n addition, it protects the collection against unnecessary supply of cultures which will not be used properly.

3 . Postal regulations The following details were kindly supplied by the G.P.0.t “Perishable Biological Substances in the Overseas Postal Service Regulations prescribed by the Universal Postal Convention signed at Vienna, 1964,coming into force on 1st January, 1966.

1. Perishable biological substances must be packed as described below, and may only be sent in letter packets to and from recognized laboratories. The names and addresses of the laboratories both sending and receiving the item must appear on the outer box and also on any outer wrapping. The address of the sending laboratory should be distinct from the address to which the packet is sent, and it should preferably be to the left of and at right angles to the name and address of the addressee. 2. Perishable biological substances consisting of living pathogenic micro-organisms or of living pathogenic viruses must be enclosed in a bottle or tube of glass or plastic materials with thick sides, well stoppered, or in a sealed phial. This container must be impermeable and hermetically sealed. It must be surrounded with a thick and absorbent material

t Operations and Overseas Department, Overseas Mails Division, Postal Headquarters, London, E.C.I.

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SHELTON, T. C . MITCHELL AND A. R. MACKENZIE

(cotton wool, swan’s down cloth or flannelette) wrapped round the container several times and bound both above and below it so as to form a sort of cocoon. The container so wrapped must be placed in a solid, well-fastened metal box. The absorbent material placed between the inner container and the metal box must be of sufficient quantity to absorb, in case of a breakage, all the liquid contained, or capable of being formed, in the inner container. The metal box must be made and fastened in such a way as to make any contamination of the outside of the box impossible, and it must be wrapped in cotton or spongy material and, in its turn, enclosed in a protective box in such a way as to prevent any movement. This outer protective box must be hollowed out of a block of solid wood, or made of metal or some other material of equivalent strength and construction, and it must have a well fitting lid fastened so that it cannot open in course of transmission. A violet coloured label bearing the serpent symbol and the words ‘Perishable Biological Substances,, ‘DANGEREUX’ must be affixed to the address side of the outer box as well as the outer wrapping if there is any. 3. Perishable biological substances which contain neither living pathogenic micro-organisms nor living pathogenic viruses must be packed in an inner impermeable container with an outer protective container, and with absorbent material placed either in the inner container or between the outer and inner containers. This material must be of sufficient quantity to absorb, in case of breakage, all the liquid contained, or capable of being formed, in the inner container, and the contents of both inner and outer containers must be packed in such a way as to prevent any movement. A violet coloured label bearing the serpent symbol and the words ‘Perishable Biological Substances’ must be affixed to the address side of the outer container as well as the outer wrapping. 4. With both classes of perishable biological substances, special provision, such as drying by freezing or packing in ice, must be made to ensure the preservation of substances sensitive to high temperatures. Air transmission, which entails changes in atmospheric pressure, makes it necessary that the packing should be strong enough to withstand these variations in pressure. 5. Specimens of all the containers and packing materials to be used must be submitted to the Postal Services Department in order to ensure that they comply with the above regulations. 6 . The special violet coloured labels-as prescribed in paragraphs 2 and 3, are obtainable from the Postal Services Department. Note : Packets containing perishable biological substances may be subject to customs control. It is, therefore, recommended that they should

11. CULTURE COLLECTlDNS

159

have a green customs label which is obtainable at any post office which accepts parcels for abroad. Packets containing perishable biological substances not complying with the above regulations are liable to be stopped and destroyed”. Biological specimens are sent in a special postal bag and precautions are taken in case of accidents. The necessity for these regulations is obvious, and post office workers are specially at risk if they are flouted, which is unfortunately not infrequent. Under no circumstances should dangerous material be sent by ordinary mail.

4. ImportlExport regulations Many countries have strict import control on biological specimens. These are often ignored when cultures are requested, frequently in ignorance, but in some cases licences are obtained. There is usually little difficulty, presumably due to the understanding of the officials concerned. However, regulations do exist and should be observed. It would be difficult for a service culture collection, supplying many countries as it does, to familiarize itself with the regulations and restrictions of every country supplied. We feel that this conformation with the regulations, and being informed of their rules, is primarily the duty of the person requesting the supply of cultures, although the culture collection should make efforts to obtain as up-to-date details as possible. Technically, an assurance could be demanded from each overseas customer that a licence had been arranged, or was not needed.

F. Safety precautions with pathogens Commonly used substances in bacteriological laboratories with which the usual care should be taken are explosive gases, liquids or solids; carcinogens; poisonous substances and anaesthetic gases; dangerous drugs ; radioactive compounds, etc. I n addition there are certain special precautions to be taken in a laboratory dealing with pathogenic organisms. These may be detailed under the headings Personal Hygiene, Handling of Bacteria, Laboratory Animals, Medical Precautions and Laboratory Design. T h e latter is outside the scope of this article. Reference should be made to Medical Research Council (1960) and Phillips (1961). For a fuller discussion of Laboratory safety see Darlow (this Series, Vol. 1). 1. Personal hygiene (i) Always wear protective clothing such as a laboratory coat, or preferably a gown; other special clothing should be worn when required.

160

s. P. LAPAGE, J. E. SHELTON, T. G. MITCHELL AND A. R. MACKENZIE

(ii) If the laboratory coat is badly contaminated and unsuitable for wear it should be autoclaved or disinfected. When changed, as it will inevitably have been contaminated during ordinary bench work, it should also be autoclaved or disinfected before being sent to a public laundry. (iii) Whether the same clothing is worn for work or special clothing is kept for use in the laboratory is often a personal matter forthe worker. Some workers carry the latter out and it would be advisable under certain circumstances. (iv) All kinds of hand to mouth operation should be forbidden, e.g., eating and smoking; licking gummed labels; sucking pencils or pens and mouth pipetting of any kind. (v) Nails should be kept short and the hands washed at the end of operations and very thoroughly at the end of the day and before meals. If necessary they may afterwards be treated with a suitable disinfectant. Many bacteria, but not all, die off on clean skin in 5 or 10 min. It is advisable to allow time to elapse before eating, and not to eat with the fingers immediately after laboratory work.

2. Handling of bacteria (i) Nothing infected should be put down the sink. (ii) All loops should be flamed to red heat. (iii) Used and discarded cultures should be placed in special containers, bins or buckets for autoclaving at 201b (125°C) for 20 min. (iv) The contents of discard bins should not be searched. (v) Pipettes, when finished with, should immediately be discarded into disinfectant and not laid down on the bench. (vi) Slide smears even when fixed by heat, may remain infective. Mycobacterium tuberculosis and Bacillus anthracis have been recovered from slides stained by Ziehl-Neelsen or Gram stains respectively. Therefore slides should be discarded into disinfectant after microscopy. (vii) Safety cabinets should be used for operations considered dangerous, and particularly in the handling of organisms likely to cause laboratory infections. (viii) The opening of freeze-dried ampoules is particularly hazardous. Inhalation of large doses directly to the lungs may occur from breakages if ampoules are opened carelessly, and particularly from not allowing the vacuum to fill gradually. (ix) Any operation likely to cause breakage of glass in your hand should be avoided, e.g., breaking open a freeze-dried ampoule or pushing vigorously a rubber teat on to a pipette.


161

(x) Slides or cultures should not be propped up casually on the bench, but should be placed in a rack or suitable container. (xi) When flaming a loop in a bunsen burner, if it is heavily charged, be careful that it does not scatter particles of the culture all over the bench and your clothing. This is particularly liable to happen with mycobacteria and some other organisms. Special bunsen burners can be obtained with a conical guard over the flame for such work. Aerosols containing pathogenic bacteria may be inhaled; an aerosol is easily created in the laboratory by shaking cultures, by opening screw capped containers, by any form of homogenization, and by similar operations. (xii) If a tube is broken accidentally or any other accident causing dissemination of the culture in the laboratory occurs, mop with a strong disinfectant and discard the swab. Beware of attempting any operation which may put broken glass in your fingers. Common sense must be used in dealing with accidents, e.g., if a tube breaks in the centrifuge, the whole of the inside of the centrifuge must be disinfected, as an aerosol will have been created which spreads throughout the centrifuge. (xiii) It is advisable to swab the bench down with disinfectant after operations are finished, and at the end of the day. 3. Laboratory animals The handling of animals infected with pathogenic bacteria is especially dangerous since the virulence of the organism may be heightened. Great care should be taken during post-mortem examinations, when sharp instruments are handled, to avoid any direct inoculation of a pathogen into the worker. Suitable protective clothing should be worn and rubber aprons, masks and gowns are usually advisable. Splashes may arise when carrying out post-mortems particularly if abscesses under pressure are opened. T h e eyes should if necessary be protected. A bite from an animal should be reported at once. Parasites of the animals, e.g., lice and fleas, may be contaminated with pathogenic bacteria so precautions should be taken. Animal carcasses should be autoclaved or incinerated. It is essential to wash your hands before and after handling animals and rubber gloves may be worn. Those concerned with laboratory animals should consult the references given above and standard works on the subject. 4. Medical precautions Vaccination is suggested for workers with pathogens. I n the NCTC our workers receive TAB (typhoid, paratyphoid A and B vaccine), tetanus toxoid and, on joining the staff, a tuberculin skin test followed, if they are 7

162 s.


not immune, by BCG vaccination. The list can be extended depending on the organisms handled in the laboratory and special vaccinations may be required with highly pathogenic organisms, e.g., tularaemia or rickettsia. Any laboratory injury, however minor, should be reported to a senior person and treated seriously. Cuts and abrasions should be covered and infection avoided. If an accident occurs immediate administration of antibiotics or antisera may be necessary, but medical advice should always be sought.

G. Allotment of cultures In some collections groups of cultures are allotted to particular workers for care and maintenance. At the NCIB approximately 350 cultures are allotted to each scientific worker. The number allotted depends, of course, on the nature of the cultures, the amount of work required in their conservation and particularly on whether extensive checking is undertaken. Specialization of workers within a collection is a matter for the internal organization of the department. It must not be so rigid that a general worker is lost to the collection, particularly at holiday times or in case of sickness. Nor must other workers be unable to carry out the work of a specialized worker when required. In addition, every effort must be made to see that the worker concerned keeps up to date in other fields, otherwise over-specialization may be detrimental.

H. Weekly meeting Once a week a discussion meeting is held in the NCTC, at which all members of staff are present, in which the plates of the biochemical checks, routine viability counts and viability counts of batches dried during previous weeks are examined. We keep the plates in a 22°C incubator for one additional week after work is finished on them, in order to allow any contaminants that may be present to become evident. Discussion takes place about these plates, and the accompanying records and biochemical results are compared with those of previous batches. Problems are discussed and sometimes experimentsinitiated. This meeting is found to provide a valuable quality control of the conserved cultures. The most difficult problem which arises regularly is the presence on the plates of several colonial types. This occurs particularly with some genera, notably Bacillus and Clostridium.It is often difficult to decide whether these are merely variants of the same organism, or contamination by a related organism, This is one of the difficulties of maintaining pure cultures especially with strains which may be mixed on deposition. Single colonies may be subcultured and tested for biochemical characters and compared with previous batches and with the results from other


163

colonies. Differentiation of closely related organisms may require specialized checks in a reference laboratory. Obvious gross contamination presents a different problem and is usually detected at once. Contamination is often seen better on the plates of the viable counts than on spread plates, where growth of the culture often occurs at the expense of the contaminant. Contaminants present in low concentrations may only appear on the low dilutions of the suspensions. Great experience is needed to differentiate colonial variation from contamination in some cases. IV. PRESERVATION

A. Subculture There are many ways of preserving bacteria; some species are delicate and require special methods, others will stand most methods. The traditional method of preservation is regular sub-culture. Some species require transfer after days or weeks, others after months or years. Subculture has great disadvantages, notably the following. 1. Mislabelling This is a frequent event, as after many transfers the name or designation of the culture may become so distorted as to be unrecognizable. It can be avoided by pre-printed or pre-written labels.

2. Contamination This is all too frequent however much care is taken, and many tubes regularly subcultured become contaminated, especially by Bacillus spp. 3. Inoculation with the wrong organism Transfer of a series of organisms to a series of tubes is tedious and it is difficult to achieve concentration for any lengthy period. It is easy to put the wrong organism in the wrong tube, and equally easy to put the same organism into a series of tubes which are intended for others. Errors of this nature cannot be wholly prevented, nor can the possibility of forgetting to flame the loop between cultures. One safeguard is to randomize tubes, e.g., to subculture 100 strains, the tubes from which they are to be subcultured are arranged in order and the tubes to receive the subcultures are randomized, so that the correct tube to receive each subculture is then found from the random basket in turn. This way concentration is maintained and one is less prone to error.

4. Loss of the culture This is frequent with delicate organisms often for unspecified reasons, e.g., differences in media or fluctuations in the incubator temperature may

TABLE I1

r

Maintenance by subculture in the NCTC

$

Genus

Acetobacter Actinobacillus Actinomyces Alcal&nes Bacillus Bacteroides Bordetella Brucella Chromobacterium Citrobacter Clostridium Corynebacterium Enterobacter Erystpelothrix Escherichia coli

"$

Transfer after :

2/12 1/12 1/12 6/12 1 year

Medium Malt extract+chalk Semi-solid s e w agar under paraffin oil Semi-solid serum agar under paraffin oil Lemco agar Lemco peptone agar

Notes

? m

B. breuis: 3/12 on meatless

14

peptone agar

1/52 211 2 2112 2112 6/12 1 year 2/12

6/12 2/12 6/12

Serum agar under paraffin oil Bordet-Gengou agar Glucose agar Mannitol agar Lemco agar Cooked meat medium Cooked meat medium Lemco agar 0-5% nutrient agar Lemco agar

Keep at 4°C. B. bionchiseptica: 1 year on Dorset's egg medium

P 0

Ei F r

C . acnes: 1/12 in glucose agar shake

p

5 0

5 E

Genus

Haemophilus Klebsiella Lactobacillus Locflerella Micrococcus Mycobacterium tuberculosis Mycobacterium, saprophytes Neisseria Pastewella Proteus Pseudomnas Salmonella Serratia Shigella Staphylococcus Streptococcus Streptomyces Vibrio

Transfer after:

1/12 6/12 1/52 or 2/12 1/12 2/12 2/12 6/12 1/12 1/12 3/12 3/12 2 years 2112 2 years 2-6/12 2/12 1 year 1-4/52

Medium Serum agar and peptic blood digest under paraffin oil Lemco agar Milk Soyabean Glycerol agar Gelatin Dorset’s egg medium Glycerol agar Serum agar under paraffin Lemco agar Lemco agar & strength Lemco agar Dorset’s egg medium Mannitol agar Dorset’s egg medium Lemco agar Cooked meat medium 10%glucose agar Pond-water agar

Notes Keep at 37°C

L. bulgaricus: 1 week in milk I

0

Keep at 37°C M. johnei: special medium (see Chapter I) Keep at 37°C

“X” strains: 1 year

2 C

8 r

z

c,

=! 0

Disinfectant-testingstrain of S. typhi: 1/12 on Lemco agar

S. lactis :in Soyabean

3

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s. P. LAPAGE, J. E. SHELTON, T.

G . MITCHELL AND A. R. MACKENZIE

be responsible. Large numbers of cultures may be lost if a laboratory accident occurs.

5. General It is advisable to maintain an organism in two tubes, and when the original culture is prepared both tubes are inoculated. One tube only is used to prepare any subculture for issue, the second tube is kept as reserve and never opened except when the other tube proves unsatisfactory. Whenever the second tube is opened, then it is immediately subcultured to two tubes, one of which is again held in reserve. It is advisable to subculture from the whole slope, or if from a plate to take 50 to 100 colonies; neither the mass of the growth which increases the chance of unseen contaminants, or single colonies should be used. Chances of genotypic or phenotypic variation are less by this method. If a series of subcultures are made by picking single colonies successively over the months or years, a gradual change in the character of the organism can be detected. Selection of variants is one of the grave disadvantages of subculture as a method of preservation. In all methods of subculture the tubes must be tightly sealed. This can be achieved by waxed corks or bungs, sealing the glass, etc. Caps with rubber liners should be tightly screwed but the method is risky as the occasional cap is not airtight and the culture dries up. Note that polythene bottles are not gas tight and water loss takes place through the polythene itself. Caution should be exercised with long term storage in plastic containers. Table I1 gives a list of some common bacterial genera, suggesting times of subculture and recommended media. It is derived from the records of the NCTC and from Cowan (1953), but the authors have had limited practical experience of this method of preservation as a routine.

B. Reduced metabolism and periodic transfer 1. Metabolic state of the organism Frequency of subculture can be reduced by keeping the organism in a reduced metabolic state. This can be achieved by using a medium with minimal nutrients, by layering the culture with paraffin oil and by storage at low temperatures.

2. Reduced metabolic rate An excellent medium is the Meat Extract Agar described by Kauffmann (1966). Cultures stabbed into this medium in tubes and sealed with wax corks will survive for many years at domestic refrigerator temperatures.


167

The principle of this type of medium is that reduction of the nutrients to a minimum maintains a reduced metabolic rate. Other similar media can be devised or found in the literature. 3 . Storage under liquid parafin (Lumihe and Chmrotier, 1914) This simple technique is an effective way of extending the storage life of many micro-organisms, otherwise maintained by serial subculture. After growing up the culture in an appropriate medium, which may be an agar slope, sloppy agar or broth, sterile liquid paraffin is added to cover the medium completely to a depth of at least one inch. When agar slopes are used the liquid paraffin layer should extend to at least half an inch above the upper end of the agar. Medicinal grade paraffin should be used. The paraffin covered cultures are stored at room temperature or, preferably, at 0-5°C. Many bacteria maintained by this method may survive for periods of years and a useful attribute is that the method may also be employed for the maintenance of fungi. The disadvantages of this method are the inconvenience of using liquid paraffin, the dangers of contamination if only single tubes of each strain are maintained, and the storage space required if multiple tubes of each strain are kept.

4. Storage in sterile soil This has been widely used, particularly for spore bearing bacteria. Suspensions of organisms are added to sterile soil, the mixture is dried at room temperature and stored in the refrigerator. Sterile sand, silica gel and many similar compounds have also been used.

5 . Miscellaneous methods Other methods of storage may be used such as maintenance in cell lines, or in living animals, The authors have had no experience of these or similar methods. C . Drying 1. General Drying has been used as a method of preservation for many organisms. It ranges from the simple drying of spores on threads or by other techniques, to drying under various conditions in a desiccator with or without a vacuum. Many bacteria withstand drying well whilst others are more sensitive, e.g., Neisseria, Vibrio and Pseudomonas spp. 2. Paper discs An easy method of drying cultures on paper discs, and enclosing the discs between two layers of clear plastic material, using a simple machine, has

168

s. P. LAPAGE, J. E. SHELTON, T.

G. MITCHELL AND A. R. MACKENZIE

been developed by Coe and Clark (1966). It is ideally suited for the cheap transport of large numbers of cultures by post. Coe and Clark developed the method for reference strains of Staphylococcus aureus which will remain Tiable for about six months. 3. Drying in gelatin discs This method of preserving cultures originally described by Stamp (1947), is particularly useful because specialized equipment is not required, allowing it to be carried out in almost any laboratory. A wide range of the commoner heterotrophs may be expected to survive by this technique, but there is a relative lack of data on long term preservation using it, and small variations in storage conditions, which are not readily standardized, may have a marked effect on the viability. (a) Materials. (i) Nutrient gelatin. Gelatin powder (Oxoidt or Difcol) 10% w/v is dissolved in a nutrient broth (formula 71 described in Chapter I is satisfactory) and 0*25y0w/v ascorbic acid added. It is dispensed into tubes in 2-5 ml amounts, depending on the final number of discs required. The tubes are sterilized by autoclaving at 15 lb for 15 min (121°C). If the nutrient gelatin is not to be used immediately, it is recommended that the ascorbic acid be omitted and added in an appropriate amount from a concentrated sterile solution just before use. (ii) Circles of waxed paper to fit in glass Petri dishes, or glass Petri dishes smeared with a silicone fluid (Collins, 1967). The dishes are sterilized by autoclaving at 15 lb for 15 min (121°C). (iii) Vacuum desiccator containing phosphorus pentoxide. (iv) Sterile capillary pipettes. (v) Sterile 1 oz screw-cap bottles. (b) Method. A culture of the organism to be preserved is grown on slopes of a suitable nutrient medium. A thick suspension of the grown culture is prepared in a little broth, and then added to a tube of nutrient gelatin at 30°C to give a density of 1010 or more organisms per ml. Using a sterile capillary pipette, drops of the suspension are spaced out on the sterile wax circle or siliconized glass surface. When the required number have been prepared, the complete dish is transferred to the desiccator and the air is evacuated with a vacuum pump. When dry, the discs are removed aseptically to sterile screw-capped bottles which are stored at 0-5°C. To revive a culture, a disc is removed aseptically with a sterile forceps and placed in a suitable liquid medium.

t Oxoid Division of 0 x 0 Ltd, Southwark Bridge Road, London, S.E.l.

1Difco Laboratories Inc., 920 Henry Street, Detroit 1, Michigan, U.S.A. UK agents: Baird and Tatlock (London) Ltd, Freshwater Road, Chadwell Heath, Sussex,.


169

D. Freezing 1. General Most bacteria can be readily frozen and preserved in the frozen state. This depresses the metabolic rate and at very low temperatures the available energy for biochemical reactions is very small. General principles are(i) The rate of cooling should be low down to -2O"C, and then as rapid as possible until the storage temperature is reached. (ii) The rate of rewarming should be as rapid as possible. (iii) Electrolytes should be kept to a minimum. (iv) Adjuvants such as glycerol or dimethylsulphoxide which protect during the freezing process may be added to the suspending medium. This is usually not required with bacteria but they may be needed for other materials. (v) Protection by the presence of sugars may be afforded. (vi) Bacteria may be roughly divided into frost resistant, to which the majority of common bacteria belong and frost sensitive, e.g., Neisseria or Haemophilus spp. T h e frost sensitive bacteria may stand supercooling and, if taken rapidly to a low temperature without phase change, can survive at low temperatures. 2. Storage at domestic refrigerator temperature ( + 4°C) At this temperature many bacteria will live for years, and longer than at room temperature or in the 37°C incubator. A few bacteria, e.g., Neisseria gonorrlzoeaeand Haemophilus spp. require maintenance at 37°C in subculture. 3 . Storage at lower refrigerator temperatures ( -20°C) Storage may be attained successfully at these temperatures but care has to be exercised as one is in the temperature range of eutectic mixtures with water.

4. Storage in solid carbon dioxide ( -70°C) This is preferable to -2O"C, but even at these lower temperatures there may be some loss of viability. 5. Storage in liquid nitrogen Ultra-low temperatures, attained by the use of liquid nitrogen, have been successfully employed to preserve a wide range of biological cells. The latter include(i) Fungi, including myxomycetes and rust urediospores (Hwang, 1960; Wellman and Walden, 1964; Davis, 1965; Davis etal., 1966; Goos et al., 1967).

170

s.


(ii) Bacteriophages (Clark et al., 1962). (iii) Protozoa (Diamond, 1964). (iv) Algae (Hwang and Homeland, 1965). (v) Mammalian cells (Stulberg et al., 1958). (vi) Bacteria (Ashwood-Smith, 1965 ; Bridges, 1966). This method appears to be of wide applicability and should commend itself particularly to laboratories called upon to handle a variety of types of cells. It also appears possible that by using this method many of the bacterial species which have so far proved refractory to other long term preservation techniques will be successfully handled. Some points which require consideration in connection with the use of liquid nitrogen freezing are(i) The complete apparatus required is comparable in cost to freezedrying plant and may be more expensive, particularly if automatic replenishment of refrigerant is required. (ii) Where numbers in excess of a few hundred ampoules are being considered, space requirements for storage can be considerable. (iii) In order to obtain consistent results, controlled conditions of both freezing and thawing are required (Goos et al., 1967). (iv) Unless appropriate precautions are taken there is a risk of explosions occurring on removal of ampoules from storage. A useful summary of the precautions to be taken has been given by Greenham (1967). (v) Where a major interest lies in the preparation of cultures for distribution, as in a service culture collection, the product is less satisfactory than dried preparations. As the authors lack direct experience of this method, no technique is given but details of successful methods for employment with the different cell types can be found in the appropriate references. V. FREEZE-DRYING Freeze-drying has been in widespread use for many years for the preservation of many types of micro-organisms, particularly bacteria and actinomycetes. Certain types of fungi, bacteriophages and viruses also survive in varying degrees, but preservation of filamentous fungi, algae, protozoa, mammalian cells and some bacteria is unreliable. Also known as lyophilization, this method is eminently suitable for a service collection. Large numbers of ampoules can readily be produced which are light in weight and easy to despatch. Viability and maintenance of characteristics are successful with the majority of species and genera. Two main methods of freeze-drying are used: (a) centrifugal, in which a


171

low speed centrifuge is used to overcome frothing until the material is frozen and (b)pre-freezing before connectionof the ampoulesto the vacuum system. For reviews and discussion of the more theoretical aspects of freeze-drying readers are referred to Symposium (1960)) Smith (1961)) Rey (1964), Meryman (1966) and Rey (1966). The following account is based on the methods used in our two Collections and of the viabilities we have obtained. Our experience of freeze-drying has largely been concerned with the centrifugal type, and the survival times quoted in the tables in this Chapter refer to this method, unless otherwise stated. Reference to other aspects of preservation including freeze-drying, and of methods for special groups of organisms which we do not keep will be found in the references given above and in other Chapters of this Series.

A. Principles of freeze-drying In general, it may be said that the freezing process damages living material by the formation of ice crystals, and by the presence of electrolytes in high concentrations as the ice crystals separate out. Both these processes can remove water from proteins and DNA, and damage them. The principle of freeze-drying is simple. The organism is first frozen, and then water is removed by sublimation from the ice as vapour. It should be noted that the suspension is not in the liquid state and that this preserves the structure and prevents damage. Reconstitution of freeze-dried material is rapid, as the original shape is preserved, and a very large surface presented for rehydration. B. Practice of freeze-drying Cells of the organism to be preserved are suspended in a medium designed to afford protection against freezing and drying injury. The initial freeze may be attained by various methods such as carbon dioxide and alcohol, salt and ice, or dielectric metal blocks, before subsequent drying. It may also be achieved in the chamber of an evaporative freeze-drying machine, in which the latent heat of evaporation of the water vapour coming off the material is sufficientto freeze it. Such a machine may be simple, as for example, a desiccator attached to a pump, or an elaborate piece of machinery. In a vacuum chamber, fast evaporation and rapid cooling takes place. Centrifugation may be carried out to prevent foaming as the air is drawn off during the initial vacuum. It is essential that there be a good vapour flow through the machine. As water vapour is removed from the suspension, the temperature of the latter

172

s. P. LAPAGE, J. E. SHELTON, T. G. MITCHELL AND A.

R. MACKENZIE

falls until it freezes and further drying occurs by sublimation. A desiccant is added to absorb the water vapour, such as phosphorus pentoxide or molecular sieves. In the large machines a refrigerated condenser is provided on which the water vapour condenses as ice, since the volume of water vapour subliming from a large amount of material would need very large quantities of chemicals to absorb all of it. The material may be heated to hasten evaporation, and as long as there is water in it, it will remain frozen by action of the vacuum. However, the temperature should be kept below -30°C as above this temperature eutectic mixtures may form. As soon as the material is dry the temperature will begin to rise. Note that the heating of the chamber does not affect the protein in a frozen state but hastens the evaporation, and when dry the protein can be heated within limits. The temperature in the chamber may be 60-70°C but it is advisable to cease heating when the final temperature in the ampoule is about 304°C.It should be noted that in our machines the chamber temperature is measured by a thermometer in the chamber wall and does not register the temperature in the ampoules. In some systems, after the primary dry in a machine, the material may be removed to another machine for a secondary dry to remove residual water, whilst in others the whole process takes place in one machine. I n all these discussions bound water is not considered. Methods vary and in the NCTC primary drying is achieved on a centrifugal machine with a condenser, ampoules are then constricted and removed to a secondary dryer using phosphorus pentoxide as a desiccant, and further drying overnight is carried out. After the primary drying the ampoules contain about 5-10% of water, and after the secondary drying approximately 1-2% of water, depending partly on the concentration of the sugar in the suspending fluid. Complete drying of bacteria is said to affect the viability unfavourably. The ampoules are finally sealed under vacuum which has the advantage that the vacuum in the ampoule may afterwards be tested with a high frequency tester, which readily demonstrates imperfectly sealed ampoules. In some processes, ampoules may be sealed in an inert gas such as dry nitrogen. This practice is not adopted in the NCTC as maintenance of the integrity of the ampoule cannot afterwards be tested by any simple method, such as that afforded by testing for a vacuum, and any flaws or leaks might remain undetected, However, an ampoule sealed under atmospheric pressure, and not maintaining a vacuum, is safer to open; it is also less likely to contamination with organisms by the incoming air when the vacuum is released. Centrifugal freeze-drying was developed by Greaves (1944), and commercial equipment for use with this method is available from several sources for large or small scale application.


173

The technique described here is based on the experience of the NCTC and NCIB, using the large centrifugal freeze-dryers models 3P, 30P (Fig. 10) and 30P2 (Figs. 11 and 12) manufactured by Edwards High Vacuum Ltd.? A very similar procedure is followed using the smaller model 5PS (Figs. 13 and 14) which is frequently used in laboratories not specializing in freezedrying. The capacity of the former models are in the region of 200-1140 ampoules depending on the head used, and in the Edwards 5PS a maximum of 48 ampoules.

C. The stages in freeze-drying The stages in freeze-drying will first be discussed, illustrated by reference to the large model 30P, but they are also applicable to the 3P and 30P2 models which only differ in minor detail. The discussion will bring out all the points and make reference to the small model easy. An overall flow chart of the freeze-drying process used in the NCTC is given in Fig. 6.

1. Be-drying cultural conditions These are given in detail in Chapter I. When freeze-drying is used as a method of preservation, the anticipated life of the culture is usually reckoned in years. Therefore, extra care in checking cultures for purity beforehand is a sensible precaution which helps in avoiding problems at a later date. In general, the culture to be preserved should be grown on a medium giving good growth, and allowing the cells to be easily harvested. A few agar slopes are all that is needed in the case of many common heterotrophs. Evidence is available that variations in the constitution of an apparently suitable medium may have marked effects on the viability of some organisms with respect to freeze-drying. It has also been demonstrated that the age of the culture is important, cultures which have passed the logarithmic growth phase surviving better than cultures harvested in an actively growing phase (Fisher, 1963 ;Annear, 1954). However, the most favourable stage of growth may vary for different species and may also vary with different conditions of freeze-drying. Since survival bears a proportionate relationship to the size of the initial population, relatively dense suspensions of organisms should be used for optimal results. Consequently, the number of slopes or the volume of broth used to grow the organism prior to preservation must be varied. Using Escherichia coli, two 10 ml slopes of nutrient agar should grow sufficient cells to give the required density of > 1010 viable cells per ml in 2-2.5 ml of suspendingfluid.

t Edwards High Vacuum Ltd, Manor Royal, Crawley, Sussex.

174

s. P. LAPAGE,J. E. SHELTON, T. G. MITCHELL AND A. R. MACKENZIE AMPOULES PREPARE I A B E L S

J

WASH ADD LABELS L PLUG (obror bent cotton wool I

INCUBATE

1

1

HARVEST

STERILIZING SOX

I

+ 1 AMPOULES

FILL AMPOULES rithrr

REMOVE PLUGS, PLACE IN . CENTRIFUGE HEAD

PUT

1

\7

INOCULATE CULTURE

t

C-- AUTOCLAVE

or

(choice of centrifuge head 1

PLACE IN CRUET STAN0

1

1

REMOVE PLUGS, PLACE IN CANISTER, CAP WITH GAUZE

I

FRAMF

PLACE HEAD I N DRYING CHAMBER

1

CENTRIFUGE ON

4

APPLY VACUUM

s TORR, CENTRIFUGE OFF

WHEN 0.1 TURN OF8 MACHINE REMOVE CENTRIFUOE HEAD, REMOVE AMPOULES P PLUG (non-absorbent atton wool)

1

CONSTRICT AMPOULES

4

PLACE ON SECONDARY DRYER SEAL U N ~ RVACUUM

1

‘yy;rY

1

TEST VACUUM, CHECK AMPOULES S T o L

FIG.6. Flow diagram of the freeze-drying process.

In general, Gram-positive bacteria survive freeze-drying better than Gram-negative bacteria, but many of the latter show high viabilities if a satisfactory suspending medium is employed. Preparations of spores are thought to be preferable to vegetative cells wherever possible, as in Bacillus and Clostridium, but it should be appreciated that the suspending medium may initiate germination, and therefore the time between harvesting the cells and processing must be kept to a minimum. Similarlythe viabilities of many strict anaerobes may be adversely affected by exposure to air during processing. The “spores” of aerobic actinomycetes, with rare exceptions,

175


are not comparable in resistance to bacterial endospores. However, the use of mature well-spored cultures of this group is the simplest method of obtaining even distribution of the culture material in suspension, and survival of such cultures of the order of ten or more years can usually be expected.

2. Preparation of labels Filter or blotting paper may be used for labels. Ford’s blotting paper (Ford 428 Mill, 38 lb) is routinely used in the NCTC following the batch colour coding system previously described (Section IIID 2). The following colours of Ford’s blotting paper have been tested in the NCTC and found not to be inhibitory: pink, white, violet, primrose, scarlet, brown, orange, mottle grey, moss green, blue. Labels are prepared measuring approximately 5 mm x 30 mm, since this is a convenient size for the ampoules used. NCTC cultures are identified by the number on the paper inside the ampoule, reading from the round end of the tube. Therefore, care is taken to ensure that the numbers do not commence less than 10 mm from the extreme left hand side of the label, since this area may ultimately become masked by the dried product in the ampoule. Two methods of marking the labels are used(i) The paper may be stamped with an automatic hand numbering machine having figures about 3 mm high, the machine being set to repeat the number. The ink should have a vegetable base and be tested to ensure that it is neither bactericidal nor water soluble; we use a black printer’s ink obtained from Ve1os.t The paper is then cut into the desired strips. This is the method normally adopted in the NCTC. (ii) The ink on ordinary typewriter ribbons has not been found to bc inhibitory, so that culture details may be typed on the blotting paper, and the labels then cut to size. Only one number should be stamped or typed at any one time, and any unused strips should be destroyed before stamping another number. Such a strict routine is essential to avoid getting tubes containing differently numbered strips in the same sterilizing box. Before a box is filled with tubes it is also essential to make sure that all tubes from any earlier batch have been removed. NCIB labels are prepared in a similar manner using Whatman No. 1 filter paper, and date stamping on the reverse side of the slip to indicate different batches in place of the colour coding system.

.

t Velos Office Appliances (Rees Pitchford & Co. Ltd), 80 Gray’s Inn Road, London, W.C.l.

176

s.

P. LAPAGE, J. E. SHELTON, T. G . MITCHELL AND A. R. MACKENZIE

3. Ampoules and their preparation The ampoules should be of neutral glass, and not alkaline. Soft glass is used in the NCTC and NCIB. Pyrex is stronger but is more difficult to seal requiring a higher temperature flame; it is also more dangerous to open in the case of pathogens as the force needed to file and crack pyrex ampoules is likely to lead to breakage, and to the dispersion of suspensions of viable organisms. Our ampoules are manufactured to the British Standard, B.S. 795 : 1961, and are obtained in the form of tubes from Johnsen and Jorgensen Ltd.? These have been referred to as ampoules in this chapter, but are initially used as tubes which in practice become ampoules when constricted and sealed. The ampoules suitable for about 0-1-0-2ml amounts are tubes approximately 6 mm internal dia. with a slight constriction at the mouth for gripping the manifold. They are sold by the firm as freeze-drying FRONT VIEW

sliding l i d

window with perforated holes

I00000 0 0 0 0 0

- sliding

shutter

b

FROM UNDERNEATH

perforated -false bottom

OOOO(

oooot OOOO( OOOO(

OOOO(

sliding bottom


177

ampoules. Ampoules suitable for about 1.0 ml amounts are sold by the firm as moulded, round bottom, and straight necked. These are the normal ampoule shape, not tubes, and the necks are approximately 6 mm internal dia. with a constriction at the mouth. An accurate figure for the diameter cannot be given as the tubes vary slightly. The batch of ampoules should be checked for any obvious defects, and pin holes may even be seen at the base of the tubes. There should be a slight constriction of the mouth for gripping the nipple of the manifold. Consequently, the nipples of the manifold are conical in shape to accommodate slight variations in size of the mouths of the ampoules. All tubes are soaked in 2% HCl overnight, washed in tapwater at least three times, and finally rinsed in distilled water and dried. A numbered label is then placed in each tube. The tubes are plugged with absorbent cotton wool and placed for sterilization in special metal boxes made of monel, a nickel-copper alloy, which does not rust. Each metal box, illustrated in Fig. 7, has a perforated false bottom and a removable bottom, a side window with a sliding shutter and a lid sliding in a groove. The boxes are made on the same principles as sterilizing drums used for dressings in operating theatres. The boxes, after removal of the lids and bottoms, are stacked so that the tubes are horizontal and placed in the autoclave for sterilization at 20 lb for 20 min (125°C). After autoclaving, they are dried in an incubator at 60°C. Sterilization in the autoclave is necessary because any charring of the paper or the cotton wool may make the ampoule bactericidal, and some degree of increase in bactericidal activity takes place if they are sterilized in a hot air oven. 4. Freeze-drying suspending jluids T h e two basic characteristics of a freeze-drying suspending fluid should be the ability ( a ) to maintain the suspended organisms in a viable state, and (b) to allow recovery of viable organisms without difficulty. Whilst for many micro-organisms the commonly used fluids give very high percentage survival and recovery following freeze-drying, there are others for which the requirements are not understood. The use of glucose in the freeze-drying suspending fluid followed work by Leschinskaya (1946) and Fry and Greaves (1951) who showed that the addition of 7.5% glucose to nutrient broth effected a marked improvement in the viability of the suspended organisms, but higher concentrations only resulted in greater difficulty in dehydrating the suspension. Substitution of equine or bovine sera for most of the broth led to further improvements in protective capacity. I t was subsequently shown that the effect of glucose and certain other sugars was to retain automatically about 1% of the water content, total desiccation resulting in total death of the organisms. T h e value 8

178

S. P. LAPAGE, J. E. SHELTON, T. G . MITCHELL AND A. R. MACKENZIE

of nutrient broth appears to be similar to that of glutamic acid, the protective effect of which is thought to be through the neutralizing of toxic carbonyl radicals in the glucose and in the cells themselves. Addition of a substance to form a cake is essential to obtain an elegant dried product and to protect against mechanical damage to the organisms. I n a large service collection expense has to be considered and one may not be able to use the most satisfactory material, if the cost is too great for the issue of thousands of ampoules, e.g., the large scale use of bovine albumen. Formulae for suspending fluids are given in Chapter I. Many others have been tested and some are recommended by other workers. 7.5% glucose serum is currently used in the NCTC, having replaced Mist. desiccans (Fry and Greaves, 1951), which was a mixture of one part of broth with three parts of serum with 7.5% glucose added. For the enterobacteriaceae, 7.5% glucose broth is used. The 7.5% glucose serum is made by adding 7.5 g of glucose to each 100 ml of Wellcome horse serum No. 3 (Natural clot, unheated, no preservative). The serum is first allowed to attain room temperature before addition of the glucose and when this is dissolved, the whole is Seitz-filtered using an EKS pad and finally bottled in 5 ml amounts. The use of equine or bovine sera as protective colloids is theoretically open to objection on immunological grounds, but the results are so much better than with either gelatin or dextran. The latter suffers from the disadvantage in the primary drying stage of forming a shiny, nearly impervious layer over the rest of the frozen material thereby reducing water vapour flow from the underlying suspension. Serum helps to prevent aerosolization of bacteria into the water vapour flow, and forms a spongy mass which reconstitutes rapidly with water or broth. When serum is used, exceptionally fatty batches should be avoided as they cause the formation of a “pellicle” on the top of the fluid with Mist. desiccans or glucose-serum, and result in a fairly high proportion of “wet” ampoules after the primary drying stage which have to be discarded. Rehydrated skim milk, either in single or double strength, is usually rather less effective than Mist. desiccans or glucose serum but has the same general properties and can be successfully used with a wide range of bacteria and actinomycetes. It has one major advantage over the fluids containing serum in being heat sterilizable. However, skim milk gives a more powdery and less coherent final product, and would appear potentially more dangerous in the case of pathogens.

5 . Harvesting Cultures are usually grown on three or more agar slopes of the appropriate medium in 6 x Q in. glass test tubes or, if the period of incubation exceeds


179

3-5 days, in 1 oz narrow mouth screw-capped bottles. The suspending fluid is added in about 1 to 2 ml amounts to the cultures, the surface of the slopes are gently rubbed with a Pasteur pipetteand the growth emulsified to produce as uniform a suspension as possible, suitable for filling the ampoules. I n the case of large batches, the growth frcm several tubes is pooled in a 30 ml, 1 oz narrow-mouth bottle with screw cap. Care should be taken to avoid excessive foaming, and gouging of the agar surface. The suspension obtained should be transferred to the ampoules without delay, to avoid leaching of undesirable substances from the spent medium which might affect the efficacy of the base. 2-2.5 ml of suspension should be sufficient to fill 15-20 ampoules. Cultures grown in liquid medium. Cells are harvested from broth cultures by centrifugation and the total volume of suspending medium required is added directly to the cells. Some organisms are very difficult to emulsify into a smooth suspension, e.g., Streptomyces spp; this is reflected by the difficulty of obtaining realistic viable counts from their ampoules. Shaking with glass beads may help to prepare more uniform suspensions.

6 . Filling of ampoules Particular care should be taken that all procedures involved are carried out aseptically. The use of a sterile room or enclosed area maintained specifically for the filling of ampoules is recommended. Consideration of the design of the air flow in such a room or area is beyond the scope of this chapter, as is description and detail of hoods and safety cabinets. Irradiation of the working surface for 30 min before use with an ultraviolet lamp mounted overhead, may assist in reducing the level of atmospheric contamination in the immediate area. Similarly, the wearing of a disposable face-mask is a simple precaution to reduce the risk of contamination from the upper respiratory tract. The risk of cross Contamination between ampoules of different species in the freeze-drying machine is probably fairly small, and is minimized either by the individual capping of the ampoules, or by placing ampoules of one strain only in the centrifuge head or canister which is then capped (see Section VC. 7). However, we do not freeze-dry related organisms in the same run of the centrifuge, as this might cause difficulty in detection if contamination occurred, e.g., two different Salmonella serotypes. Capping or plugging also prevents contamination of the ampoules from the air, and contamination of the machine and air from the ampoules. The latter assumes more obvious significance when pathogenic cultures are being dried. The tubes are filled with approximately 0.1-0.2 ml of suspension using

180 s.


Pasteur pipettes with long fine capillaries. It is important that the upper portion of the ampoule must not be contaminated with the suspension, in order to avoid charring which may occur during the constricting process, apart from the dangers of infection. This requires a steady hand and practice. After filling each ampoule, the cotton wool plug is replaced. The time between commencing ampoule filling and the freeze-drying operation itself should be kept to a minimum, preferably minutes, and should not exceed one hour. Suspending fluids may act as growth media, and it is possible that prolonged periods of contact prior to freezing may lead to renewed growth of the culture or to the germination-of spores. Alternatively, the high concentration of carbohydrates and of other organic constituents in the suspending fluid may prove deleterious to certain autotrophs. Filling machines would be desirable but we have not had experience with them. Filling under a hood should be used for dangerous pathogens. We fill with approximately 0-1-0.2 ml amounts and make no attempt to measure accurately the volume in the ampoules. This is because we are interested in the viability of the organisms from the point of view of obtaining successful revival, not from the actual numbers present in the ampoule. In practice, the number of organisms in 0.1 ml x 108 =1 x 107 and

0.2 ml x 108 =2 x 107

is very small from our point of view. The position in the preparation of vaccines is entirely different.

7. Loading centrifuge head The machine will hold a variety of heads and the head suitable for the purpose required is chosen. The head for the 0.1-0.2ml ampoules will hold 380 tubes in a single tier, therefore permitting a maximum holding of 1140 ampoules in a three tier centrifuge plate assembly. The centrifuge head suitable for the 1.0 ml ampoules holds 120 ampoules, and with a second tier, a maximum holding of 240 ampoules is possible; a third tier cannot be added as these ampoules are longer than the others. In our routine process, one of the heads of the centrifugal freeze-drying machine has been adapted to hold eight metal canisters (see Fig. 8). This permits comparatively small batches of cultures to be dried together when desired, and avoids the labour of capping every ampoule since the canister can be capped as a whole. Each canister will hold a maximum of 18 ampoules, and it is our practice never to dry more than one culture in the same canister. The ampoules are contained within the canister in a special stand referred to as the cruet stand (see Fig. 8).

181

11. CULTURE COLLECTIONS G A U Z E COVER

AMPOULES

PERFORATED PLATE OF CRUET STAND

FIG.8. Cruet stand and canister. This contains the ampoules during centrifugation in the freeze-drying machine. A specially adapted centrifuge head is needed to hold the canisters.

The normal procedure is to place the ampoules in the cruet stand immediately after filling, with the cotton wool plugs still in position. When filling of the batch is completed, the absorbent cotton wool plugs are removed and discarded into disinfectant, the necks of the tubes flamed, and the entire stand placed in the previously flamed canister. The mouth of the canister and tubes are again lightly flamed before the canister is covered with a sterile cap made from six thicknesses of gauze. Loose fitting cotton wool plugs can be used in each ampoule, but this is inadvisable as they may on occasion be centrifuged to the bottom of the ampoule. At this stage the canisters are ready for loading into the primary drying chamber, and there should always be an even number of canisters for counterbalancing in the centrifuge head. It is not necessary for the canisters to be accurately balanced before loading, as the weight of the ampoules and canisters is negligible compared to that of the centrifuge head. However, the same number of ampoules are put into pairs of canisters which are

182 s.

P. LAPAGE, J. E. SHELTON, T. G . MITCHELL AND A. R. MACKENZIE

matched when loading. The canisters are inserted into the centrifuge head at an angle of approximately 60", and the head is then ready for placing in the vacuum chamber. Ampoules are loaded directly into the centrifuge head only when comparatively large batches, consisting of 100 to 200 ampoules, are to be dried. The head is placed on a stand so that it is at an angle of approximately 60" and can be turned on the centre boss. The plugs are removed from the filled tubes, discarded into disinfectant, and the ends of the tubes flamed before the tubes are loaded into the centrifuge head. Care is taken to counterbalance the tubes by virtue of their relative positions in the centrifuge head. When loaded, a specially made circular metal frame is screwed on to the centrifuge head and covers the tubes (Fig. 9). After lightly flaming the frame, centrifuge head and necks of the tubes, the whole is covered with a sterile

BASE PLATE

WITH GAUZE COVER IN POSITION

.

.

A

IGA COVER

FIG.9. Centrifuge head. This diagram illustrates the position of the ampoules in the centrifuge head and the method o f covering with gauze to prevent contamination,


183

cover made from six thicknesses of gauze. The head is then ready to be put into the primary drying chamber.

8 . Operation As mentioned before, a flow diagram of the major stages in the method is given in Fig. 6. A diagrammatic representation of the Edwards model 30P primary drying machine is given in Fig. 10 and photographs of the 30P2 in Figs 11 and 12. In practice, these models consist of a chamber which can be heated, in which a large centrifuge head can be placed. From this chamber is a duct leading to a condenser chamber, consisting of a refrigerated unit for the condensation of water. From the refrigerated condenser is a lead to the vacuum pump which is an oil-operated pump. Pirani gauges, which measure the vacuum, are situated between the drying chamber and the cooler, and the cooler and the pump. The first gauge effectively measures the water vapour pressure coming off from the chamber, and the second the efficiency of the condenser and the pump. The centrifuge runs at 500 rpm at a fixed speed. (a) Operation of model 30P (Edwards High Vacuum Ltd.) (i) The machine is turned on 40 min before use for the following preparative operations: (a) The refrigerator is started and the temperature should be allowed to fall until the gauge records -50°C before the machine is used; the minimum for use is -30°C. (b) The heater is turned on, the temperature is set manually between 30 and 60"C, and from then on the heater is thermostatically controlled at the pre-set temperature. (c) The Pirani vacuum gauges are switched on. (ii) The centrifuge head loaded with ampoules or canisters is put into the machine and the central spindle locked in place above the head. (iii) The underside of the lid of the drying chamber and the O-ring seal is wiped with a fluff free material such as linen or wash leather; do not use an ordinary cloth in order to avoid fluff. T h e lid is then replaced. (iv) The surplus water is drained out, and then the drain cock and the air release cock are closed. (v) The centrifuge is turned on and all controls and taps are checked. The pump is then turned on, and when the Pirani gauge between the vacuum chamber and the condenser registers below 0.1 torr, the suspensions should be frozen; this takes approximately 15 min.

184 s.


centAfugo head

FIG.10. Principles of Centrifugal Freeze Dryer: Edwards model 30P.

(vi)

(vii)

(viii) (ix) (x)

The centrifuge is then turned off. (1 torr approximately equals 1 mm Hg.) The primary drying is allowed to proceed for 23-4 h with 200-300 ampoules, but for longer when this number is exceeded. During this time heat is applied to the walls of the vacuum chamber and approximately 95% of free water is removed. The temperature of the ampoules is finally raised to about 35-4OoC, but should not exceed this. When the Pirani gauge gives a reading of at least 0.09 torr, though preferably below this to a level of approximately 0.01 torr, then 15 min is allowed to elapse and the readings are taken again. The heat is turned off and air is allowed to enter the vacuum chamber via the air release cock. The air is released slowly into the vacuum chamber in order to avoid a hurricane in the chamber. The head of the centrifuge is removed from the machine, and then the ampoules are removed and constricted.

Note: the thermometer registering the heat in the chamber of the machine is attached to the outside of the drum, so that it records only the temperature of the metal drum and not that inside the ampoules. (b) Operation of model 5PS (Edwards High Vacuum Ltd.). The methods for the preparation of ampoules, suspensions and for filling are the same as those described for the model 30P machine. The machine is illustrated in Figs. 13


185

FIG. 1 1 . Photograph of Centrifugal Freeze Dryer: Edwards model 30P2. The front and side panels of the machine have been removed to display the parts. (Reproduced by permission of Edwards High Vacuum Ltd.)

and 14. The total procedure for operation of the machine may be summarized as follows(i) Load the centrifuge head. (ii) Unplug the tubes.

186 s.


FIG.12. Photograph of control panel of Edwards model 30P2. (Reproduced by permission of Edward High Vacuum Ltd.).

(iii) Cap each ampoule with a strip of sterile surgical gauze folded over the top and stapled at both sides. (iv) Charge the unit with PzO5. (v) Remove the bell jar, place the centrifuge motor on the base plate, assemble the loaded centrifuge head on the motor, and replace the belljar. (vi) Switch on the centrifuge. (vii) Apply the vacuum. (viii) Centrifuge for 10-15 min, or until the vacuum reaches 0.1 torr. (ix) Maintain the vacuum for about four hours, which constitutes the primary dry. (x) Unload the dryer, take off the bell jar and remove the centrifuge head. (xi) Plug thetubeswith non-absorbent wool maintaining an angleof 45". (xii) Constrict the ampoules. (xiii) Remove the centrifuge and replace it with the secondary plate manifold, and place the ampoules on this. (xiv) Apply the vacuum. (XV) Dry for a further four hours. (xvi) Check each ampoule for leaks using a high frequency tester. (xvii) Seal off under a vacuum, or an inert gas such as nitrogen.

187


-GLASS BELL-JAR

-CENTRIFUGE

I

G

Y

G

A

1

U

G

HEAD

E

FIG.13. Principles of Centrifugal Freeze Dryer: Edwards model 5PS.

9. Removal and constriction of ampoules, 30P and 5PS The centrifuge head is removed and placed on a stand maintaining the ampoules at an angle of 45". The gauze covers are loosened and the tubes are removed. A plug of non-absorbent cotton wool, previously sterilized by autoclaving in situ in similar-sized tubes, is placed into the tube. Nonabsorbent cotton wool is used for these plugs since absorbent wool would take up water vapour and freeze to a solid, and not allow free evaporation from the material. The top of each plug, having been exposed to possible contamination, is cut off to leave approximately 15 mm inside the ampoule. The plug is pushed part of the way down the tube by means of a ramrod, which has a fixed stop so that the plug cannot be pushed too far down the tube (Fig. 15). The tube is then constricted about half an inch above the plug in an oxygen plus coal-gas flame. This is a skilled process and takes practice.

188 s. P. LAPAGE, J. E. SHELTON, T.

G. MITCHELL AND A. R . MACKENZIE

FIG. 14. Photograph of Centrifugal Freeze Dryer: Edwards model 5PS. The centrifuge is in position on the machine and the manifold unit has been placed on top for display. '(Reproducedby permission of Edwards High Vacuum Ltd.)


189

METAL SAFETY DISC

SCREW TO ADJUST

FIG.15. Ramrod. Alternatively, a fan-tail coal-gas burner may be used, or an automatic constricting machine. A useful machine for this purpose is available from Edwards High Vacuum Ltd., but the machine may require modification to prevent charring of the cotton wool plugs.

10. Secondary drying After constriction, the necks of the ampoules should be checked for cracks or wisps of adherent cotton wool; cracked ampoules should be rejected and any cotton wool removed or burnt off. The ampoules are carefully fitted with a gentle twisting movement on to the nipples of the secondary dryer. The principles of the secondary dryer are illustrated in Fig. 16 and a photograph in Fig. 17. Fig. 18 shows an ampoule on the nipple. The machine is charged with phosphorus pentoxide, of a technical grade, in the tray provided at the bottom, and the vacuum pump is turned on. If desired, the phosphorus pentoxide can be allowed to cake by exposure to the laboratory air for 10 min. The pressure of the vacuum system, isolated from the manifolds by closing their taps, is then read on the Pirani gauge and should be equal to or below 0.01 torr. The first manifold is then opened slowly, and the pressure is checked to ensure that no undue increase occurs, the next manifold is then opened and the others in turn until all are connected to the vacuum

190 s. P. LAPAGE,

J. E. SHELTON, T. G . MITCHELL AND A. R . MACKENZIE

4 ampoule

NIPPLES TO HOLD AMPOULES

nipple

ARM IS INDEP

N IIB

FRONT VIEW

AMPOULES THE ON

PlRANl GAUGE

TAP

.:............. .. ... ... . .................... .. . . .. ....... .. ....

905 IN TRAY

*

-END

PLATE

"1 TO PUMP

FIG.16. Principles of the secondary dryer.

system. Alternatively, the first manifold can be opened until the vacuum has dropped to at least 0.01 torr, then closed and the next manifold opened, the vacuum allowed to drop and the manifold then closed. This is done successively to each manifold separately. Finally, all manifolds are opened to the vacuum system together, The advantage of this method is that any leak into the system will be isolated to one manifold. The constriction processes should be as short as possible to minimize delay in transferring ampoules from the primary to the secondary dryer, since exposure to air has a detrimental effect on the viability of the partially dried cultures. On occasions when delay is unavoidable, the ampoules should be replaced in the primary dryer or into a vacuum desiccator connected to a vacuum pump. If there is a leak into the vacuum system, suspected ampoules are checked


191

FIG. 17. Photograph of the secondary dryer. (Reproduced by permission of Edwards High Vacuum Ltd.)

with a high frequency tester. A puncture will be shown by a long thread of white light which appears to connect the puncture to the tip of the probe. A satisfactory vacuum is shown by a pale blue or violet glow in the ampoule. Sparing use of the high frequency tester should be observed, as each test may kill some cells. The ampoules are left on the secondary dryer for 18 to 20 h in order to reduce the moisture content to about 1%.

11. Sealing and checking of ampoules The ampoules, in situ on the secondary dryer, are first checked for maintenance of vacuum with a high frequency tester, and are then sealed with an

FIG.18. Detail of ampoule on nipple. (Reproduced by permission of Edwards High Vacuum Ltd.)


193

oxygen coal-gas flame or a coal-gas cross-fire burner. Sealing too close to the body of the ampoule will result in dimpling. The final product in the ampoule should be a light powder and not a glassy substance. The causes of glassiness are not fully understood, but in the case of Mist. desiccans it has been attributed to an increased concentration of glucose, either in the preparation of the suspending fluid or due to supercooling.

12. Storage Ampoules are stored in the NCTC in the drawers of wooden cabinets. These drawers, which are divided by wooden dividers into compartments and are cork lined, are well-fitting so that no light enters. The ampoules are kept at room temperature due to lack of other facilities. The room temperature in the building fluctuates from about 5 to 40°C. Experience in the NCIB has shown that viability is better maintained at 4°C than at room temperature. Ampoules should be protected from light, and preferably stored in the dark. Unpublished examination in the NCTC has shown light to be harmful, and fluorescent light particularly is more damaging than the usual tungsten bulbs, yielding higher drops in viable counts on exposure.

13. Opening of ampoules Identify the culture by the number on the paper inside the ampoule, reading from the round end of the tube (see Fig. 19). Make a file mark on the ampoule about the middle of the cotton wool plug, and apply a red-hot glass rod to crack the glass. Care should be taken in opening the ampoule as the contents are in a vacuum. Allow time for air, filtered by the plug, to seep into the ampoule; otherwise when the pointed end is snapped off the plug will be drawn to one end. Hasty opening may release fine particles of dried organisms into the air of the laboratory. The plug may be impregnated with dried culture, and should be regarded

FREEZE - DRIED SUSPENSION

F I L E HERE AND APPLY RED HOT G L A S S ROD

FIG.19. Opening of ampoule.

194 s. P. LAPAGE, J. E.

SHELTON, T. G . MITCHELL AND A. R. MACKENZIE

as dangerous to handle and removed with forceps. Flame the open end of the tube and insert a sterile cotton wool plug (e.g., the plug from a Pasteur pipette). The discarded plug and the pointed end of the ampoule should be autoclaved. About 0.5 ml broth should be added to the ampoule, and the contents carefully mixed to avoid frothing. Most of the organisms will be on the number paper. According to the growth requirements, the broth suspension should be subcultured to suitable medium ; the addition of blood improves the growth of many organisms. When the organism is an aerobe, plate out a loopful; the paper may be lifted on a wire loop or with forceps and placed on the surface of a solid medium. Suspensions of anaerobes should be transferred to cooked meat or other appropriate media. Incubate both the broth and surface cultures at the optimal temperature for growth for several days. It is possible for an occasional contaminant to enter the ampoule when the vacuum is released, and this is more likely to occur if there is no plug in the ampoule. 14. Re-hydration and recovery Suitable recovery media for many organisms are given in Chapter I. It is advisable to include a solid medium when possible to detect aerial contamination if this occurs during the opening process. I t should be noted that some organisms appear to need haemin or catalase after freeze-drying, and our experience has shown that viable counts are much higher on blood agar than on nutrient agar with these organisms, e.g., Pseudomonas and Vibrio They should therefore be revived on blood agar. Little work has been done on the optimal revival media for most species, but as rich a medium as possible is recommended. Organisms may take time to revive, and frequently they are not in optimum physiological condition, requiring several subcultures before they fully regain their characteristics. This is quite noticeable in some cases, e.g., with the serological properties of some strains of Salmonella. It is important that suitable media and growth conditions are used. A culture collection frequently receives complaints that its freeze-dried cultures have failed to revive, but on investigation it is found that the conditions for revival were unsuitable for the organism concerned.

VI. OTHER METHODS A. Separate freezing and drying for pathogens This method is adopted in the N C T C for highly dangerous organisms, to avoid the risk of contamination of the machine in the event of any breakage


195

of ampoules occurring during centrifugation, which is consequently avoided in the method. In practice, it is used for organisms such as Pasteurella pestis, Franciscella tularensis, Mycobacterium tuberculosis, Loeflerella mallei, and Bacillus anthracis. The principle of the method is that the organisms are first frozen by a mixture of crushed dry ice, and are then dried without centrifugation. The tubes are filled with a suspension of the pathogen as earlier described. When a batch of tubes is filled, the original plugs of absorbent cotton wool are discarded into disinfectant, and are replaced by lightly packed nonabsorbent cotton wool plugs which have been previously sterilized by aurociaving in similarly sized tubes. The tops of these plugs are then cut off, and the plugs are pushed part way down the tube to a pre-determined level using the ramrod previously described (see Fig. 15). The tubes are then laid in a cardboard box at a slight angle, and packed with dry ice and frozen. The canisters, also, are cooled with dry ice. The tubes, when frozen, are placed in the cruet stands, which are then put into the chilled canisters containing a little dry ice. This maintains the tubes in the frozen state until they are ready for transfer to the drying chamber. I t is essential that the temperature of the condenser chamber is below -30°C. The cruet stands are then removed from the canisters and are placed on the bottom of the machine; no centrifugation takes place. A vacuum is applied for two and a half hours. T h e tubes are then removed, constricted, and put on the secondary dryer overnight. Subsequent procedure for sealing and testing of ampoules follows the normal method.

B. Sordelli’s method This method was described by Professor A. Sordelli of Buenos Aires in 1934 to Dr. St John Brooks, the Curator of the NCTC at that time (Rhodes, 1950a, 1950b). It was adopted in the N C T C and proved very useful in the early days of the Collection, as many cultures were kept by this method rather than by regular subculture. In 1949 it was supplanted by large scale freeze-drying, although Sordelli tubes are still prepared of each new culture.

Method T h e complete method is illustrated in Fig. 20. The cultures are incubated on a solid medium for a suitable period of time, When good growth is obtained, a heavy inoculum from the solid growth is emulsified in a loopful of horse serum deposited on the inner wall of a small (8 x 60 mm) tube which fits into a larger (10 x 150 mm) tube. A small amount of phosphorus pentoxide is placed in the bottom of the larger tube, using a glass rod and funnel to avoid touching the sides of the tube. Identification data are written on the outside of the inner tube with indian ink. T h e inner tube is

196 s. P. LAPAGE, J. E. SHELTON, T. G.

MITCHELL AND A. R. MACKENZIE

COTTON WOOL PLUG HOLDS SORDEUI TUBE IN POSITION IN OUTZR

\

c

OUTER TUBE CONSTRICTED

OUTER TUBE SEALED OFF UNDER VACUUM

FIG.20. Sordelli’s method. then placed in the outer tube, and is held in position above the phosphorus pentoxide by the cotton wool plug of the inner tube. The outer tube is constricted, and when cool, it is attached to a vacuum pump such as a Hyvac pump. A vacuum is applied and after about five minutes, the tube is sealed at the constriction. The vacuum is checked with a high frequency tester. The cultures are stored in the dark at room temperature. Rhodes (1950a) found, with many bacterial genera, that a high proportion of organisms in the tubes were viable for up to 10-12 years. However, some species did not survive for long periods, and for further details the reader is referred to the original paper.


197

The great advantage of the method is that it can be readily done in a small laboratory as it requires no elaborate equipment. I t is not, however, suitable for the drying of large numbers of ampoules of a culture, but relatively large numbers of cultures can easily be dried to yield one or two ampoules of each. The method is akin to freeze-drying since, by the application of a vacuum, an instantaneous freeze is obtained of the small amount of culture emulsified in the serum. It is important that the inner tube is supported above the phosphorus pentoxide so that the culture is not in the region of the chemical, otherwise the heat generated by absorption of water by phosphorus pentoxide may kill the culture.

c. Ldrying This method of preservation was described in a series of papers by Annear (1954; 1956a; 1956b; 1957; 1958; 1962). He placed small drops of culture on sterile peptone starch plugs and dried them under a vacuum. Following this he developed a technique in which the peptone starch plug was replaced by cotton wool fibres. From the temperature measurements quoted by Annear it is evident that in his system no freezing occurs, hence it is a process of direct drying from the liquid state, or L-drying. Published results have shown the technique to be successful with various bacteria, yeasts and viruses. The technique described here is a modification of that of Annear, and has been developed in the NCIB; the filter paper strip identifying the ampoule is used as the “carriel” in place of cotton wool fibres. Experience with this method has confirmed the work of Annear, since a number of sensitive species of bacteria have yielded viabilities between 10 and 100 times those which are obtained with freeze-drying. The method allows us to dry

FIG.21. Diagram of L-drying apparatus. A: Rotary vacuum pump. B :Phosphorus pentoxide trap. C : Saunders’ valve. D : Manifold with ampoules attached. E : Water bath. F: Thermocouple. G: Temperature recorder.

198 s. P. LAPAGE,

J. E. SHELTON, T. C . MITCHELL AND A. R. MACKENZIE

FIG.22. Photograph of L-drying apparatus.

successfully organisms which would otherwise have to be maintained in active culture.

1. Materials (i) Rotary vacuum pump with moisture trap. (ii) (iii) (iv) (v)

Single-sided manifold. Saunders’ valve. Water bath with glass side to accommodate the manifold. Temperature monitor such as a thermocouple and potentiometric recorder. (vi) Sterile, straight-sided and capillary pipettes, ampoules, identifying slips, face masks, burners, secondary stage dryer, phosphorus pentoxide and high frequency tester. These items are similar to those described for freeze-drying. The arrangement of the pump, manifold, water bath, recorder and valve are shown in Figs. 21 and 22. 2. Method (i) A dense suspension of the culture is prepared in a suspending fluid, such as Mist. desiccans. T h e ampoules are filled using a sterile capillary pipette, taking care that none of the suspension touches


199

the upper portion of the ampoules. T h e quantity of suspension placed in each ampoule should be kept to a minimum, with an absolute maximum of 5 drops (about 0.1 ml). It is preferable to use a smaller amount of a denser suspension if possible, in order to avoid a prolonged de-gassing period or difficulty in maintaining the temperature. (ii) Phosphorus pentoxide is placed in the moisture trap of the pump and allowed to cake by exposure to the laboratory air for 10 min. The lid of the trap is replaced and, with the valve to the manifold closed, the pump is switched on. (iii) The ampoule plugs are clipped off neatly, leaving approximately 15 mm inside the ampoules and the plug is then pushed down using the ramrod. (iv) The ampoules are attached to the manifold nipples and lowered into the water bath at 20°C to a depth of 40-50 mm. (v) The valve to the manifold is opened wide for about half a second, to remove most of the air from the system, but without causing violent removal of dissolved air from the suspensions; the valve is then closed. (vi) The valve is then opened again very slowly until the first small bubbles of air are formed on either side of the filter paper strips in the ampoules. The process of de-gassing is controlled by manipulation of the valve, and care is taken to avoid foaming which would lead to a loss of the suspension on to the plug in the ampoule. De-gassing is normally completed within 5 min. (vii) When de-gassing is complete, the valve is opened further to allow drying to take place. This is the most critical part of the process, and the temperature of the contents of the ampoules is dependent on such factors as the number of ampoules being dried, the pump capacity and the thickness of the ampoule walls. Preliminary tests should therefore be carried out on the proposed system to check the temperature characteristics, Alternatively, a temperature monitoring device should be connected to a control ampoule on each occasion. By adjusting the valve it is possible to control the temperature of the ampoule contents so that it does not fall below 10-12°C. It is not known whether there is a critical temperature for liquid drying of different species, but there is evidence that, with delicate organisms, the results may not be better than those of freeze-drying if the suspension is allowed to freeze at any stage. The recommended temperature of 10-12°C offers some safety margin above the freezing point and at the same time allows reasonably rapid drying. The temperature of the water bath remains

200 S. P.

LAPAGE, J. E. SHELTON, T. G . MITCHELL AND A. R. MACKENZIE

relatively constant during the process and offers no guide to the conditions within the ampoules. (viii) The ampoules appear superficially dry after approximately 15 min, but drying is continued for a further 10 min after this stage has been reached. (ix) The ampoules are then removed from the manifold, constricted as described under freeze-drying, and the process is then completed by attachment of the ampoules to the secondary stage dryer used for freeze-drying in exactly the same manner.

VII. METHODS FOR BACTERIA UNSUITED T O FREEZE-DRYING With certain species of bacteria, the survival rate resulting from freezedrying is too low to permit satisfactory maintenance by this method. Largecelled bacteria, particularly if Gram-negative, appear relatively susceptible to injury by freeze-drying, and at the present time the suspending media in general use do not offer adequate protection. It must be admitted that satisfactory alternatives are not always available and there is a need for further research on this topic. Whenever available, liquid nitrogen freezing is probably the method of choice for long term preservation of “difficult” cultures. I n its absence, satisfactory results may be obtained, in many instances, by L-drying. Even freeze-drying may offer useful results by using a much denser suspension of cells than is normally employed, by minimizing delay between the various stages and by subsequently storing at a low temperature. If freezedrying of a dense suspension of cells is employed, and some viability is obtained, it should be appreciated that the survivors may not be fully representative of the original culture as only a small fraction of the original population may survive. When such procedures fail or are unavailable, it may be necessary to resort to active culture maintenance. When relatively large numbers of cultures are involved, serial culture is seldom practicable or desirable, and the chosen method should aim at as long an interval as possible between subculture. Serial cultures in stabs or sloppy agar are usually preferable to slopes, and in some cases the increase in longevity is dramatic. When the medium contains carbohydrate, the addition of a little chalk is often of definite value. Table I11 gives a list of organisms poorly preserved by freeze-drying and possible methods of maintenance are suggested.

TABLE I11 Maintenance of bacteria which are poorly preserved by freeze-drying Organisms Azotobacter agilis Azotobacter insigne Azotobacter macrocytogenes Cytophaga aurantiaca Cytophaga fernlettans Cytophaga hutchinsonii Cytophaga salmonicolor Cytophaga succinicans Cytophaga spp. (filamentousforms) Derxia gummosa Halobacterium spp. Myxobacteriu (fruiting types) Saprospira grandis Saprospira thermalis Spirillum spp. Sporocytophaga myxococcoides Vibrio spp.

Maintenance L-dry L-dry L-dv L-dry. Alternatively, maintain in active culture in liquid Dubos’ salts solution plus cellulose and subculture 6 monthly Stab cultures under liquid paraffin As for C. aurantiaca Stab cultures under liquid paraffin Stab cultures under liquid paraffin L-dry L-dry Active culture, subculture 1-3 month intervals Active culture on large slopes in well-stoppered tubes, kept in the refrigerator. Subculture 6month intervals L-dry. If maintained in active culture, must be subcultured at frequent intervals-very 7 days As for S.grandis L-dry. Also maintain well under liquid paraffin As for C . aurantziaca. Can also be successfullyfreeze-dried L-dry. May be preserved by freeze-dryingbut viabilitiesup to 100 times better by L-drying

cl

s

i+

202

S. P. LAPAGE, J. E. SHELTON,

T. G. MITCHELL AND A.

R. MACKENZIE

VIII. STABILITY OF CHARACTERS Changes in characteristics after regular subculture are well known. T h e degree of change of characteristics after freeze-drying is not so clearly demarcated. In the majority of cases where we have received complaints of change of characteristics these have been found to be due to something other than the freeze-drying. However, Shigella cultures show minor antigenic changes after freeze-drying, and after many batches tend to reach a serologically degraded state. Salmonella cultures may not show full antigenic development on first recovery from the ampoule but this is usually fully developed after a few subcultures. It is of course possible that the freeze-drying process may select frost resistant organisms, and possibly organisms with other special properties enabling them to survive. Jennens (1954), dried a paracolon in the antiserum to it and was unable to demonstrate any antigenic change. A case could perhaps be made for drying the original culture sent by the depositor, as well as the subculture checked by purity and other tests, if the facilities of a collection were able to deal with this.

IX. VIABILITY

A. General Viability is a relative term. It must be stated on what medium the organism is recovered. Some organisms require special growth factors on recovery from the freeze-dried state. The counts of Vibrio or Pseudomonas on blood agar are 10-or 100-fold greater than they are on nutrient agar. Therefore, if regard is paid only to one medium then the estimate of viability may be false. I n view of this, and the fact that one cannot know if recovery conditions are optimal, it is impossible to speak of an absolute viability. On occasions, freeze-dried cultures may appear non-viable because the cultural conditions are more critical for the recovery of small numbers of viable cells from the dried state than for the normal transfer of actively growing cultures of the same organism. This is a common problem with autotrophs and anaerobes. Similarly, the time required before obvious growth is present on recovery from the freeze-dried state may be much longer than normal. In the case of DesuZfovibriogigas, a slow growing organism, recovery in one test took three months due to the small number of viable cells present, instead of the normal 10-14 days. In general, it may be said that the higher the initial count on the suspension used for drying, the higher the survival rate of the bacteria which can be expected. Difficult organisms to freeze-dry which are maintained in the N C T C


203

include : Haemophilus, Neisseria, Cafipylobacter and Streptobacillus moniliformis. I n general, Gram-positive organisms survive freeze-drying better than Gram-negative organisms as was shown by Steel and Ross (1960).

B. Viable count technique Estimates of viability are obtained from colony counts, based on a modification of the method of Miles and Misra (1938) (see also Postgate, this Series, Vol. 1). We use nutrient broth routinely as a diluent, in some cases other fluids may be preferable. Decimal dilutions are made by adding 0.1 ml of the suspension used for freeze-drying to 0.9 ml of diluent, using a fresh pipette for each dilution. Freeze-dried cultures are however first resuscitated in the ampoule with 1 ml of the diluent for approximately 10 min before using this suspension as the 10-1 dilution. Routinely, dilutions are only made to 10-6 in the N C T C for practical reasons. Dilutions are carried out using dropping pipettes which are calibrated to deliver drops of 0.02 ml, i.e., 50 drops per ml. These pipettes are made from ordinary glass tubing in the usual way, and the ends are cut off square to give the required pipette diameter using a metal gauge plate with a hole in it. For details of the size of the hole see Fildes (1931). It is most important that the glass used for making the pipettes is chemically clean, therefore the glass tubing is first soaked in 2% HCl, then washed with tapwater and rinsed in distilled water. The pipettes are designed so that when held vertically, they deliver 0.02 ml drops of diluent when the delivery rate is 40-45 drops per minute (see Fildes, 1931). Plates of a suitable agar medium are dried sufficiently to absorb drops of 0.02 ml in 15 to 20 min. The plates are inoculated by adding drops of diluent from a height of 1 to 2 cm with the pipette held vertically. Three drops from each of the six dilutions are seeded on to the same plate in numbered sectors, one sector per dilution. After a suitable period of incubation, the growth resulting from each dilution is recorded as followsC :confluent growth. SC :semiconfluent growth. UNC: colonies so densely packed as not to be practicable to count. Mean: the mean number of colonies per 0.02 ml drop, calculated from the three drops. The pattern of results scored in this way are compared with former and subsequent counts, so that it is not necessary to calculate the absolute bacterial count,

204

S. P. LAPAGE, J. E. SHELTON, T. G. MITCHELL AND A. R. MACKENZIE

C. Estimation of shelf life The principle of estimation of shelf life is that survival for periods at elevated temperatures is measured, and extrapolation is made to the shelf life at the storage temperature. Mention must be made of the boiling test (Greaves, 1960) and of accelerated storage tests (Grieff and Rightsel, 1966; 1967; 1968). The authors have had no experience of either, and the reader is referred to the original work.

D. Survival of bacteria by freeze-drying 1, NCIB results and notes on Table IV Table IV gives the known survival times for some species of bacteria when preserved by the centrifugal freeze-drying method described in this chapter. In some cases the time shown is only a fraction of the probable maximum storage life of the organism, as experience has been limited in many instances to relatively short periods. I n general, organisms have only been listed where observation periods have been for at least five years. Whilst in some cases small variations in the method of freeze-drying will not lead to marked changes in shelf life, it must be appreciated that with organisms which are intrinsically more sensitive to freezing or drying injury, attempts to use the times shown in the table as guides to probable results with other methods might have disastrous consequences. The suspending fluids used for the results shown in the table were either glucose broth, glucose serum or Mist. desiccans, as described in Chapter I (Formulae Nos. 112, 113 and 114). Unless otherwise indicated, storage has been at room temperature, which does not attain more than 25°C in the NCIB. Storage at ambient temperatures above this limit might be expected to lead to poorer survival times. As a general rule, storage of the dried cultures at low temperatures will lead to extended shelf life. Refrigerated storage is a useful precaution to adopt for a small proportion of the ampoules of any batch, and is particularly recommended when an organism of unknown shelf life is being investigated. It is advisable to test and re-dry cultures at regular intervals in order to ensure that strains which behave differently from the expected pattern for that species are not lost. The interval chosen must depend on individual circumstances but it is suggested that apart from checking immediately after drying, each strain should be tested and re-dried at a maximum of half the known storage life of the culture.

205


TABLE IV Minimum storage life of bacteria preserved by centrifugal freeze drying and held at 20-25°C Storage life (years)

Organism ~

Acetobacter aceti Acetobacter ascendens Acetobacter estunense Acetobacter lovaniense Acetobacter mesoxydans Acetobacter pasteurianum Acetobacter peroxydans Acetobacter rancens Acetobacter xylinum Acetomonas oxydans Agrobacterium radiobacter Agrobacterium tumefaciens Arthrobacter aurescens Arthrobacter citreus Arthrobacterglobiformis Arthrobacter pascens Arthrobacter simplex Arthrobacter terregens Arthrobacter tumescens Arthrobacter ureafaciens Azotobacter beijerinckii Azotobacter chroococcum Azotobacter vinelandii Bacillus macroides Brevibacterium ammoniagenes Brmibacterium linens Cellulomonas biazotea Cellulomonas bibula Cellulomonas cellasea Cellulomonas$mi Cellulomonasflavigena Cellulomonas gelida Cellulomonas rossica Cellvibriofulvus Cellvibrio vulgaris Chlorobium limicola Chromatium spp. (small-celled) Clostridium acetobutylicum Clostridium nigrijicans Clostridium pectinovorum Clostridium scatologenes Corynebacterium mediolanum

Comments

~

9 10 9

8 10

10 14 15 10 14

6 6 6 6 6 6 6 6 6

6 6 6 6

Should be stored at 0-5°C if possible Should be stored at 0 4 ° C if possible Should be stored at 0 4 ° C if possible

10

6

6 6 6

6 6

6 6 6 10

10 10 10 10

Should be stored at 0 4 ° C if possible Should be stored at 0-5°C if possible See Desulfotomaculum

10

7

6

206


TABLE IV-continued Organism Cytophaga johnsonii Cytophaga marinoflava Desulfotomaculum nigrificans Desulfotomaculum orientis Desulfotomaculum ruminis Desulfovibrio africanus Desulfovibrio desulfuricans Desulfovibriogigas Desulfovibrio salexigens Desulfovibrio vulgaris Flavobacterium deerorans Flavobacterium halmephilum Kurthia zopfii Lactobacillus acidophilus Lactobacillus bifidus Lactobacillus brevis Lactobacillus buchneri Lactobacillus casei Lactobacillus cellobiosus Lactobacillus delbrueckii Lactobacillusferment; Lactobacillusfructovorans Lactobacillus helveticus Lactobacillus hilgardii Lactobacillusjugurti Lactobacillus lactis Lactobacillus leichmannii Lactobacillusparvus Lactobacillusplantarum Lactobacillus salivarius Lactobacillus viridescens Leuconostoc dextranicum Leuconostoc mesenteroides Microbactmumfm Microbacterium lacticum Micrococcus denitrificans Myxococcus xanthus Nocardia calcarea Nocardia cellulans Nocardia coeliaca Nocardia rugosa Pediococcus acidilactici Pediococcw cerevisiae Pediococcus pentosaceous Propionibacteriumarabinosum

Storage life (years) 5 6 10 8 8 8 8 4 8

10 6 9 10 15 10 14 12 12 12 10 12 10 12 12 12 12 10 10 12 10 8 10 10 6 6 6 5 6 6 6 6 10 30

10 10

Comments

Should be stored at 0-5°C Should be stored at 0 4 ° C

Should be stored at 0 4 ° C

Should be stored at 0-5°C

rr.

207

CULTURE COLLECTIONS

TABLE IV-continued Organism

Propionibacteriumfreudenreichii Propionibacterium intermedium Propionibacteriurnjensenii Propionibacteriumpentosaceum Propionibacteriumpetersonii Propionibacteriumpituitosum Propionibacteriurn rafinosaceum Propionibacterium rubrum Propionibacteriumsanguineum Propionibacteriumshermanii Propionibacteriurn technicum Propionibacteriumthoenii Propionibacteriumwentii Propionibacteriumzeae Protaminobacter alboflavus Protaminobacterruber Pseudomonas aeruginosa Pseudomonas arsenoxydans Pseudomonas convexa Pseudomonas denitrificans Pseudomonas desmolyticum Pseudomonasjluorescens Pseudomonasfragi Pseudomonas indoloxidans Pseudomonas lemonnieri Pseudomonas multophilia Pseudomonas nigrifaciens Pseudomonas oleovorans Pseudomonas ovalis Pseudomonas oxaliticus Pseudomonas reptilivora Pseudomonas ribofivina Pseudomonas rubescens Pseudomonas stutzeri Pseudomonas synxantha Pseudomonas testosteroni Rhodopseudomonas capsulata Rhodopseudomonasgelatinosa Rhodopseudomonaspalustris Rhodopseudomonas spheroides Rhodospirillumrubrum Sporosarcina ureae Streptomycesantibioticus Streptomycesaureofaciens Streptomycescinnamomeus

Storage life (years) 10 10 10 10 10 10 10 10 10 10 10 10 10 10 6 6 6 6

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

10 10

8

Comments

208 s. P.

LAPAGE, J. E. SHELTON, T. G. MITCHELL AND A. R. MACKENZIE

TABLE IV-continued Organism

Streptomyces erythraeus Streptomyces fradiae Streptomycesgriseus Streptomyces lavendulae Streptomyces noursei Streptomyces olivaceus Streptomyces phaeochromogenes Streptomy cespolychromogenes Streptomyces rimosus Streptomyces venezuelae Streptomyces vinaceus Streptomyces viridifaciens Thiobacillus concretivorus Thiobacillus denitrificans Thiobacillus neapolitanus Thiobacillus novellus Thiobacillus thiocyanoxidans Thiobacillus thiooxidans Thiobacillus thioparus Thiopedia rosea

Storage life (years)

Comments

10 10 10 10 10

10 10

10 10

10 8 8 6 6 6 6 6 6 6 6

Should be stored at 0 4 ° C Should be stored at 0 4 ° C Should be stored at 0-5 "C Should be stored at 0-5°C Should be stored at 0 4 ° C Should be stored at 0 4 ° C Should be stored at 0 4 ° C Should be stored at 0 4 ° C

2. N C T C results and notes on Table V Table V gives the results obtained for the viability of freeze-dried ampoules of many bacterial species, based on the experience and methods of the NCTC since the introduction of freeze-drying in 1949. Viability is determined by plating samples from serial dilutions (see Section IXB). Using 0.02 ml volumes, the highest dilution to yield a colony count of 10 or more from a series of decimal dilutions is recorded as the logarithmic count value. For example, if the 10-4 dilution gave a mean colony count of 12, then the mean logarithmic count was scored as 4. Dilutions giving less than 10 colonies were discounted, as it was considered that these results would not necessarily be reproducible with another ampoule. In practice, therefore, the penultimate dilution yielding growth was usually scored as the logarithmic count value, since the last dilution frequently gave less than 10 colonies. Thus, the actual viability of an organism is often one logarithm higher than that recorded. As we only count dilutions up to 10-6, when the highest dilution still gave confluent growth, semiconfluent growth or uncountable colonies (see Section IXB), the logarithmic count has been scored as 6 + . Thus, the

XI. CULTURE COLLECTIONS

209

initial reduction in numbers after freeze-drying is unknown in some cases. Especially in some of the earlier records, the counts have been scored as + + , + or and there is no accurate quantitative information available. Such counts have been recorded in Table V as AL = alive. The results are presented as follows-

+

+ +

Column 1: Name of organism. The names of the organisms are listed alphabetically by genus and species. Column 2: No. of strains. The total number of strains of each bacterial species used in the analysis is given. Column 3 : Mean log. count. The mean logarithmic value of the count of the suspension before drying (BD) is given, followed by the mean value for the logarithmic count of the reconstituted material in a sample ampoule immediately after drying (AD). Column 4: Mean log. count after various storageperiods (yews). The mean logarithmic count value obtained for each organism during various storage periods after freeze-drying is given. Thus, viability is recorded after 3 months 6 months, 1 year, 5 years, 10 years, 15 years and 18 years. I n general, if a count was done for a year not given in the table, then this count has been scored for the nearest preceding year in the table, e.g., a count at 12 years is scored in the 10 year column. Figures in parentheses denote the number of strains counted at each period, and the average count value has been estimated from these. This does not necessarily equal the total number of strains quoted in Column 2, since the interval chosen between viable count determinations varies with each culture. Column 5 : Comments. The organisms are graded as given below to enable easy reference to the suitability for preservation of a given species by freeze-dryingE: Easy to freeze-dry giving a good survival rate; batches should be satisfactory. M: Moderately easy to freeze-dry. Batches should be satisfactory with attention to detail, but care is needed as some batches may give a poor survivalrate. D: Difficult to freeze-dry and many batches can be expected to give a poor survival rate. Other methods of preservation may be more successful. The values for the logarithmic counts are expressed as an average of the results obtained for all the strains and all the batches of a given organism. An inherent disadvantage of this method of expression, is that, in some instances, the figures may represent a more favourable survival rate than may actually exist. Batches with low counts which have been included in 9

210


these earlier count values may have been discarded to be replaced by new batches; such poor batches were omitted from subsequent count determinations as, if either a very low count or complete loss of viability had been expected, then in practice the batch might have been discarded or the count never done. On the other hand, batches yielding low initial counts immediately after freeze-drying have been included and may show correspondingly low counts throughout their shelf life. This gives a bias towards lower survival rates. However, the overall effect tends to represent the survival time of the best batches, i.e., under the optimal conditions. A more comprehensive record of results was, however, considered to be beyond the scope of this chapter. Reference to the table will provide guidance as to the success or failure rate which might reasonably be expected from freeze-drying a given bacterial species, paying attention not only to the absolute survival times but to the logarithmic drop in count values. For simplicity, we have graded the species as described above. It must, however, be stated that our temperature of storage, 041"C, is far from the optimal, and better survival could be expected after storage at 4-10°C. We have found that the suspension in which the organism is dried makes some difference to the survival. Therefore, many earlier counts where the organisms were dried in horse serum alone or in other substances are not as satisfactory as the later batches when more successful suspending fluids were used. Where this was obvious we have discounted batches with unsatisfactory media. Our results are not, of course, entirely due to the effects of freeze-drying since they are dependent on other factors such as the suitability of the predrying medium, the cultural conditions of the organism, and also on the recovery media and methods for estimating viability.

X. LIST OF SOME CULTURE COLLECTIONS For the benefit of readers a list of some Culture Collections of microorganisms is appended in Table VI (p. 222). Such a list cannot pretend to be comprehensive but it includes some of the important collections in the world. Our list is based largely on that given in Clark and Loegering (1967). For convenience, these Culture Collections are arranged by the countries in which they are situated rather than by the type of organisms they maintain. The name of the Collection is given followed by the address and the kinds of organisms preserved. The I.A.M.S. Section on Culture Collections is assemblingaWorld Directory of Culture Collections which will be published.

TABLE V

NCTC: viability of freeze-dried bacteria Mean log. count Nameof Organism

Acinetobacter anitratus Acinetobacter l w o f i Actinobacillus actinomycetemcomitans Actinobacillus equuli Actinobacillus lignieresii Actinomyces bovis Actinomyces israelii Actinomyces odontolyticus Aerococcus virihns Aeromonas liquefaciens Alcaligenes dknitrificans Alcaligenes faecalis Alcaligenes viscosus Anaerobic cocci Bacillus alcalophilus Bacillus alvei Bacillus anthracis Bacillus brevis Bacillus carotarum Bacillus cereus

No. of , -A- ( strains BD

11 3 2 7 5 5 7 2 15 4 1 3 1 17 1 4 7 3 1 9

Mean log. count after various storage periods (years), with no. of strains counted in parenthesis :

AD

>

A

3/12

6/12

1

5(2) 6+(10)

5

10

6(11) 6(1)

5(9)

3(2) 3(7) 3(5) 4(5) 3(7) 4(2) 5(15) 5(4) 3 5(3) 4

3(1) l(3) 2(1) 4(1) 2(6)

3(17) 3 5(4) 4(7) 5(2) 5 5(8)

..

15

18

l(1)

..

.. ..

E E

..

.. ..

M

M E

E M E E E

l(1)

M M .. M 2(1) M

..

..

.. .. .. ..

5(13)

4(5)

5(1) 3 l(1)

..

.. .. .. ..

..

..

4 3(16) 1 4(4) 4(6) 5(1)

..

4(4)

Comments

.. .. ..

..

..

4(2)

.. .. ..

E

E

.. E

.. .. .. ..

5(1) E

..

2(1)

E E

2

TABLE V-continued Meanlog. count

NameofOrganism

Bacillus cisculans Bacillus coagulam Bacillus freudemkchii Bacillus laterosporus Bacillus lentus Bacillus lichenifwmis Bacillus loehnisii Bacillus maceram Bacillus megaterium

N0.0f & strains BD AD

h,

m

Mean log. count after various storage periods (years), with no. of strains counted in parenthesis A

3/12

6/12

1

>

5

10

15

18

Comments

.. .. .. ..

.. E .. E .. E .. E .. E 2(;) 2(1) E .. .. E .. .. E Dead(1) . . E 15 year count of

5 3 1 3 1 14 1 4 6

batch having low Bacillus mycoides Bacillus pantothenticus Bacillus pasteurii Bacillus polymyxa B a c a ~ pumilus h Bacillus sphaericus Bacillus stearothennophilus Bacillus subtilis Brrcteroidesfragilis Bacteroides melaninogmktnn Bordetella bronchiseptiur

..

4 4 1 3 5 5 2 9 3 3 11

..

.. .. ..

..

3(2)

.. ..

6+(11) 6+(11)

..

initialcounts

..

E 3(4) E

.. .. .. .. ..

M

.. ..

M

E E E E l(1) E

..

D 2 batches of different strains dead after 1 year E

F % " L(

TABLE V-continued Mean log. count Name of Organism

N0.0f strains

Bordetella parapertussis 3 Bordetella pertussis 8 Brucella abortus 12 Brucella melitensis 7 Brucella neotomae 4 Brucella ovis 1 Brucella suis 7 Chrdacterium lidum 3 C h r d a c t e r h m typhijlavum 1 C h r d a c t e r i u m violaceurn 15 Citrobacter ballerupensis 8 C i t r h c t e r freundii 8 Clostrkiium aerofoetidum 1 Clostria'ium bifemrentuns 10 Clostridium botulimm type A 8 Clo&um botulinum m e B 4 Clostridium botulimnn type C 3 Clostridium botulinum type D Clostridium botulinum type E Clostridium butyricum Clostridium chauvoti

. C l O d u mfauax

1 2 2 3

1

Mean log. count after various storage periods (years), with no. of strains counted in parenthesis

BD

AD

3/12

\

A

( p A >

6/12

1

5

10

15

..

.. 5(2) 6(1)

.. ..

..

.. ..

..

..

..

18

Comments

3(1) E .. E 5(1) E .. E .. E

..

M

4(1) E .. M .. E .. E 2(2) E

l(1) E .. E .. E .. E 3i2) 3(1) E .. .. M 2 batches of different strains dead after 8 years 1 .. M 3(1) .. M . . .. E . . Dead(1) D

2i2)

..

..

..

..

..

..

E

t3

z

1'ABLE V-continued Mean log count

N0.0f \-*( NarneofOrganism

strains

Clostridium oedematiens type C 4 Clostridium oedematiens type D 4 Clostridium putrefaciens 1 Clostridium putrificum 1 Clostridium septicum 11 Clostridium skatalogenes 1 Clostridium sphenoides 1 Clostridium sporogenes 6 Clostridium tertium 2 Clostridium tetani 29 Clostridium tetanomwphum 4 Clostridium welchii type A 25 Clostridium welchii type B 4 Clostridium welchii type C 5 Clostridium welchii type D 3 Clostridium welchii type E 1 Clostridium welchii type F 1 Camamonas percolans 3 Corynebacterium acnes 1

'd

A

BD

AD

3/12

6/12

1

5

10 -

Clostridium histolyticum 4 Clostridium oedematiens type A 7 Clostridium oedematiens type B 3

v)

Mean log. count after various storage periods (years), with no. of strains counted in parenthesis I

15

18

..

..

.. ..

..

..

..

..

.. .. ..

..

..

..

.. ..

.. ..

..

..

2(3)

..

..

*. ..

..

Comments E E M 1 batch dead after 5 years D

2(1) D .. E .. E .. E .. E .. E 5(1) E

..

E

cd P

.$ U

m

k

B

2z 9 0

E E

.. E .. E .. E

.. E 2(2) E .. E .. E

.. ..

E M

$ m

TABLE V-continued Mean log. count

N0.0f Nameof Organism

strains

Corynebacterium bouis Corynebacterium diphthiae Corynebacterium equi Corynebacterium jimi Corynebacterium jibvidum Corynebacterium haemolyticum Corynebacterium hofmunii Corynebacterium murium Corynebacterium ovis Corynebacterium pyogenes Corynebacterium renale Corynebacterium segmentosum Corynebacterium ulcerans Corynebacterium viscosum Corynebactmiurn xerosis Enterobacter aerogenes

Enterobacter cloacae Erysipelothrix insidiosa Escherichia alkalescens Escherichia coli Escherichia dispar Flavobacterium meningosepticurn

r p A - - >

BD

AD

Mean log. count after various storage periods (years), with no. of strains counted in parenthesis r * > 3/12 6/12 1 5 10 15 18

..

1 56 5 1 1 3 3 3 7 7 2 1 11 1 6 2 14 7 10 166

.. E

..

3(5) E 5 ~ )5~ E

4[4)

6(2)

.. .. .. ..

..

..

..

3(1)

..

4

..

4(2)

..

..

6

6

6

5

..

E .. E .. E .. E

4(1) 3(4) 4(4) 4(2)

..

3(6) 3(2)

E E E

..

..

E

..

6+

3(1) E .. E .. E 3(1) E 3(1) E

E E

..

6+

.. E .. E

.. .. ..

5(1)

8

1

Comments

E

.. E

0

C

E 0

El


Name of Organism

No.of , -A- ( strains BD

Mean log. count after various storage periods (years), with no. of strains counted in parenthesis \

A

f

AD

3/12

6/12

1

5

10

.. ..

..

l(1)

.. .. ..

::.. J

3(10)

1(1)

l(3) E Later batches give higher counts E .. E .. E

HaemopMus canis

4(2)

2(3) Dead(1) 1(1) Dead(1)

..

..

Klebsiella aerogenes Klebsiella edwardsii Klebsiella ozaenae Klebsiellapneumoniae Klebsiella rhinoscleromatis Kurthia zopjii Lactobacillus acidophilus Lactobacillus odontolyticus Listeria monocytogenes

1 1 14 2 4 10

6

5 6+(14) 6+(2) 6(4) 6(10)

6 4 6(9) 6(2) 5(4) 5(10)

l(1)

2

.. ..

.. ..

i(3) 2(2)

.. .. ..

..

..

.. ..

1

MorD (strains differ)

..

..

.. .. .. ..

..

E E . . E Later batches

..

..

3(2) 3(2) 5(5)

Comments

..

4(3)

Haemophilus gallinarum Haemophilus haemolyticus Haemophilus influenzae Haemophilus parainjluenzae Haemophilus suis Hafnia alvei

18

..

Haetmphilus aegyptius Haemophilus aphrophilus

..

15

2(1) 3(2) 4(6)

.. ..

4(1)

.. .. ..

give higher counts M M E

TABLE V-continued Mean log. count NameofOrganism

Loeflerella mallei Loeffleella pseudomallei Moraxella bovis Moraxella lacunata Moraxella liquefaciens Moraxella nonliquefaciens Mycobacterium balnei Mycobacteriumfortuitum Mycobacteriumjohn&

Mycobactmum marinum Mycobacterium phlei Mycobactmum rhodochrouc Mycobacteriummegnuatis Mycobactmum tuberculosis, avian variety bovine variety human variety murine variety Mycobacterium ulcerans Mycobacterium xenopk Neisseria catarrhalis

,-A-( N0.0f strains BD

3 9 3 2 1 1 4 8 1

2 5

Mean log. count after various storage periods (years), with no. of strains counted in parenthesis \

h

AD

3/12

6/12

1

5

10

15

18

..

5(1) E 4(1) E .. M .. M .. M .. M .. E .. E . . D 5 and 10 year counts on batch with low initial counts .. E .. E 5(1) E .. E

4(2) 4(1)

.. ..

.. .. .. .. ..

3 11

4(2) 3(1) 3(2)

5

..

3

5 3

1 1 4

3il)

.. .. .. ..

..

Comments

M

.. M 2(1) M .. M .. D .. E .. E

CI CI

0

s

i! 0

E;;

3

TABLE V----continued

NameofOrganism Neisseria fivescens Neisseria gonorrhoeae Neisseria mingitidis Neisseria pharyngis Nocardia asteroides Nocardia blackwellii Nocardia caviae Nocardia congolensis Nocardia cuniculi Nocardia farcinica Nocardia mudurae Nocardia pelletieri Pasteurella haemolytica Pasteurella pestis

Pasteurella pseudotuberculosis Pasteurella septica Polysepta pedis Proteus inconstans Proteus mirabilis

N0.0f strains

Mean log. count after various storage periods (years), Mean log. with no. of strains counted in parenthesis count &I A BD AD 3/12 6/12 1 5 10 15 18

2(2) l(1) .. Dead(4) . . .. 3(21) Dead (1) Dead (1) 1(1) 1(1) 4(2) .. ..

3

7 28 2 2 1 1 1 1 1 2 2 2 10

18 22 1

10 10

\

..

.. ..

.. .. ..

..

5 3

.. .. .. ..

..

2(1)

..

6(18) 6(18) 6(22) 6(22) 4 4 6(10) 6(10) 6+(10) 6+(10)

.. .. ..

.. ..

3(6)

.. .. ..

..

..

6(W W2) 4

6(8) 6+(10)

4(1) 4(6) 3 3(8)

500

..

..

..

..

.. .. .. .. ..

..

.. .. .. .. ..

.. .. ..

..

..

.. .. .. ..

..

.. .. .. .. ..

Comments

E D m

M E

R

E E E

E E E E

E M E 10 year count on early batches with low initial count

E E E

E E

?

P

TABLE V-continued Mean log. count Nameoforganism

Proteus morganii Proteus rettgeri Proteus vulgaris Pseudonwnas aeruginosa Pseud0mona.s alcaligenes Pseudmonas chlororaphis Pseudomonas diminuta Pseudomonasfluorescens P s A n a s graveolens Psnrdomonas iodinum Pseudomoms mucidolens PseudomoMs ovalis Pseudomoms syncyanea Pseud0mona.s sp. (achromogenic)

Salmonella abortusbovii SalmonelIa abortmovis Salmonella paratyphi A Salmonella pullorum Salmonella sendui Salmonella typhi Salmonella typhisuis Salmonella spp. Serratia marcescens

N0.0f strains

Mean log. count after various storage periods (years), with no. of strains counted in parenthesis r

BD

AD

3/12

,

h

( p A -

6/12

1

5

10

15

8 8

.. ..

10 16 1

..

..

.. .. .. .. ..

1 1 3 1 1 1 2 1

4 2 1 12 1 1 19

5 340 15

.. ..

.. ..

6+

5 6(2) 4

..

18

..

.. .. .. .. ..

.. .. ..

..

.. ..

..

..

.. ..

4(2)

.. 3(5) ..

..

Comments

E E E E E E E E E E E

E E

.. E

.. ..

.. ..

E

E E

E 3 .. E 1(2) E 3(5) 3(1) 2(3) E 5(232) 4(104) 4(55) E 3(71 E

..

..

CI

w

TABLE V-continued Mean log.

Nameof Organism Shigella boydii Shkellu dysmteriae Shigella fixneri Shigella sonnei Shigella spp. Staphylococcus afmnmtans Staphylococcus aureus Staphylococcus lactis Staphylococcus roseus Staphylococcus saprophyticus Streptobacillus nwntil~oonnis Streptococcus agalactiae Streptococcus b m k Streptococcus durans Streptococcus dysgalactiae Streptococcus equi Streptococcus equisimilis Streptococcus faecalis Streptococcus faecium Streptococcus hominis Streptococcus liquefaciens Streptococcus pneumonioe Streptococcus pyogenes

28 35 43 7 25 16 73 9

6+(28) 6+(38) 6(43) 6+(7) 6+(25) 6(16) 6(60) 6(20) 6(9)

11 1 13 3 4 4

6(11) 4 5(13) 4(3) 4(4) 5(4)

4 4 6 3 3 3 41

5(4) 5(4) 5(6) 4(3) 5(3) 5(3) S(41)

20

1

5

AD

cd l

3/12

6/12

.. .. ..

.. .. .. ..

..

..

..

.. .. ..

..

.. .. ..

..

1

5

10

15

.. 4(i6)

.. ..

4(4) 5(30) 56) 4(1) 5(3)

i(4) .. .. .. .. ..

..

.. .. .. ..

46)

18

*

r

A.

r-

( p A \

BD

P


count

N0.0f strains

0

Comments

2


N0.0f NameofOrganism

Streptococcus sanguis Streptococcus uberis Streptococcuszooepidemicus streptococcus zynogenes Streptococcus :unnamed GOUPS A-H and K-T inclusive streptomyces buccalis Streptomyces graminis Streptomyces griseus Streptomyces homnis Streptomyces hortonensis Streptomyces listeri Streptomyces pellethi Streptomyces s o d * Streptomyces upcottii Vibrio alcaligenes Vibrio choleraeasiaticae Vibrio eltw Vibriofetus Vibrio metchnikovii

strains


BD

AD

3/12

6/12

1

5

10

5(2) 5(2) 5(2) 5(3)

5(3) 5(4) 5(4) 5(3)

4(3) 5(4) 5(4) 5(3)

4(2) 5(4)

4(49) 2 2(2) 3 2 2 3 2(2) 3 1 6(3) 5(14) 5(9) 3(3)

4(54) 1

3(50) 1

..

..

3 1 1 1 2(2) 2 1 5(3) S(14) 5(9) 4(1) 2

3 1

3

3 4 4 3

5(3) 5(4) 6(4) 5(3)

5(3) 5(4) 5(4) 5(3)

5(2) 5(2) 5(2) 5(3)

54

5(54)

5(54) 3 3(3) 3 2 3 3 2(2) 3 2 6+(3) 6(14) 6(9) 3(3) 5

4(51)

1 3 1 1 1

1 2 1 1 3 14 9 3 1

4

3(3) 3 2 3 4 3(2) 3 2 6+(3) 6+(14) 6+(9) 4(3) 6+

1

A

..

.. .. .. ..

3

.. .. ..

6;s) ii3)

..

.. 2 3(2) 3

.. 2

.. 2(2) .. .. ..

6(5)

.. 3(3) ..

4

..

4(4)

4(3)

..

i(i)

..

0

48) 4(3) 3(1) 1

15

18

.

..

*

41)

..

3(8)

..

..

..

.. .. .. .. ..

3(2) 5(1)

.. ..

Comments

E E 3(2) E 4(2) E

..

3(6) E .. E .. E .. E .. E .. E .. E .. M .. E .. M .. E .. E 50) E .. D .. E

w Y

TABLE VI List of some Culture Collections of microsrganisms (see text on p. 210)

E N P

country Argentina

Australia

Brazil

Brazil

Canada

Culture Collection

Address

Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Department de Microbiologia Bacteriology Department, Directory of Collectionsand University of Queensland, List of Speciesmaintained in Brisbane, Queensland. Australia Catalogue available:Her Majesty's Stationery Office, London, England Instituto de Micologia, Universidade Federal de Pemambuco, Recife Faculdade de Medicina da Collectionof Fungi Universidade de S I o Paulo, Departamento de Microbiologia e Imunologia, Caka Postal 2921, SZo Paulo Directory of Collectionsand Canadian Committee on Culture List of Speciesmaintained in Collectionsof Microorganisms, Canada (52 separate collections Division of Applied Biology, National Research Council, listed) Sussex Drive, Ottawa, Ontario. Catalogue available: Her Majesty's Stationery Office,London, England Collection of Micro-organisms

'd

Most recent catalogue

Organisms maintained

1966

Bacteria and fungi

1966

Bacteria, rickettsia, viruses (animal, insect, bacterial, plant), algae, fungi, yeasts, protozoa, metazoa

1966

Fungi

1961

Fungi pathogenic for man and animals

1967

Algae, bacteria, fungi, protozoa, viruses (animal, avian, insect, bacterial, and plant), and yeasts

r

P

TABLE VI-continued

Country

Culture Collection

Mold Herbarium and Culture Collection Czechoslovakia CzechoslovakCollections of Microorganisms (1 3 separate collections listed) Canada

France France Hungary India

IdY

Japan

Netherlands

New Zealand

Institut Pasteur National Museum of Natural History The Hungarian National Collectionof Medical Bacteria Directory of Collectionsand List of Speciesmaintained in India La Collegione dei Lieviti Vinari

Address


University of Alberta, Edmonton, Alberta J. E. PurkynE University ti. Obrhch miru 10, Bmo


1966

Yeast and moulds

1964

25, Rue du Dr. Roux, Paris, 1 Se 12, Rue de Buffon, Paris

1966 1966

Bacteria (including entomogenous, animal-pathogenic, dairy), fungi (includingwood-destroying, and pathogenic and saprophytic yeasts), viruses for animals, plants, and bacteria Bacteria and bacteriophages Fungi

National Institute of Public Health, Budapest Her Majesty’s Stationery Office, London, England

1964

Bacteria (medical)

1960

Algae, protozoa, bacteria, fungi (including yeasts), viruses (animal and bacterial) Yeasts

Instituto de Microbiologia Agraria e Tecnica dello Universiti di Perugia Japanese Federation of Culture Institute of Applied Microbiology, CollectionsUFCC) (1 1 Tokyo Additional member collections) editions Centraalbureau voor 20 Javalaan, Baam Schimmelcultures Directory of Collections and Her Majesty’s Stationery Office, List of Speciesmaintained in London, England New Zealand

1957

1962 {1966 1968

H

2 c 0

0

r

G

c)

3

Bacteria, fungi (includingyeasts) and human and animal viruses

1968

Fungi and yeasts

1968

Bacteria, fungi, viruses (animal and plant), and yeasts

E w

TABLE VI-continued country

Culture Collection

Address



N

%

m

w

Poland

Polish Collectionof Microorganisms

Roumania

(CNIC) National Collection CantacuzinoInstitute

Switzerland

International Center for Information on and Distribution of Type Cultures Her Majesty's Stationery Oflice, Directory of Collectionsand List of Speciesmaintained in London, England United Kingdom, Ghana and Trinidad Collectionof Fungi and Sub-departmentof Medical Yeasts pathogenic to man and Mycology, London Schoolof Hygieneand Tropical Medicine, animals Keppel (Gower) Street, London W.C.I. Forest Products Research Collectionof Wood-rotting Laboratory, PMces Risborough, Fungi Aylesbury, Bucks Ferry Lane, Kew, Surrey CommonwealthMycological Institute Collectionof Fungus Cultures

U.K.

U.K.

U.K.

U.K.

Polish Academy of Sciences, Ludwik Hirszfeld's Institute of Immunology and Experimental Therapy, Wroclaw, ul. Chalubibkiego 4 Institute of Microbiology, Parasitologyand Epidemiology, Spl. Independenfei, 103, Bucurqti 19,Avenue Ctsar R o n , Lausanne

-

Bacteria +I

1960

Algae, bacteria, fungi,protozoa, viruses (animal, bacterial and plant),

9

and yeasts

3

Fungi and yeasts (pathogenicto

r

manandanimals)

0

Er

* s* ?

Fungi (wood-rotting)

1968

Fungi, including phytopathogens

F

-

TABLE VI-continued

0

Country U.K. U.K.

U.K. U.K. U.K.

U.K. U.K.

U.S.A.

Culture Collection Culture Collectionof Algae and Protozoa National Collection of Dairy Organisms National Collection of Industrial Bacteria National Collection of Marine Bacteria National Collection of Plant Pathogenic Bacteria National Collection of Type Cultures National Collection of Yeast Cultures American Type Culture Collection

Address


1966 Botany School, University of Cambridge 1961 National Institute for Research in Dairying, Shinfield, Reading Berkshire supplement 1965 Torry Research Station, 1966 135 Abbey Road, Aberdeen Terry Research Station, 135 Abbey Road, Aberdeen 1965 Plant Pathology Laboratory, Ministry of Agriculture, Fisheries and Food, Harpenden, Herts 1958 Central Public Health Laboratory, Colindale Avenue, London N.W.9 1963 Brewing Industry Research Foundation, Lyttel Hall, Nutfield, nr. Redhill, Surrey supplement 1967 12301 Parklawn Drive, Rockville, 1966 Maryland

Organisms maintained Algae and protozoa Bacteria (dairy)

Bacteria Bacteria (marine) Bacteria (phytopathogenic)

c)

0

r r m

c)

Bacteria (medical and veterinary)

2 0

3

Yeasts (other than pathogenic)

Bacteria (general, including phytopathogens), fungi (including yeasts, plant rusts, and other phytopathogens), viruses (human, animal and bacterial),cell lines (human and animal), protozoa, and algae

p3

!2

tQ tQ

TABLE VI-continued Country

Culture Collection

Q\


Address

v)


cd

r

U.S.A.

Culture Collection of Algae at Indiana University

Indiana University, Bloomington, 1964 (Am.J. Bot. (1964), Indiana

U.S.A.

Fungal Genetics Stock Center

U.S.A.

IMRU Culture Collection

U.S.A.

International Collection of Phytopathogenic Bacteria

U.S.A.

(NRRL) Culture Collection

U.S.A.

Quartermaster Culture Collection

U.S.S.R.

Tarassevich State Control Institute Medical Biological Preparations All-Union Collection of Microorganisms

Dartmouth College, Division of Biological Sciences, Hanover, New Hampshire Institute for Microbiology, Rutgers, The State University, New Brunswick, New Jersey Department of Bacteriology, University of California, Davis, California Northern Utilization Research and Development Division, U.S. Department of Agriculture, Peoria, Illinois Quartermaster Research and Development Center, U.S. Army, Natick, Massachusetts Institute for Medical and Biological Products, 41 Sivzev Vragek, Moscow, G-2 Department of Type Cultures, Institute of Microbiology, U.S.S.R. Academy of Sciences,Profsouznaya str. 7a, Moscow

Algae

51,1013-44.)

U.S.S.R.

1961

Neurospora and Aspergillus

-

Bacteria

-

Phytopathogenic bacteria

-

Yeast, moulds and bacteria, especially of use in fermentations

-

Fungi (especiallydeterioration)

1959

Bacteria, protozoa

1964

Fungi, yeasts, bacteria and actinomycetesnon-pathogenic for man and animals


227

ACKNOWLEDGEMENTS

Our acknowledgements are due to the present and previous staff of the NCTC and NCIB, on whose work throughout the years we have drawn for much of the material in this chapter. The permission of the Director of Torry Research Station to quote from the official records of NCIB and NCMB, which are Crown Copyright, is gratefully acknowledged. We wish to thank Dr. W. A. Clark for permission to use the table in Clark and Loegering (1967), as a basis for Table VI. We would like to thank Mr. P. J. Fisher for advice on the section on freeze-drying, Mr. R. Rudge for help with the preparation of the table of viability of bacteria in the NCTC, Mr. W. Clifford for translating our sketches into finished illustrations and photographs, and Edwards High Vacuum Ltd, for the photographs in Fig. 11, 12, 14, 17 and 18. Our thanks are also due to Miss Brenda Pentland and Mrs. Barbara Buckton for typing the manuscript through its many stages; without their help it would never have been finished.

REFERENCES Annear, D. I. (1954). Nature, Lond., 174, 359-360. Annear, D. I. (1956a).J. Hyg., Cumb., 54,487-508. Annear, D. I. (1956b). J. Path. Bact., 72, 322-323. Annear, D. I. (1957).J. appl. Bact., 20,17-20. Annear, D. I. (1958). Aust. J. exp. Biol. med. Sci., 36, 1-4. Annear, D. I. (1962). Aust. J. exp. Biol. med. Sci., 40, 1-8. Ashwood-Smith, M. J. (1965). Cryobiology,2, 3 9 4 3 . Bacteriological Code (1966). 1nt.J. syst. Bact., 16,459-490. (Edited by The Editorial Board of the Judicial Commission of the International Committee on Nomenclature of Bacteria.) Bridges, B. A. (1966). Lab. Pract., 15, 418-422. Clark, W. A., Horneland, W., and Rlein, A. G. (1962). Appl. Microbiol.,10,463-465. Clark, W. A., and Loegering, W. Q. (1967). A. Rew. Phytoputhol.,5, 319-342. Coe, A. W., and Clark, S. P. (1966). Mon. Bull. Minist. Hlth., 25,97-100. Collins, C. H. (1967). “Microbiological Methods”, 2nd ed. Buttenvorths, London. Cowan, S. T. (1951). In “Symposium-Freezing and Drying”. The Institute of Biology, Tavistock Square, London. Cowan, S. T. (1953). Lab. Pract., 2, 641-643. Cowan, S. T. (1968). “A Dictionary of Microbial Taxonomic Usage.” Oliver and Boyd, Edinburgh. Davis, E. E. (1965). Mycologia, 57, 986-988. Davis, E. E., Hodges, F. A., and Goos, R. D. (1966). Phytopathology, 56,1432-1433. Diamond, L. S. (1964). Cryobiology, I, 95-102. Fildes, P. (1931). Syst. Bact., 9,174-183 (Medical Research Council, Her Majesty’s Stationery Office, London.) Fisher, P. J. (1963). J. appl. Bact., 26, 502-503. Fry, R. M., and Greaves, R. I. N. (1951). J. Hyg., Camb. 49, 220-246. Goos, R. D., Davis, E. E., and Butterfield, W. (1967). Mycologia, 59, 58-66.

228 s.


Greaves, R. I. N. (1944). Nature, Lond., 153, 485-487. Greaves, R. I. N. (1960). In “Recent Research in Freezing and Drying”. (Ed. A. S. Parkes and A. U. Smith.) Blackwell, Oxford. Greenham, L. W. (1967). J. med. Lab. Technol., 24, 223-224. Grieff, D., and Rightsel, W. A. (1966). In “Lyophilization, Recherches et Applications Nouvelle” (Ed. L. Rey). Hermann, Paris. Grieff, D., and Rightsel, W. A. (1967). Cryobiology, 3,432444. Grieff, D., and Rightsel, W. A. (1968). Appl. Microbiol., 16, 835-840. Hwang, S. W. (1960). Mycologiu, 52, 527-529. Hwang, S. W., and Horneland, W. (1965). Cryobiology, I, 305-311. Jennens, M. G. (1954).J. gen. Microbiol., 10, 127-129. Kauffmann, F. (1966). “The Bacteriology of Enterobacteriaceae.” Munksgaard, Copenhagen. Leschinskaya, E. N. (1946). A m . Rew.sov. Med. (Feb.), pp. 210-215; Reviewed in Publ. Hlth. R e . Wash. (1947), 62,211-213. Lumiere, A., and Chevrotier, J. (1914). C. r. hebd. Skunc. Acud. Sci., Paris, 158, 1820-1 821. Martin, S. M. (Ed.) (1963). “Culture Collections: Perspectives and Problems.” Proceedings of the Specialists’ Conference on Culture Collections, Ottawa, 1962. University of Toronto Press, Toronto. Martin, S. M. (1964). A. Rev. Microbiol., 18, 1-16. Medical Research Council, (1960). “Safety Precautions in Laboratories.” Medical Research Council, London. Meryman, H. T. (Ed.) (1966). “Cryobiology.” Academic Press, London and New York. Miles, A. A., and Misra, S. S. (1938).J. Hyg., C u d . , 38, 732-748. Phillips, G. B. (1961). “Microbiological Safety in U.S. and Foreign Laboratories.” U.S. Army Chemical Corps., Biological Laboratories, Fort Detrick. Rey, L. (Ed.) (1964). “Aspects Thkoretiques et Industriels de la Lyophilisation.” Hermann, Paris. Rey, L. (Ed.) (1966). “Lyophilisation Recherches et Applications Nouvelle.” Hermann, Paris. Rhodes, M. (1950a). J. gen. Microbiol., 4,450-456. Rhodes, M. (1950b). Trans. BY.mycol. Soc., 33, 35-39. Smith, A. U. (1961). “Biological Effects of Freezing and Supercooling.” Monographs of the Physiological Society No. 9. Edwin Arnold, London. Sneath, P. H. A., and Skerman, V. B. D. (1966). 1nt.J. syst. Buct., 16,l-133. Stamp, Lord (1947).J. gen. Microbiol., I, 251-265. Steel, K. J., and Ross, H. E. (1963). J. uppl. Buct., 26, 370-375. Stulberg, C. S., Soule, H. D., and Berman, L. (1958). Proc. SOC. exp. Biol. Med., 98,428-431. Symposium (1960). “Recent Research in Freezing and Drying” (Ed. A. S. Parkes and A. U. Smith). Blackwell, Oxford. Wellman, A. M., and Walden, D. B. (1964). Cun.J. Microbiol., 10, 585-593.

CHAPTER 111

Design and Formulation of Microbial Culture Media E. Y. BRIDSONAND A. BRECKER Oxoid Limited, London, England Basal Ingredients of Culture Media . A. Protein hydrolysates . B. Meatextract . C. Yeastextract . D. Maltextract . E. Agar . F. Gelatin . G. Bile salts and ox bile . H. Carbohydrates used in culture media I. Minerals, chelates, and buffers . J. Silicagelmedia . 11. Use of Constituents in Media . 111. Interactions of Ingredients, Incompatibility in Culture Media A. Precipitation . B. Insoluble matter of organic origin . C. pH . D. Browning reactions . E. Photosensitivity . F. Solubility IV. Sterilization of Culture Media . A. Heat sterilization . B. Cold sterilization . C. Chemical sterilization . References . I.

.

.

.

. . . . . . . . . .

. .

. . .

. .

. . . .

.

229 229 249 252 256 257 266 268 271 273 278 280 284 284 285 285 286 286 286 286 287 289 290 292

I. BASAL INGREDIENTS OF CULTURE MEDIA

A. Protein hydrolysates 1. Sources of protein Plants produce proteins, animals concentrate them. The protein content of raw materials utilized for culture media may vary from animal products with 50-90% of dry weight to some cereals containing less than 1% of protein,

230

E. Y. BRIDSON A N D A. BRECKER

A wide variety of sources may be used to provide protein hydrolysates; plant proteins are normally concentrated by prior alkaline extraction to make an economical process. A list of protein sources, most of which have been used to produce hydrolysates at some time, is given belowMeat (fresh frozen) Meat meals (dried, pulverized) Fish (fresh) Fish meals (dried, pulverized) Casein Ge1atin Keratin (horn and chicken feathers) Groundnut meal Soya meal Micro-organisms (yeasts and Escherichia coli) Cottonseed Sunflower seed Fresh meat, either as carcase muscle or offal (liver and heart) is widely used for high quality peptones. Dried meat may also be used in the form of meat meal. It is important that meat dried for this purpose is processed at temperatures below 50°C until most of the moisture is removed, the temperature may then be raised to drive off the residual water. The initial drying is preferably carried out in vacuo to ensure rapid drying which prevents putrefaction. Overheating may cause loss of labile amino-acids and other factors. Fish protein suffers from its strongly characteristic smell and the presence of fish oils which are prone to rancidity. Casein (milk protein) is extracted from skim milk by acid precipitation. The quality of caseins from various sources varies and caseins with low lactose content should be used. Gelatin, extracted from collagen protein, may be hydrolysed by steam as well as acids or enzymes. It is high in proline and hydroxyproline residues. The amino-acid analysis of gelatin peptones usually reveals little or no sulphur-containing amino-acids. For most organisms gelatin peptone shows little growth stimulating effect. Keratin is the principal constituent of wool, hair, horns, nails, and hooves of mammals, the feathers of birds, and the scales of fishes. It is high in proline and cystine (up to 8% w/w protein has been recorded for each amino-acid) but deficient in lysine. The mechanical strength and chemical inertness of keratin is due to strong disulphide bonding. These bonds must be broken by reducing agents or by heating before keratin is susceptible to enzymic hydrolysis. There is little information on the hydrolysis of keratin as a peptone

111. DESIGN

OF MEDIA

23 1

source although mixed protein hydrolysates may contain keratin in the raw materials. Brewer and McLaughlin (1957) reported that they had produced a peptone from chicken feathers and found it to be perfectly satisfactory. T h e use of micro-organisms as a source of protein is discussed by Pirie (1963) and Ingram (1963). Recent work on the growth of organisms on hydrocarbons has stimulated greater interest in this source of protein. Ground-nut meal, soya meal, cottonseed, and sunflower seed are all oil-bearing products. The oil is expressed from the raw material, in a press, and the residue may contain up to 40% protein. The residual protein will vary in quality according to the processes and processing temperatures. Lysine in particular may be degraded.

2. Methods of hydrolysis Proteins are polymers of approximately twenty amino-acids, linked by peptide bonds, which have the general structure-

R-CH-COOH

I

NH2 Hydrolysis of proteins into peptides and amino-acids is accomplished by(i) Acid hydrolysis. (ii) Enzymic hydrolysis. Acid hydrolysis is carried out at high temperatures using mineral acids, e.g., hydrochloric or sulphuric acid. A typical procedure isTo approximately 1000 litres of 15% w/w hydrochloric acid in a hydrolysis vessel, 800 kilograms of protein (such as casein) is added. T h e mixture is stirred and heated, usually by direct steam injection, until the temperature reaches llO"C, and the pressure 5 lb/sq. in. At this temperature hydrolysis is complete in about 18 hours. Shorter periods of time suffice in high-pressure units where the temperature rises to 160°C. T h e vessel and stirrer must be protected by acid-resisting material to prevent extensive corrosion. Acid hydrolysis yields amino-acids and the hydrolysate must be neutralized before further processing. This neutralization process increases the salt content of the hydrolysate to 3 0 4 % w/w. The neutralized hydrolysate is decolourized, filtered ina multiplate filter press, and the filtrate evaporated in O ~ C U Oto a syrup containing approximately 85% total solids. The syrup may be stored and it is ultimately spray-dried to a white powder,

232

E. Y. BRIDSON AND A. BRECKER

TABLE I The common amino-acids found in proteins Name and Symbol ~~

Glycine

-

GlY)

Structure $H&OOH

Name and Symbol L-Cysteine

Structure

y1

(CyS)

I

CHzCHCOOH

I

L- Alanine

NH2

1.-Methionine (Met)

SCH3

I

CH CH CHCOOH

L-Valine

z NHZ I

L-Aspartic acid (Asp)

L- I soleucine

I

CHzCHCOOH

I

NHZ

L-Leucine

(Leu)

H3C>CHCH~CHCOOH H3C I

L-Glutamic acid (Glu)

COOH

I

CH2CHZCHCOOH

I

NHZ

L-Serine L-Lysine

(Lys)

CHzNHz

I

CH$CY)zCHCOOH

I

L-Threonine

NHZ

L-Tryptophan (Try)

CH,CHCOOH

I

I

L-Proline

I

L-Phenylalanine ((Phe)

CH2CHCOOH

I I

OH.

H

L- Arginine

\-

L-Tyrosine

(Tyr)

CHSHCOOH

L- Asparagine

4Hz 4CHvoo OH

L-Glutamine L-Histidine

(His) H-N

/N

I/

NHz

233

111. DESIGN OF MEDIA

Analysis of Acid-hydrolysed Casein Total Nitrogen Amino Nitrogen AN/TN ratio Sodium Chloride Ash Moisture Amino-acid Analysis Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Serine Pro1ine Threonine Tryptophan Tyrosine Valine

(% wlw casein) 8.3 6.4 77.2 38.0 39 3.5

(% wlw protein) 2.4 1.4 3.7 0.3 4.2 1 *o 0.7 2.7 3.5 3.7 1.7 0.7 9.6 4.1 2.5 absent (destroyed) 3.1 4.1

Enzymic hydrolysis of protein may be carried out by a variety of proteolytic enzymes (proteases). T h e production of peptone on a large scale normally involves the use of papain, trypsin or pepsin. Ficin and bromelain are also effective enzymes but at the moment they are too expensive for large scale hydrolysis. Microbial proteases are today assuming greater significance in peptone production. The mould proteases, produced by strains of aspergilli or streptomycetes, are particularly active in the crude culture liquors. Proteolysis on a large scale must be closely controlled to obtain maximum yields and a standard hydrolysate. The most important feature of a bacteriological peptone is its performance as a sustaining nitrogen source for the growth of bacteria. Chemical analysis can only be used as an approximate guide in evaluating the characteristics of a peptone. Meat is finely chopped and minced, and other proteins are suspended in water before digestion. A slurry of the protein material is pumped to large, steam jacketed, stirred vessels (Fig. 1). These stainless steel vessels may vary from 300-1000 gallons capacity. Base or acid is added to bring the pH of the protein to the optimum for the particular enzyme to be

234


FIG.1. Digestion pans for peptone production. These pans project down into the floor and have a capacity of 4000 litres. They are continuously stirred and thermostaticallycontrolled.

used, i.e., pH 2.0 for pepsin, pH 8.5 for trypsin, pH 6.5 for papain. As soon as the optimum temperature of the digest is reached the enzyme is added. The proportion of enzyme to substrate protein is calculated to yield the highest amount of hydrolysed protein within an acceptable processing time. Digestion is allowed to take place, usually with more enzyme being added at intervals. The digestion period may be hours or days and the pH must be controlled throughout this time. Digests which take place at about 37°C for long periods must be protected from bacterial spoilage by the addition of volatile preservatives such as toluene and chloroform. Chloroform must be dispersed throughout the digest by vigorous stirring or agitation, 0.25-0*5% v/v is usually sufficient to preserve a digest. Toluene is also dispersed but its primary function is to cover the surface of the liquor. Both preservatives may be used together but approximately half the volume of toluene is used compared with chloroform. These preservatives are removed during the later heating and evaporating stages of the process. Separation of the soluble nitrogenous liquor from the protein/enzyme slurry is effected by large-scale filtering or by continuous centrifugation. The dilute peptone solution is then rapidly concentrated at low temperatures (at or below 40°C) using large evaporators or plate heat exchangers

111. DESIGN

OF MEDIA

23 5

FIG.2 Peptone manufacture. The upper illustration shows a multiplate filter and pressure vessels. The lower illustration shows a continuous two-stage concentrator which, by plate heat-exchangers, evaporates dilute filtrates to thick syrups.

(Fig. 2). The peptone syrup, containing 67-75% total solids, may be stored for a period whilst awaiting further processing which may include changing the pH, precipitation of excess alkaline earth metals or further filtration to

236


obtain an optically bright solution. A final concentration to a dried product may be carried out by spray-drying or by drying in thin layers in vacuum ovens. 3. Properties of enzymes used in the preparation of peptones (a) Pancreatin (trypsin, chymotrypsin). Trypsin activates trypsinogen to produce further trypsin. This autocatalytic reaction proceeds rapidly in the presence of Ca++ ions. Trypsin is not inhibited by the common sulphydryl (-SH) reagents nor by mild oxidation. It is inhibited by soyabean inhibitor, wheat inhibitor, lima-bean inhibitor and ovomucin. Thus a tryptic digest of soya protein would not be successful unless the inhibitor was removed or neutralized. Trypsin is stable below pH 6.0, chymotrypsin is stable below pH 7.0. Both enzymes are destroyed autocatalytically above these pH values. The optimum pH values for maximum activity against proteins are pH 7-9. (b) Pepsin. Activation of pepsinogen to pepsin is produced by acid pH values between pH 1 and pH 4. The optimum pH for activity against proteins is about pH 1-8. Above pH 4,pepsin is relatively stable and most stable at pH 5.0-5.5. The enzyme does not hydrolyse esters or amides of a-amino-acids; in contrast to trypsin or chymotrypsin, pepsin shows a broad specificity for bonds with adjacent aromatic amino-acids. (c) Papain (chymopapain).Papain is activated by reducing agents and inactivated by oxidizing agents. Iodine, peroxides, oxygen, iodoacetate, methyl bromide are all inactivators of papain and are all capable of modifying the essential -SH groupings in some way. Papain (papaya) is stable at pH 5-0 but becomes unstable below pH 3.0 and above pH 11.0 (L’ineweaver and Schwimmer, 1941). Apart from its relative tolerance of p H values during protein hydrolysis, papain is quite stable at high temperatures when compared with other proteolytic enzymes. Thus a papain digest of muscle is commonly carried out at 70°C. Papain has a broad specificity and it is a very active protease. (d) Ficin (jigs). This enzyme resembles papain in many respects. It is activated and inhibited by the same reagents. Ficin is stable from p H 3.5 to pH 9.0. Its optimum pH for hydrolysing proteins varies with the substrate; casein, either pH 6-7 or 9.5; gelatin, pH 5.0 (Whitaker, 1957). Ficin is relatively thermostable showing optimum activity at 62~5°C but it is completely inactivated at 80°C. (e) Bromelain (pineapple). This enzyme shows close similarity to papain and ficin. The activators and inhibitors of this enzyme are those of papain and ficin. The optimum pH for protease activity is in the range pH 6-8.


237

Bromelain is active at 55°C but the activity falls sharply as the temperature rises above this value. (f) Microbial Proteases. A very large number of different protease enzymes are secreted by micro-organisms. No clear-cut classification of them is yet possible. 4. IdentiJcation of peptone fractions and amino-acids The nitrogen requirements of bacteria, grown by artificial means, were first met by the addition to media of such naturally occurring substances as blood, urine and other body fluids. Naegeli (1880) was probably the first to use egg albumin, which he called “peptone”. However, it was later discovered that peptones, obtained by partial digestion of proteins, furnished organic nitrogen in a more readily available form. The action of peptone as a supply of growth factors for bacteria is very complicated and the supply of peptides in certain amino-acid combinations may be the most critical part of this action. Nevertheless, determinations of the simpler nitrogen contents of peptone partly characterizes digests. A typical analysis would include total nitrogen, total and primary proteose nitrogen, free amino-acid nitrogen, and amino-nitrogen. Total Nitrogen-determined by Kjeldahl technique (see Herbert, Phipps and Strange, this Series, Vol. 5). Total Proteose Nitrogen-the peptone is precipitated by saturating the peptone solution (2% w/w) with zinc sulphate. The precipitate is filtered off and washed several times with saturated zinc sulphate solution to remove non-protein nitrogen. The separated insoluble material is then dissolved in dilute (1 : 1) sulphuric acid with the aid of heat. The nitrogen content of the sulphuric acid is determined by the Kjeldahl technique. Primary Proteose Nitrogen-the peptone is precipitated with half saturated zinc sulphate. That is, equal volumes of peptone solution and saturated zinc sulphate solution are mixed and allowed to stand at room temperature overnight to complete the precipitation. The precipitate is washed with half saturated zinc sulphate solution by centrifuging with several different changes of solution. The insoluble material is then dissolved in dilute (1 : 1) sulphuric acid and the nitrogen content estimated by the Kjeldahl technique. Secondary Proteose Nitrogen-is normally calculated arithmetically by subtracting the primary proteose value from that obtained for total proteose nitrogen. Peptone Nitrogen-equal volumes of peptone solution and cold 20% aqueous tannic acid solution are mixed. The tubes are placed at 4°C for 30 min to complete the precipitation. It is important not to have an

238


excess of tannic acid, as this will dissolve the tannic acid-peptone complex. T h e tubes are centrifuged and the precipitate washed twice with cold 5% w/v tannic acid solution. T h e washed precipitate is then dissolved in dilute (1 : 1) sulphuric acid and the peptone nitrogen estimated by the Kjeldahl technique. Free Amino-acid Nitrogen-a 2% w/v solution of peptone is precipitated with phospho-tungstic acid in the following manner: to 10 ml of peptone solution a 20 ml volume of 5% w/v aqueous phospho-tungstic acid is added slowly while stirring. The vessel is allowed to stand at room temperature for 30 min. Diatomaceous earth is used to aid filtration of the precipitate and the precipitate washed with 1% phospho-tungstic acid. 5% Phospho-tungstic acid is used to check the filtrate for complete precipitation of the protein intermediate products. If this test is negative, the filtrate volume is concentrated, measured, and the free amino-acid nitrogen determined by the Kjeldahl technique. Ammonia Nitrogen-free ammonia can be determined by the modified Van Slyke and Cullen aeration method. T h e peptone solution is made alkaline and the liberated ammonia is blown into a boric acid solution, where it is then determined by titration (Hawk, Oser and Summerson, 1947). (See also Herbert, Phipps and Strange, this Series, Vol. 5.) Amino Nitrogen-determined by formol titration. A 25 ml sample is titrated to pH 8.0 with N/10 alkali, 12.5 ml of 37% w/w formaldehyde is added and the titration repeated, using a p H meter to p H 8.0. A correction is made for the formaldehyde blank. T h e calculation is based on the nitrogen equivalent of protein. (See also Herbert, Phipps and Strange, this Series, Vol. 5.) By adjusting the ratio of enzyme to substrate and altering the period allowed for proteolysis, it is possible to vary the size of the polypeptide chains to some extent. If as a result of this process a peptone is obtained which contains a high proportion of large polypeptides the description proteose-peptone may be used. An approximate measure of the hydrolysis process may be obtained from the peptone analysis of nitrogen content. T h e example of a casein peptone analysis shown in Table I1 is representative. A more accurate and satisfactory method of assessing hydrolysis is by use of a Sephadex ion-exchange polymer (G25)(Fig. 3). Fractions collected may be examined by measuring the absorption of the aromatic amino-acids at 280 nm. Assuming that the distribution of these amino-acids is random between the various fractions present in the peptone, quantitative estimations of the content of each fraction can be made. Characteristic distributions of peptides in digests are observed when they are examined in this way (Fig. 4). (Sephadex Handbook, Pharmacia, 1967.)

239


TABLE I1

Casein peptone analysis Tryptic hydrolysed casein (%) Total Nitrogen Amino Nitrogen Amino N/Total N ratio

12.9 6.6 51.2

Acid hydrolysed casein (%)

8.3 6.4

73 - 0

The nitrogen content is expressed as % w/w of hydrolysate and the difference in values between the two products is a reflection of the higher salt content of the acid hydrolysate.

FIG. 3. Separating column for fractionating protein digests. The Sephadex column is to the right of the apparatus and the circular table carries tubes collecting aliquots of the column effluent.

240


Fractionation of peptic digest of fresh meat on Sephadex G-25

Fractionation of papaic digest of fresh meat on Sephadex G-25

a,

0

u 10

20

Froctionation of poncreatin digest of fresh meot on Sephadex G-25 N

g e

1

2.0-

0 C

5: n a

O-

8

W

IO-

50

60

(b)

(0)

E c

30 40 Fraction no.

Fraction no

Fractionation of pancreotin digest of casein on Sephadex G-25

W

;2 0 i51: 3 10-

rn - A

10

20

30

40

50

60

Fraction no (d)

FIG.4. Fractionation on Sephadex G-25 at 280 nm: (a) Papaic digest of fresh meat; (b) Peptic digest of fresh meat; (c) Pancreatic digest of fresh meat; (d) Pancreatic digest of casein.

Alternatively biuret or ninhydrin reagents may be added to the fractions and the absorption values measured (see Herbert, Strange and Phipps, this Series, Vol. 5). Fractions isolated from digests may be individually hydrolysed with acid. T h e hydrolysed fractions can then be examined by paper chromatography (Block et aE., 1958) or other supports or by use of an automatic aminoacid analyser (Moore and Stein, 1951, 1954). Most of the techniques available for investigating peptones are described by Moss and Speck (1966). A combination of bioautographic techniques and chemical fractionation was made in an attempt to identify specific bacterial growth factors in enzyme-hydrolysed casein.


241

Biologically active components may be detected by laying strips of paper, cut from chromatography sheets, on to pre-seeded agar plates. T h e medium used is deficient in nitrogen and the nitrogen fractions required by the organisms are exhibited by growth in those zones after incubation (Kennedy etal., 1955). This technique of bioautography may be used for other substances and other separation techniques. Henry et al. (1967) described a method using thin-layer chromatography. The seeded agar layer was poured on to the chromatogram plate and, after incubation for 24 hours, the agar surface was flooded with 1% neotetrazolium and the plate allowed to develop in the dark. Reduction of the tetrazolium salt occurred where growth had taken place.

5 . Analysis of peptones Although large scale preparation of protein digests helps produce standard peptones, batch to batch variations do occur. As discussed earlier in the description of peptone manufacture, biological variation in the protein substrate and the enzyme preparations will contribute to differences between types of peptone and batches of the same peptone. Analyses of peptones therefore list mean contents of the various factors. Quality control is concerned with the examination of batches and the elimination of material not conforming to standards set up within the mean figures. Apart from fractionation of peptides, it is not practical to analyse peptone for peptide content. The peptone is usually hydrolysed with acid or alkali and a total amino-acid analysis carried out. This information is of limited value but does illustrate general differences between peptones ; especially between animal and plant proteins. Total nitrogen is commonly quoted at the head of any list of peptone analysis but this figure includes nitrogen contributed from any source, not only from amino-nitrogen. Knowledge of the distribution of nitrogen as proteoses, smaller peptides, etc., is often useful. Identical organisms, when grown on solid media containing peptones which differ in these distributions, will often exhibit variations in colony size, appearance, haemolysis, etc. Amino-acid nitrogen is a useful figure in the nitrogen analysis. A simple ratio of amino-acid nitrogen to total nitrogen value is commonly expressed for a peptone. This ratio expresses roughly, the degree of hydrolysis undergone by the protein. Nitrogen distribution of peptones was described by Hook and Fabian (1943) in their chemical analysis of commercial peptqpes. T h e summary of examples shown in Table I11 is taken from their table.

242


Large differences in nitrogen distribution may not be revealed by a simple ratio of amino-nitrogen to total nitrogen, A peptone N/total N ratio is helpful in these circumstances.

6 . Moisture Moisture content of peptones is of importance because above 5% WIW microbial growth could take place during storage. A high moisture content favours chemical transformations in the peptone, especially at high ambient temperatures. A sensitive indicator of moisture rising to critical levels is the clumping together of peptone powder and eventual solidificationwithin the bottle. Darkening of colour and change in pH are indications of degradation of peptone.

7. Ash The ash content of peptone is composed of sodium chloride, phosphates, sulphates, silicates,and metal oxides. Acid-insoluble ash is usually composed of silicates only. Silica is present in the muscle of bovidae and other grazing animals; it comes from grass and fodder. Peptone ash rises during pH changes in processing and is highest in acid-hydrolysed protein, unless special low salt processes are used. 8. Salt (as NaCl) The salt content of peptone reflects the sodium chloride content although it is recognized that other sodium or potassium salts may be present. Salt is estimated as sodium chloride and any rise in salt content is also detected in the peptone ash. Very acid digests (e.g., pepsin hydrolysates) or acid hydrolysates require substantial pH control in processing. This neutralization causes high sodium chloride values in such peptones. 9. Phosphate

Phosphate is a useful substance to measure in peptone. It not only acts as a buffer but plays an important part in bacterial metabolism. It is important that highly buffered peptones are not used in carbohydrateindicator media. In these circumstances, small changes in pH caused by fermentation will be masked. 10. Tracemetals Peptones vary widely in trace metal content. Spectrographic analysis i\

TABLE I11 Nitrogen distribution of peptones

Peptone A B C D

E F G Average values of 24 different types of peptone

Total N

Prim. Sec. Prot. N Prot. N Peptone N

15-72 13-73 13.55 15-24 15.83 14.48 11.48

0.07 0.19 0.19 5-27 0.00 1-71 0.19

13.90

0.66

From Hook and Fabian (1943).

0.45 1-91 3-63

Amino-acid N (Sorenson)

Ratio Ratio Amino-acid Peptone N/Total N N/Total N

0.37 6-79 2-13

9.69 8.33 7-99 1.04 12-53 10.55 2-65

3.27 2.54 2.69 1.74 1.74 1.79 3.81

20.806 18.5% 19.8% 12.8% 12.8% 12.4% 22.2%

61.3% 60.5% 59.0% 6.8% 79.2% 73 04 23 7;

2-78

7.41

2.59

18-704

..

8-00

U +I U

8 F

244


shows that calcium, potassium, soclium, magnesium, and silicon are present between 100-1000 ppm in all peptones. The following metals have been detected in various concentrations in differing peptonesSilver Barium Cobalt Chromium Copper

Iron Lithium Manganese Molybdenum Nickel

Lead Tin Strontium Titanium Vanadium

Zinc Aluminium Rubidium

Sykes (1956). Kempner (1967) estimated the trace metals in nutrient broth by X-ray fluorescence, emission-spectroscopy and neutron analysis. A few of the minor components of trace metals in peptone are of greater importance than the others. Copper, zinc, lead, and silver show toxic effects in quite small amounts. Lead and silver should either be absent or present at less than 10 ppm in peptone. Iron and copper may arise in peptone as a result of manufacturing processes. The use of stainless steel equipment alleviates this form of metal contamination occurring. The iron content of peptones may be critical for protein hydrolysates used in toxin production media and is usually supplemented with a known excess of iron in the media. Wide fluctuations in the natural metal content of the peptone would make the supplementations valueless. Copper is more directly toxic to micro-organisms but toxicity cannot be correlated with the total copper content. The inhibitory effect of copper is more marked at pH 6 or less than at higher pH values. Copper has been reported to be toxic in open containers (plugged with cotton wool) and less toxic in screw-capped containers or in the presence of reducing agents. Dubos (1930) found the toxicity of copper to be related to the 0 - R potential of the medium. Lankford et al. (1958) were not able to support this hypothesis. Neither were they able to confirm the suggestion of Woiwood (1954) that CuS is the toxic substance in peptones.

1 1. Fermentable carbohydrates Peptones used in carbohydrate fermentation studies should not contain fermentable carbohydrates. Tests are made by inoculating peptone solutions with yeasts, E. coli or Aerobacter aerogenes cultures. After 48 h incubation at optimal pH and temperature, the peptone is examined for evidence of gas production or acid pH products. Plant peptones and yeast extracts are usually high in carbohydrate


245

content. Meat peptones may contain traces of glycogen but arc normally high in utilizable carbon compounds such as lactate. Liver peptone may contain high levels of glycogen.

12. Lipids Lipids estimated as ether-soluble extracts are normally low in peptones. When present in excessive amounts (above 1%) they interfere with the clarity of the peptone solution. 13. Vitamins The vitamin content of peptone is seldom reported and culture media for organisms requiring excess vitamins are normally reinforced. Only yeast extract or yeast peptones are considered to be substantial donors of water-soluble vitamins to culture media. Enzyme-hydrolysed plant and meat peptones have levels of thiamine, riboflavin, niacin, pyridoxine, and pantothenic acid approximately one-tenth of those reported for yeast extract. Acid-hydrolysed peptones show values between one-tenth and one-hundredth of their enzyme-hydrolysed counterparts. Most acid-hydrolysed peptones are treated with activated carbon at some stage, during which vitamins are absorbed. 14. Microbiological evaluation A microbiological evaluation of a peptone uses a group of organisms covering a wide range of growth factor requirements and measures the growth of each one. A simple solution of peptone (1-2%) adjusted to pH 7.2 may be used. More usually a glucose-mineral salt basal medium is used to test the unknown peptone. Gram-positive, Gram-negative, aerobic, and anaerobic organisms are used. The growth of these organisms is measured against a standard peptone selected for test purposes. It is important of course, to select standards which correspond to the group of test peptones, i.e., meat peptones are compared with meat peptones, plant with plant and enzymic peptones with enzymic peptones. Growth of the test organisms is measured by changes in the absorbance of the medium over 24-48 h growth. A more convenient growth-indicating system uses the Bonet-Maury Biophotometer (Fig. 5). This instrument measures the growth of the organism by means of a selenium photo-electric cell and increasing absorption or decreasing transmittance of light is recorded. The optical cell in which the organism grows is mounted in an incubator unit and the growth

246


FIG. 5. Bonet-Maury and Jouan Biophotometer. Upper illustration shows incubator/absorptiometer chamber on right with automatic growth density recorder on left. Lower illustration shows white-capped cuvettes in situ between condenser lens and selenium photocells. The central light source and mechanism for adjusting the iris aperture to each cell can also be seen.

constantly suspended in the medium by means of a magnetic stirrer. A unique feature of the instrument is that six cells are recorded simultaneously.

247


TABLE IV

Identification, sources and characteristicsof peptones used in microbiological growth media Substrate

Hydrolysis

Name

Uses

Casein

Enzyme (pancreas)

Trypticase (B.B.L.l) General cultivation of microCasitone (Difcoz) organisms especially from milk, Tryptone (Oxoidg) water, or natural environAmber E.H.C. (Amber4) ments. The high tryptophan N-Z Amine (Sheffields) content induces prompt indole N - 2 Case (Sheffield) reactions. The constant aminoacid peptide composition of the hydrolysate favours its use for standard culture media. Used in media for toxin production. High content of amino-acids and small polypeptides.

Casein

Acid

Acidicase (B.B.L.) Widely used in media for toxin Casamino-acids (Difco) production. In spite of deficiCasein hydrolysate encies in cystine and tryptophan it is used as an amino(Oxoid) HY-Case (Sheffield) acid supplement for culture media. It is a constant hydrolysis product containing free amino-acids, useful for standardization.

Meat

Enzyme

Myosate (B.B.L.) The mixture of peptides present Peptone (Difco, Oxoid) in meat digests is suitable for Proteose Peptone the growth of most chemo(Difco, Oxoid) organotrophic bacteria, especiAmber MPH, OM-HAP ally those of medical imporOM (Amber) tance.

Meat

Pepsin

Thiotone (B.B.L.) Peptamin (Difco) Peptone P (Oxoid)

A U.S.P. specified peptone for sterility testing. It is recommended for the growth of streptococci and also for detecting hydrogen sulphide-forming organisms.

Gelatin

Enzyme

Gelysate (B.B .L.)

Peptone of low nutritional value.

Milk

Enzyme

Peptonized Milk (B.B.L., Difco, Oxoid)

Used in culture media for lactic acid bacteria.

248

B. Y. BRIDSON A N D A. BRECKEK

TABLE IV (cont.) Substrate

Hydrolysis

Soya

Enzyme

Lactalbumin

Enzyme

Liver

Enzyme

Cotton-seed Protein

Enzyme

Blood

Enzyme

Mixed or unknown

Enzyme

Name

Uses

A plant peptone containing a high content of carbohydrate. Micro-organisms grow quickly in soya-peptone media but may exhibit a rapid decline in viability. It should not be used in fermentation studies. A highly nutritious peptone for Lactalysate (B.B.L.) lactobacilli. Useful for indole Lactalbumin Hydrotesting because of its high lysate (Difco) tryptophan content. Used in Edamin (Sheffield) tissue culture media as an amino-acid supplement. Used for the cultivation of Liver Digest (Oxoid) protozoa, pathogenic and saprophytic fungi, bacteria and mycoplasmas. Amber CTPH (Amber) Like soya peptone, this is a carbohydrate-containing plant Proflo, Pharmamedia (Traded) peptone. Used in the fermentation industry and for largescale growth of organisms. Amber 0.M.-BHY Chiefly a hydrolysate of serum (Amber) proteins plus haem pigment. Biosate (Casein Yeast) More than one peptone is now B.B.L. commonly specified in culture Polypeptone (Casein media formulations. Manufacturers now compound mixed Meat) B.B.L. Milk Protein peptones in optimal proportions for rapid growth, and Hydrolysate (B.B. L.) other desirable reactions. PepTryptose (Difco, Oxoid) Neopeptone (Difco) tones manufactured from unProtone (Difco) specified substrates are offered Mycological Peptone by primary manufacturers. The lack of details about the sub(Oxoid) strate in no way detracts from the excellence of the peptones. Phytone (B.B.L.) Soytone (Difco) Soya Peptone (Oxoid, Sheffield) Amber HSP (Amber)

+

+

1. Baltimore Biologicals Ltd., Baltimore, Maryland, U.S.A. 2. Difco Laboratories, Detroit, Michigan, U.S.A. 3. Oxoid Ltd., Southwark Bridge Road, London, S.E.I. 4. Amber Laboratories Inc., Milwaukee, Wisconsin, U.S.A. 5. Sheffield Chemical, Nonvich, New York, U.S.A. 6. Traders Protein Division, Fort Worth, Texas, U.S.A.


249

A full description of the instrument and its applications is presented by Coultas and Hutchinson (1962). Simple metabolic reactions of organisms are tested to detect unsatisfactory peptones. Indole production, Methyl Red and Voges-Proskauer reaction, H,S production are examples (see Holding and Collee, this Series, Vol. 6). Bernard and Lambin (1961) used these methods for testing peptones together with a measurement of the concentration of phenol required to inhibit the growth of Staphylococcus auras for 24 h. The amount of phenol required varied between the peptones. General descriptions of methods used in testing peptones are presented by Kheshgi and Saunders (1959), Desbordes and Ninard (1962) and Pluschkart (1964). Hook and Fabian (1943) also prepared complex culture media and measured recovery of organisms and production of gas. Tests must also be made in solid media and for this, peptone-agar media plates are prepared. On agar plates colonial characteristics such as size, pigment, surface characteristics, and haemolysis of erythrocytes can be determined. Bacterial growth recovery tests using the Miles-Misra surface drop test are also used (Miles and Misra, 1938; see also Postgate, this Series, VOl. 1).

B. Meat extract T h e term “meat extract’’ is not specific and different types of extract can be prepared according to the quality of meat, the length of time and the temperature used during extraction. The longer the time period used for extraction the greater the amount of gelatin in the extract. High temperature extraction will have a similar effect and the extract will set to a jelly on cooling. Liebig considered that 34 parts of trimmed meat should yield 1 part of extract. In modern commercial practice “meat extract” is a by-product from the manufacture of corned beef and involves the immersion of chopped beef in boiling water for a short time. This short period of immersion is sufficient to allow the readily soluble bases and inorganic salts to go into solution without the extraction of more than small quantities of gelatin. Approximately 50 parts of trimmed meat are required to produce 1 part of extract, containing 17% of moisture. The use of commercial concentrates of meat extract in place of fresh meat infusions in culture media, was quickly adopted when these became available, as results showed equivalent effects in the majority of cases. The commercial product “Lab-Lemco” is now synonymous with meat infusion, in the preparation formulae of culture media.

250


In manufacture, fresh meat is trimmed free from fat and sinew and then minced and loaded into perforated baskets. The baskets are lowered into tanks of water and steam heated. Beef and water are brought to boiling point and allowed to simmer for sufficient time to secure extraction of the soluble solids. The liquor is run off and passed through a centrifige to separate fat and insoluble matter. The centrifugate is filtered through presses in the presence of a filter-aid such as diatomaceous earth. This imparts a brilliance to the solution as well as removing finely suspended matter in suspension. Finally the filtrate is concentrated to remove most of the water as vapour until the soluble solids in the concentrate reach 75430%. In this condition the concentrate is self-preserving and is packed as a thick paste into jars. Wood and Bender (1957) and Bender et al. (1958) carried out extensive analyses of ox-muscle extracts and Table V is based on their reported figures. The major difference between the two extracts was the higher aminoacid content and reducing-sugar content in the fresh extract. The darker colour of the concentrated commercial extract was evidence of a Maillard reaction taking place between these two substances. The water soluble vitamins of the B group are present in meat extract with the exception of thiamine which is broken down during manufacture. The following figures are based on the Society for General Microbiology report (Sykes, 1956). Creatine and creatinine are the organic bases found in largest amounts in meat extracts. The creatine content of muscle is high and only small amounts of creatinine are found. In stored, concentrated meat extracts there is a slow conversion of creatine to creatinine (Bender et al., 1958). The sum of creatine plus creatinine is usually reported as total creatinine and is roughly similar in fresh and stored extracts. Meat extracts for bacteriological use should be free from toxic materials and low in content of metals such as copper and zinc. The extract should be free from fermentable carbohydrates, if it is to be used in indicator carbohydrate media. The high buffer-phosphate content of meat extract usually prevents acid/alkali indicator changes unless a high concentration of carbohydrate is present in the medium. At a concentration of 0.3-0.5% the meat extract should support the growth of fastidious organisms, when combined with peptone in suitable proportions. The extract can be considered as an aqueous solution of peptides and amino-acids, nucleotide fractions, organic acids, minerals and some vitamins. Its function can therefore be described as complementing the nutritive properties of peptone by contributing minerals, phosphates, energy sources and those essential factors missing from peptone.

111. DESIGN

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TABLE V Analysis of ox-muscle extracts

Component

Amino-acids a-alanine aspartic acid glutamic acid glycine isoleucine leucine lysine methionine phenylalanine serine, threonine & asparagine tyrosine taurine valine Peptides carnosine anserine imidazole peptide Guanidines creatine creatinine methylguanidine guanidine

y i Fresh

yo Commercial

extract

extract

1-27 0.25 0.78 0.25 0.24 0.63 0.07 0.21 0.40 2-59 0.21 1a67 0.39

1-48

4.86 1.12 trace

9.45 0.75

.. .. ..

0.09 0.09

..

0.01 0.11

..

0.36

..

4.15 0.84 2.10

absent absent

5 a40 6.18 -0.1 -0.1

1*46

2.57

Organic Acids lactic acid glycollic acid succinic acid

23 *04 2-34 0.88

16.40 1-10 1.42

Carnitine Urea Ammonia Inorganic matter

3.45 0.68 0.37 27.50

3.7 0.12 0.47 33.0

(8.95% K ; 7.3% Pzo5) Colouring matter Reducing sugar (as glucose)

15.80 2.10

absent

Purines hypoxanthine (free and combined)

20.55

252


TABLE VI Water soluble vitamins of the B group present in meat extract Vitamin (ug/g)

Fresh Beef (pg/g) Meat Extract (pg/g) ~~

Cynanocobalamin (Vit B12) Thiamine (Vit Bi) Riboflavin (Vit B2) Nicotinic acid Pyridoxine (Vit BB) Pantothenic acid Choline

0.3-1.0 0 ‘9-3 * 0 1 *8-3.5 24-1 02 0 * 77-4 0 4.9-15 760

-

0.2 0-1 30-35 1000-1 200 5 25 1500

The following is a typical analysis of meat extract for bacteriological useTotal solids Ash Chlorides as NaCl Phosphate as P205 Total nitrogen Amino nitrogen Ether soluble extract Calcium Magnesium Sodium Potassium Iron Copper Lead Tin Zinc

76.9% 19.1% 6.2% 4.3% 9.2% 1.5% 0.1% 0.05% 0.2% 4.9% 5.8% 51 PPm 1 5 PPm 2 PPm 10-20 ppm 25 PPm

C. Yeast extract Yeast extract is described (U.S. Pharmacopoeia XVIII) as a peptonelike substance derived from cells of Saccharomyes. The substance is available to the laboratory worker, either in a spray-dried powder or a paste form. It may be prepared by autolysis or plasmolysis of the yeast cells. The first commercial yeast extracts were prepared by a patented process of Wahl-Henius in 1894. Since that time, many patents have been granted for various extraction processes. Brewers’ yeasts are commonly obtained as a suspension of 25% w/v solids and may be stored at low temperatures, to retain viability, for up to two weeks. The yeast may be filtered through a fine screen and then washed to remove the beer. Hop resins are solubilized by raising the pH to

111. DESIGN

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253

6.5-7.0 and removed by further washing. It is important that the yeast cells should be kept alive during these processes to prevent the loss of cell constituents during washing and filtration. Bakers' yeasts will not require de-bittering and can be obtained either in a slurry, delivered by tankers, or as compressed yeast in blocks. The extraction process may be carried out by autolysis of the yeast cells by the enzymes present in the cells. The yeast is held in large autolysing vessels, continuously stirred and thermostatically controlled. T h e temperature is slowly raised to 48"-50"C and held at this temperature for some hours. After autolysis the yeast enzymes are inactivated by raising the temperature above 75°C. Extraction by plasmolysis is carried out by holding the yeast in strong sodium chloride solution. The osmotic effect of the NaCl causes plasmolysis of the yeast cell and cell constituents diffuse out through the cell wall. Such extracts contain 3 0 4 0 % w/w sodium chloride. T h e extracted liquor is separated from the cell membranes by continuous centrifugal separators or filter presses. Concentration of the extract must take place quickly to prevent microbial spoilage and at low temperatures (less than 37°C) to preserve heat-labile constituents. Further filtration, using filter aid, may be required. Some extracts produce large quantities of free tyrosine which must be filtered off to obtain a bright filtrate. Powdered yeast extract may be obtained from spray-dried liquor or it may be shelf-dried in vacuum ovens. Yeast extract is widely used in the food industry and at this stage it may be bleached, various salts may be added to it, caramel colouring may be added or spice or vegetable extracts put into the extract before final concentration to the familiar paste. These additions may have untoward effects on bacteria if the extract is added to culture media. Bacteriological yeast extracts should be free from such additives. Yeast extract is basically a mixture of amino-acids and peptides, watersoluble vitamins, and carbohydrates. The carbohydrates of the yeast are chiefly glycogen and trehalose, and these substances undergo enzymic hydrolysis to glucose during the extraction process. Other polyhexoses, mainly glucan and mannan remain in the cell wall and are removed with the insoluble cell material. From the bacteriological viewpoint, the extract differs in relation to its origin. When made from brewing yeast, the extract may contain hop resins which, if not removed, are inhibitory to the growth of organisms. The extract prepared from brewers' yeast is dark brown in colour. Extract prepared from bakers' yeast is free from the influence of hop resins and is much lighter in colour. It is worth recalling that standards of hygiene and aseptic procedure

254


10

z

20

c- 3 0

2

5?

40

50

60

7

70

80 90

IOU

.1

2 Hours of incubation

3

4

FIG.6. Recording of growth of organisms in yeast extracts, as obtained on BonetMaury and Jouan biophotometer. Brewers’ yeast extract-1 . Staphylococcus aureus var. Oxford, 2. Escherichia coli, 3. Streptococcus faecalis; Bakers’ yeast extract4. S. aureus var. Oxford, 5 . E. coli, 6 . S t . faecalis.

in the preparation of bakers’ yeast have to be of a very high order so as to render the end product suitable for bread manufacture (Harrison, 1967). Unfortunately, the same cannot be said for brewing yeast. The authors believe that extract prepared from bakers’ yeast is to be preferred to the brewers’ product. The illustration (Fig. 6) shows comparative absorption readings obtained with E. coli, S. aureus (Oxford strain), and Streptococcusfaecalis when grown in 0.1Y’ yeast extract. The duration of lag phase of the test organisms is shorter in bakers’ yeast extract than in brewers’ yeast extract. Chemical analysis, however, shows a similar picture for extracts from the two sources, as can be seen in Table VII. TABLE VII

Chemical analysis of yeast extracts used in the growth experiments recorded in Fig. 6 Bakers’ yeast extract Moisture Ash Acid insoluble ash Phosphorus as Pz05 Total nitrogen Form01 nitrogen Colour (Tintometer readings)

Red Yellow

30% 8.35 0.26 1-98 9.97 2.91 1.2 5.0

Brewers’ yeast extract

30% 10.0

0.13

4-0 7.6 4-5 3.4 14.0

255


Analytical Tables A General Analysis of Yeast Extract Paste Total nitrogen Amino nitrogen Chlorides as NaCl Moisture Phosphates as Pz05 Carbohydrates Purine nitrogen Fat Sodium Potassium Calcium Iron Magnesium Copper zinc Manganese Cobalt

(g/100 g) 7 *5-10* 5 3.4-4.8 0.07-1 *3 30 3.8 8.2 0.27 trace 5.6 3.0 0.01

0.005 0-2

0*005 0.005 0~0005 0*0005

Range of Values of Vitamin Content of Yeast Extract (CLg/g) Thiamine 18-40 Riboflavin 18-1 50 Nicotinic acid 300-1250 Pantothenic acid 20-1 00 Pyridoxine 25-35 Folk acid 5-1 0 Inositol 1000-1 700 Choline 1000-2000 Biotin 0 * 5-1 a 0 p-Aminobenzoic acid 6 Vitamin Biz 0.01

Amino-acid Composition ( g / lOOg) of Yeast Extract Alanine 3.4 Arginine 2.0 Aspartic acid 4.5 0.45 Cystine Glutamic acid 6.7 Glycine 2.3 Histidine 1.2 Isoleucine 2.3 Leucine 3.0 Lysine 3.5

256

E. Y. BRIDSON AND A. BRECKER Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

0.7 1.7 1.7 2.3 2.3 0.5 1.6 2.5

D. Malt extract Malt extract is a water-soluble extract of malted barley. T h e malting process takes place in three stages: steeping, germination and kilning. During steeping, the grain absorbs water, increasing its content from approximately 12 to 45% and at the same time becomes oxygenated. Germination begins and is only allowed to develop to the stage where rootlets form with slight acrospires development. As slow germination occurs the endosperm enzyme systems split non-soluble compounds of high molecular weight into soluble components of lower molecular weight. The duration of germination determines the nature of the malt required. At the completion of the germination stage, the green malt is kilned. Kilning dries the product by reducing the moisture from approximately 42% down to 8% and it “cures” the malt by fixing in the grain those properties of colour, flavour and friability that have developed during germination. Malt extract is then prepared from malted barley as follows: The grain is cracked in a mill and the grist so formed is extracted with warm liquor to produce wort. The latter is filtered and evaporated to a thick syrup containing 80% total solids or alternatively it is dried under vacuum and packed as a powder. Wort is a primary reagent used in the brewing industry; it is the fermentation of wort by yeast that yields alcohol. Brewers realize that small differences in wort composition may have significant effects on the resulting fermented brew. Therefore control of worts and measurement of changes between batches is of immense concern to them. Variation in wort quality will not only be caused by differences in barley species but also by the condition of the harvested grain. Mashing temperatures and the duration of steeping will cause differences unless the conditions are strictly controlled. Additive cereals other than barley and the use of gibberellic acid also give rise to alterations in wort, specifically the total nitrogen and and amino-nitrogen content. MacWilliam (1968) has published an extensive review of the composition of wort and the following analysis is based on this report.

257


The major constituents of wort are(i) Carbohydrate 90-92y0 of wort solids. (ii) Nitrogenous materials 6 5 % of wort solids, Other components include lipids, fatty and organic acids, phosphates, sulphur compounds, and inorganic constituents. The carbohydrate components of wort include the hexoses, glucose and fructose, the disaccharides, maltose and sucrose, the trisaccharide, maltotriose, and polymers of glucose, dextrins. T h e nitrogenous components include proteins, peptides, amino-acids, ammonia and amines, purines and pyrimidines and vitamins. The amino-acid analysis varies from wort to wort but in all the material examined proline is present in very high proportion (50% of total amino-acids). T h e vitamins present in wort are biotin, folic acid, inositol, nicotinic acid, pantothenic acid, pyridoxine, riboflavin, and thiamine. Potassium represents 12-15% of the inorganic constituents of ash of malt. Sodium and calcium are present in small amounts. Calcium, iron, zinc, and magnesium being present in small amounts in all tested worts. For use in culture media, malt extract should be free from diastatic activity. This will be ensured by a steam sterilization process. If on the other hand the extract is to be filter sterilized, then an extract that is non-diastatic should be selected. Malt extract is a convenient source of wort for use in media required for the cultivation of moulds and yeasts. Because of its acid pH and high level of reducing sugars, it should not be superheated during sterilization as this will lead to darkening in colour due to Maillard reactions between sugars and amino-acids. pH drift below 5-0 will lead to hydrolysis of agar with soft gel formation. A typical analysis of dried malt extract is as followsMaltose Hexoses (glucose and fructose) Sucrose Dextrin Other carbohydrates Protein Ash Moisture pH (10%solution)

52.2% 19.1% 1.8% 15.0% 3.8% 4.6% 1.5% 2.0%

5.5

E. Agar Agar is a gel-forming polysaccharide which is extractable with hot water from several species of red seaweeds (agarophytes). Commonly used 11

25 8


species includeGelidium amansii Acanthopeltisjaponica Gelidium subcostatum Gracilaria confervoides Gelidiumjaponicum Ceramium rubrum Gelidiumpacijicum Ceramium boydenii Pterocladia tenuis Campylaephora hypnaeoides Araki (1958) lists the following components of agarso42-(existing in half-ester form associated with Ca, Mg, and other metallic ions) D-Glucuronic acid Pyruvic acid L-Galactose D- Galactose 3,6-Anhydro-~-galactose Agar can be considered as a mixture of at least two polysaccharides: agarose (ca. 70%) and agaropectin (ca. 30%). Agarose contains less ash, sulphate, and uronic acid than agaropection. Agarose is composed almost entirely of D-galactose and 3,6-anhydro-~galactose; a very small quantity of L-galactose is present. Araki (1958) suggests that the structure of agarose is a 1-3 and 1 4 linked linear polymer"

0

CHzOH

Agar is easily hydrolysed with acids because the 3,6-anhydro-a-~galactoside linkage is very sensitive to acid cleavage. Agaropectin may be a mixture of polysaccharides although its chief components would be D-galactose and 3,6-anhydro-~-galactose. The sulphate esters are assumed to reside on the sugar residues through the half-ester links. Red seaweeds capable of yielding a gel-forming extract are harvested in many parts of the world. The major sources of supply today are Spain, Japan, Portugal, Morocco, America, Korea, New Zealand, and Denmark. Agar is also being produced in Russia but little, if any, finds its way into the Western world. The list of geographical areas capable of producing agar is by no means exhausted and as production fails, or is limited for one reason or another, so new supplies come into being to meet a world demand, e.g., Argentine and Mexico are currently developing industries of their own. The extraction of agar from the weed follows a basic pattern. The weed

259


is harvested once a year from the sea, and is roughly hand sorted from brown seaweed, shells, debris, etc., and dried. The weed is not washed with fresh water but dried with salt water residues. This prevents the weed subsequently becoming too dry when it is baled and stored in silos. Humidity is important during storage of the dried weed as this is believed to affect the gelling quality of the agar. In the extraction of agar (Wood, 1946), the dried weed is first washed to remove weed pigment, salts, and foreign matter, and then extracted by boiling or autoclaving in weakly acid solution at p H 6.5 to produce a 1-27, solution. The hot liquor is strained from weed residue and clarified by centrifugation or filtration. I t is then run into containers which are frozen at -10°C for a period of 24 h. During the freezing process the agar comes out of solution and water soluble impurities pass into the ice. The freezing process is most important for the purification of agar and the more care and time given to this stage the better the quality of the final product. The ice-agar mixture is thawed by spraying with water at ambient temperature, when the ice separates leaving the agar as a soft matt absorbing about 3-4 times its weight of water. T h e matt is finally dried in hot air before milling and grinding. T h e foregoing summary will differ in detail according to local technology (see Table VIII) but the broad principles serve to illustrate the main TABLE VIII American process of agar manufacture (Chapman, 1950) Agrophyte (Sun-dried, unbleached)

+r-

Fresh water Waste water, s a l t s t foreign matter

Spent weed

I

-

4 Agarophyte

-1

and water

Pressure cooker in water

rp

Crude hot agar so1ut’ ion

Filter Sludge

-

Washing tank (12-24 h)

I

1

Storage tank

260


1 1 p=--

Filter press

Storage tank

I

4

Gelchopper

Chopped agar

i - 7 1 Ice can (2 days) I-__-

1

Chopped agar gel 1 I Brine tank (14°F.) in ice can

--

1

Fresh water Waste water and soluble impurities

Th;%

tank

I--

Freshwater Waste water and + Wet flakes soluble impurities

-i

I

Hot air agar flakes

Dehydrator

I

I

Fresh water Waste water

Hot air Moist ari-

--i ~

Agar flakes and 1yo hy,’,chlorite

1

Bleaching tank

Bleached agar flakes in water

I

Washing tank

-Rakes1

Wet bleached agar ~

Agar and 20% moisture

Dehydrator


26 1

sources of impurities that interfere with the preparation of bacteriological media. If the agar is improperly clarified, then subsequent gels will contain insoluble matter, consisting of mineral and vegetable debris that have been left associated with the agar. For every ton of agar produced, 10 tons of water are employed in the extraction process. This contributes calcium and magnesium salts to those already present in the product. T h e presence of these salts is responsible for the precipitates that develop when agar media are autoclaved or remelted. Soluble phosphates which are natural ingredients of meat and yeast extracts and peptone react with calcium and magnesium salts to form insoluble phosphates. Due to the viscosity of the agar solution, the development of insoluble phosphates often results at first in an opalescence of finely divided matter. On standing, or more generally on remelting, the insoluble “haze” contracts to form a flocculent precipitate. During the processing of the agar and more particularly during the drying of the product, contact may be made with iron equipment which can be a source of rust. The iron content of some commercial agars may be sufficiently high (150-200 ppm) to impart a grey-brown hue to the product Iron salts will produce insoluble “phosphates” in culture media in the same way as calcium or magnesium. Dark agar equally can be due to residual weed pigment left in incompletely processed products. The quality of the gel is important for the successful conclusion of many bacteriological techniques. Thus the agar should be capable of remaining molten and fluid at 40’45°C after cooling from boiling point. This is necessary to allow the inoculatioa of test samples, and still permit mixing and distribution into dishes without premature setting. The gel at ambient temperature must have consistent qualities of strength at the recommended concentration. Of the methods which have been put forward, that of Jones (1956) is recommended. This consists in the determination of the “Grade Strength” or weight of test agar required to give a similar gel to that of an arbitrary standard. T h e determination is carried out using the apparatus illustrated (Fig. 7). Agars which produce a satisfactory gel at a concentration required for solid media may not be suitable for the weak gels, e.g., 0.1-0.2% required for semi-solid media. The ability to form a continuous gel at these low concentrations may be limited by syneretic properties which result in shrinkage of the gel. When this occurs, a solid core of agar reflecting the contours of the container may occur floating in a volume of free liquid. This should not be confused with a strata of very strong agar gel, overlayed with liquid medium due to incomplete mixing or unequal cooling on a heat conducting surface. Some commercial agars may contain substances which are toxic to

262


FIG.7. B.F.M.I.R.A.Agar gel testing apparatus. The cubical plastic box containing agar can be seen in the centre of the instrument, just beneath the rotatable spade. The beaker, suspended on the left of the agar box, collects the flow of water and its weight eventually turns the spade through a measured deflection.

bacteria. These may consist of bleaching agents, fatty acid residues (Ley and Mueller, 1946) or trace metals, e.g., copper derived from processing equipment. Measured inocula, e.g., 0.02 ml from decimal dilutions of test organisms added to broth and to broth solidified with test agar will reveal the presence of inhibitors if colonies do not develop on the agar from inocula which have produced growth in the broth from the same dilution. Conversely, heat resistant thermophiles may be associated with agar and resist autoclave times and temperatures usually employed for bacteriological culture media, e.g., 15 min at 121°C. Their presence probably stems from the flora that develop under anaerobic conditions during storage of the dried weed prior to extraction of the agar.

111. DESIGN

263

OF MEDIA

It should be recognized, however, that the presence of viable organisms in agar media following sterilization for 15 min at 121°C is not conclusive evidence that heat-resistant organisms are present in the agar. This apparent contradiction stems from the fact that agar is a very poor conductor of heat and heat penetration is slow. The charts show the temperatures recorded inside a thick-walled and a thin-walled bottle containing 500 mls of 1.2% agar solution during an extended period of autoclaving. In the thick-walled bottle the agar did not reach 121°C until 40 min after the autoclave chamber and 24 min after a control bottle containing water. Thus an agar medium "sterilized" in a thick-walled container for 15 min after the chamber thermocouple indicated 121°C would only reach 100" and any heat resistant spores present would survive and germinate on storage. In the case of the thin-walled bottles, the agar reached 121°C 27 min TABLE IX Chart showing heat penetration in Medical Research Council of Gt. Britain transfusion bottles (thick-walled) containing (a) water (b) 1.2% agar Temperature "C 1

Time (min)

Autoclave chamber

500 ml water

500 ml agar

18 21

18 18 26 46 59 70 79 86 92 100 107 111 114 116 118 119 120 121 122 122 122

~~~~

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60

22 63 105 118 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

44 70 90 103 111 116 119 121 122 123 123 123 123 123 123 123 123 123 123

-

-

264


after the autoclave chamber and 6 min after the control bottle of water. A sterilization cycle of 15 min after the chamber attained 121°C would mean that the agar reached 117°C for 1-2 min. Clearly, inadequate sterilization may give rise, on incubation, to growth of heat resistant organisms derived from sources other than the agar itself. Clarification of the agar in solution after extraction from the weed may or may not be efficient. Cellular debris from the weed, together with mineral matter may remain in the hot agar solution and become associated with the final product. A drop of a hot solution from such agar in water, placed on a slide, will, when examined microscopically reveal the presence of cells from plant tissue, amorphous particles probably of sand origin, and sometimes fragments of diatoms. Black specks, hairs, and other dirt fragments are generally of factory origin and derive from the surfaces of drying equipment, plant and containers and from the atmosphere. TABLE X Chart showing heat penetration in 20 oz screw-cap bottles (thin-walled) containing (a) water ( b ) 1.2% agar ~~

Temperature "C A

I

Time (rnin)

Autoclave chamber

500 ml water

23 55 100 116 120 121 123 123 123 123 123 123 123 123 123 123 123 123 123 123 123

-

500 ml agar .

~~~

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60

>

23 28 56 83 100 109 114 115 117 119 120 120 121 123 123 123 123 123 123 123 123

-

-~

23 28 56 72 82 90 97 107 112 115 117 119 119 120 121 122 122 122 122 122 122

265


TABLE XI Agar analyses

Analyses obtained from commercial samples of agar of different geographical origins (Table XI) serve to illustrate some of the differences in mineral content, gel strength, and acid radicals to be found in these agars. Purification for bacteriological and immunological purposes can bring about a marked diminution of mineral content as illustrated by the analyses of commercial samples given in Table XII.

TABLE XI1 Agar analyses American agar purified Ash Acid insoluble ash Sulphate Chloride Calcium Magnesium Total nitrogen Iron Grade strength Recommended concentration

1.6% 0.14% 0.69% 0.09% 0.39% 0.08% 0.02% trace 145 1.3-1 ' 5 %

"Ionagar" No. 2

Oxoid agar No. 1

1.9% 0.24% o.55yo 0.08% 0.24% 0.49% 0.18% 125 ppm 175

0.9% 0.1504 0.6% 0.1%

1 .O%

0.9%

o.oso/, o.01yo 0.2% 10 PPm 190

266


In addition to its gel-forming properties, agar is important as a clear substrate through which diffusion phenomena can be observed. Precipitin lines at the point of reaction between antigens and antibodies in agar gels have long been observed by workers using agar gels to demonstrate serological reactions (Elek, 1948). Since the introduction of antibiotics, sensitivity tests employing agar gel-diffusion techniques have become widespread. Diffusion of large-molecule antibiotics and chemotherapeutic drugs through agar can be impeded according to the nature of the agar employed. This is particularly applicable to the slowly diffusing antibiotics and chemotherapeutic agents, e.g., tetracyclines and sulphonamides and can lead to erroneous assessments of activity. Bechtle and Scherr (1958) described a test using a few drops of a large molecule dye, Saffranin, placed in a hole in the gel to measure the diffusion rate in agar gels over a period of 2 and 4 h. A modification used by the writers to determine diffusion characteristics of different agars is to extend the diffusion time at room temperature to 24-48 h. The coloured areas of diffusion vary from small restricted zones showing rings of dye concentration to large uninterrupted zones of uniform colour gradient. Agar gel diffusion under the influence of an electric current employed in electrophoretic techniques will depend upon the voltage applied and heat generated in the gel. Kawerau in a personal communication has shown that the presence of contaminating electrolyte in the agar is detrimental and advises the complete removal of chloride and sulphate ions from the gel. The contamination of agar with mineral impurities has often lulled bacteriologists into a disregard for the mineral requirements of bacteria when formulating culture media. Thus agar, which is frequently regarded as an inert substance required to act as a gelling agent, is in fact a variable source of calcium, magnesium and other inorganic ions. With the introduction of pure forms of agar this source of mineral will be minimized and more attention to mineral supplements of known composition will need to be made. T o avoid chemical precipitation during heating, the mineral supplements should be added from sterile solution just prior to pouring the agar media at 5Oo-55"C.

F. Gelatin Gelatin is prepared from bone, but before this can be achieved fat and mineral, mainly phosphate, have to be removed. Before World War 11, degreasing was carried out by solvent extraction by exposing the bones to vapour from boiling benzene.

111. DESIGN

OF MEDIA

267

Today, modern technology employs a cold process without the use of solvent. Crushed bones are fed in a stream of cold water into an “impulse renderer” which separates the fat almost instantaneously. Separation of the degreased bone is thereafter by gravity and the fat is skimmed off from the water. The defatted bone is next demineralized in dilute acid to yield a low-ash bone matrix, termed “ossein”. T h e spent acid is neutralized with lime and provides di-calcium phosphate as a by-product for animal feeds, fertilizers and pharmaceutical industries. The phosphate-free ossein is then ready to be processed for the extraction of gelatin. Hide as well as ossein can be utilized as raw material for the extraction of gelatin and the process that follows applies to both substrates. The materials are washed and treated either with lime or acid depending on the type of gelatin required. In the lime process, the ossein or hide is held in a cold lime suspension for several weeks prior to extraction. Acid treatment takes on average 5 days before extraction and the properties of the final gelatin are determined largely at this stage of the process. Extraction is carried out by low heat-treatment and the gelatin is released as a weak solution which is filtered, sometimes de-ionized, refiltered, and the filtrate allowed to gel by chilling. The gel is cut into cubes or extruded into “noodle” shaped shreds prior to drying. The dried product is finally sold as granules or as a powder. Gelatin has been employed since the introduction of laboratory culture media for the solidification of nutrient broth. It has been displaced by agar largely because of its low melting point, circa 3Oo-35”C, which limits its suitability for incubation to temperatures of 20”-25°C and below. In more recent years, gelatin has become a diagnostic reagent for determining proteolysis by liquefaction of nutrient gelatin. Alternatively, it may be combined with agar, as in Chapman’s medium when gelatinase activity is revealed by absence of protein precipitate around colonies when a suitable development reagent, e.g., sulphosalicylic acid, is added to the culture medium. Not all grades of commercial gelatin are suitable for the above purposes. Many samples of gelatin contain inhibitory concentrations of sulphite which have been added during manufacture as preservatives against microbial spoilage. Failure of organisms to grow in nutrient gelatin, or the presence of minute colonies, can often be attributed to this defect. Compatibility with agar is not always satisfactory. Gelatins possessing a high Bloom strength (jelly strength) often produce surface distortions when mixed with agar media, giving a “scum-like” appearance. This is associated with the differences in setting properties of the agar and the gelatin. A gelatin of low Bloom strength is generally more suitable for

268


admixture with agar if physical distortion of the surface of agar plates is to be avoided. The low melting characteristic of gelatin can be overcome by treatment with formalin for 24 hours, followed by copious washing to remove formaldehyde residues. Formalized gelatin does not melt below boiling point but remains susceptible to liquefaction by gelatinase activity. Kohn (1953) and later Lautrop (1956) showed the advantages of using formalized gelatin into which finely divided charcoal had been incorporated, for the rapid detection of gelatinase. Liquefaction of discs of formalized charcoal gelatin releases the charcoal into the culture medium and provides an early indication of enzymic activity. Such discs can be added to a variety of liquid and solidified media for incubation over a wide range of temperature. A current development in the use of gelatin for quantitative microbiology is the use of high-grade nutrient gelatin for the culture of organisms in milk and dairy products. In this technique, micro-colonies are allowed to develop in the nutrient gelatin. Colonies are fixed with an acid-formalin mixture and the gelatin is liquefied. T h e suspension of fixed colonies in the molten gelatin is then passed through an automatic counter of the type used to enumerate blood cells. In this way, very rapid and accurate counts of bacteria in milk become possible (Tolle et al., 1968). For this semi-automated procedure, gelatin of very high optical clarity is required.

G. Bile salts and ox bile Bile derivatives are used in culture media to differentiate between those bacteria which are adapted for survival in the gut and those which cannot live in that environment. By increasing the concentration of bile, or fraction of bile, selective media may be prepared, which are capable of differentiating groups of bile tolerant organisms. Bile salts may also be used indirectly as metabolic indicators in carbohydrate media. Thus the precipitation of bile acids in broth or the halo of precipitated bile around colonies in agar, will indicate active acidproducing organisms. Ox bile, as a by-product from abattoirs, was the first bile additive to culture media (MacConkey, 1908). Variation in quality and seasonal changes in the characteristics of bile caused the search for more refined derivatives. Bile pigments have no particular value in culture media; they may even cause confusion in media with indicator dyes. Therefore, the separation of bile salts from crude bile was a logical step to more refined reagents. Bile is produced or secreted by liver cells and it drains, by ducts, into

269


the upper small intestine. In most birds and mammals a gall-bladder is present, in which the secreted bile is stored and concentrated. The contents of these bladders are collected in abattoirs. Bile contains bile pigments and bile salts, mucin, protein, lipids, phospholipids, traces of enzymes, and inorganic ions. Bile salts are composites of bile acids, conjugates of bile acids, and bile alcohols. Apart from species-specific differences in bile composition, bacterial action in stored bile will give rise to changes in bile acids. I t is not surprising therefore, that differences can be demonstrated between batches of bile salts in their performance in culture media (Fig. 8).

:$:

Solvent front

0

-Io

0

0

0 0

I

I

I

I

I

I

7

8

9

10

I1

12

FIG. 8. Reproduction of thin-layer chromatograms of hydrolysed bile salts. Full circles represent spots visible in ordinary light. Broken circles represent spots visible only in U.V. light: 1 . Cholic acid; 2. Sodium deoxycholate; 3-5, Producer A (3 different batches); 6. Producer B; 7-12, Producer C (6 different batches).

Freshly collected ox bile is largely a mixture of bile acid conjugates. Glycine and taurine conjugates may be separated but commercial preparations labelled “sodium taurocholate” or “sodium glycocholate” are usually mixtures of the two. The preparations will also contain unknown quantities of unconjugated bile acids and sodium deoxycholate.

Manufacturing methods O x bile-freshly collected, or preserved ox bile, is filtered, concentrated in vuacuo at low temperatures, and dried, either in vacuum ovens or spray dried.

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The reconstituted bile has a typically brown-green colour and contains all the components of bile listed above. Bile salts-bile is added to a large excess of acidified alcohol and the bile acids are separated as a white insoluble precipitate. The bile acid may be filtered, neutralized, and dried but usually further washing processes are carried out with methanol or fat solvents before final drying. Bile acids are more inhibitory to bacteria in the unconjugated state. Such bile acids may be prepared by hydrolysis of bile salts in strong alkalis under pressure at temperatures of 120”-160°C for 6 to 8 hours. T h e alkaline solution is made acid and the insoluble bile acids collected, washed, and dried. Another characteristic of bile acids is the ease with which they can be precipitated by acid-production of metabolizing bacteria. The “halo” of bile acids seen around bacterial colonies in certain bile salt media is of diagnostic significance. Bile salts may be presented as a fine powder or as coarse lumps. T h e colour of the dried product may vary from white to brown. T h e reconstituted material should be clear in a 2% w/v solution in distilled water. I t should give neither a surface scum in culture media nor should it produce a deposit of insoluble material. The bile salt used in culture media should affect neither the colour of indicator dyes nor should it prevent typical colour changes in the medium. A chemical analysis of bile and bile salt preparations can only indicate the presence of adulterants or inert material. Thin-layer chromatography on silica gel plates is the most convenient and useful analysis. A true test of the ability of a bile derivative to function satisfactorily in culture media is to make tests with known organisms, in the specific media, using standard satisfactory bile salts as controls. I t must be stressed that because a bile salt gives a satisfactory performance in one medium formulation, it does not follow that it will do the same with other formulations. Burman (1955) described a method of evaluating bile salts in MacConkey broth. This test was based on the recovery of E . coli from river water in the presence of various concentrations of bile salt (0.2% w/v to 0.5% w/v). T h e other constituents of the medium remained constant and a bile salt of known performance was used as a control. Comparative trials of bile salts in MacConkey agar and desoxycholate-citrate agar were described in the Public Health Laboratory Service Report (1967). Solid media prepared with differing bile salts were tested by the Miles-Misra surface drop test. The growth recovery of various enterobacteria was recorded for each medium. A method of computation of the growth recovery figures was devised to smooth differences arising from other causes between the participating laboratories.


27 1

Collins (1967) describes a more general method of evaluating agar media which is based on earlier descriptions by Stokes (1955). Bile salts have the ability to modify the toxicity of triphenyl methane dyes (Harvey, 1956). Thus bile salt/brilliant green dye combinations are commonly found in culture media formulations. I n brilliant-green bile (2%) broth, critical evaluations of the medium must be carried out by(i) testing the ability of the broth to enrich the growth of coliform organisms at 30°C and 37°C. (ii) confirming that the medium will allow gas formation by E. coli type I only at 44°C. (iii) ensuring that the toxicity of brilliant green to mesophilic sporing organisms has not been reduced by the bile to a level where growth of these organisms will be allowed. (iv) testing that these abilities are maintained in the presence of milk proteins as well as in water. T h e exact function of bile in media has not been clearly defined. Attempts have been made to formulate media in which bile has been replaced with synthetic detergents “Teepol” [Teepol 610 (B.D.H. Ltd)] (Jameson and Emberly, 1956) and “Tergitol” [Tergitol-7 (Union Carbide and Carbon Corp.)] (Chapman, 1947). Other workers have used glutamic acid-based media to replace the conventional bile media (Folpmers, 1948; Burman and Oliver, 1952; Gray, 1964; Collingwood, 1964; P H L S Report, 1968). Liefson (1935) investigated bile and isolated deoxycholic acid as the most inhibitory fraction of bile useable in culture media. T h e action of deoxycholate could be modified by other ingredients in culture media, such as sodium chloride and phosphate. But his most striking discovery was the enhancement of its action by straight chain fatty acids, i.e., acetate, propionate and butyrate. Thus although deoxycholate media are used for the isolation of coliform organisms generally, deoxycholatecitrate media are much more selective because citrate behaves as acetate in its action on deoxycholate. T h e satisfactory performance of these latter media is based on their inhibition of the normal coliform flora of the human intestine and enhancement of the pathogenic invaders, i.e., salmonellae, shigellae and vibrios.

H. Carbohydrates used in culture media Carbohydrates constitute more than one-half of the organic matter upon the Earth. T h e greatest part of plants is built of carbohydrates while animal tissue contains rather small amounts of them. Sugars, starches, cellulose, hemicelluloses, pectins, numerous gums, and mucilages are all carbohydrates. Simple sugars such as glucose and fructose are found in

272


honey and fruits. They may be combined in disaccharides, sucrose being the most common example. The complex carbohydrates are polymers of the simple sugars (or their derivatives) and are present in plants as cellulose, hemicellulose, and pectin. They may act as reserve materials, such as energy stores, e.g., starch in plants. They may also be waste products of plants such as some of the gums. Glycogen is the energy reserve polysaccharide of many animals. The carbohydrates commonly used in culture media are-

Pentoses Hexoses Disaccharides Trisaccharides Polysaccharides Alcohols Glucosides

Arabinose, xylose, rhamnose Glucose (dextrose), fructose (laevulose), mannose, galactose Sucrose (saccharose), maltose, lactose, trehalose Raffinose Starch, inulin, dextrin, glycogen Glycerol, erythritol, adonitol, mannitol, dulcitol, sorbitol Salicin, aesculin.

Further general information on carbohydrates may be obtained from textbooks such as Pigman (1957). For specialized information the published volumes of Advances in Carbohydrate Chemistry should be consulted.

Precautions in use Carbohydrates used in culture media must be tested to ensure their identity and freedom from adulterants. Simple saccharides may be tested by chromatography, using known standards to compare their Rf values. Polysaccharides may have to be hydrolysed with acids before testing in this way. Microbiological tests utilizing the fermentation reactions of known organisms are often used to ensure correct reactions in culture media. Carbohydrate solutions for biochemical tests with micro-organisms, e.g., specific fermentation tests, should be sterilized by filtration. Normally 10% w/v solutions of the carbohydrate are prepared in distilled water and passed through a sterilizing filter without heating. Some of these solutions are very acid and p H correction of the solution is advisable before filtration. Heating solutions of carbohydrates may cause hydrolysis of complex “sugars” or formation of oxidation products. Concentrated solutions are particularly prone to decomposition when heated. Decomposition is also greater when the pH of the carbohydrate solution is above 7.0. Thus dextrose is stable to autoclaving when buffered with acid-citrate solution.


273

Evidence of heat decomposition is acid p H drift or darkening of the colour of the solution. Sugars such as glucose, arabinose, galactose, and mannose (i.e., those containing free carbonyl groups) may form addition products with sulphites. These products form slowly and are called hydroxysulphonates or hydrosulphonic acids. Carbohydrates may complex readily with phosphates to form brown pigmented solids or solutions. These complexes may be inhibitory or stimulatory to the growth of micro-organisms (Finkelstein and Lankford, 1957; Sargeant et al., 1957; Lankford and Sargeant, 1958). T h e reaction is particularly evident at high temperatures and at alkaline pH values. T h e most important browning reaction in culture media is the Maillard reaction. Aldehydes, ketones, and reducing sugars will combine readily with amino-acids, peptides, and proteins to form brown melanoidins. A Maillard reaction may cause essential amino-acids to be altered so that they are not available for metabolism. Lysine is especially vulnerable to complexing in the Maillard reaction. Optimum conditions for the occurrence of the Maillard reaction are (i) a fairly low water content, (ii) a p H between 7 and 10, (iii) high temperature. The reaction does not occur in the absence of water or at low water activity. Prevention of Maillard reactions in culture media requires attention to the following precautions(i) Do not allow concentrated solutions of sugars and peptones to lie at the bottom of vessels during the autoclaving process. Peptones should be completely dissolved before heating. Sugars such as dextrose should preferably be dissolved in warm solution with thorough mixing to ensure that layering of the concentrated solution does not occur at the bottom of the vessel. (ii) Do not attempt to autoclave high dextrose/peptone media in double or triple strength solutions anticipating dilution after heating. (iii) Autoclave on the principle of maximum temperature for minimum period and remove the media from the autoclave immediately after the cycle; cool rapidly. (iv) Avoid alkaline pH above 8.0. (v) Use the smallest feasible volumes of media to prevent excessively long autoclaving times.

I. Minerals, chelates and buffers 1. Minerals Some inorganic mineral salts are essential for the growth and metabolism of all living cells. Many metallic cations affect micro-organisms; the type 12

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B. Y . BRlDSON AND

A. BRECKER

of effect depends upon their concentration. Low concentrations of ions are usually stimulatory and high concentrations are usually inhibitory. Most bacterial inorganic requirements are met by the addition of K+, Mg2+, Mn2+, Fez+ Or 3+ and Po43-, and SO& ions to purified media, when complex N sources are used. Albert (1958) listed the following essential trace metals for cells: Cu, Co, Zn, Fe, Mg, Mo, Mn, and Ca. Mineral salts are always present in bacterial culture media prepared from complex ingredients (Kempner 1967). Agar will provide minerals to culture media, the amount donated depending on the agar characteristics such as ash content, presence of chelating material in the agar, and the degree with which Ca2+ and Mg2+ are bound to the polysaccharide. Thus the simple addition of agar to a satisfactory broth may produce a copious precipitate upon autoclaving. Other common media constituents supply adequate amounts of mineral salts (Sykes 1956). Nevertheless Marshall and Kelsey (1960) supplemented their complex medium with MgS04, MnS04, FeS04, and a large amount of glycerophosphate; with improved growth of some organisms. Their medium had the following inorganic constituents (mg per 100 m1)Ca

c1

cu Fe Mg Mn P K Na S

0.6

340 0.006 5.7

30 0.5 118

204 167

4.6

Basal media designed to test peptones, extracts or other N sources are traditionally compounded from a mineral salt, dextrose and buffer, such asN source K2HP04 Dextrose

MgSO4.7H20 FeS04.4HzO MnS04.4H20 Water

0.2 g 0.2g 1.0g 0.02 g 0.001 g 0.001 g 100 ml

Mn2+ and Fez+ may be omitted if it is considered that sufficient ions are derived from the glassware or other constituents. Powell and Errington (1963) compiled a mixture of trace elements containing all the metals listed by Albert (1958). Toxin production by Clostridium tetani and Corynebacterium diphtheriae


275

is controlled by critical Fe levels (Pappenheimer, 1947; Mueller and Miller, 1954). Harris and Richards (1968) tested the effects of Fez+, Cr3+, Caz+, Mg2+, and Mn on the recovery of phenol treated E. coli. They found that iron and chromium phosphate ((floe" produced in the medium may have had physical effects such as absorption of toxic factors. It is often overlooked that the homely procedure of “phosphating” culture media, i.e., making the medium alkaline (pH 8-5) and heating to boiling point, is in fact an efficient method of removing alkaline earth metals. Calcium and magnesium are precipitated as the insoluble phosphate salts in the form of a very finely divided “floc”. Culture media treated in this manner may not only be deprived of the alkaline earth metals, if the insolubles are filtered off, but absorption of other factors may occur. Iron is adsorbed on to the phosphate “floc”, possibly fatty acids may also be absorbed (Mueller and Miller 1954). Phosphate precipitation was described by Donald et al., (1952) as a method of treating media to remove metals. Critical studies on metal dependence can only be carried out on metal depleted media and it is extremely difficult to design culture media that will demonstrate dependence on trace elements required in minute quantities, such as Co. 2. Chelates Chelating agents (sequestrants) react with metals to form complexes which, depending on the stability of the metal complex, tend to alter the properties and effects of metal in a substrate. Examples of natural sequestrants are poly- and hydroxy-carboxylic acids, polyphosphoric acids, amino-acids, and various macromolecules (porphyrins, peptides, and proteins). Ethylenediaminetetraacetic acid (EDTA) is probably the best known and most widely used synthetic sequestrant. Carboxylic acids such as citrate, succinate, tartrate or acetate are often added to culture media to complex Ca2+, Mg2+, and Fez+ 01 3+, to prevent insoluble metal phosphate compounds forming and to provide a soluble reserve of cations which may be utilized by the organisms (Meynell and Meynelll965). Cystine, histidine, and glycine are examples of amino-acids that will also act as cation chelating agents in culture media (Meynell and Meynell 1965). Although EDTA is an excellent chelating agent for Mg2+ and Ca2+ at p H 7.0, it is seldom used in culture media formulations. This is because of the toxic effects of EDTA possibly resulting from binding these essential metals so that they are no longer available to the organism (Adam, 1959;

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E. Y. BRIUSON AND A. BRECKER

Meynell and Meynell, 1965);for the use of EDTA see Evans, Herbert and Tempest (this Series, Vol. 2). Albert (1958)discussed the dissociation and stability constants of metal binding agents and suggested that these agents will only exhibit toxicity towards organisms if their stability constants for vital metals are greater than those of the chelating sites on the organism. The stability constants of sequestering agents are significantly influenced by changes in pH and can be determined only within strictly defined systems. Extrapolation of such constants is of limited value in predicting the performance of sequestrants in other complex media. The effect of sequestrants on the growth of organisms often varies between genera and species. Satisfactory levels for one organism may be inhibitory for another, therefore care must be taken to test the effect of a chelate, at the chosen level, on all the organisms that may be grown in a particular culture media.

3. Buffers Peptones are significant buffering agents and meat or plant extracts may contain sufficient inorganic materials to provide substantial buffering capacity. However, micro-organisms which are capable of de-aminating or de-carboxylating amino-acids may considerably alter the pH of a medium during their metabolism. Very many buffers have been utilized in culture media at various times but the choice is limited to those compounds which are effective at the optimum pH value (see Munro, this Series, Vol. 2). Phosphate compounds are most widely used because, apart from their buffering action and suitable pK values, they are an essential nutrient. Phosphates, however, show the disadvantage of sequestering alkaline earth metals and forming an insoluble complex. Glycerophosphate does not exhibit this drawback. Meynell and Meynell (1965)point out that most buffers are effective for pH- pK 1. Acetate with a pK of 4.7 is therefore useful over a range of pH 3-8-5.6.Citrate-phosphate buffer complex has the following pK values: 3.1,4.8,5.4,1.9,and 6.7.This most useful buffer will therefore be effective over a pH range 2.2-7.6. Mallette (1967)examined the carboxylic acid compounds available as buffers for bacteriological systems. The pK values of 23 such compounds are listed in a valuable table; some of the buffers having as many as six pK values, 3,6-endo-Methylene-l,2,3,6-tetrahydrophthalic acid (EMTA) was chosen as the most suitable buffer for studies of bacterial nutrition and endogenous metabolism.


277

A buffer system of bicarbonate + COz is used in tissue culture media. It has the advantage of being poised about the most commonly required pH values and COz is often a growth stimulatory compound. It has the disadvantage, however, that one component is in the gaseous phase and the growth environment must be closed in plugged tubes or special incubators. Hutner et al. (1958)examined the buffers that can be used in vitamin assay media and suggested that the following four compounds could be incorporated in the mediaTris (hydroxymethyl) aminomethane Quadrol ( N , N , N‘, ”-tetrakis (2-hydroxypropyl) ethylene diamine) (Wyandotte Chemical Corp., Michigan, U.S.A.) Pyromellitic acid (Benzene-l,2,4,5.-tetracarboxylicacid) Succinic acid Tris buffer tends to antagonize potassium competitively therefore media containing this buffer should have ample potassium. Lewis (1966) reported other tris compounds “mono-Tris” (British Drug Houses, Poole, Dorset, U.K.) and “bis-Tris” (British Drug Houses, Poole, Dorset, U.K.)with pK values of 7-83 and 6-46respectively. These compounds showed advantages over Tris buffer with a pK value of 8.18. Good et al. (1966)reported a new range of buffers which were specifically developed for biological systems. These buffers are water soluble, have low binding capacities for divalent cations and have pK values from 6-8. Two of these buffers were superior to the other tested buffers in cell-free systems : N-trishydroxymethyl methyl-2-amino ethane sulphonic acid (TES), pK = 7.14. N , 2-hydroxymethyl piperazine-”-ethane sulphonic acid (HEPES), pK = 7.31. Good et al. divided the characteristics of a buffer into two parts: (i) maintenance of pH; (ii) side effects on biological systems. The second part was measured using in vitro cell systems or mitochondria1 preparations. In such preparations the two buffers mentioned showed higher rates of protein synthesis and maintained particularly active and stable mitochondrial preparations, when compared with other buffers. Good et al. (1966)also mentioned the very significant effect of temperature on the pK values of buffers. Thus a tris buffered system, adjusted to pH 7.8 at room temperature, may be at pH 8.4 in the cold room and at pH 7-4at 37°C. Williamson and Cox (1968)tested both TES and HEPES in the serial passage of continuous cell lines. Both were found to be satisfactory and free from toxicity but the buffer HEPES showed overall best performance.

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Good et al. (1966) include in their paper an excellent introductory section on the problems of buffering systems in biology. J. Silica gel media The use of inorganic gel media in microbiology is often mandatory for carbon nutritional studies since appreciable growth on silica gel, unlike that on agar, can only result from the utilization of specific organic compounds as sole sources of carbon (Marshall et al., 1960). Nitrifying bacteria, as an example of fastidious bacteria, may be isolated more readily on silica gel media than agar media. Kingsbury and Barghoorn (1954) reviewed the early work on silica gel media carried out in the 1890’s. The basic technique was to add aqueous sodium silicate solution to hydrochloric acid. The acidified solution was then dialysed until all chloride ions had been removed. The remaining silica sol (Si02) was stable to sterilization, selected nutrients could be added to it and a gel induced by the addition of one drop of concentrated sodium chloride. Hanks and Weintraub (1936) modified this technique to a “gel in plate” method. Equal volumes of sodium or potassium silicate were added to an inorganic acid, or mixture of acids so that the end pH was close to 7.0. It is possible to adjust the combinations of these ingredients so that gelling occurs very shortly after they are poured into the dish. Nutrient materials must be added before this stage and sterilization may be carried out in the dish. Alternatively, nutrients may be allowed to diffuse into the gel after the plate has been prepared. Thin layers of gel must be poured to prevent cracking of the gel. The plates are sterilized at 15 lb pressure (121°C) for 15 min. Cooling the autoclave must be carried out as carefully as with agar media to prevent cavitation or blow-holes in the gel. The sterilized plates must be stored in plastic bags to prevent excessive drying. Ingleman and Laurel1 (1947) substituted silicic esters for the sodium silicate and hydrochloric acid. Ortho-silicic acid tetramethyl esters, Si(OCH&, or ortho-silicic acid tetraethyl esters, Si(OCzH&, may be used. The methyl esters may be dissolved in water but the ethyl esters must be dissolved in alcohol to give a clear gel. An outline of the method described by the authors is as follows: Equal volumes Si(OCzH& and ethyl alcohol are mixed together. Into this solution is poured 6 volumes of water, the water being added in portions, with thorough mixing. Boiled water may be used to prevent excessive bubble formation. Gels of varying rigidity can be prepared by varying the proportions of ester and water. The gel is slightly turbid and it must be centrifuged to obtain clear silica gel. At this stage, it is ready to be poured into dishes or tubes. Sterilization is then carried out at 120°C for 30-40 minutes and during


279

this time the gel sets firm, clear as glass. T h e usual precautions must be taken on cooling to prevent cracking or splitting of the gel. Water must now be poured over the plates to allow the ethanol to diffuse out of the gel. T h e water is then replaced with a nutrient solution and left for some time so that the nutrients may soak into the gel. Further sterilization may be carried out at this stage. The plates or tubes must be stored in the presence of water vapour to prevent drying out. Smith (1951) modified the ion-exchange column method, first proposed by Taylor (1950) to remove sodium ions from sodium silicate. I n his paper, Taylor described a preparation method in which alkali and electrolytes were added to SiOz. T h e speed of setting and rigidity of gel were controlled by, (i) pH, the optimum being 6-0-6.5, (ii) electrolyte concentration, being directly proportional to gel strength, (iii) temperature, (iv) silica concentration. Smith examined the various methods of ionexchange procedures and the effect of various electrolytes. H e considered that the ion-exchange resins, Amberlite IR 105, IR 120, and Zeo-Karb 215 were preferable to the resin Soucol, recommended by Taylor (1950) (Amberlite resins and Zeo-Karb resins-British Drug Houses Ltd., Poole, Dorset). Temple (1949) used a highly purified colloidal silica preparation marketed under the trade name of Ludox (du Pont). T h e material consists of a 30% aqueous solution of colloidal silica stabilized with a little alkali. This solution may be diluted to 10% silica, nutrients dissolved, p H adjusted and the medium autoclaved in dishes or tubes. During the sterilization process, gelation occurs. T h e resulting medium is not clear but cloudy and translucent. Kingsbury and Barghoorn (1954) modified the Ludox preparation of Temple by passing the colloidal silica through an ion-exchange column to remove the residual NazO. In their experiments a combination of strong base exchange resin (Amberlite IR-120) and a weak acid exchange resin (Amberlite IR-45) was used. This combination was chosen on the assumption that it would not retain silica but would remove other highly active negative ions and all possible positive ions. Clear gels can be prepared by this method but the clarity of the medium will depend on its formulation and the final pH. Pramer (1957) used several silica starting materials and examined the effects of silica content, pH, temperature, concentration, and nature of ions present. All the silica materials were processed by ion-exchange techniques to obtain SiOe. Pramer found that silica gel containing 1.5% Si02 and ~ / 3 0NaCl gelled most rapidly at p H 6.0-7.0. It gelled in 18 minutes at 55°C and 265 minutes at 5°C. Syneresis was much greater when the sol was gelled and incubated at elevated temperatures. Syneresis

280


can be a great problem with silica gel plates and only absorbent pads in the lids of the plates may control it. The original papers should be consulted for details of the methods available to prepare silica gel plates.

11. USE OF CONSTITUENTS I N MEDIA To grow micro-organisms in the laboratory, whether on a small or large scale, a nutrient environment or culture medium is required which will satisfactorily supply all the factors demanded by the organism for multiplication. Culture media have been described for the successful cultivation of plant and mammalian cells, protozoa, fungi, and bacteria. A non-cellular culture medium for viruses has not yet been formulated. At first sight the various micro-organisms have little in common with each other and this impression is reinforced by the many thousands of diverse culture media described for their cultivation (see Lapage et al., this Volume, p. 1). Nevertheless there are certain things held in common by all cells and through the biochemical events that occur a uniformity of nutrition is apparent. All micro-organisms, except viruses, contain about 80% water. Bacteria, yeasts, and unicellular algae contain about 40-50% dry-weight protein. Organisms that have a complex structure, such as fungi and seaweeds, contain about half this quantity of protein. The structural polysaccharide components of the cell walls make up a larger proportion of the dry weight. The nutrient environment may be divided into(i) The physical environment-temperature, humidity, atmosphere. (ii) The chemical environment-those chemical compounds supplied in culture medium to permit growth of micro-organisms. The physical factors are discussed elsewhere in this series and only the chemical environment is considered here. The chemical compounds used to cultivate micro-organisms may be grouped in the followingway(i) Protein components-peptides, amino-acids. (ii) Vitamins. (iii) Minerals-metals and inorganic ions. (iv) Components of genetic material-purine and pyrimidine derivatives. (v) Energy source. Other substances may be added to this list according to the particular purpose of the culture medium. Buffer salts, indicator dyes, Eh reducing substances, and selective agents are commonly present in various combina-


28 1

tions. Solidifying substances such as agar, gelatin, inspissated protein or silica gel are often used. Items (i) to (iv) in the list may be considered as microbiulgvowthfuctors. The cell requirement for these various factors depends on the presence of a functioning synthetic pathway; if the cell can synthesise a required compound then there is no need to supply it in the culture medium. T h e energy source required by cells varies widely and the cells may be classified as-

(i) Photosynthetic-demanding only light as an energy source. (ii) Chemo-lithotropic-deriving energy from oxidation of inorganic molecules (iii) Chemo-organotrophic-requiring organic carbon compounds as energy sources, which often act as a carbon source as well. T h e nutrient requirements of the photosynthetic and chemo-lithotrophic organisms are usually simple as these organisms possess wide, synthetic pathways. Solutions of metal salts, inorganic ions, simple nitrogen, and carbon sources are all that is required. T h e chemo-organotrophic organisms exhibit a wide spectrum of nutrient requirements. Thus E. colimay be successfully cultivated on a simple mineral mixture, utilizing ammonia as a nitrogen source and dextrose as an energy source. The transplanted mammalian cell may require the whole gamut of amino-acids, vitamins, nucleic acid fractions, minerals and an unidentified factor from serum before it will begin to multiply. T h e undefined culture media used to cultivate the chemo-organotrophic bacteria contain extracts, infusions or digests of natural substances, as rich sources of the factors needed by the cells. Wide variation is possible in the content, or proportions, of the factors required in these natural substances. Therefore an understanding of the manufacturing processes involved in producing them and an analysis of their content is necessary, if they are to be used intelligently. Some of these products defy the efforts of chemists to supply an adequate analysis and only certain key factors are available for comparative purposes. Most of the major constituents of bacterial culture media have been discussed under their separate headings and analyses have been given, wherever these are available, or have been thought to be useful. The assembly of the complex constituents into culture media that will prove to be of value for the isolation, enrichment or selection of microorganisms follows fairly simple rules. First consider the nitrogen basis of the medium. This should supply adequate amounts of all the natural protein amino-acids, usually as small polypeptides. Peptones in 1-2% w/v concentration are usually able to

282


do this but consideration should be given to the source of the hydrolysed protein and its method of hydrolysis. Some proteins naturally lack certain amino-acids, or hydrolysis may lead to destruction or non-availability of amino-acids to the organism. Supplementation may be required ; either with specific amino-acids or by the addition of peptone from another source. The carbohydrate admixture of plant protein digests have been mentioned and thought should be given to the desirability of adding carbohydrate to the medium in this form. Excellent bacterial growth responses have been reported from the use of plant extracts. High carbohydrate content plays a considerable part in this response but a medium designed for bacterial longevity would not contain such factors. The nitrogen base may require supplementation with meat, yeast or plant extracts. I t would be as well to define the fraction of the extract that is supplying the essential or stimulatory factors. Broadly speaking, extracts may supply(i) (ii) (iii) (iv) (v)

Carbohydrates as energy sources. Inorganic ions (including metals and phosphates). Purines and pyrimidines. Essential vitamins. Supplementary amino-acids and peptides.

Simple substitution of the extract with these factors may pinpoint the growth requirement, although the crude extract at 0 5 1 % w/v addition may be cheaper, more convenient or give better yields than the specific component. The energy source used in culture media may be the mixture of aminoacids provided by the peptone. Some organisms, however, require carbohydrate substrates and the growth of many organisms is improved by the addition of simple carbohydrates to the medium. 0.2% w/v dextrose is usually sufficient to promote growth without causing a marked lowering of p H from the acid fermentation products. The growth stimulatory properties of complex carbohydrate mixtures, such as malt extract or corn-steep liquor may be due to a diauxic growth effect. The simple sugars are utilized first, followed by the more complex carbohydrates being broken down and utilized sequentially. 0.5% w/v of such extracts is usually sufficient although fungi may require much higher concentrations. The mineral, inorganic ion content of culture media is often ignored in complex media containing peptones and extracts. However, supplementation of media with magnesium, manganese or iron often improves growth, pigmentation or haemolysis.


283

Very fastidious organisms may require media enriched with blood, serum or fresh yeast extract, if their demand is for labile growth factors such as glutathione or NAD. Micro-organisms may be equally demanding for factors affecting their growth environment such as low redox potential, high C02 or complete absence of 0 2 . Quite apart from the nutritional basis of culture media design is the incorporation of selective indicator substances. Thus a nutrient medium will often contain selective chemicals such as bile salts, aniline dyes, antibiotics or toxic chemicals. Selenium and tellurium salts, azides, and lithium salts are examples of the latter. Excessive quantities of carbohydrates plus a p H indicator will be added to media to detect fermentation or oxidation of specific “sugars”. It must be recognized, however, that the deliberate addition of selective compounds to culture media may profoundly affect even those organisms for whom the medium is specifically designed. Nutritional shortcomings in the medium may not be detected until the inhibitory factor is added. Or the inhibitory factor may interfere with the normal metabolic pathways and subsequent metabolism may call for other growth factors. Inhibition of bacterial growth in culture media may be caused by toxic factors which arise during preparation and heating of culture media. Wright (1933) described toxic factors which arise during the preparation of peptones. Proom et al. (1950) described inhibitory substances arising in agar media which resembled peroxides. Taylor (1957) described the inhibition of thermophilic bacteria on the surface of agar media. This toxic effect could be overcome by the inclusion of 1% soluble starch in the medium. Starch, charcoal, or heated blood are commonly added to media to counteract toxic factors. It is postulated that agar contains inhibitory fatty acids and absorption of these substances occurs on starch or charcoal (Ley and Mueller, 1946). A similar absorption function of charcoal in culture media occurs in the charcoal-agar medium of Alwen and Smith (1967). This medium is a simple nutrient agarCharcoal-Agar Medium (Alwen and Smith, 1967) 1g Peptone Meat extract 10 g NaCl 5g Agar 12 g Water 1 litre pH 7 . 2

to which 1% w/v activated charcoal “Darco G-60” (B.D.H., Poole, Dorset) is added prior to sterilization. T h e authors describe their treatment of the charcoal, before use in the medium, by refluxing with 2~ HC1 for 3 hours,

284

E. Y. BRIDSON A N D A. BRECICER

followed by distilled water washing until excess H ions were removed. Strains of Proteus spp. will grow on this charcoal agar without swarming because of the absorption of a possible “negative-chemotoxic agent”, a substance produced by Proteus and causing the swarming phenomena (Lominski and Lendrum, 1947). Brumfitt (1959) showed that Haemophilus spp. and Bordetella spp. gave increased growth in the presence of both charcoal and heated blood. Lankford et al. (1958) discussed the toxic factors which arise in culture media at some length. The most significant factor was, in the author’s opinion, the formation of glucose-phosphate interaction compounds during heating. These compounds would cause a prolonged lag-phase in a test organism or prevent germination of viable microbial spores. A peptone-copper complex was also listed among the inhibitory factors which arise in culture media.

111. INTERACTIONS OF INGREDIENTS, INCOMPATIBILITY IN CULTURE MEDIA Incompatibility in culture media generally announces its presence by a visual change in the appearance of the substrate. Precipitates, opalescence, colour changes, loss of gel strength or the separation at the surface of the medium of low density fractions all indicate a chemicaI or physical change in the composition of the medium. Less noticeable, unless monitored by the presence of coloured indicators, is pH change. Loss of volatile constituents is even more difficult to detect if unaccompanied by pH change or other measurable properties.

A. Precipitation One of the most common causes of precipitates developing in media following a heating process, is the reaction between di- and tri-valent metals and soluble phosphates in the medium. The presence of either of these reactive groups may be intentional, i.e., as deliberate ingredients. More frequently the metals may be there as impurities in other reagents and the phosphates derive from peptone, meat and yeast extract, and similar organic matter of biological origin. Release of metals and phosphates from their carriers is often gradual with the result that primary heating may produce no more than an opalescent haze, whereas secondary heating or alternatively prolonged and excessive primary heating will give rise to a precipitate. Common metals associated with manufactured reagents include calcium and magnesium derived from hard water residues used in production, and iron, zinc, copper and tin arising from the use of contaminating plant and equipment.


285

Reactive anions and cations are frequently added intentionally to culture media as diagnostic reagents. Common examples are the use of soluble bismuth salts with sodium sulphite to demonstrate reduction by salmonellae and soluble iron salts in the presence of sulphur compounds to indicate sulphide production by anaerobes. Progressive reaction occurs on overheating with the formation of a dark brown to black stain, followed by black precipitates. The minimum heating compatible with sterilization is necessary to prevent chemical decomposition of these media. Divalent metals such as calcium and magnesium precipitate in the presence of carbonate-the latter arising from the breakdown of bicarbonate buffer in the medium. Tightly closed containers to minimize loss of carbon dioxide or alternatively the aseptic addition of mineral salt solutions after sterilization helps to prevent irreversible chemical reaction with precipitation. Alkaline earths and trivalent metals react with bile salts to form insoluble bile complexes. Calcium is particularly prone to give rise to an insoluble ester which frequently appears as a surface film in liquid bile salt media or on agar plates.

B. Insoluble matter of organic origin Opalescence and insolubles, which are not due to chemical incompatibility, often appear following heat treatment. Large-molecule peptides in extracts of animal and vegetable protein, e.g., meat, yeast, and malt extracts may give rise to insoluble deposits on standing, following heat treatment. Insoluble haze in such solutions often produces clumping on storage, probably due to electrostatic forces, with the formation of a deposit and clearing of the supernatant liquor. A cause of opalescence not prone to separation is formation of insoluble soaps brought about by the reaction between divalent cations and fatty acids. Digests of fresh meat, fresh bile, and the use of fresh animal glands, e.g., pancreas, all contribute fatty acids to the medium. I n the presence of minerals of hard-water origin, insoluble soaps are formed which are often difficult to remove by filtration at elevated temperatures. Storage overnight at 4°C generally produces a physical separation which can then be followed by decantation, skimming or filtration.

C. pH p H drift due to acid hydrolysis is not uncommon, particularly when sugar-containing media are overheated or superheated in concentrated solution. Acid p H drift in these circumstances can lead to precipitation of bile acids, softening of agar gels and change of colour in indicators present in the medium.

286


D. Browning reactions A common example of interaction of ingredients, during heat processing, is the Maillard-type browning reactions. These arise from the reaction of carbonyl groups, generally from reducing sugars, with the amino groups of proteins and amino-acids. Coloured condensation products are formed so that significant quantities of carbohydrate and amino nitrogen may be rendered inactive. A high temperature-short time sterilization process may prevent or reduce the reaction but separate sterilization of carbohydrates and nitrogenous compounds may be necessary. Alkaline drift often follows breakdown by heat of heat-labile substances such as urea which decomposes, liberating ammonia and carbon dioxide. The former produces a marked alkaline reaction in the medium. Another factor sometimes responsible for alkaline pH drift, particularly on storage of agar media, is the presence of alkali in some forms of soda glass. Slow leaching of alkali from the glass will produce a pH change in agar media which will result in a colour change if a sensitive indicator is present, e.g., phenol red.

E. Photosensitivity Chemicals such as selenium and tellurium and their salts are light sensitive and if left exposed to bright light will produce red or black precipitates. Heat will accelerate this process. Dyes, e.g., brilliant green and basic fuchsin are light sensitive: brilliant green will slowly fade on exposure to light, whereas freshly reduced basic fuchsin (colourless) will become strong magenta unless protected from light. Both reactions are irreversible.

F. Solubility Concentration by evaporation or depression of the temperature in a refrigerator will cause the solubility product of sparingly soluble substances to be exceeded, with consequent separation. This factor is not common when single strength media are prepared but may occur when concentrated stock solutions are involved. Cystine is typical of this class of reagent. IV. STERILIZATION OF CULTURE MEDIA A culture medium, to be of value in the study of microbial growth, must be sterile. Since these media are seldom derived in sterile form, unless they are living tissue preparations, a terminal process of sterilization is necessary before use or storage.


287

For most purposes the definition of sterility, that the medium is free from micro-organisms after an adequate incubation time at the appropriate temperature, is sufficient. I t must be borne in mind, however, that the strict criterion of sterility is freedom from all microbial life. Many tests at various temperatures and atmospheric conditions would be necessary to judge this requirement, with incubation times up to one year or more. Russel and Gilbert (1964) have described the requirements of sterility testing. Sterilization of culture media is largely carried out by two processes(i) Heat treatment-for various times and temperature. (ii) Cold treatment-by filtration through material capable of holding back bacteria or by chemical processing. Heat sterilization of culture media varies widely in times and temperatures. The volume of medium to be sterilized will control the time interval required to heat the medium to the specified temperature for the required time.

A. Heat sterilization Heat sterilization by steam under pressure is the most commonly used procedure for culture media. The temperatures used vary from 115°C to 126°C or even 134°C. A typical sterilization cycle at 121°C may be divided as shown on the following page. Autoclaves with long heat-up-cool-down times should not be used for culture media. These instruments are common causes of over-heating because little account is taken of the first and last stages of the autoclave cycle, when the heating process is described. The total heat input into the medium may be seen in Fig. 9.

Time ( m i n )

FIG.9. A schematic graph of the total heat input into the medium,

r

1

STERILIZATION CYCLE Autoclave chamber heat up time

Room temperature [

1-

m -4

m

121°C

E

Ez

Fluid heat penetration time 1il.C

121+ Time at 121°C [121"C 121"C] Cooling down time < l0O0C] [121"C

111.

DESIGN OF MEDIA

289

If the autoclave cannot be altered, it is possible to compute the total heat input into the system and allow for a shorter sterilization cycle. The fluid heat penetration time must be pre-determined by means of thermocouples placed in the most suitable part of the vessel. The absolute centre of the vessel may not be the best place for the thermocouples. A considerable difference may be observed between the upper and lower portions of fluid in a large vessel. This difference is particularly aggravated if large masses of agar are allowed to gravitate to the bottom of the vessel before autoclaving. Pre-steaming large volumes of agar media before autoclaving to ensure solution of the agar is the only satisfactory method of sterilizing such media. In 4 litres of agar medium, with the agar lying undissolved at the bottom of the vessel, the temperature of the centre of the agar mass failed to reach 121°C after the medium had been held at that temperature for 1 h. The following time intervals for fluid heat penetration 121°C in glass bottles of media are given for guidance only500 ml bottle 18 min 1,000 ml bottle 22 min 2,000 ml bottle 27 min 5,000 ml bottle 37 min It will be appreciated that these times depend on the geometry of the container as well as on the volume. Short, wide vessels require longer times for heat penetration than tall, thinner vessels. It is possible to heat up very large volumes of fluid very quickly if the surface area of the fluid presented to the steam is very large. It is on this principle that heatexchangers are constructed. A 4 litre volume of medium in a rectangular vessel of not more than 24 inches in depth will heat up in approximately the same time as a 100 ml volume in a round bottle. The exposure time of the medium at the specified temperature is usually based on the thermal death time of Bacillus stearothermophilus spores at neutral pH. T o this calculated holding time, a 50% increase is added as a safety margin. The M.R.C. recommended exposure times are-

3 rnin at 134°C 10 rnin at 126°C 15 rnin at 121°C (M.R.C. Working Party, 1959). For further details, see Sykes, this Series, Vol. 1.

B. Cold sterilization It is not always desirable to heat process a culture medium and it is absolutely undesirable in those media containing heat-labile constituents.

290


I n these circumstances, filtration is the method of choice to obtain sterility, where sterility implies freedom from bacteria and fungi. Details are available in the articles by Sykes and Mulvany, this Series, Vol. 1. C. Chemical sterilization At first sight the concept of chemical sterilization of culture media would appear to be against all the accepted precedents in microbiology. Chemical sterilants are associated with phenolics, mercurials, etc., the persistent anti-microbials. However, the chemical sterilant sought is one that has an actively microbiocidal short-lived phase before changing into an inert compound. The principle is that the chemical agent is added to the culture medium, it is hydrolysed to a biocidal compound and then further hydrolysis leads to the residual inert chemical. An ideal substance, which acts in this way, to leave a perfectly satisfactory medium at the correct pH, is still being sought. Two compounds only approach these criteria ; 8-propiolactone and pyrocarbonic acid diethyl ester (Bayer).

1. 8-propiolactone 8-propiolactone (BPL) is a colourless liquid of specific gravity 1.146 with a vapour pressure of 10 mm Hg at 51"C, molecular weight 72, boiling point 155°C and freezing point - 334°C. It is liquid at normal temperatures and pressures but it is usually stored at 4°C to prevent the formation of harmful vapour. The vapour of BPL is inflammable in air at a concentration greater than 8% v/v and it is intensely lachrymatory. The concentrated solution is corrosive and should not be allowed to touch the skin (Palmes et al., 1962). It is miscible with most organic solvents and soluble in water to 3% v/v. BPL is an alkylating lactone compound. The lactone ring opens on hydrolysis and ,%substituted propionic acids are formed. 0 CH2-CH2-C=O

+ HOH -+

II

HO-CH2-CH2-C-OH

1 , 1 I t is during the ionic reactions occurring in the hydrolysis phase that biocidal activity is shown. I t is of great importance therefore to have some idea of the length of time of hydrolysis, as the end product, 8-propionic acid, is quite harmless. The figures in Table XI11 are quoted by Hoffman and Warshowskey (1958). Although pure, concentrated BPL is stable for long periods, it reacts

111. DESIGN

29 1

OF MEDIA

TABLE XI11 BPL hydrolysis time (Hoffman and Warshowskey, 1958) Temperature

10°C 25°C 50°C 75°C

Half-life of hydrolysis period (min)

1,080 210 20

5

readily with hydroxyl, carboxyl, sulphydryl, amino, and phenolic groups. Phillips (1952) suggested that the activity of the alkylating sterilizing agents could be explained by supposing that the alkyl radicals could replace reactive H atoms and thus alkylate -SH, -COOH, -OH and -NH2 groups. Such reactions would cause irreversible death as the blocking action of these alkyl groups can be removed only with drastic chemical treatment. This proposed mechanism is supported by the fact that bacterial spores are relatively easy to kill with these alkylating compounds. T h e usual disinfecting compounds principally attack the -SH radical which is much less accessible in spores. The alkylating agents, however, can act on any of the alternative groups listed above. Culture media broths are prepared in the normal manner and BPL added to them in approximately 0.2% v/v concentration. T h e hydrolysis product usually lowers the p H of the medium and subsequent correction must be made. Toplin (1962) discusses such a procedure for continuous cultivation processes. Tacquet et al., (1963) measured the activity of BPL against microorganisms and of the sterilizing effect of BPL in Lowenstein-Jensen medium. Himmelfarb (1961) described the use of BPL in the sterilization of culture media containing carbohydrates, thus preventing significant loss of glucose during autoclaving. The chemical sterilization of blood and plasma by BPL is described by LoGrippo and Rupe (1957). Medium must be stored at appropriate temperatures to allow the full effectiveness of the hydrolysis stage. It must be remembered that, subsequent to complete hydrolysis of BPL, the culture medium is as vulnerable to microbial infections as is any sterilized medium.

2. Pyrocarbonic acid diethyl ester No comparable experience of pyrocarbonic acid diethyl ester (PKE) in culture media is recorded. Nevertheless, this compound has been used to sterilize carbonated beverages for many years. It is manufactured by Bayer under the trade name “BAYCOVIN” and

292


the hydrolysis products are alcohol and carbon dioxideC~H~0-CO-OCO-OC~H~ + HzO +-2CzH50H + 2C02 As with BPL, the microbiocidal activity occurs during the hydrolysis phase. This phase is approximately 8 h at 20°C and 16 h at 10°C. BAYOCOVIN is a colourless liquid with a weak, fruity odour. It is corrosive, in the concentrated state and must be kept away from skin, mucous membranes and eyes. It is soluble in alcohol and most organic solvents but only 0.6% v/v soluble in water. The low solubility in water means that PKE must be mechanically dispersed in droplet form in the solution to be sterilized. The greatest activity of PKE is at or below pH 4.5, although activity is evident up to pH 8.0. PKE is bactericidal and fungicidal in concentrations ranging from 0.01 to 0.1% v/v. It will not show great activity in the presence of many organisms, preferably the bacterial load should be less than 500 organisms/ml. Practically all the published work on PKE is reported in the German literature and Messrs. Bayer will supply an extensive bibliography. Molin et al. (1963) published a valuable paper in which they investigated the effect of PKE on yeasts, fungi, and foodstuffs. This paper, together with the technical information given by Messrs. Bayer Ltd., should be consulted before attempting to use BAYCOVIN for sterilization purposes. REFERENCES Adam, K. M. G. (1959).J. gen. Microbial., 21, 519-529. Albert, A. (1958).In “The Strategy of Chemotherapy” (Ed. S. T. Cowan & E. Rowatt). C.U.P. Cambridge, England. Alwen, J., and Smith, D. G. (1967).J.appl. Bact., 30, 389-394. A.O.A.C. (1955) “Association of Official Agricultural Chemists 1955. Official Methods of Analysis”. p. 307. Honvitz, E. D. Washington. Araki, C. (1958).Proced. 4th Int. Congr. Biochem.,Vienna., 1, 18-24. Balls, A. K.,and Hoover, S. R. (1937).J. biol. Chem., 121, 737-745. Bechtle, R. M., and Scherr, G. H. (1958).Antibiotics Chemother., 8, 599-606. Bender, A. E., Wood, T. and Palgrave, J. A. (1958).J . Sci.Fd Agric., 9, 812-817. Bergkvisk, R. (1963).Acta chem. scand., 17, 1521-1551. Bernard, J., and Lambin, S. (1961).Annlspharm. Fr, 19, 557-579. Block, R. J., Durrum, E. L., and Zweig, G. (1958).In “A Manual of Paper Chromatography and Paper Electrophoresis”, pp. 110-169. Academic Press, New York. Brewer, J. H., and McLaughlin, C. B. (1957)Bact. Proc., 60. Brown, F., Cartwright, B., and Newman, J. F. E. (1965). Nature, Lond., 205,

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Finkelstein, R. A. (1958). “Chemical Factors in Culture Media which Influence Bacterial Growth Initiation”. School of Aviat. Med. U.S.A.F. Texas. 1-1 3. Report No. 58-86. Lamanna, C., and Mallette, M. F. (1959). In “Basic Bacteriology”, pp. 471-474. Williams and Wilkins, Baltimore. Lautrop, H. (1956). Acta path. microbiol. scand., 39, 357-363. Lewis, J. C. (1966). Analyt. Biochem., 14, 495496. Ley, H. L., and Mueller, J. H. (1946). J. Bact., 52, 453-460. Liefson, E. (1935). J. Path. Bact., 40, 581-599. Lineweaver, H., and Schwimmer, S. (1941). Enzymologia, 10, 81-86. Lochhead, A. G., and Chase, E. E. (1943). Soil Sci., 55, 185-195. LoGrippo. G. A., and Rupe, C. E. (1957). In “Hepatitis Frontiers” (Ed. F. W. Hartman), pp. 371-385. Little, Brown & Co., Boston. Lominski, I., and Lendrum, A. C. (1947). J. Path. Bact., 59, 688-692. MacConkey, A. T. (1908). J. Hyg., Camb., 8, 322-328. MacWilliam, I. C. (1968). J. Inst. Brew., 74, 38-54. Mallette, M. F. (1967). 3. Bact.,94, 283-290. Marshall, J. H., and Kelsey, J. C. (1960). J. Hyg., Camb., 58, 367-372. Marshall, K. C., Whiteside, J. S., and Alexander, M. (1960). Soil. Sci. SOC.Am. Proc., 24,61-64. Meynell, G. C., and Meynell, E. (1965). In “Theory and Practice in Experimental Bacteriology”, pp. 239-240, C.U.P., London. Miles, A. A., and Misra, S. S. (1938). J. Hyg., Lond., 38, 732-734. Molin, N., Satmark, L., and Thosell, M. (1963). Fd Technol., Lond., 17, 797-801. Moore, S., and Stein, W. H. (1951). J. biol. Chem., 192, 663-680. Moore, S., and Stein, W. H. (1954). J. biol. Chem. 211, 895-900. Moss, C. W., and Speck, M. L. (1966).J. Bact., 91, 1098-1104. . M.R.C. Working Party (1959). Lancet, i, 425-435. Mueller, J. H., and Miller, P. A. (1954). J. Bact. 67, 271-277. Naegeli, C. (1880). Akad. Wiss. Munich, 10, 227-367. National Formulary (1965). Twelth Edn. Nomoto, M., and Narahashi, Y. (1959).J. Biochem., 46, 653, 1481, 1645. Nomoto, M., Narahashi, Y., and Murakami, M. (1960). J. Biochem., 48, 593-600. Osgood, G. (1967). Filtrat. Sepn., 4, 327-337. Palmes, E. D., Orris, L., and Nelson, N. (1962). Am. ind. Hyg. Ass. J., 23, 257-264. Pappenheimer, A. M. (1947). J. biol. Chem., 167, 251-259. Phillips, C. R. (1952). Bact. Rm., 16, 135-143. Phillips, A. W., and Gibbs, P. A. (1961). Biochem. J., 81, 551-556. Pigman, W. W. (1957). In “The Carbohydrates, Chemistry, Biochemistry & Physiology”, Academic Press, New York. Pirie, N. W. (1963). Rec. Adv. Fd Sci., 3, 87-99. Plushkat, H. (1964). Pharmazie, 19, 270-273. Powell, E. O., and Errington, F. P. (1963).J. gen. Microbiol., 31, 315-327. Pramer, D. (1957). Appl. Microbiol., 5, 392-395. Prescott, S. D., and Dunn, C. G. (1959). In “Industrial Microbiology”. 3rd Edn. McGraw-Hill, New York. Proom, H., Woiwood, A. J., Barnes, J. M., and Orbell, W. G. (1950). J. gen. Microbial., 4,270-276. Rainbow, C., and Rose, A. H., Eds. (1963). “Biochemistry of Industrial Microorganisms”. Academic Press, New York.


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Reed, G. (1966), In “Enzymes in Food Processing”, pp. 126-128. Academic Press, New York. Report (1966) “Routine Control of MacConkey Agar and Desoxycholate-Citrate Agar”. Report of a Working Party of the Public Health Laboratory Service. Mon. Bull. Minist. Hlth, 25, 289-299. Report (1968) “Comparison of MacConkey Broth, Teepol broth and Glutamic acid media for the enumeration of coliform organisms in water”. Public Health Laboratory Service Standing Committee on the Bacteriological Examination of Water Supplies. J. Hyg., Camb., 66, 67-82. Rubbo, S. D., and Gardner, J. F. (1965). “A Review of Sterilization and Disinfection”. Lloyd-Luke, London. Russel, A. D., and Gilbert, R. J. (1964). Mfg. Chem., 35, 42-46. Salatem, L. T., Gantz, C. S., and Gray, P. P. (1963). Proc. Am. SOC. Brew. Chem. 74. Sargeant, T. P., Lankford, C. E., and Trader, R. W. (1957). Buct. PYOC.,55 (abstract). Smith, W. K. (1951). Proc. SOC. uppl. Bact., 14, 139-146. Stokes, J. E. (1955). In “Clinical Bacteriology”, pp. 250-257. Arnold, London. Sykes, G. A. (1956). Ed. “Constituents of Bacteriological Culture Media”. SOC. gen. microbiol. Special Report. p, 3. Taquet, A., Tison, F., and Polspoel, B. (1963). Annls Inst. Pusteur Lille, 14, 139-1 43. Taylor, C. B. (1950). J. gen. Microbiol., 4, 235-237. Taylor, M. M. (1957). J. gen. Microbiol., 16, ix. Temple, K. L. (1949). J. Bact., 57, 383. Toplin, I. (1962). Biotechnol Bioeng., 4, 331-340. Tolle, A., Zeidler, H., and Heeschen, W. (1968). Milchwissenschuft., 23, 65-68. Varley, H. (1957). In “Practical Clinical Biochemistry”. 4th Edn.. Heinemann, London. Whitaker, J. R. (1957). Fd Res., 22, 483-493. Whitaker, J. R. (1959). “Properties of the Milk Clotting Activities of Ficin”. Fd Technol. Lond., 13, 86-92. Whitaker, J. R. (1961). Wallerstein Labs Commun., 24, 4-20. Williamson, J. D., and COX,P. (1968). J. gen. Virol., 2, 309-312. Woiwood, J. (1954). J. gen. Microbiol., 10, 509-520. Wood, F. E. J. (1946). Council of Sci. & Industrial Res. Bull. No. 203. Wood, T., and Bender, A. E. (1957). Biochem. J., 67, 366-373. Wright, H. D. (1933). J . Path. Bact., 3, 257-282.


CHAPTER IV

Quantitative Relationships Between Growth Media Constituents and Cellular Yields and Composition D. W. RIBBONS Defartment of Biochemistry, University of Miami School of Medicine, and Howard Hughes Medical Institute, Miami, Florida, US.A . I. Energy Source

.

.

11. Carbon and Energy Source 111. Nitrogen and Sulphur IV. V. VI.

.

Potassium, Magnesium and Phosphorus Sodium

.

Trace Elements

References

.

.

.

. . . . . .

299 299

300 301 302 303 303

This Chapter is intended to provide a guide to calculating the yield of cells expected from media of known composition. From a knowledge of the approximate elemental compositions of microbial cells, a medium can be quickly assessed for possible nutrient deficiencies or for which element is likely to be the growth-limiting nutrient. T h e data provided can only be related to media of known composition, and do not take into account the influence of physical conditions of culture, e.g., temperature, p H or the requirements for oxygen and carbon dioxide. These parameters are discussed elsewhere in this series (Brock and Rose, this Series, Vol. 3B; Stouthamer, this Series, Vol. 1; Willis, this Series, Vol. 3B). The yield of the population may be limited by restricting individually the supply of almost every major nutrient; this is often difficult to demonstrate for “trace nutrients”. Provided that conditions of pH, agitation, temperature, toxic products, etc., in the growth milieu are not those that limit growth, then a strict stoicheiometry usually exists between the yield of a population and some growth-limiting nutrient ; this is characteristic of the micro-organism and also of the physical cultural conditions. It is

298

D. W. RIBBONS

also apparent that this stoicheiometry extends to many of the other nutrients in the medium which are supplied in excess. T h e exceptions to the latter cases seem to be the utilization of carbon and energy sources after growth has ceased; thus a quantitative relationship does not exist between cell yield and carbon source, energy source, and oxygen supplied for aerobes when these nutrients do not limit growth, e.g., they are consumed in the absence of growth. I n addition many of the other elements required for growth are transformed by one micro-organism or another into “secondary metabolites” or storage compounds, and this will destroy the relationship between cell yield and nutrients utilized. The literature contains many examples of average elemental compositions of micro-organisms which do not specify, even if they were known, the exact environmental conditions that were used to provide the microorganisms. They can at best provide only an indication of cellular composition. Table I reproduces the values given by Luria (1960) for Escherichiu coli. I t is evident however from the extensive studies at the Microbiological TABLE I Elementary composition of Escherichia coli (from Luria, 1960)

% drywt Carbon Nitrogen Phosphorus Sulphur Ash (total) Fixed salts (non-extractable by water after heat killing) Free salts (extractable)

50 15,10.3 3.2 1.1 12-75 7 * 25 5.5

yo fixed salt :k free salt Sodium Potassium Calcium (as CaO) Magnesium (as MgO) Phosphorus (as P205) Sulphur (as so&) Chlorine Iron (as Fe2S03) Manganese Copper Aluminium

fraction 2.6 12.9 9.1 5.9 45.8 1.8 0.0

3.4

fraction 19.8 9.9 13.8 2.0 41 * 3 4.4 7.4 trace (20 PPm) (80 PPm) (100 PPm)

IV. GROWTH MEDIA CONSTITUENTS AND CELLULAR YIELDS

299

Research Establishment, Porton that the gross composition of various cellular constituents varies widely with conditions of growth-see for example Herbert (1961), Tempest and Dicks (1967). Nonetheless it is a useful starting point for a discussion of the cell yields that might be expected from various nutrient concentrations.

I. ENERGY SOURCE There is a good correlation between the amount of ATP formed during the catabolism of carbon and energy sources, and the cell yield (Bauchop and Elsden, 1960; Stouthamer, this Series, Vol. 1). The cell yields of different micro-organisms differ considerably when provided with the same amount of energy source (Table 11). Thus Zymomonas mobilis can achieve TABLE I1 Correlation of yields of cells from energy-source limited media+

Organism

Cell yield, Y Energy source ( g dried cells/mole substrate)

Saccharoniyces Glucose cereaisiae Zjmonzonas mobilis Glucose Lactobacillusplantarzraz Glucose Aerobacter aerogenes Glucosef Streptococczts faecalis Glucose Arginine Streptococciisfaecalis Propionibacteriz~m Glucose pentosaceu?n

18.8-21 * O 8.0-9.3 18.8 26.1 20-32 10.2 37.5

+

For discussion of the significance of these values see Stouthamer, this Series, Vol. 1 ;Bauchop & Elsden, 1960; Senez, 1962. f This value was obtained in simple salts media and the glucose was also utilized as the major carbon source (see Stouthamer, this Series, Vol. 1, for details).

only one fourth the cell density of Propionibacterium when provided with the same amount of glucose in the growth medium.

11. CARBON AND ENERGY SOURCE When the energy source provided is used to a large extent for cell synthesis, as is most often the case during aerobic growth in simple salts media, then such great differences of cell yield in relation to carbon and energy source supplied are not so apparent (Whitaker and Elsden, 1963; MacKech-

300

D. W. RIBBONS

nie and Dawes, 1969; Stouthamer, this Series, Vol. 1). The yield of microbial cells per g atom of carbon and energy source will vary with ( a )the oxidationreduction state of the carbon source (Gunsalus and Shuster, 1961; Senez, 1962; Mayberry, Prochazka, and Payne, 1968); (b) the degree of its polymerization, e.g., when a Cz unit such as acetate is utilized as sole source of carbon, more energy is required for its incorporation into cell constituents than equivalent amounts of a C6 unit as glucose (Whitaker and Elsden, 1963); (c) the pathways of its metabolism ;( d )growth rate (Tempest, Herbert, and Phipps, 1967); and (e) various physical parameters of cultivation. Table I11 illustrates the yields of cells obtained from a variety of carbon and energy sources during aerobic growth in minimal media. TABLE I11 Aerobic growth yields on various carbon sources in simple-salts media Carbon and energy source Pse-udomonas ClzBt Pseudomonas CizBt Pseudomonas CizBt Pseudomonas CizBt Pseudomonas aeruginosal Psnidomonas aeruginosat Psnidomonas aeruginosax Aerobacter aerogeness Aerobacter aerogenesl

Cell yield, Y (g dried cells/mole substrate)

Benzoate Succinate and acetate Succinate Acetate Glucose Gluconate 2-Ketogluconate Glycerol (growth rate 0.24 h-1) Glycerol (growth rate 0.004 h-l)

86.8 72,2 42.3 23.5 77 75.5 66 36.8 14.7

t Date from Mayberry, Prochazka & Payne (1968). Data from MacKechnie & Dawes (1969).

5 Data calculated from Tempest, Herbert & Phipps (1967). 111. NITROGEN AND SULPHUR The nitrogen and sulphur contents of microbial cells are usually between 6-13% and 0-7-1.3~0respectively. The yield values of micro-organisms, when grown on a limiting N source such as NH4+ or limiting S source such as SO42-, are a variable function of growth rate in continuous culture (?'empest and Dicks, 1967). The variation in microbial mass is due, in those instances investigated, to the deposition of storage materials such as glycogen or poly-/3-hydroxybutyrate. Yields based on total protein synthesized are however more constant, and these approach the values expected for an average N content or S content of protein of 16 and 1.5% respectively. Batch cultures in defined media show similar yield (as total

301


TABLE IV Variations in the protein and nucleic acid contents of Aerobacter aerogenes (from Tempest & Dicks, 1967, expressed as g/100 g dried bacteria) Growth rate NH4t-limited h-1 protein KNA

0.1 0.2 0 .4 0.8

61.5 64.2 69.4 69.3

7.5 10.0 13.6 18.3

SO&-limi ted DNA

protein

RNA

DNA

3.8 3.8 4. 0 4. 0

68.9 72.9 72.7 70,4

7.3 10.6 14.4 16-9

2.7 3.0 2. 9 2.3

mass) variations with carbon source in the presence of excess of NH4+ or SO42-. At high growth rates during N limitation, more N is used for RNA synthesis and consequently less N is used for the synthesis of other nitrogenous constituents. Data showing the variation of protein and nucleic acid contents of Aerobacter aerogenes are shown in Table IV. The quantitative relationships between growth yields and the utilization of other sources of nitrogen and sulphur are less clear. For example, when nitrate is the nitrogen source several factors such as nitrite toxicity or Nz evolution complicate the stoicheiometry. This is also discussed by Stouthamer (this Series, Vol. 1). IV. POTASSIUM, MAGNESIUM AND PHOSPHORUS These three elements are considered together because of their interrelationship, amounting to their presence in stoicheiometric proportions in microbial cells. These elements are usually supplied as K+, Mg2+ (or as chelate) and PO&. Phosphate is occasionally supplied as an ester. The ratio of potassium tomagnesium to phosphorus in K+-limited cultures is shown in Table V. Clearly the cellular contents and hence requirements TABLE V Potassium, magnesium and phosphorus contents of K+-limited-Aerobacter aerogenes organisms, grown at different dilution rates (from Tempest & Dicks, 1967, pH 6.5, t = 35", expressed as g/100g dried bacteria)t Dilution rate (h-l)

0.1 0.2 0.4 0. 6 0.8

K 0.81 1*08 1 *40 1.54 1 *60

Mg

P

0.12 0.16 0.19

1.31 1a69 2.17 2.30 2.56

0.22

0.24

t Average molar ratios of the five sets of values for Mg : K

: P = 1 : 4.25 : 8 -22.

302

D. W. RIBBONS

of these elements are a function of the growth rate, but the molar ratio of one to another is constant, i.e., K : Mg : P is 1.0 : 0.25 : 2.0. Tempest and Dicks (1967) further showed that this relationship, which is independent of growth rate, extends to the ribosomal R N A content of the cells (see Table IV) ; i.e., when rates of protein synthesis are high, at high growth rates, the ribosomal R N A and cellular contents of K, Mg and P are high, but all four are in proportion. It is obvious then that the growth yields of micro-organisms on K, M g or P as growth-limiting nutrient are greatly dependent upon growth rate and can vary by factors of more than 2 when the growth rate varies between 0.1 and 0.8 h-1. The above data were obtained with A . aerogenes. A rather higher K requirement is evident in the Gram-positive Bacillus subtilis. This is related to the content of cell-wall polymeric acids, teichoic or teichuronic acids. During phosphate limited growth of B. subtilis the character of the cell-wall polymers alters ; teichuronic acids completely replace the phosphorus-containing teichoic acids, and higher K : P ratios are observed (Tempest, 1969). Reports (Rouf, 1964; Webb, 1966) that the Mg contents of Grampositive bacteria are greater than Gram-negative bacteria have been resolved by Tempest, Dicks and Meers (1967). Growth of B. subtilis in Mgz+-limited continuous cultures showed that the Mg content of the cells was dependent on growth rate and almost identical to those of A . aerogenes grown under similar conditions. T h e earlier reports that the Mg contents of Grampositive bacteria are greater than Gram-negative bacteria is probably due to a greater capacity of Gram-positive bacteria to absorb Mg2f although A . aerogenes, at least, has a greater affinity for this cation. Gram-positive bacteria are unable to grow at Mg2f concentrations in the media less than 2 pg/ml, because they cannot assimilate such low concentrations. On the other hand Mg at this level is quantitatively absorbed by Aerobacter, Salmonella and Pseudomonas. Webb (1967) has shown that the presence of Mn2-+in the media reduces the Mg concentration required for growth of Gram-positive micro-organisms but this is not a general phenomenon (Tempest, 1969).

V. SODIUM Bacteria, except in special circumstances, have no absolute requirement for sodium like that of yeasts and fungi (Herbert, Strange, and Phipps, this Series, Vol. 5 ) . Several of the analyses showing high sodium contents in bacteria reflect the harvesting and washing treatments of cells prior to analysis, when sodium readily enters the cells and the potassium content is lowered.


303

VI. TRACE ELEMENTS There is little available data on the relationships of trace element content of media and growth yield, mainly because of the practical difficulties of purifying all reagents and cleaning of apparatus. The data provided in Tables VI and VII show the elemental compositions of several microbial species. They can be used only as a guide in formulating media for the growth of micro-organisms since the presence of many of these elements have no known physiological significance and the compositions given reflect the conditions of growth and subsequent treatment of the cells in preparation for analysis. TABLE VI Inorganic elements in vegetative cells and spores of bacteria (from Rouf, 1964)

yo dry wt of cells and spores Organism Escherichiacoli Micrococcus roseus Sphaerotilus natans Bacillus cereus B . cereus?

MgO PzO5 KsO

so4

NazO SiOz CaO Fez03 ZnO

9.83 7.46 4.89 7.42 2.81

2.58 2.39 1.72 2.09 2.11

0.01 0.85 0.10 0.75

1.04 1.99 0.95 1.72 0.95

1.42 1.11 0.20 5.52 2.19

0.09 0.14 0.05 0.06 0.05 1.05

0.02 0.10 0-23 0.04 0.08

0.03 0.02 2.60 0.03 0.04

0.01 0.03 0.03 0.03 0.32

j- This entry refers to spores; the rest are vegetative cells.

REFERENCES Bauchop, T., and Elsden, S. R. (1960).J. gen. Microbiol., 23,457. Gunsalus, I. C., and Shuster, C. W. (1961). In “The Bacteria” (Ed. R. Y. Stanier and I. C. Gunsalus), Vol. 2. Academic Press, Inc., New York and London. Herbert, D. (1961). In “Microbial Reaction to Environment”. Symp. SOC.Gen. Microbiol., 11, 391. Luria, S. (1960). In “The Bacteria” (Ed. R. Y. Stanier and I. C. Gun~alus),\~ol. 1, p.1. AqacKechnie, I. G., and Dawes, E. A. (1969).J. gen. Microbiol., 55, 341. Mayberry, W. R., Prochazka, G. J., and Payne, W. J. (1968).J. Bact., 96, 1424. Rouf, M. A. (1964).J. Bact., 85, 1545. Senez, J. C. (1962). Bact. Rev., 26, 95. Tempest, D. W., and Dicks, J. (1967). In “Microbial Physiology and Continuous Culture” (Ed. E. 0. Powell, C. G. T. Evans, R. E. Strange and D. W. Tempest). H.M.S.O., London, p. 140. Tempest, D. W., Dicks, J., and Meers, J. L. (1967).J. gen. Microbiol., 49,139. Tempest, D. W., Herbert, D., and Phipps, P. J. (1967). In “Microbial Physiology and Continuous Culture” (Ed. E. 0. Powell, C. G. T. Evans, R. E. Strange and D. W. Tempest). H.M.S.O., London, p. 240. Tempest, D. W. (1969). In “Microbial Growth” Symp. SOC. Gen. Microbiol., 19,87. Webb, NI. (1967).J.gen. Microbiol., 51,325. Whitaker, P., and Elsden, S. R. (1963).J. gen. Microbiol., 31, xxii.

w

0

-P

TABLE VII Inorganic elements in vegetative cells and spores of bacteria (from R o d , 1964)

Organism

p.p.m. dry wt of cells and spores Bz03 A1203 Ti02 Cr203 MnO NiO CuO SrO AgzO SnOz BaO

PbO

v205

kfOO3 CO

12

see note+

4

-

4

-

7

-

15 8 4

11

-

C

3

2 7 6 4 2

4 0 . 6

5

7 18 9 31 3 43 4 937 0.4

0.7 8 1.0 4 2.0 10 0.6 0.1 0.1

14 30 5 6 0.1

Esclterichia coli

4 0 4 4

2

Micrococcus roseus Sphaerotilus natans Bacillus cereus B . cereus1 Minimal detectable amount

25

9 7 2 8 1 53 620 3 0.5 3 3 1560 0 - 5 0.1 0.7

-

8 -

3

64 36 31 29

-

t None detectable. 1This entry refers to spores; the rest are vegetative cells.

5

5

5 5 2 5 0.6

0.1

-

5 0.5

-

0 . 2 0.5

€ P, m

m

2m!

CHAPTERV

Enrichment Cultures of Prokaryotic Organisms H. VELDKAMP Microbiological Laboratory, State University, Groningen, The Netherlands I.

Introduction

.

.

. . . . . .

309 310 331

Isolation of Prokaryotic Organisms . . A. Filtration as a means of separating microbes from mixed . populations B. Separation of motile bacteria from mixed populations . . C. Microbial morphology as an aid in isolation procedures .

345 348 350 353

.

355

11. Enrichments in Closed Systems A. The liquid enrichment culture B. Population dynamics and enrichments in natural habitats 111. Enrichments in Open Systems . A. The homo-continuous culture system B. The hetero-continuous culture system IV.

305

. .

.

References

.

340 341 343

I. INTRODUCTION Natural milieus like soil, leaf surfaces or ocean waters are always occupied by diffcrent kinds of micro-organisms. These organisms are able to coexist in the same habitat because each type of organism has its own functional status in the ecosystem in which it takes part. The more heterogeneous a natural milieu, the more possibilities there are for harbouring organisms of different types. In a richly manured garden soil, conditions may vary from millimeter to millimeter, enabling organisms of many different kinds to grow under the conditions they prefer. Waters of the open ocean form a much more homogeneous milieu, and consequently harbour a less diverse community of microbes. And the nutritionally poor conditions and high temperature of the effluent from a hot spring in Yellowstone Park allow only two organisms to grow there: one blue-green alga and one filamentous bacterium (Brock, 1968). When one wants to separate a special type of organism from a complex 13

306

H. VELDKAMP

community different possibilities exist. One of these is to create a liquid growth medium whose physicochemical conditions are such that the desired organism may be expected to grow faster than other components of a mixed population. This medium is subsequently inoculated with a sample from an environment in which the desired microbe is likely to occur. When the inoculum does contain the organism, and growth conditions are appropriate, its numbers will soon become much larger than those of other microbes. The organism forming this dominant population can then be isolated in pure culture by commonly applied isolation procedures. This technique, the enrichment culture technique, led Winogradski and Beijerinck in the first decades of this century to the discovery of a great many ecological niches. As a result of their initiative, in a relatively short time the different metabolic types that we now know to exist among microbes were discovered by studying enrichment cultures of many different kinds as far as physicochemical conditions of the growth medium is concerned. Two main types of enrichment cultures are applied. The first and most generally applied one is the closed system. The inoculum is placed in a shallow layer of liquid exposed to the required gas mixture, or in a filled stoppered bottle (anaerobic conditions) and the development of microbial growth is studied. Samples are taken for microscopic examination and for seeding appropriate solid media to allow the desired organism to form colonies which are spatially separated from those of other microbes. The technique thus applied has one disadvantage characteristic for any closed system. As soon as the growth medium for the enrichment is inoculated, the organism that starts growing changes the growth conditions by consuming ingredients and excreting metabolic products. And, as growth conditions change, growth rates change. Sometimes, when the stationary growth phase of the organism that came to the fore is reached, a more or less stable end phase is obtained. Frequently, however, conditions are then favourable for the development of another microbe present in the mixed inoculum. Opportunities for such successions are greater when higher concentrations of metabolizable substrates are used. Concentrations generally chosen for the carbon and energy source for chemo-organotrophs, for instance, are often much too high, resulting in excessive acid or alkali production leading to unfavourable conditions for the organism to be enriched. Thus, in this type of enrichment culture, conditions are changing continuously and one should be alert in order not to miss the stage in which the desired organism is dominant. The phenomena described above do not occur when use is made of an open system, as for instance the chemostat. In such a system fresh medium

V. ENRICHMENT CULTURES OF PROKARYOTIC ORGANISMS

307

is introduced at constant speed and culture liquid is removed at the same rate. When a mixed population of micro-organisms is inoculated into the chemostat, selection is determined by the way in which growth rates of the components of that population depend on the concentration of a chosen growth-limiting substrate. This concentration in turn depends on the applied dilution rate. The organism with the highest growth rate at the dilution rate applied becomes dominant, and slow or non-growing organisms are washed out. The characteristics of the chemostat are such that organisms which do not get a chance in any liquid growth medium applied in closed system can be allowed to become predominant. Details about application of the closed and open type of enrichment culture will be given in Sections I1 and 111, respectively. T h e enrichment culture technique serves many purposes, and has been successfully applied by various microbiologists. When an industrial microbiologist wants to isolate an organism that produces a useful exoenzyme, he may proceed by adding to the basal medium described in Section IIA the substrate for the enzyme as carbon (or carbon and nitrogen) and energy source. A series of such growth media is inoculated with inocula from different sources and subsequently the organisms that come to the fore can be isolated by making streak plates, using a solid medium of similar composition. When the enzyme in question has to be applied under special physicochemical conditions, growth conditions chosen for the enrichment are often adapted to those in which the enzyme has to be applied. However, the latter conditions need not necessarily be optimal for growth of the enzyme-producing organism. Sometimes a quite different approach may work. When descriptions are available of organisms that release a desired metabolic product, it may be useful to determine whether these organisms have metabolic or taxonomic properties in common. If so, it may be rewarding to select for such properties in enrichment cultures. Industrial experience has shown that selection for properties which nobody could think of being related in any way to the desired property, often yields organisms that upon screening show an appreciable number of “positives”. T h e microbial physiologist who wants to make a comparative study of a certain dissimilatory process in various organisms should apply the enrichment culture method to obtain such organisms. By varying in his enrichment factors as pH, pOz, pCOz, temperature, etc., he will be able to isolate different organisms, each of which carries out the particular process under specific environmental conditions. Whether the organisms thus enriched are of any quantitative ecological importance in a natural milieu generally does not particularly interest the physiologist. However, such data are of

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interest to the ecologist, and he therefore would make a dilution series of a sample from a natural environment and use this as inoculum for a series of enrichment cultures. Or he would add small amounts of the substrate to be studied to samples of the natural environment and incubate these under conditions as closely related to the natural environment as possible. T h e ecologist is, furthermore, often not merely interested in selection towards a certain substrate. In nature there is not only a selection depending on the kind of substrates available, but also towards the concentration of these substrates. Different organisms come to the fore at different substrate concentrations. This type of selection should be studied in an open enrichment system, such as the chemostat, as will be described in Section 111. In the search for pathogens that form a minority population in a mixed flora, the enrichment culture technique has proven to be of great value to the medical and to the food microbiologist. From the above it is clear that the ways in which the enrichment technique is applied are different, depending on the purpose for which it is used. All enrichments have the following points in common, however. T h e water content of the medium or environment studied should be high enough to make proliferation of the desired organism possible. This does not only hold for the water content as such. Some water containing environments are “physiologically dry” because of the high concentration of solutes (e.g., salt brines) and allow the development of only a limited number of microbes (cf. Eimhjellen, 1965). Water content is particularly important when enrichments are carried out in soil. Depending on the amount of water in the soil, quite different aspects of the microflora may be favoured. The growth medium should furthermore contain the elements and energy source needed by the microbe in order to reproduce itself, and these should be present in a form available to the organism. Finally, the organism to be enriched should be present in the inoculum. Though microbes often do survive conditions that do not allow proliferation, samples for inoculation of enrichments should generally be taken from milieus favouring growth of the organism to be isolated. When applying the enrichment-culture technique, a certain minimum knowledge about physicochemical conditions of natural environments is indispensable. I t would not make sense for instance to study the natural flora of ocean waters (to a large extent obligate psychrophiles growing at very low concentrations of organic substrates) by cultivating samples in 1 yopeptone at 30°C. Direct microscopical examination of samples taken from natural environments has confronted microbiologists already for decades with organisms


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that never show up in any enrichment culture (Winogradski, 1949; Nikitin, 1965; Hungate, 1966; Gray et nl., 1967). As pointed out by La Rivihre ( 1 9 6 9 the enrichment of these organisms requires knowledge of their physiology; but, conversely, this knowledge can only be obtained by studying the organism in pure culture. This presents a vicious circle, which the present status of microbiology is still unable to break. This holds for the curious organisms that we can distinguish morphologically, and also for the certainly still greater number of organisms with a less exotic appearance.

11. ENRICHMEN'I'S I N CLOSED SYSTEMS Natural milieus form heterogeneous open systems in which substrates for microbial growth generally become available as a flow of low rate and low concentration. The mere taking of a sample of ocean water and placing this in a flask changes conditions to those characteristic of the closed system. The flow of growth-limiting substrates for certain components of the mixed microbial population changes ; growth rates change and metabolic products begin to accumulate. The overall result of the complex of processes is a change in population densities within the mixed population of microbes (cf. Jannasch, 1967a). A large fraction of the original population in the sample consists of chemo-organotrophs deriving its selective advantage in the nutritionally poor ocean water from a relatively high growth rate at low substrate concentrations (Jannasch, 1967b). This group of organisms will be dealt with in Section I11 on open systems. Many of these microbes have relatively low growth rates when there is a large surplus of nutrients. They will never come to the fore in the usual batch enrichments, even in a simple mineral growth medium with 0.5-1 yoof a carbon and energy source. But it is not only that a large fraction of naturally occurring bacteria do not develop in the usual batch enrichments. These are also to a large extent irreproducible, especially those for chemo-organotrophs. Generally speaking, the more extreme the conditions of the artificial milieu in the enrichment, the higher is the chance that time and again a specific organism will become dominant. Since there are, for example, only a limited number of bacteria that can derive energy from the oxidation of ammonia, and only a few photosynthetic bacteria which can utilize light of wavelength > 1000 nm, enrichments for these organisms will often give the same result. However, a great many different chemo-organotrophs can develop in yeast extract-glucose medium, and it is impossible to predict which of these will become dominant in a special case. This will depend to a large extent on the composition of the flora present in the inoculum. In this respect

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it should also be realized that in most cases it is not just one organism that starts to multiply when the medium is inoculated. This means that chemical changes in the first instance are due to the metabolic activities of a mixed population, and it is thus not only the initial chemical composition of the medium, but also the uncontrollable changes brought about by the mixed inoculum, that determine which of the components present in that inoculum will eventually become dominant. It may also occur that metabolic activities of a dominant organism changes the medium in such a way that ultimately other organisms come to the fore. A classic example is the growth of Azotobacter in media devoid of combined nitrogen. This organism excretes amino-acids, which gives other organisms present in the mixed inoculum a chance to proliferate. Such secondary developments can be prevented to a large extent by frequent transfers. The type of batch enrichment most generally used is the liquid culture inoculated with a sample taken from a natural environment. A disadvantage of this method is that the organism that becomes dominant need not necessarily play a quantitatively important role in the natural milieu. For these and other reasons, enrichments are sometimes carried out in the natural milieu. Examples of both methods will be given below.

A. The liquid enrichment culture One of the attractions of the closed system liquid enrichment culture is that only simple apparatus is needed. In fact, conical flasks, stoppered bottles and some cheap chemicals handled by imaginative minds were the only ingredients needed to reveal the astonishing diversity of metabolic types that we now know to exist among prokaryotic microbes. Liquid media that have to be exposed to air or other gas mixtures generally are applied as a shallow layer in a conical flask. Completely filled stoppered bottles are used for anaerobic enrichments in which no special gas atmosphere is required. In some exceptional cases more elaborate equipment may be convenient, or even necessary, as will be indicated below. For the enrichment of a great many organisms a basal medium of the following composition can be appliedBasal medium

KzHPOi

MgS04.7HzO FeS04.7HzO CaCla Trace elements solution (see below) Distilled water

l P

0.2 g 0.05 g 0.02 g 0.1 ml 1 litre


31 1

The requirements for carbon, nitrogen and energy source depend on the type of organism to be enriched, and will be dealt with in Section IIA.5. Trace elements generally are present in sufficient amount as impurities in the chemicals applied and in the inoculum. T o avoid any possibility of a trace-element deficiency, a mixture of these elements often is included in enrichment media. A suitable trace element solution is that of Hoagland, as modified by Pfennig (1965a). The following amounts of trace salts are dissolved separately in distilled waterTrace elements

AlC13 KI KBr LiCl MnClz .4Hz0 H3B03 ZnClz cuc12 NiClz coc12 SnC12.2HaO BaClz NazMo04 NaVOs. HzO Selenium salt

1g 0.5 g 0.5 g 0.5 g 7g 11 8 1g 1g Ig 5g 0.05g 0.5 g 0.5 g 0.1 g 0.5 g

Before mixing the constituents together, adjust the pH of each solution to below pH 7.0. The total final volume is 3.6 litres. Adjust the pH of the final solution to pH 3 4 with HCI. The flaky yellow precipitate that is formed after mixing transforms after a few days into a white precipitate. Before use, mix the solution thoroughly. Pfennig, who successfully applied the above mixture in enrichment procedures for phototrophs (cf. Pfennig, 1967) included EDTA (final concentration 25 mg/litre) as a chelating agent. Trace elements should not only be present, but also available. Availability of trace elements needs special attention in marine environments, since at the pH of sea water (8.0-8.3; cf. Harvey, 1960) their salts are extremely insoluble. It has been shown for instance that growth of phytoplankton was favourably influenced by addition of chelators to ocean waters that allowed only limited growth of algae (cf. Provasoli, 1963). And it thus appears that growth of these organisms is to a large extent dependent on the presence of organic compounds that have the ability to form soluble metal complexes. In this respect it should be mentioned that EDTA may affect growth of marine bacteria unfavourably. The growth rate of marine Pseudomonas

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species decreased considerably when 50 mg/litre of EDTA was included in the growth medium, and the cells showed decreased refractility. Normal growth rates and refractility were not obtained until the EDTA concentration was lowered to 5-10 mg/litre (W. Harder, personal communication). Stanier et al. (1966) reported good growth of Pseudomonas species in a heavily chelated basal medium enriched with organic substrates. A nonchelated mineral base had to be used, however, for chemolithotrophic growth at the expense of H2. Sometimes a copious precipitate is formed during heat sterilization of a growth medium. When the medium is filtered and re-sterilized, it should be realized that trace elements may show co-precipitation and then are removed by this procedure. Most marine organisms have an absolute requirement for Na+, which has a transport function (Drapeau et al., 1966); it is supplied as NaCl (2-3 yo w/v, final concentration) Carbon dioxide is not only needed by those organisms that use it as the sole source of carbon, but also by various chemoheterotrophs. It is not usually added to aerobic cultures, but should be included in growth media for anaerobes unless it is known that the microbe to be enriched has no C02 requirement. It is generally added as NaHC03 which is sterilized separately by filtration (under positive pressure) in 5-10 yo solution. When the final desired p H of the medium is approximately 7.0, it is convenient to use KH2P04 as the phosphorus source and add enough NaHC03 to reach the required pH. Apart from the ingredients mentioned above, enrichment media for a large variety of microbes should contain growth factors. These requirements may range from one vitamin (e.g., B12, which is needed by many aquatic organisms; cf. Droop et al., 1959; Mulder and van Veen, 1965; Pfennig and Lippert, 1966) to an extremely complex mixture of vitamins, amino-acids and other factors (e.g., lactic acid bacteria; cf. Guirard and Snell, 1962). Growth-factor requirements can often be met by including a small amount of yeast extract (0.001-0-05 %) in the enrichment medium. I n media for the enrichment of highly exacting organisms, much higher amounts are used (0.5-1%). It should be emphasized that yeast extract does not contain all the growth factors that may be needed. Vitamin BIZ and ferrichromes for instance are not present in yeast extract. Sometimes unusually large amounts of a complex nutrient are needed. I n such cases the need for the complex nutrient may not be a normal growth-factor requirement. Clostridium tyrobutyricum for instance was found to be able to ferment lactate only in the presence of an unusually large amount of yeast extract. Barker found that the active component in yeast extract was acetate. T h e organism needs two molecules of acetate


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to form butyrate; one is formed from lactate, the other should be supplied in the growth medium (cf. Barker, 1956). I n fact, this is a process that occurs not uncommonly in low-grade silage. It sometimes happens that an organism, after becoming dominant in an enrichment culture, shows very poor growth on transfer to fresh medium. This may mean that in the first culture a growth factor was present, either introduced with the inoculum or produced by one of the components of the mixed population. In such cases addition to the growth medium of a sterile sample of the first culture or of an extract of the natural environment may be useful. These additions can best be sterilized by membranefiltration after centrifugation. Most enrichment media can be heat sterilized. Among the components that should be sterilized separately, either by heat or filtration, is glucose (cf. El-Ghazzawi and Schmidt, 1967). When this sugar is heat sterilized in complex media, it gives rise to products that may influence bacterial growth ; growth of Cytophaga strains is often completely inhibited, whereas the effect on growth of Lactobacillus and Propionibacterium .strains may be favourable. Clostridium aceticum would only grow on glucose when this sugar was heat sterilized in the growth medium, or in the presence of phosphate. This appeared to be due to the fact that 25% of the glucose was converted to fructose during heat sterilization; and it was this sugar that was utilized by the organism (El-Ghazzawi and Schmidt, 1967). Sometimes ingredients used to prepare enrichment media may contain toxic compounds. Filter paper and agar may, for example, contain substances that inhibit growth of cellulose-decomposing cytophagas (Fihraeus, 1947; Imschenetski, 1959). The agar used in such cases should be repeatedly washed with distilled water; or a commercially available purified agar should be used. Filter paper can be purified by immersion and autoclaving in dilute alkali or in a slightly alkaline basal salts solution. The paper is then washed with distilled water and dried at 50°C (Fihraeus, 1947). It should finally be mentioned that even tap water has in some cases been shown to contain toxic substances that may inhibit growth of certain microbes. Growth inhibitory substances may purposely be included in media to prevent growth of unwanted microbes. A classic example is the use of bile salts (0*15-0.5% w/v; cf. Difco Manual, 1963; Oxoid Manual, 1965) in enrichment media for Esclierichia coli that have an inhibitory effect on growth of the closely related Aerobacter aerogenes. I n enrichments for Gram-negative bacteria, growth of Gram-positives may be inhibited with penicillin (1 pg/ml). Polymyxin B (5 pg/ml) can be used to prevent growth of Gram-negatives (cf. Goldberg, 1959). Growth of fungi in cultures of bacteria and actinomycetes can conveniently be

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prevented by inclusion of actidione (50 pglml) and nystatin (50 pglml) (Williams and Davies, 1965). When growth of protozoa has to be prevented in enrichments for bacteria, the antifungal antibiotic protomycin (5 pglml) might be promising (cf. Gottlieb and Shaw, 1967; Korzybski et al., 1967). It has the same potency as the antiprotozoan antibiotic puromycin, but shows little activity towards bacteria.

1. p H value of enrichment media (a) p H as a selective factor. Beijerinck (1913; cf. Beijerinck, 1921-1940) described the studies of enrichment cultures as a new branch of science: microecology. Baas Becking (1934) pointed out that its basic principle could be given by two rules(i) Everything is everywhere. (ii) The environment selects. He illustrated these rules by the example of Sarcina wentriculi, which Goodsir in 1842 discovered in human stomach contents. In 1911 Beijerinck (cf. Beijerinck, 1921-1940) confirmed Goodsir's observation and also succeeded in revealing the presence of this organism in garden soil by incubating a sample under similar conditions (anaerobiosis,low pH, temperature 37°C). The experiment can still be repeated as follows. Malt extract is heat-sterilized in an Erlenmeyer flask and after rapid cooling acidified to pH 2.0 with a M solution of either HCl, H3P04 or HzSO4. A 50 ml stoppered bottle is completely filled with this medium, inoculated with ca. 5 g of soil or mud, stoppered and incubated at 37°C. Excessive gas formation is indicative of the presence of S. ventriculi, which forms large packets of cells to be found in the sediment. Even though the above conditions are to a large extent selective for the enrichment of Sarcina (Mucor may also develop), growth of this organism is not restricted to a low pH value. It shows growth over pH range 1-9. At higher pH values, however, Sarcina is rapidly outgrown by other organisms. S. wentriculi is not particularly sensitive to oxygen. After a few transfers in the above medium, the organism can easily be obtained in pure culture by making a dilution series in neutral, or slightly acid, melted agar (cf. Canale-Parola and Wolfe, 1960). The organism is, however, very sensitive to an unknown toxic product which it produces. Actively fermenting liquid cultures should be transferred at 1-2 day intervals. CanaleParola and Wolfe (1960) developed a method of keeping stock cultures viable for a period of 2 months. For this purpose a sample of cell-packets is deposited in a depression made in the dry surface of a thick layer of agar. pH changes in enrichment cultures are often responsible for a succession


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of microbial populations, each of which shows development in a restricted p H range. A striking example of such a succession is encountered in silage and sauerkraut production, which will be described in Section IIB.4. It should be realized, however, that these successions are often not due to a mere pH effect. The concentration of undissociated organic acids also plays an important role. Under acid conditions weak organic acids (e.g., acetic acid) are toxic for many microbes, whereas at similar p H values strong inorganic acids (e.g., HC1) have relatively little effect. This is due to the fact that undissociated molecules of organic acids enter the cell more easily than the corresponding ions, and once penetrated, cause damage by changing the internal pH. Similar considerations hold for basic substances under alkaline conditions. The consequence is that it may not be possible to indicate accurately the p H range for growth of an organism. This depends on the buffering capacity of the growth medium. In a weakly buffered medium growth may occur at a lower pH than in a well buffered medium, owing to the concentrations of undissociated organic acids. Beijerinck's imaginative approach in applying the enrichment culture technique also revealed the existence of a microbe that can be selected at high pH values: a urea-decomposing Bacillus. The method was more recently applied by Wiley and Stokes (1962) as follows. 1 g soil sample was suspended in tap water, pasteurized (10 min at SOOC), and inoculated in a 100 ml conical flask containing 30 ml of the following medium: yeast extract, 1%; urea, 5%; pH 9.0. The incubation temperature was 30°C. The authors isolated B. pasteurii on a solid medium containing: yeast extract, 2% w/w; (NH&S04, 1% w/w; 0.13 M Tris buffer (pH 9.0); in distilled water. These ingredients were heat sterilized separately, since a medium in which all components were sterilized together did not support growth. A pure culture study showed that not merely an alkaline pH, but also the presence of an ammonium salt was essential for the oxidative metabolism of the organism. When unpasteurized inocula are used, other urea-decomposing organisms may also develop (e.g., Mimococcus ureae), and even pasteurized inocula applied in aerobic cultures may not give exclusive development of B. pasteurii, since the spores of Sarcina ureae have about the same heat resistance as those of urea-decomposing bacilli (MacDonald and MacDonald, 1962; Kocur and Martinec, 1963). The classic examples given above merely illustrate the selective effect which pH may have. When a pH gradient is made of a large variety of growth media, and the series is inoculated with soil samples, the variety of microbes that come to the fore at different pH values clearly shows that pH is an important selective factor under natural conditions.

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(b) p H changes in enrichment cultures. According to Schlegel and Jannasch (1967) “it is the aim and the art of the enrichment culture technique to control those selective conditions that quickly and reproducibly lead to the predominance of the population of one special organism, thereby facilitating its isolation”. pH changes in the closed system enrichment culture are the rule rather than the exception. This means that the period in which the p H is optimal for the organism to be enriched, is often limited. Among factors causing a decrease in p H are(i) Application of (NH&S04 as nitrogen source. (ii) Production of organic acids by heterotrophic bacteria. (iii) Production of inorganic acids by autotrophic bacteria.

A pH increase can be caused by(i) (ii) (iii) (iv)

Utilization of K N 0 3 as nitrogen source. Ammonia formation during amino-acid decomposition. Utilization of salts of organic acids by heterotrophs. Utilization of inorganic compounds as electron acceptor in anaerobic respiration.

Inclusion of buffers in enrichment media to prevent p H changes is generally of limited value. Phosphate buffers are usually applied in concentrations not exceeding 0.03 &I. Larger amounts of phosphate may not be tolerated by the particular organism and have moreover the disadvantage of easily giving rise to precipitates of insoluble phosphates (e.g., MgNH4P04). T h e Tris buffer, which is mainly used in media that tend to become alkaline, has the disadvantage of being organic and providing a carbon-nitrogen source. When excessive acid formation is to be expected, CaC03 may be included in the enrichment medium which keeps the p H from falling below ca. 6.0. However, CaC09 is not very effective in stationary cultures. And when the culture is placed on a shaking machine, bacterial growth cannot easily be followed macroscopically. The only way of keeping the pH constant at a desired value is to make use of an automatic titrator (cf. Munro, this Series, Vol. 2). T h e culture liquid should in this case be constantly agitated to ensure homogeneous conditions. This method of p H regulation has the disadvantage of being expensive and requiring rather complicated culture vessels. There are, however, a few simple ways in which p H changes can be kept within reasonable limits in many cases. The first is to use as little as possible of the compound whose conversion is mainly responsible for the p H change. T h e concentrations of ingredients applied in enrichment cultures very often are much higher than is strictly needed to obtain a dominant population of the desired organism. Secondly, in order to prevent secondary growth, it is


317

good practice to transfer frequently and as soon as there is visible growth. This is very important in the application of enrichment techniques in closed systems. I t was, in fact, the secret of Pasteur’s work with enrichments. He never worked with pure cultures. By choosing the right environmental conditions, however, and by frequently transferring his cultures, he maintained conditions in which one organism by far outnumbered others. It was these cultures that led him to the conclusion that a special type of fermentation was caused by one specific microbe.

2. Redox conditions in enrichments (a) Aerobic enrichments. An enrichment culture is generally considered aerobic when the culture liquid is applied as a shallow layer exposed to air. It should be realized, however, that aerobiosis generally is limited to the very surface of the culture; and even there the concentration of dissolved oxygen may become very low in stationary cultures, when a pellicle develops. The following example may illustrate this. A mineral medium devoid of combined nitrogen and enriched with glucose is inoculated with a small soil sample and poured in a Petri dish to form a layer of ca. 5 mm thickness. In this medium, nitrogen-fixing bacteria will develop. After a few days incubation at 30°C cells of Axotobncter will float on the surface, and at the bottom nitrogen-fixing clostridia develop, as can be judged from the butyric acid smell such cultures often have. The growth of Clostridizim at a distance of only a few millimetres underneath the liquid surface means that conditions there must be strictly anaerobic. I n order to obtain better aeration conditions, enrichments may be made in Erlenmeyer flasks which are placed in an incubator-shaker. This, however, does not always have a favourable effect on aerobic organisms. Some acetic acid bacteria, for instance, grow best in stationary culture in a pellicle that is formed at the surface. Other bacteria may show a considerable lag or do not start growing at all when the concentration of dissolved oxygen is too high. This may hold for bacteria that are strict aerobes. The early growth of Thiobacillus thioparus, for instance, often develops much better in stationary than in shaken culture (Postgate, 1966). Once growth develops, the culture may conveniently be further incubated on a shaking machine. Similarly, enrichments for Spirillum strains often do not show any development at all when cultivated on a shaker, whereas growth starts readily in stationary culture. Thus, aeration is a more subtle variable than is often thought, like so many other factors involved in microbial growth. It is therefore always good practice, especially when the properties of the organism to be enriched

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are largely unknown, to set up several enrichments simultaneously under various conditions. When a gradient of dissolved oxygen concentrations is needed in a series of enrichments, it is necessary to use oxygen electrodes. The Mackereth electrode (Mackereth, 1964), for example, is very dependable. Different constant oxygen concentrations can be obtained with the device developed by MacLennan and Pirt (1966). (b) Anaerobic enrichments. Exclusion of oxygen from enrichment cultures offers possibilities for growth of facultative and obligate anaerobic chemoorganotrophs, phototrophic bacteria and even some chemolithotrophs with denitrifying capacities. Specific growth requirements of each group are dealt with in Section IIA.5. Unless the anaerobic enrichment medium should be exposed to a special gas atmosphere, this type of enrichment can best be performed by using a stoppered bottle. The medium to be used is rapidly cooled after autoclaving, and ingredients that have to be sterilized separately are added before inoculation. The most common of the latter ingredients are a sugar or other heat-labile carbon and energy source, NaHC03 and a

H

FIG.1. Diagram of a Hall tube for anaerobic culture in fluid media. (Reproduced by permission of Springer Verlag.)


319

reducing agent. A very convenient way of keeping purified cultures of strict anaerobes in liquid culture is to make use of Hall tubes (see Fig. l), as modified by Barker (1936). It is good practice to purify the initial enrichment culture by several successive transfers before initiating the isolation procedure. In this respect it is of importance to realize that it is often easier to obtain optimal growth conditions in the initial enrichment than in successive cultures. When enriching for facultative anaerobes or denitrifying bacteria, the presence of trace amounts of oxygen is not critical, since these are utilized by the organisms to be enriched. In a first enrichment culture of organisms that develop only at relatively low Eh values (e.g., methanogenic bacteria), small amounts of oxygen remaining in the medium are of no importance; the soil or mud used as inoculum generally contains an appreciable amount of organic matter, as well as facultative anaerobes that will quickly remove the last traces of oxygen. On further purification of the obligate anaerobes, however, due attention should be given to the redox conditions of the growth medium. For sulphate-reducing bacteria, the medium should be poised at an Eh value more negative than about -100 mV (Postgate, 1966); and growth of methanogenic bacteria may not be obtained unless the redox potential is in the range in which benzyl viologen (Eo’, pH 7, -359 mV) is reduced (Smith and Hungate, 1958). The habit of the latter organisms of growing in the sediment of enrichments may well be due to favourable local redox conditions. On purifying methanogenic bacteria in liquid media, a sediment (e.g., CaC03, shredded asbestos, sterile mud; cf. Barker, 1936) is often included. When working with strict anaerobes of unknown optimal growth conditions, the inocula should not be too small when transferring cultures, e.g., 10%. It should be realized that mere anaerobiosis does not necessarily mean that the redox potential of the medium is sufficiently low (see Hungate, this Series, Vol. 3B). A relatively high potential can also be caused by oxidizing agents other than oxygen, as for example nitrate. For the enrichment of organisms that only develop at low Eh values, a reducing agent is always included in the growth medium, often together with a redox indicator. The most commonly applied indicators are resazurin (,To’, pH 7, ca. -30 to -40 mV), indigocarmine (,Yo’, pH 7, -123 mV) and for the more exacting organisms, benzyl viologen (Eo‘, p H 7, -359 mV); the concentrations used are 0.00010.0005% w/v. Reducing agents become toxic when a certain critical concentration is exceeded, which may be rather low, and depends on the organism under investigation. The toxicity level is often pH dependent. For instance, a strain of the phototroph Thiocapsa was observed to grow well over a pH range of 7.0-8-5, and to tolerate 11 mg/litre of undissociated H2S. The concentration of NazS.9HzO that can be applied therefore

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depends on the pH of the culture (H. van Gemerden, personal communication). In enrichments of some strict anaerobes a more rapid development is obtained when the bottle to be used is prepared ca. 24 h before inoculation. This ensures better redox conditions. Sulphide included in enrichments for photolithotrophs not only serves as a reducing agent, but also as an electron donor for photosynthesis. A variety of such organisms may coexist in a natural habitat by occupying different niches characterized by such factors as light intensity and wavelength, and concentration of electron donor. The range of acceptable sulphide concentrations at a certain pH may be rather narrow and this may create special enrichment problems, especially in closed systems. When an organism grows only at very low sulphide concentrations, a continuous supply is needed in order to obtain a substantial number of cells. This may be provided by sulphate-reducing bacteria (cf. Section ZIB.5), by a sulphide-containing layer of agar (cf. Section IIIB.2) or by supplying the culture with small amounts of sulphide when necessary (Pfennig, 1965a). When enriching for chemo-organotrophs that can grow anaerobically, it should be recognized that not all of them can grow anaerobically under all conditions, and, conversely, that some of the facultative anaerobes may fail to grow aerobically. Examples are as follows. Some hetero-fermentative lactic acid bacteria (e.g. Lactobacillus brevis) are unable to ferment glucose anaerobically, but do so aerobically. These organisms ferment glucose through the hexosemonophosphate pathway, but since they are unable to reduce the acetic acid formed, they need oxygen (or another external electron acceptor) to accomplish the first oxidative steps of glucose breakdown. Thus, anaerobic enrichments for lactic acid bacteria with glucose as carbon and energy source would never yield L. brevis, unless an external electron acceptor is provided (Buyze, 1955; van den Hamer, 1960). An alternative example is provided by the propionic acid bacteria. Early attempts in Kluyver’s laboratory to isolate these organisms from bottle enrichments, by making streak plates that were incubated aerobically, failed. I t was not until van Niel (1928) prepared agar shake tubes that colonies of these organisms developed. Propionic acid bacteria grow under aerobic conditions only when a heavy paste of cell material is smeared on an aerobic agar surface. Although use of the stoppered bottle has revealed the existence of many ecological niches, it is not suitable for the enrichment of all anaerobes. Especially when a special gas atmosphere is needed (e.g., C02-H2) more complicated equipment is required (for preparation of gas mixtures see


32 1

Skerman, 1967). The most convenient equipment for enriching such anaerobes should have the following facilities: removal from, or addition of gas to, the system and sampling of culture liquid without disturbing anaerobic conditions; quantitative measurement of gas formation or uptake; collection of liquid samples in a vessel containing the required gas atmosphere. I n some cases, a provision to keep the p H constant during fermentation is desirable, although here again p H changes may be prevented by transferring frequently. Technical details about handling anaerobes are given by Hungate, Willis, Barnes and Hobson (this Series, Vol. 3B). 3. Temperature as a selective factor Temperature is an important selective factor in enrichment cultures, as it is in nature. An example of the latter was given by Sieburth (1967), who studied the seasonal selection of estuarine bacteria by water temperature. Throughout the year samples were taken from Narragansett Bay, Rhode Island, the temperature of which varies from -2°C (JanuaryFebruary) to 23°C (August-September). Dilution series of the samples were seeded on a "non-selective" nutrient agar, incubated at a range of temperatures and the developing colonies counted. The results showed that the growth temperature range of the dominant bacterial winter populations was roughly between 8 and 20°C, whereas the dominant summer populations showed a growth temperature range 20"-33°C. Thus a growth temperature of 20°C was maximal in February, but minimal in September for the dominant populations. The seasonal selection of thermal types by water temperature occurred in all bacterial genera demonstrated. Thus, if samples had been taken during winter and used as inocula for enrichment cultures incubated at 30"C, they would have given a dominant population of an organism belonging to an ecologically unimportant fraction of the natural mixed population. But even when the culture had been incubated at 15"C, not all members of the naturally occurring dominant populations might have been given a fair chance. A high percentage of the winter population in Narragansett Bay consists of obligately psychrophilic bacteria (Sieburth, 1967). It has been shown that these organisms, which commonly occur in cold natural environments, are often rapidly killed when exposed to temperatures above 20°C (cf. Harder and Veldkamp, 1968; Brock and Rose, this Series, Vol. 3B). Thus, if these organisms are to be given a chance in enrichment cultures, the inocula should be kept at a temperature below 20°C before use. The temperature of incubation of an enrichment culture may act as a

+

322

H. VELDKAMP

nutritional variable, either by affecting the composition of the growth medium, or by inducing metabolic lesions through which an organism may become auxotrophic for a certain compound. Temperature affects the composition of the growth medium with respect to the concentration of gaseous compounds. Sinclair and Stokes (1963) showed that maximum cell numbers in stationary cultures are obtained between 10" and 20°C with organisms having an optimum temperature for growth above 20°C. This appeared to be simply due to the increased solubility of oxygen and therefore to its less rapid depletion at the lower temperatures. Borek and Waelsch (1951) found that a culture of Lactobacillus arabinosus exposed to air grew well at 35°C without phenylalanine, though at 37°C the organism did not grow at all unless phenylalanine was included in the growth medium. Similarly, a requirement for aspartic acid could be induced by cultivating the organism at 39°C. The profound effect of small temperature changes on the synthetic abilities of L. arabinosus could be traced to associated changes in C02 concentration. When the atmosphere above the medium was enriched with C02, abundant growth was observed at 39°C without phenylalanine or aspartic acid. Another way in which temperature may affect the result of an enrichment is the induction of a metabolic lesion. This phenomenon may be encountered when an organism is cultivated near or beyond its maximal or minimal temperature for growth. It has for instance been shown repeatedly that thermophiles grown at their lower temperature extreme and mesophiles cultured at temperatures above the optimum for growth may require an exogenous supply of a metabolite that is produced at reduced rate or not at all at the extreme temperature (Langridge, 1963; Farrell and Rose, 1967). Extra requirements exhibited at or near temperature extremes need, however, not necessarily be metabolites, as was shown by Goldman et al. (1963) for Pediococcus homari. At 40°C this organism showed a mandatory requirement for NaCl whereas at the optimal growth temperature (30°C) this salt was stimulatory only. Stanley and Morita (1968) showed that the maximum growth temperature of marine bacteria may be highly dependent on the salinity of the growth medium. Vibrio marinus for instance had a maximal growth temperature of 21~2°Cat a salinity of 35%,, but could not grow at temperatures above 10.5"C at a salinity of 7%,, which was the lowest salinity at which growth occurred. Thus, microbial competition in estuaries may be expected to be profoundly influenced by the temperature-salinity relation. From the above it is clear that even small temperature changes may profoundly affect competitive processes among microbes. As an example of this the observation may be mentioned of Conover (1956; cf. Provasoli,


323

1958), who found that a temperature difference of only 1 ~ 5 ° Cdetermined which of two species of diatoms became predominant in a mixed population either under natural or laboratory conditions. 4. Light as a selective factor for enrichments Among the photosynthetic bacteria the purple sulphur bacteria and the green bacteria are obligately phototrophic. They occur in marine as well as fresh water environments which are anaerobic and contain sulphide which is used as electron donor. The fact that among these organisms a considerable ecological diversity is encountered is at least partly due to differences in their behaviour towards light. Among the various organisms are differences in preference for wavelength as well as light intensity. Both factors act as selective agents in nature and can also be applied for the enrichment of specific organisms in the laboratory. A few examples may illustrate this. Stanier and Cohen-Bazire (1957), in dealing with the role of light in the microbial world, describe the completeness with which the available wavelengths are utilized by various groups of phototrophic organisms. The 01 bands of the plant chlorophylls (including the prokaryotic blue-green algae) and the chlorophylls of green and purple bacteria show maximum in viva absorption at or near 680,750 and 880 nm, respectively. As indicated by Stanier and Cohen-Bazire (1957), the only segment of solar irradiance not used by any then known organism was in the infrared region between 950 and 1100 nm. It has recently been shown that this niche between light absorption of the chlorophyll of commonly occurring purple bacteria and light absorption by water is, in fact, occupied. In an elegant enrichment experiment, using infrared light of wavelength > 900 nm as the selective agent, Eimhjellen et al. (1967) revealed the existence of a sulphur purple bacterium which has a chlorophyll with in viva absorption in the far infrared (1017-1020 nm). T h e chemical composition of the growth medium used (Pfennig, 1965a) was not essentially different from that commonly used for isolating several other purple bacteria. Non-sulphur purple bacteria with similar chlorophyll absorption characteristics had already been isolated by Eimhjellen et al. (1963) and Drews and Giesbrecht (1965, 1966). T h e above organisms are thus able to harness the same fraction of solar irradiance, but their occurrence is restricted to environments in which appropriate organic electron donors are available. The use of wavelength as a selective agent can also be applied to enrichments of other phototrophs. Enrichment media in which an organic electron donor is included usually give rise, when illuminated with daylight or incandescent light, to the development of non-sulphur purple bacteria. As suggested by C. B. van Niel (1956, personal communication), selective

324

13. VELDKAMP

enrichment of the equivalent of the Athiorhodaceae among the green bacteria should be possible by making use of light of a wavelength of around 750 nm, which would exclude purple bacteria. Preliminary experiments (H. Veldkamp, 1956, unpublished work) confirmed the truth of this suggestion. The light intensity applied to enrichments for phototrophs may also affect competitive processes. Chlorobium practically always becomes dominant in cultures having a high sulphide concentration (0.2 % w/v NazS.9Hz0) which are incubated at temperatures of 20"-3O"C, and are continuously exposed to a light intensity of 50-200 ft cdls. Pfennig and Cohen-Bazire (1967) showed that for the enrichment of other green bacteria (e.g., Pelodictyon) not only a lower sulphide concentration (0-03yo) and temperature (10"-20°C) should be applied, but also a low light intensity (5-15 ft cdls) is required with daily alternation of light and dark phases. As an example of the various preferences for light intensity encountered in fresh water purple sulphur bacteria, growth conditions as recommended by Pfennig (1965a) are listed below(i) Light intensity 10-30 ft cdls (25 W incandescent lamp at a distance of 45-25 cm from culture); daylight or incandescent light applied in day-night rhythm (e.g., light phase 16 h, dark phase 8 h); temperature, 15"-20°C; pH 6.5-6.8. Organisms that may develop : Chromatium okenii; Chromatium weissei; Thiospirillum jenense (0.04%w/v NazS.9HzO) ; Thiodictyon. (ii) Light intensity 30-70 ft cdls (25 W incandescent lamp at a distance of 25-15 cm from culture); continuous illumination or alternations of light and dark phases; temperature, 20-25°C; p H 6.8-7.2. In this case, Chromatium ecarmingii may develop. (iii) Light intensity 70-200 ft cdls (60 W incandescent lamp at 35-15 cm from culture); continuous light; temperature, 25-30°C; pH, 6.6-7-2. Organisms that may develop are: small Chromatium species; Thiocystis, Thiocapsa, Rhodothece, Amoebobacter. The medium used for these enrichments is that given by Pfennig (1965a); to obtain the concentrations of sulphide and COz prescribed, one should include 0.07% w/v NazS.9Hz0, and 0.43% w/v NaHC03. In order to exclude the development of green bacteria, the cultures are, in the initial stages of enrichment, placed in a water bath and exposed to infrared light (using a filter with transmission > 800 nm). After a few transfers, they are further incubated in daylight or incandescent light as described above. The above examples show how the subtle application of wavelength


325

and light intensity has revealed ecological niches that would never have been discovered when prescriptions for the enrichment of phototrophs which have been used for decades are adhered to. An adverse effect of light was encountered in the aerobic chemoorganotroph Hyphomicrobium aulgare (Hirsch and Conti, 1964a, b). The production of swarmer cells by this organism was inhibited by incandescent light of approximately 300 ft cdls. Hirsch and Conti (1965) therefore recommend that enrichment cultures of Hyphomicrobium be kept in the dark.

5 . Enrichment of dzflerent groups of prokaryotic organisms in liquid media T h e literature on enrichment methods is vast and widely scattered. A wealth of information on early results is to be found in the collected papers of Beijerinck (1921) and Winogradski (1949). Early work of Beijerinck and contemporaries was well summarized by Stockhausen (1907). A short survey of the history of enrichment cultures was given by Veldkamp (1965a). A general discussion of enrichment cultures was given by Hungate (1962) in his extensive treatise on microbial ecology. Enrichment methods for soil bacteria were reviewed by Pochon and Tardieux (1967). Selection of mutants and results obtained with enrichments in open systems were summarized by Schlegel and Jannasch (1967). Nutritional aspects of enrichment techniques were dealt with by Hutner (1962). Many selective media for organisms that are of interest in connection with public health, food industries, etc., are commercially available. For details on media composition and for references to relevant literature the reader is referred to the Difco Manual (1963), and the Oxoid Manual (1965), as well as to the regularly appearing supplements. Valuable information on growth media for the enrichment of various microbes and recent literature including cultivation techniques was reported by Skerman (1967). Stanier et al. (1963) most elegantly summarized a large amount of information on enrichment methods. Several of their Tables are given below. The following references listed include all groups of prokaryotic organisms discussed at the symposium (Gottingen) on enrichment cultures (Schlegel, 1965), and only give a fair impression of available methods and data relevant to enrichment procedures. As far as possible, recent papers were included. The reader is referred to these papers and to other Chapters in this Volume for further information. Tables I and I1 deal with chemo-organotrophs, Table I11 with chemolithotrophs, and Table IV with phototrophs. The references listed below are also divided into those for chemo-organothrophs, those for chemolithotrophs, and those for phototrophs.

w

N

TABLE I Enrichment conditions for chemo-organotrophic bacteria Special environmental conditions

Additions to basal medium, g/litre A

f

Common Features

Basal medium MgS04.7Hz0 KaP04 FeS04.7H20 CaCk Mnch. 4H20 NaMoO4.2H20 Distilled water

Organic

Inorganic

o\

>

Non-fermentable None 0.2 g substrate, e.g. ethyl alcohol, 4 -0 1.og 0.05 g Non-fermentable NH4C1, 1.0 substrate, e.g., ethyl alcohol, 4.0 0 -02 g 0.002 g 0.001 g Non-fermentable NaNOs, 3.0 substrate, e.g., ethyl alcohol, 4.0 1 litre

& Atmosphere

pH

Organisms enriched

Air

7* 0

Azotobacter

Air

7.0

Aerobes, e.g., PseudomoMs

None (stop- 7 -0 pered bottle)

Denitrifying bacteria, e.g. some species of

None (stop- 7.2 pered bottle) None (stop- 7.4 pered bottle)

Desuvovibrio Methobacterium omelianskii

Pure Na

Clostridiumpasteurianumand

Pseudomonas Em'ronnzent In the dark : temperature 25"-30°C

From Stainer et al. (1963).

Non-fermentable NH4C1, 1.0 substrate, e.g., ethyl alcohol, 4.0 Na2S04 5 -0 Non-fermentable NH4C1, 1.0 substrate, e.g., ethyl alcohol, 4 - 0 NaHC03,l-0 CaCOs, 5 - 0 Fermentable substrate, CaCOs, 5.0 e.g., glucose, 10-0 Fermentable substrate, NH4C1, 1.0 e.g., glucose, 10-0

7 -0

None (stop- 7.0 pered bottle)

related species Fermentative bacteria, e.g., Aerobacter

z

8

E CCI


327

TABLE I1 Some complex media for enrichment of organotrophs Additions g/litre

Special environmental conditions

Preferred choice of inoculum

None

pH 7.0; aerobic

Soil

None None

pH 7.0; aerobic pH 7.0: anaerobic

Pasteurized soil Pasteurized soil

Urea, 50.0

pH 8 * 5 ; aerobic

Pasteurized soil

Glucose, 20.0

pH 2.0-3.0; anaerobic pH 6 . 5 ; anaerobic

Soil

Glucose, 20 .O

Glucose, 20 * O pH 7 . 0 ; aerobic or anaerobic CaC03,20*0 Glucose, 20 a 0 pH 7.0; anaerobic CaC03,20.0 Sodium pH 7.0; anaerobic lactate, 20 .O Ethanol, 40.0 pH 6 * O ;aerobic

Organisms enriched Aerobic amino-acid oxidizers Bacillus spp. Amino-acid fermenting clostridia Alkali-tolerant ureadecomposing bacilli (Bacilluspasteurii) Anaerobic Sarcina spp.

Plant materials, milk Soil or sewage

Lactic acid bacteria

Pasteurized soil

Sugar fermenting clostridia Propionic acid bacteria

Swiss cheese Fruits, unpasteurized beer

Common components areYeast extract KHzPOi or KzHP04 MgS04 Distilled water From Stanier et al. (1963).

Coliform bacteria

Acetic acid bacteria

10 g 1g

0-2g

1 litre

REFERENCES FOR CHEMO-ORGANOTROPHS Frateur, 1950; Prescott & Dunn, 1959; De Ley & Kersters, 1964 Actinomycetes Waksman, 1959; Ntiesch, 1965; Williams & Cross, this Series,Vol. 4. Arthrobacter Jensen, 1952; Mulder & Antheunisse, 1963; Ensign & Wolfe, 1964; Veldkamp, 1965c; see also Section IVC Azotobacteriaceae Becking, 1961 (BeGerinckiu); Jensen, 1965 Bacillus Smith et ul., 1952; Claus, Acetobacter

1965

Bacteroides Bdellovibrio

Post et al., 1967 Stolp & Starr, 1963; Stolp, 1965; see also Section IVA.3 Beggiatoa Faust & Wolfe, 1961; Scotten & Stokes, 1962; Pringsheim, 1967 Brevibacterium Mulder et al., 1966 Carbon monoxide Kistner, 1953 oxidizing bacteria Caulobacter Stove-Poindexter, 1964; Hirsch & Conti, 1965; Stove, 1965

328

H. VELDKAMP

REFERENCES FOR CHEMO-ORGANOTROPHS-continued Cellulolytic soil bacteria (aerobic) Cellulolytic bacteria (anaerobic) Chitinolytic organisms Clostridium

Winogradski, 1949; Went & De Jong, 1966; Imshenetski, 1967; see also Cytophaga and Cellvibrio (Section IVB.4) Hungate, 1950; Skinner, 1965 see Section IIB.1

Kutzner, 1965; Postgate, 1965 (Cl. nigrificans); ElGhazzawi, 1967 (Cl. aceticum); Willis, this Series, Vol. 3B; see also Section IIB.2, IIB.3 Jensen, 1952; see also Coryneform bacteria Arthrobacter, Nocardia (Actinomycetes) and Mycobacterium Stanier, 1942; Veldkamp, Cytophaga 1965b Denitrifying Verhoeven, 1952 (Bacillus); bacteria Verhoeven et al., 1954 (Micrococcus denitri’cans) ; Valera & Alexander, 1961 ; Woldendorp, 1963 Desulphovibrio Posteate. 1965.1966 Enterobacteriaceae Difco Manual, 1963 ; Oxoid Manual, 1965 ;Kampelmacher, 1967; Harvey & Price, 1967 Halophilic Larsen, 1962; Eimhjellen, bacteria 1965; Raymond& Sistrom, 1967; Gibbons, this Series, Vol. 3B Hydrocarbon Fuhs, 1961 ; Foster, 1962; oxidizing Overbeck, 1965 (methane) ; bacteria Foster & Davis, 1966 (methane); Davis, 1967 Hyphomicrobium Hirsch & Conti, 1964a, b, 1965; Zavarzin, 1960, 1961 Lactobacteriaceae Barnes, 1956 (enterococci); De Man, et al., 1960 (Lactobacillus); Kenner et al., 1961 (enterococci); Stamer et al., 1964; Whittenbury, 1965; see also Section IIB.4 Mulder & Van Veen, 1963, Leptothrix 1965; see also Section IIB.4 Leucothrix Harold & Stanier, 1955; Brock, 1966 Methane oxidizing see Hydrocarbon oxidizing bacteria bacteria I

,

Barker, 1936 (Methanosarcina, Methanococcus, Methanobacteritrm; Stadtman & Barker, 1951 (Methanococcus); Smith & Hungate, 1958 (Methanobacterium); Bryant et al.,1967 (Methanobacillus) Mycobacterium Lukins & Foster, 1963; (saprophytic) Hirsch, 1965; see also Hydrocarbon oxidizing bacteria Myxobacteria see Cytophaga, Sporocyto(non-fruiting) phaga Myxobacteria Kiihlwein & Reichenbach, (mainly 1965; Peterson, this Series, fruiting) Vol. 3B Nevskia Babenzien, 1965 Pectinolytic Dowson, 1957 (cf. Skerbacteria man, 1967); Stewart, 1962; Wieringa, 1963; see also Section IIB.3 Pseudomonas Stanier et al., 1966 Psychrophilic Brock & Rose, this Series, bacteria Vol. 3B Photobacterium Spencer, 1955; Bukatsch, 1965 Propionibacterium Van Niel, 1928 ; Prescott & Dunn, 1959 Rhizobium FBhraeus, 1957 (summarized by Skerman, 1967) Rumen bacteria Hungate, 1966; Hobson, this Series, Vol. 3B Sarcina Canale-Parola & Wolfe, 1960 (S. ventriculi); see also Section IIA.1 and Methanogenic bacteria Sphaerotilus Mulder & Van Veen, 1963, 1965; Rouf & Stokes, 1964 Spirillum Giesberger, 1936; Rittenberg & Rittenberg, 1962 ( S p . volutans) ; Jannasch, 1965a; see also Sections IVA.1, B.l and B.5 Spirochaetaceae Veldkamp, 1965d; Canale(saprophytic) Parola et al., 1967; see also Section IVA.2 and B.2 Sporocytophaga Stanicr, 1942; Veldkamp, 1965b Staphylococcus Difco Manual, 1963; Oxoid Manual, 1965; Giolitti & Cantoni, 1966 Thermophilic Brock and Rose, this Series, bacteria Vol. 3B

Methanogenic bacteria


329

T A B L E 111

Enrichment conditions for some chemolithotrophic bacteria Special environmental features Additions to medium, g/litre

Common features

Basal medium MgS04.7HzO K2I-IP04 FeS04.7H2O CaClz MnClZ. 4 H 2 0 NaMo04.2HzO Distilled water Environnient In the dark ; temperature, 25"-30" C

Atmosphere p H

Organism enriched

NH4C1, CaC03,

1* 5 5.0

Air

8 . 5 Nitrosomonas

0 . 2 g NaN02, 1 .o g 0.05 g NH4C1, 0.02 g 0.002 g 0.001 g 1 litre NI-bCl,

3.0

Air

8 . 5 Nitrobacter

1.0

85 % Hz 10% 0 2 5 % COz

7a 0

7 ' 0 Thiobacillus

1.0 7 .O

Air

NazSz03.7HzO

NH4N03,

3.0

None 7 .O (stoppered bottle)

NazSz03.7Hz0, 7 .O NaHCOs 5.0

Hydrogen bacteria

Thiobacillus denitrificans

From Stanier et a1 (1963).

REFERENCES F O R C H E M O - L I T H O T R O P H S Hydrogen oxidizing bacteria Iron and manganese oxidizing bacteria

Methane oxidizing bacteria Nitrifying bacteria

Schatz & Bovell, 1952; Eberhardt, 1965; Collins, this Series, Vol. 3B Leathen et al., 1956 (Ferrobacillus) ; Kucera & Wolfe, 1957 (Gallionella); Kinsel, 1960 (Ferrobacillus) ;Wolfe, 1964; see also Thiobacillus ; and Sphaerotilirs, Leptothrix (chemo-organotrophs) see Hydrocarbon oxidizing bacteria (chemo-organotrophs) Gould & Lees, 1960 (Nitrobarter); Skinner & Walker, 1961 (Nitrosomonas); Wat-

son, 1962, 1963 (marine nitrifying bacteria); Soriano, 1963 (Nitrosococcus); Bock, 1965 (Nitrosomonas, Nitrobacter); Collins, this Series, Vol. 3B Colourless sulphur Temple and Colnier, 1951 (Thiobacillus ferrooxidans) ; bacteria Vishniac and Santer, 1957 (Thiobacillus); La Rivikre, 1963 (Thiovuktm), 1965; Postgate, 1966; Wieringa, 1966; London & Rittenberg, 1967 (Thiobacillus perometabolis); Collins, this Series, Vol. 3B

w w

0

TABLE IV Enrichment conditions for photosynthetic micro-organisms Additions to medium, gfitre A

r

Common features Basal medium MgS04.7HzO

mpoi

FeS04.7H20 CaCh MnC12.4Hzo NaMoOi .2H20 NaCl Distilled water

0-2g

Organic None

1-og 0.01g None 0.02 g 0.002g O-OOlg None 0.5g 1 litre None

Environment Constant illumination; temperature, 25 "-30 O C

From Stanier et al. (1963).

Special environmental features >

Inorganic

P Atmosphere

PH

Organisms enriched

None

Air, or air+5 % C02 6 -0-8 *O Blue-green algae

NaN03 or NHdCI,

Air, or air +5 % C02 6 -0-8 *O Green algae

1.0

NH4C1, 1.0 None (stoppered bottle) NazS.9H20, 2.0 NaHC03, 5.0 NH4C1, 1.0 None (stoppered NazS.9 HzO, 1 .o bottle) NaHC03 5.0

Sodium malate, 5.0 NHiCl 0-5 Yeast extract

1 *o None (stoppered bottle)

7-5

Green sulphur bacteria

8 -0-8.5

Purple sulphur bacteria

7 -0-7 * 5 Non-sulphur purple bacteria

x

sr

E 5


33 1

REFERENCES FOR PHOTOTROPHS Van Niel, 1931 ; Drews, 1965:Drews & Giesbrecht, 19"' (Rhodopseudomonns) Whittenbury & McLee, 1967 (Rhodopseudomonos); Carr. this Series, Vol. 3B Chlorobacteriaceae Pfennig, 1965a;b, 1967; Carr, this Series, Vol. 3B Cyanophyceae Allen, 1952; Pringsheim, (blue-green 1954; Koch, 1965; Wieralgae) inga, 1968; Allen & StanAthiorhodaceae

ier, 1968,Carr, this Series, Vol. 3B Rhodomicrobium Duchow & Douglas, 1949; Hirsch & Conti, 1965; Pfennig, 1967 Thiorhodaceae

Van Niel, 1944; Pfennig, 1965a,b, 1967; Postgate, 1966; Eimhjellen et nl., 1967; Carr, this Series, Vol. 3B

B. Population dynamics and enrichments in natural habitats Induced population changes in natural environments may lead to enrichment of a species or group of specialized organisms. This type of enrichment has the advantage that the prevailing conditions more closely simulate those that occur in the natural ecosystem than is often the case in liquid enrichments. This means that chances are better that the organisms that come to predominance actually play an important role under natural conditions. The examples given below of chitin decomposition in soil and of enrichment in soil of nitrogen-fixing bacteria show how it is possible to reveal niches directly in the natural habitat. In other words, the functional status of microbes can be discovered through induced population changes within the ecosystem. A typical example of the effect of changing the physicochemical conditions in a natural environment is the preparation of silage. This illustrates how such a change may lead to a succession of bacterial populations, eventually resulting in the predominance of an organism initially present in very low numbers. It is also possible to induce an increase in cell numbers within various coexisting bacterial populations simultaneously. As will be shown below, this situation is created in the Winogradski column. It enables the microbiologist to obtain a better insight into the kind of organisms coexisting in a special habitat. Once the components of the ecosystem are recognized, a study can be made of the different niches by transferring organisms to sets of artificial environments. This should eventually lead to an understanding of optimal conditions for growth and achievement of successful enrichment under defined conditions. 1. Enrichment of chitin-decomposing microbes in soil When finely powdered chitin is added to soil samples subsequently kept water saturated and aerated in a soil percolator, a rapid increase can be observed in the number of chitinovorous bacteria. Chitin-decomposing

332

H. VELDKAMP

actinomycetes, however, never develop in these water-saturated soils. When chitin particles are added to the same soils, and the samples are kept relatively dry, chitinovorous actinomycetes invariably become dominant. In most cases the dominant population is formed by Streptomyces, but members of the genera Nocardia and Micromonospora may also develop (Veldkamp, 1955). Actinomycetes undoubtedly play an important role in the decomposition of chitin in the top few inches of soil under field conditions. Liquid enrichments clearly fail to elucidate this and similar processes. Treatment of soils with chitin has been shown to be effective in controlling plant pathogenic Fusarium and Rhizoctonia species. T h e effect of chitin is probably due to antibiotic activities of the developing chitinovorous actinomycete flora, rather than to the effect of chitinase production (Potgieter and Alexander, 1966; Henis et al., 1967). Experimental details of the above experiments are the following. Powdered chitin (particle dia. 80-300 pm) is added to air dried soil (5-10 mg/g). The mixture is subsequently screened through 2 mm mesh. T o prevent clogging in percolator experiments soil aggregating substances are added (1 mg/g air dried soil). Carboxymethylcellulose is added to sandy soils and krilium (Monsanto Chemical Co., St. Louis, U.S.A.) to clay soils. T h e model 1 soil percolator of Jefferys and Smith (1951) has been found useful (see Fig. 2). For other types of perfusion apparatus, see Gray and Parkinson (1967). In experiments with relatively dry soils, the moisture content of sandy soils is adjusted to 10% w/w, that of clay soils to 17% w/w; 60 g samples are placed in stoppered conical 500 ml flasks. Controls without added chitin are run simultaneously. All samples are incubated at 28"C, and dilutions of each sample are seeded on a mineralchitin agar at regular intervals. Chitin serves in the agar medium as carbon and nitrogen source for both the dominant bacterial and actinomycete populations. Preparation of chitin and chitin agar is described by Stanier (1947), Veldkamp (1955) and Skerman (1967). Exoskeletons of shrimps form the material of choice in the preparation of chitin. They are easier to purify and grind than the usually recommended shells of crabs and lobsters.

2. Enrichment of Azotobacter in soil Azotobacter occurs in neutral soils of temperate regions. Though their numbers in any soil are relatively low, the organisms can easily be enriched because of their ability to develop under aerobic conditions in media that lack combined nitrogen. Easy as it is to enrich Azotobacter, its isolation in pure culture may at times be very difficult and time consuming. This holds especially for liquid enrichments. Culture media with organic

V. ENRICHMENT CULTURES OF PROKARYOTIC ORGANISMS AIR OUTLET

n

333

I

(SAUPlE BIOWN OW@BVClO3lNG IN L W i l E I i

+AIRLIFT TUBE

GLASS WOOLSOIL TUBE-

FACED BUNG -COVER SLIP -BUNG WITH NOTCHED FACE -FLAT

Il

1

GLASS W 0 O L - k

6 -I

INCHES

SCALE FOR ENLARGED DETAILS

ZNLARGED DETAIL OF

vAI;vz

FIG.2. Diagram of soil percolator (Jefferys and Smith, 1951). In this type of percolator waterlogging of the soil is minimized by the valve which automatically controls the amount of percolate reaching the soil. The percolator can be operated either on a low pressure of compressed air (or other gas) at the air inlet, or a low vacuum applied at the outlet. After placing the soil sample in the soil tube, the air supply is started, and the percolation fluid is introduced into the reservoir. When some of this liquid has reached the soil, the resistance of the soil to the air flow increases and as pressure increases, the valve is closed. It opens again when enough percolate has been carried by the airlift tube to the reservoir, to give a sufficient pressure decrease. (Reproduced by permission of the Society for Applied Bacteriology.)

sources of carbon and energy and devoid of combined nitrogen, frequently give rise to development of Azotobacter when inoculated with a sample of a neutral or slightly alkaline soil (cf. Jensen, 1965). The cells of Azotobacter can easily be recognized microscopically. As soon as the first cells appear on the liquid surface, a transfer should be made. Azotobacter cells excrete amino-acids (Jensen, 1954), and this gives rise to a rapidly developing

334

H. VELDKAMP

secondary flora. Organisms belonging to this flora become easily attached to the slime layer which surrounds Azotobacter cells. Oncethis has happened, purification of Azotobacter on agar media should not be tried, because in many cases it will be unsuccessful. Another liquid enrichment should be made, or even better, enrichment of Azotobacter directly in soil should be performed. This method, introduced by Winogradski (cf. Jensen, 1965) enables Axotobacter to form colonies on the surface of the soil it inhabits. The procedure is as follows. A soil sample of sufficient size to fill a small Petri dish (1 cm deep, 5 cm dia.) is supplied with 1-2 g of both CaC03 and mannitol. After mixing, 4 drops of a 10% solution of both KzHP04 and MgS04.7HzO are added. Subsequently, enough water is added to prepare a soil paste that is not water saturated. If the soil is too wet, development of isolated Axotobacter colonies is prevented, and growth of the anaerobic Clostridiurn pasteurianum may reach the surface. The mixture is put in the bottom half of the Petri dish and the surface is smoothed with a knife. The dish is then placed (without lid) into a larger Petri dish on a wet filter paper, which ensures a moist atmosphere. The lid of the large dish should not touch the soil surface. After 2-3 days' incubation at 30°C, the mucoid colonies of Axotobacter develop on the soil surface. Colonies of other organisms may also appear, but since the available combined nitrogen becomes rapidly depleted, these colonies generally develop slowly and remain small. Azotobacter can be purified on mannitol agarMannitol agar 10 g 1g 0.5 g 20 g

Mannitol K2HPO4 MgS04.7H20 Agar Trace element solution (see p. 31 1) Distilled water

0 . 1 ml 1 litre pH 7.0

Axotobacter can utilize various carbon and energy sources (cf. Brown et al., 1962). Jensen (1965) recommends ethanol (ca. 1 yo)which is utilized by all Azotobacter species. Molybdenum, which is essential for nitrogen fixation, is practically always present in sufficient amounts in the soils used. I n some exceptional cases, however, the presence of Azotobacter in a soil cannot be revealed unless molybdate is added (van Niel, 1935). Clostridium pasteurianum hardly ever fails to occur in considerable numbers in the lower anaerobic parts of the Winogradski soil plates. It can easily be isolated as follows. A 25 ml portion of the following medium-


33s

Medium Glucose

KzHPOj MgS04.7HzO

30 g 0.5 g 0.5 g

CaC03 Trace element solution (see p. 3 1 1 ) Distilled water

20 g 0.1 ml 1 litre

is boiled in a 100 ml conical flask. A small soil sample is deposited in the boiling liquid, and this is subsequently cooled under the tap. T h e flask is incubated at 37°C in a desiccator in a Nz atmosphere. T h e soil sample used need not necessarily be derived from the Winogradski soil plate. Nitrogen-fixing clostridia are much more common in arable soils than Asotobacter, and practically any soil sample treated as described above will give rise to a predominance of Cl. pasteurianum. Purification can be achieved on a homologous agar medium under Nz. In contrast to vegetative cells, the spores of Clostridium are not damaged by exposure to oxygen.

3. Enrichment of Clostridium pectinovorum in potato tissue Some naturally occurring bacteria live on or near the sterile tissues of plants and animals and grow very rapidly and profusely within these tissues when given a chance to invade them. Notorious examples are the clostridia commonly occurring in soils, among which Cl. tetani and Cl. welchii may rapidly develop when introduced in deep wounds of animals. Similarly, Cl. pectinovorum comes to the fore when a mixed population of soil microbes is introduced in the tissue of a fresh potato. This can be shown as follows. A potato is washed under the tap and subsequently stabbed once or twice with a knife. I t is then placed in a beaker, and enough water is added just to cover the potato; the beaker is covered with a watch-glass and incubated at 37°C. The oxygen that might be introduced into the tissue is consumed by its cells. I n the anaerobic environment Clostridium rapidly starts to decompose the pectin in between the plant cells. T h e tissue is thus macerated. When the tuber floats, due to profuse gas formation, the water is poured out of the glass; the potato is washed and dissected. Microscopic examination of the tuber contents invariably shows the typical pleomorphic clostridial cells; among these, spore-bearing spindle-shaped cells are nften encountered. Isolation can easily be achieved as follows. A sample of the macerated tissue is inoculated into yeast extract-glucose broth and after pasteurization (10 min at 80°C in a water bath) the culture is incubated at 37°C

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H. VELDKAMP

in Nz atmosphere. A pure culture can be obtained by streaking a sample on yeast extract-glucose agar and incubating under N2. Processes similar to those described above occur during the retting of flax in hot water.

4.Enrichment of lactic acid bacteria in anaerobic plant material A typical example of the enrichment principle is provided by the microbiological preservation of plant material. The surface of plant leaves forms a typical aerobic environment, and the observation (Gibson et al., 1958) that the microbial flora of grass leaves consists mainly of obligate aerobes is therefore not surprising. Lactic acid bacteria occur on fresh grass only in very small numbers. One hundred lactobacilli in 2 x lo6 herbage bacteria should be considered as an unusually high number (Gibson et al., 1958). Still, when grass is cut and placed in a container, the microbial population that becomes dominant generally consists of Lactobacillus. T h e dominance of homofermentative lactobacilli is preceded by a succession of microbial populations. T h e sequence is mainly determined by the acid tolerance of the respective organisms. It should be emphasized that the effect of acid on microbial growth is not only due to hydrogen ion concentration, but also to the concentration of undissociated organic acids. A rapid dominance of lactic acid bacteria is favoured by a high sugar content and low buffering capacity (low protein content) of the plant material. When acid production and p H decrease are insufficient, butyric acid bacteria may develop. This does happen occasionally in the ensilaging of grass, but never occurs, for instance, in sauerkraut production. T h e latter process forms a typical example of the sequence of events that occurs in anaerobic plant material. A brief description will be given below. White cabbage is sliced and NaCl (2 g/lOO g of cabbage) is evenly distributed throughout the plant material, which is subsequently placed in a 250 ml porcelain pot or beaker. A stoppered bottle filled with water is placed as a weight on top of the shredded cabbage. As much air as possible is thus removed, and the brine to be formed will completely cover the cabbage. This is then incubated at 2Oo-25"C. The succession of bacterial populations can be shown by seeding samples diluted in sterile tap water on the following agarAgar Yeast extract Glucose CaC03 Agar Distilled water


337

In addition, Gram stains (Skerman, 1967) made of brine samples at regular intervals of time will clearly show the successive appearance of populations of Gram-negative rods and Gram-positive cocci and rods. After pressing the shredded cabbage into the container, the oxygen is rapidly consumed by respiration of the plant tissue. The viability of the plant material then rapidly decreases. The cells become permeable and a solution of sugars, amino-acids, vitamins and minerals is released. This process is enhanced by the presence of NaCl. T h e cabbage contains 3-4.5 % of sugar, mainly sucrose. Examination of the freshly sliced cabbage reveals the presence of the mixed flora originally present on the living plant. It consists mainly of obligate aerobes. Among the organisms capable of anaerobic growth, those belonging to the Amobacter group as well as Erwinia herbicola (cf. Graham and Hodgkiss, 1967; Deley, 1968) are clearly more numerous than lactic acid bacteria. In the competition among the fermentative organisms, Aerobacter easily comes to the fore, This phase of the fermentation is characterized by gas and acid production by Gram-negative rods. These organisms, however, disappear almost as rapidly as they develop and are replaced by lactic acid bacteria. Within the latter group of organisms, there is, however, also a succession of populations. Heterofermentative cocci (Leuconostoc) appear first and are followed by heterofermentative and homofermentative lactobacilli, respectively. The final dominant population is generally formed by Lactobacillus plantarum. As described earlier, some heterofermentative lactic acid bacteria are not able to ferment hexoses without an external electron acceptor. A typical example are those lactic acid bacteria in the sauerkraut fermentation that need for each fructose molecule to be fermented two additional fructose molecules as electron acceptor, according to the equation-

3 fructose + 2 mannitol

+ 1 lactic acid + 1 acetic acid + 1 COz

This explains the occurrence of mannitol in sauerkraut. T h e only microbes that can grow under the acid conditions (pH 3.54.0) created by fermentative bacteria in anaerobic plant material are yeasts. These are not generally encountered, however, probably because of lack of sugar in the final product. An exception is found in leaven, which mostly contains a mixed flora of Lactobacillus and yeasts. Probably growth of lactobacilli in this material is retarded by low p H and/or concentration of undissociated acids, whereas yeast growth is possible through slow hydrolysis of starch by plant enzymes. The lactic acid bacteria occurring on cabbage and other plants can of course also be enriched by inoculating a piece of a leaf in a stoppered bottle containing a complex growth medium. T h e succession of different 14

338

H. VELDKAMP

populations resulting in highly selective growth conditions for lactic acid bacteria is, however, most elegantly demonstrated by enrichment within the plant material. The processes described above can also readily be demonstrated in other plant material. Leaven for instance can easily be produced as follows. Grind rye grains, add tap water and put the resulting paste in a beaker. Incubate at 20°-25”C. An entirely different enrichment is obtained when 20 g of flour is added to 100 ml of water in a beaker and the mixture is boiled for several minutes. When, after cooling, the excess of water is poured off, and the flour is incubated at 30”C, Bacillus subtilis invariably develops on the surface, and a starch-decomposing Clostridium becomes dominant within the flour. Incubation at 55°C results in development of thermophilic sporeformers. The above enrichment experiments are, because of their simplicity and illustrative value, useful for educative purposes.

5 . Coexistence and interdependence of microbial populations : the Winogradski column From an ecological point of view, it is often desirable to obtain an insight into the composition of naturally occurring ecosystems. Though direct observation of organisms occurring in a natural habitat may be useful, their numbers may be rather low and direct studies are therefore very time consuming and difficult to interpret. In such cases it would be very convenient to have available in the laboratory a habitat-simulating system in which the population densities of ecologically important organisms are higher than those generally occurring in nature. Especially when changes in the physicochemical conditions in the closed system are relatively slow, this would provide a tool for careful study of coexisting microbial populations. Once several components of the ecosystem are recognized and the localization thereof in the heterogeneous milieu has been established, the system provides a basis for the enrichment of specific organisms under more rigorously defined growth conditions. A study as described above of a habitat-simulating system in the laboratory can be made by making use of the “Winogradski column” (Winogradski, 1888, cf. Winogradski, 1949). This mimics the habitat formed by anaerobic muds and the natural waters by which they are covered. In canals, lakes and similar environments, sedimentation of organic debris and animal life in the mud provides the microbial flora in the muds with substrates for fermentation and anaerobic respiration. End products of fermentation serve as substrates for methanogenic and sulphate-reducing bacteria. When light penetrates the anaerobic water layers covering the mud, photolithotrophic organisms (e.g., Chromatium, Chlorobium) develop,


339

which utilize the sulphide produced by Desulfovibrio as electron donor for photosynthesis. When the level of soluble organic substrates in this anaerobic environment is sufficiently high, photo-organotrophic microbes (e.g., Rhodospirillum, Rhodopseudomonas) may develop. During the night, the sulphide concentration immediately above the mud surface increases, whereas a decrease can be observed during the day when in the light the photosynthetic bacteria reduce the sulphide level. The depth of the anaerobic water layer above the mud may show considerable seasonal changes. In lakes with a summer stratification the dimensions of the anaerobic zone are much larger in summer than in winter. T h e habitat described above may be simulated, and the density of several naturally occurring microbial populations may be increased as follows. A mud sample is mixed with CaS04 (e.g., 3 parts of mud to 1 part of CaS04.2HzO) and some organic substrate, preferably insoluble (e.g., filter paper). The sample is placed at the bottom of a tall glass cylinder (e.g., 500 ml graduated cylinder) to a height of ca. 10 cm and covered with natural water. T h e cylinder is exposed to diffuse daylight at 18"-25"C. Provided there are no nitrogen, phosphorus or growth-factor limitations (many phototrophs need vitamin B12; Pfennig and Lippert, 1966), a sequential increase of microbial populations occurs as follows. A population increase of fermentative organisms in the mud, owing to the introduction of organic material, results in an increased formation of end products of fermentative processes. This in turn increases the rate of sulphide production by Desulfovibrio. The increase in the rate of sulphide production subsequently induces an increase in population densities of phototrophs (e.g., Chlorobium, Chromatium). The Winogradski column beautifully illustrates the coexistence and interdependence of ecological niches occurring in the same habitat. Within the column there is a gradient of nutrients and metabolic products diffusing from the mud upwards and of oxygen penetrating from the liquid surface downwards. This induces a stratification of microbial populations, and provides indications for optimal growth conditions of various organisms. Above the anaerobic zone there is an area in which both oxygen and sulphide occur, which enables development of populations of chemolithotrophic sulphur bacteria (e.g. Thiobacillus). When marine mud and sea water are used, specific marine organisms like Thiovulum may develop in this zone. Blue-green algae, diatoms, green algae and flagellates may develop in those parts of the column that become depleted of hydrogen sulphide. As pointed out by Pfennig (1965a), the distribution of many phototrophic bacteria in seemingly similar habitats is not as regular as is often assumed. Therefore, the kind of phototrophs that develop in the Winogradski column, depend on the source from which the inoculum is derived.

340

H. VELDKAMP

In some cases phototrophic bacteria do not develop. This may occur when a relatively high light intensity is applied and diatoms or other oxygen-producing algae develop against the glass wall. Then the mud locally turns brown owing to conversion of ferrous into ferric iron compounds. An intermediate between the habitat-simulating Winogradski column and the niche-simulating stoppered bottle with defined growth medium is a Winogradski column in which development of certain phototrophs is prevented. These columns therefore have a more selective character than that described above. One of the possibilities is to expose the column to light of a limited wavelength range (Schlegel and Pfennig, 1961). Temperature is another selective factor that greatly influences the equilibrium existing between microbial populations. At temperatures above 33"C, sulphate reduction may become retarded (Schlegel and Pfennig, 1961). This results in an accumulation of fermentation products. Growth of the photolithotrophic bacteria and of chemolithotrophs is then restricted by lack of sulphide, and a rapid development of non-sulphur purple bacteria and other heterotrophs can be observed.

111. ENRICHMENTS I N OPEN SYSTEMS Exhaustion of nutrients and accumulation of metabolic products are inherent to the closed culture system, I n open enrichment systems, fresh medium is continuously provided and at the same time, metabolic products are removed. All naturally occurring ecosystems are of the open type. Two types of open enrichment systems have been applied, the heteroand the homo-continuous culture. An example of a hetero-continuous system is a soil column through which a nutrient-containing solution is passed. The organisms in the heterogeneous milieu that are best fitted to the prevailing conditions increase in numbers. Since most cells of the newly developed population(s) are retained in the system, a steady state is not reached. Selection in such systems may closely resemble naturally occurring processes. The heterocontinuous culture system is, however, of a complexity that makes a rigorous analysis of selective factors difficult. Much better defined growth conditions are obtained in the homocontinuous culture. Of the two types available, the turbidostat and the chemostat, the latter system has been used almost exclusively in enrichment experiments. A review of the scant literature on enrichment in homo- and heterocontinuous cultures was given by Schlegel and Jannasch (1967). A brief outline of both types of enrichment techniques will be given below.


341

A. The homo-continuous culture system Principles and practice concerning the chemostat are dealt with by Tempest (this Series, Vol. 2), and therefore only a few remarks relevant to competition will be made here. Theoretical details about selective processes in the chemostat are given by Powell (1958), Maxon (1960) and Pfennig and Jannasch (1962). I n brief the principles are the following. As pointed out by Herbert (1958): “The key to the mode of action of the chemostat, lies in the way in which the specific growth rate p, depends on the concentration of a limiting growth substrate (s) in the culture medium”. T h e concentration of the growth-limiting substrate in the chemostat (s) depends on the dilution rate ( D ) , according to the steady-state equation (Herbert et al., 19.56)-

where /lmax is the maximum specific growth rate, K , is the saturation constant, numerically equal to the substrate concentration at which 11. = &pmax and D is the specific dilution rate, or flow rate divided by culture volume. A thorough discussion of the growth rate of micro-organisms as a function of substrate concentration was given by Powell (1967), who also derived an equation that fits the available experimental data best. For reasons of simplicity, the relation between ,u and s as approximated by Monod will be given here (Monod, 1942, 1950)-

When two organisms are competing for a growth-limiting substrate in the chemostat, the result will depend on the relationship of p to s in both strains. If relations exist as given in Fig. 3(a), organism A will grow faster than B at any value of s. When, however, the saturation curve of organism A cuts the curve of organism B (Fig. 3b), the result of the competition depends on the concentration of the growth-limiting substrate. And since this concentration in the chemostat is determined by the dilution rate (equation 1), organism A will come to the fore at dilution rates which are lower than a certain critical value, whereas organism B will become predominant at the higher dilution rates. There is one substrate concentration, however, for which the corresponding growth rates of A and B are equal (Fig. 3b). When this substrate concentration is maintained in the chemostat, both organisms will stay together, irrespective of the ratio in which they occur.

342

H. VELDKAMP

P

P

(a)

B

'

A rnax B max

P max A max

I

;

K $ K$

K$

K$

S

p-s relationship of two organisms A and B. (a) < KsBand pFLmaxA > (b) Ks-4< K p R and p m a x A < ,umaxB (cf. Pfennig and Jannasch, 1962; Schlegel and Jannasch,1967).

FIG.3.

pmax';

There is one other case conceivable, in which two organisms stay together in the chemostat, and that is when cross-feeding occurs. In such a case of syntrophy, the two interdependent organisms will also stay together. The above is theory, but in practice it often occurs that many organisms do stay together in the system. Slow-growing organisms for which the applied dilution rate is larger than their growth rate are washed out of the liquid phase. They may stay, however, in considerable numbers within the system by sticking to the walls of the culture vessel. When wall growth occurs, the culture is heterogeneous, and is not the ideal homo-continuous system. However, in most cases this does not seriously harm the enrichment procedure. Only at extremely low dilution rates and concentrations of the growth-limiting substrate in the inflowing growth medium may wall growth give erratic results. The considerations given above also hold when in a pure culture maintained at steady-state conditions a mutant arises that grows faster than the parent strain. Details about selection of variants in such cultures are reviewed by Schlegel and Jannasch (1967). As pointed out by Pfennig and Jannasch (1962), natural conditions that most closely resemble those in the chemostat, are found in the oceans. Jannasch (196513, 1967a, b) carried out enrichment experiments in the chemostat with marine microbes. He actually found that the theoretical possibilities as described above do occur in nature. Samples of ocean water containing similar microbial populations were inoculated into chemostats run at different dilution rates. The media employed consisted of minerals (excess), and a growth-limiting carbon and energy source (e.g., lactate). The result was that at different dilution rates, different


343

organisms became predominant. These organisms were isolated in pure culture, mixed again, and re-inoculated into chemostats. Again, organisms isolated originally at low dilution rates came to the fore under similar conditions. At high dilution rates those organisms became predominant that had originally been isolated under similar conditions. T h e technique appeared to be suitable for reproducible enrichment for bacterial species of low substrate specificity. T h e reason for the reproducibility appears to be that growth conditions are perfectly defined. Successful or unsuccessful competition for the growth-limiting substrate is based upon the particular growth parameters of the individual species of a mixed flora under given culture conditions. In the chemostat one can exactly choose conditions favouring a particular organism of low substrate specificity. Jannasch (1967b) carried out his selection experiments with organisms occurring in samples of sea water and was able to repeat them at will; we have obtained similar results (H. Veldkamp, unpublished work). Details about threshold concentrations of growth-limiting substrates are given by Jannasch (1967a). Many devices for chemostats have been described. In our experience a small fermenter (500 ml working volume), is very suitable for enrichment purposes. T h e culture volume can be kept constant by an internal overflow tube, which also serves as air outlet. Sterile medium is most conveniently added to the culture at a constant rate by a metering pump (e.g., Micropump, series 11; F. A. Hughes Co. Ltd, Epsom, England). The use of fritted glass filters for introducing finely divided gas bubbles in the culture should be avoided in the chemostat. These become easily clogged on prolonged operation by bacterial growth. Technical details of the chemostat are described by Evans, Herbert and Tempest (this Series, VOl. 2). Though application of the chemostat for enrichment purposes has the disadvantage that elaborate equipment is required, the unique properties of the system allow study of the regulation of microbial numbers as it often occurs in nature. No other system provides equal possibilities for the study of naturally occurring competitive processes under conditions as well defined as in the chemostat.

B. The hetero-continuous culture system

1. Enrichment of organisms of the Sphaerotilus-Leptothrix group Filamentous iron bacteria were enriched by Mulder and van Veen (1963, 1965) in Erlenmeyer flasks through which a flow of iron-containing soil extract (artificial ditch water) was passed. The apparatus used, shown in Fig. 4,consists of five sets of two 300 ml flasks in which the organisms

344

H. VELDKAMP

W

JjL

.-

I

r

____

1

-3

91

FIG.4. Apparatus for growing iron bacteria in running artificial ditch water: 1 , 2, inlet for tap water; 3, steel cylinder containing ironstone soil; 4, Seitz filter; 5, manifold; 6a, upper Erlenmeyer flask, with 7, inlet for gas mixture and, 8, inoculation tube; 6b, lower Erlenmeyer flask with 9, culture outlet. (From Mulder and Van Veen, 1965 ; reproduced by permission of Gustav Fischer Verlag.)

are grown, and a steel cylinder (ca. 1.5 m long, ca. 20 cm dia.) containing ironstone soil. T h e soil was collected from a length of ditch containing large masses of ensheathed iron bacteria. This soil was supplemented with 1-2 g of ferric carbonate/kg, and the organic matter content was 20%. After an incubation period of about 3 weeks, during which the soil was kept saturated with water to reduce ferric iron, tap water was introduced at the bottom of the cylinder. After percolating through the soil, the water was sterilized by Seitz filtration and then introduced dropwise into the upper Erlenmeyer flasks. After passing through the lower flasks, the water was collected in a plastic drain exposed to air. The upper flasks, which were inoculated with fresh water samples containing filamentous iron bacteria, were aerated with a sterile gas mixture consisting of 1% 0 2 , 5% COz and 94% N2, which bubbled slowly through the solution. Pure cultures, originally obtained by streaking samples on appropriate agar media, were also grown in the apparatus described above. This thus served not only for enrichment purposes, but also for morphological studies of pure and crude cultures under conditions simulating the natural habitat.

2. Enrichments in H2S-02 atmosphere Colourless sulphur bacteria like the marine Thiovulum, which need H2S as hydrogen donor should be enriched in a medium with low concentrations of oxygen and sulphide. Since HzS is autoxidizable, such


345

organisms should be enriched in a milieu that provides a continuous supply of both 0 2 and H2S. Wijler (cf. La Rivikre, 1963, 1965) successfully created such conditions as follows. A layer of decaying algae (e.g., Ulwa) is placed in a jar of 5-10 litres capacity. This layer serves as a continuous source of H2S, and a slow trickle of seawater introduced near the decaying algae provides a continuous 0 2 supply. The jar is kept in the dark at 15°C. On the surface of the algae, Thiothrix and Beggiutou may develop, and the typical veils of the highly motile Thiovulum develop above the bottom at places where 0 2 and H2S concentrations are optimal for its development. Purification of Thiovulum has to be performed under similar growth conditions. This difficult task has been successfully achieved by La Rivihre (1963, 1965) by using a closed system in which the culture medium was placed above a sulphide-containing agar layer. T h e medium was locally aerated with a slow current of air by means of a device that prevented turbulence. Thus, within the system a redox gradient was obtained and was made visible by the indicator thionine (Eo' at pH 8, -t30 mV).

3. Continuous flow method in soil microbiology An apparatus for studying microbiological processes in soil by a continuous flow method has been described by Macura (1961, 1964, 1966). T h e apparatus has been used for studying nitrification and decomposition of glucose and glycine in soil. I t can conveniently be used for enrichment procedures in which a continuous supply of fresh medium and removal of metabolic products is required.

IV. ISOLATION O F PROKARYOTIC ORGANISMS The most commonly applied method of obtaining a pure culture of an organism developing in an enrichment culture is to let it form isolated colonies on or in a solid medium. When the cells are grown on the surface of agar, it is often desirable to limit swarming of motile contaminants. Therefore the plates should be dried, either before inoculation (e.g., 2 days at 37"C), or after inoculation by placing a piece of filter-paper moistened with glycerol in the lid of the inverted Petri dish. After incubation overnight the paper is removed; the bottom half of the dish is placed on a flamed sterile surface, and the lid is cleaned, dried and flamed. The agar concentration used in plate cultures is generally 1-2%. Sometimes a soft agar is to be preferred, however, as for instance when aerobic chemo-organotrophic spirilla are to be isolated (cf. Section IVB. 1). When the isolation procedure is to be carried out on a solid medium of well-defined composition and free from organic material, silica gel may be

3 46

H. VELDKAMP

used. Its preparation has been described by Skerman (1967) and by Codner (this Series, Vol. 1). It often happens that the organism to be isolated does not really form a dominant population in the enrichment. Colonies of such an organism can often be obtained by seeding many plates instead of one or two. T h e number of plates to be made depends on the population density of the organism. 'The incorporation of antibiotics in agar media to prevent growth of unwanted organisms has been successfully applied in various isolation procedures. Examples are the following. Could and Lees (1960) purified Nitrobacter obtained from enrichments by streaking successively on matromycin-nitrite agar and terramycin-nitrite agar. T h e final concentration of these antibiotics was 100 ,ug/ml. Williams and Davies (1965) isolated actinomycetes from soil by applying the dilution-plate technique. T h e medium used (starch-casein agar) was made highly selective by incorporating the antibacterial antibiotics polymyxin B sulphate (5 ,ug/ml) and sodium penicillin (1 pg/ml), as well as the antifungal antibiotics nystatin (50 ,q/ml) and actidione (50 ,ug/ml). Plating methods and other isolation methods commonly applied in soil microbiology were reviewed by Casida (1967). The isolation of anaerobic bacteria is generally carried out by preparation of dilution shake cultures. The first of a series of ca. ten tubes, containing a melted agar medium kept at 40°45"C, is inoculated with a sample from the enrichment. A dilution series is then made by pouring ca. 1 ml from one tube into the next and mixing the contents by turning tubes once or twice upside down. After mixing, the tubes are rapidly cooled under the tap. To prevent entrance of air, the surface of the solidified agar is generally covered with paraffin (1 part of solid to 3 parts of liquid paraffin). When the medium contains a reducing agent (e.g., 0*05-0*2%w/v NazS.9H20) it may be more convenient to avoid the paraffin plug, and to close tubes with a rubber stopper (preferably black rubber); the trapped oxygen is removed in the oxidation of the reducing agent in the upper agar layers (0.5-2 cm). Another way to obtain anaerobic conditions in a culture tube is the following. Push the cotton plug into the tube and place a plug of absorbent cotton on top of this. One or two drops of each of a 2040% wlv solution of KzC03 and pyrogallol are then introduced, and the tube is closed by a rubber stopper. This method may also be convenient for keeping pure cultures on slants of organisms that cannot be kept in agar stab culture because of excessive gas formation (e.g., Clostridium). T h e slants ("Burr; slants") are incubated upside down. When the organism to be isolated produces a reducing agent it is not


347

always necessary to prevent diffusion of air into the agar. I n the procedure for isolation of Desulfovibrio described by Postgate (1965), 4 ml aliquots of a medium containing 0.01% w/v of each of sodium ascorbate and sodium thioglycollate are dispensed in sterile tubes, inoculated and incubated without exclusion of air. Colonies of Desulfovibrio develop in the depths of the agar. T h e first colonies that develop after transfer of inocula from the enrichment culture to a solid medium should never be considered pure, even if only one colony type develops. At least one transfer should be made by suspending part of a well isolated colony in a suitable sterile diluent, and streaking a second plate from this suspension; or by preparing another series of shake cultures. If necessary, this should be repeated until plates or tubes are obtained that show only one colony type. Colonies thus obtained should first be examined with a low-power objective and in wet mount before preparing a stock culture. When this is to be done with colonies of anaerobes grown in shake tubes, the agar column can be conveniently set free by the method of Giesberger (1947). A mild current of air or nitrogen is blown through a capillary pipette pushed between the agar column and the tube wall to the bottom of the tube. The agar is thus blown out and collected in a sterile Petri dish. Colonies can then be sucked into a Pasteur pipette and transferred. This method is not suitable for highly exacting anaerobes. Such organisms can best be cultivated in roll tubes, and special care should be taken not to expose the cells even briefly to oxygen. The methods required to handle these organisms are described by Hungate (this Series, Vol. 3B). Even when the usual precautions are taken to ensure the isolation of a single organism, it may occur that the “pure culture” obtained consists of two organisms. This may easily happen when synergism is involved. A striking example is Methanobacterium omelianskii, isolated in 1940 by Barker. The culture of this organism which has been used for years in metabolic studies was shown to be composed of a symbiotic association of two species of bacteria (Bryant et al., 1967). One organism produced H2 from ethanol, which was used by the other organism for the reduction of COz to methane, at the same time keeping the pH2 low enough to make Hz production from ethanol possible with a favourable free-energy change. It frequently occurs that dilutions have to be made of samples from enrichment cultures, or that material from colonies has to be suspended in a diluent before transfer. Sterile tap water is often used for this purpose. Straka and Stokes (1957) found, however, that organisms like aerobic spore formers and pseudomonads may die very rapidly, within minutes, when suspended in water or saline (0.85% w/v NaCl). Bacterial losses were avoided by using 0.1% w/v peptone water as the

348

H. VELDKAMP

diluent. T h e best way to avoid the risk of killing sensitive microbes during transfer is to use the growth medium as a diluent. Several microbes do not grow on or in solid media (e.g., Thiospirillum, Thiovulum). T h e purification of such organisms, which should be carried out in liquid media, is especially difficult when the population does not become dominant in the enrichment. I n such cases, special tricks should be applied as making use of the motility of the organism (Thiospirillum; Pfennig, 1965a), or carrying out a single cell isolation (cf. Johnstone, this Series, Vol. 1). Application of special methods may also be required in other cases in which commonly used methods for isolation fail or are not applicable. A few examples will be given below.

A. Filtration as a means of separating microbes from mixed populations Filters with pore sizes that do not allow passage of most prokaryotic organisms have successfully been used for separating microbes having extraordinarily small cell diameters. I n this way Anderson and Hefferman (1965) separated marine organisms belonging to the following genera: Spirillum, Leucothrix, Flavobacterium, Cytophaga and Vibrio. Additional examples are the following.

1. Isolation of spirilla Canale-Parola et al. (1966) isolated thin spirilla (0.25-0.30 p m dia.) by this method as follows. Sterile cellulose ester filter discs (Millipore; average pore size 0.45 pm) were placed on “non-selective” solid nutrient media. A typical composition isSolid nutrient medium Peptone Yeast extract (Difco) Tween 80 K2HP04 Agar Tap water

5g

0.5 g 0.02 g 0.1 g 10 g 1 litre

A sample of 0.05 ml of pond or stream water was deposited in the centre of each disc. After incubation at room temperature for 1-5-5 h, the discs were removed, and incubation was continued. After several days, growth was observed in the agar spreading from the centre of the plate outwards. T h e semi-transparent areas of growth appeared to be due to a thin spirillum that had moved through the fine-pored filter. When streaked on homologous media, the organism yielded isolated colonies from which pure cultures could be obtained by standard procedures.


349

2. Isolation of spirochetes Canale-Parola et al. (1967) separated free-living anaerobic spirochetes from black muds by a method based on the same principle. T h e mud samples were suspended in aqueous 0.02% w/v NazS.9H20. The suspensions were filtered through Whatman No. 40 filter paper, and filtrates were passed through Millipore filter discs (pore diameter 0.45 pm). Aliquots of 1 ml were then used to inoculate stoppered bottles containing a “non-selective” anaerobic growth medium. After 5-7 days’ incubation at 30°C many bottles showed a predominant population of thin spirochetes. Pure cultures were obtained by use of dilution shake cultures, and the organisms were maintained in paraffin-layered stab culturesStab culture medium

5s 2s

Glucose Yeast extract Peptone Sodium thioglycollate Distilled water

2g

0.5 1 litre

pH 7.0-7’3

Direct isolation from natural environments of aerobic spirochetes by filtration had been achieved already in 1914 by Wolbach and Binger; they obtained the first pure culture of Leptospira bijexa by passing water from a pond through a Berkefeld-V filter. 3. Isolation of bacteria which are parasitic upon other bacteria (Bdellovibrio Pseudomonas) Stolp and Petzold (1962), when isolating bacteriophages from soil, discovered the existence of a tiny parasitic bacterium, requiring living Gram-negative bacteria for their propagation. The generic name Bdellovibrio was assigned to this group of organisms, and extensive details about isolation, host organisms and selection of saprophytic mutants were given by Stolp and Starr (1963) and Stolp (1965). The organisms occur in soil and fresh water. Mitchell et al. (1967) showed, moreover, that the lethal effect of seawater on Escherichia coli is mainly due to the activities of Bdellovibrio. The width of Bdellovibrio cells is 0.3 p m and they can therefore pass through Millipore filters of 0.45 p m dia. However, by far the greater part of naturally occurring organisms are firmly attached to host cells. And thus it is essential that the parasites be detached before filtration procedures are applied. Soil samples should be vigorously shaken and water samples thoroughly stirred for 1 h. Since the population density of Bdellovibrio is relatively small, large samples are generally applied (e.g., 500 ml of canal water, or 500 g of soil suspended in 500 ml of tap water). After decantation, filtration or centrifugation to remove coarse particles, the suspension

350

H . VELDKAMP

is submitted to step-wise filtration through Millipore filters with pore diameters of 3 , 1.2, 0.8, 0.65 and 0.45 pm. Subsequently 0.5 ml of filtrate, 0.5 ml suspension of test organism (containing ca. 109 cells/ml) and 4 ml of the following medium are mixedMedium Yeast extract (Difco) Peptone (Difco) Agar (Difco) Tris buffer, 0 . 0 5 pH ~ 7* 5

3g

0.6 g 6g 1 litre

T h e mixture is poured onto a solidified nutrient medium of similar composition, but containing 18 g of agar/litre. Lytic spots due to phages reach a maximum size within 24 h ; after 3 days the slowly extending parasite plaques appear. The plates are incubated at the optimum temperature of the host cells. Postgate (1967) showed that parasitism on bacteria is not restricted to Bdellovibrio. A non-filterable Pseudomonas parasitic on Azotobacteriaceae was discovered by him in soil enriched with Azotobacter chroococcum cultures. Filtered soil extracts yielded no plaques with Azotobacter vinelandii, but a positive result was obtained with unfiltered extracts. These appeared to contain a Pseudomonas specifically parasitic to Azotobacteriaceae.

B. Separation of motile bacteria from mixed populations The isolation of motile organisms is often facilitated when they are allowed to move away from the mixed population in which they occur. This isolation method is of special value when the population density of the organism in question is relatively low. It has been applied in purification procedures for flagellated eubacteria as well as for spirochetes and organisms with gliding motility. Cellulolytic cytophagas form an example of the latter group. They decompose cellulose only when in direct contact with it. Therefore, Jensen (1940) recommended for the isolation of these cytophagas, a weak 1% w/v agar gel containing an evenly dispersed suspension of fine cellulose particles. This allows the organism to penetrate the agar and to migrate towards their food supply, and away from localized eubacteria. Further details about enrichment and isolation of cellulose, chitin and agar-decomposing cytophagas are given by Veldkamp (1965b). Other examples of the separation of motile organisms are the following.

1. Separation of chemo-organotrophic spirilla in soft agar The enrichment conditions applied for these organisms generally are not very specific, and thus the spirilla often form only a minority population in the enrichment. Surface colonies of spirilla are often difficult to distinguish from those of other organisms. But colonies submersed in a


35 1

soft agar are generally very characteristic, since they are more diffuse and extend much more rapidly than those of other motile organisms (e.g., Pseudomonas). The isolation procedure is as follows. A drop of enrichment medium is placed on a glass slide. Next to this a drop of fresh liquid medium is placed, and both drops are connected through a narrow liquid bridge. Since the spirilla generally are the fastest moving organisms in the mixed population, a certain initial enrichment of spirilla occurs in the drop of fresh medium (Giesberger, 1947). By means of a capillary pipette a sample is taken, and a dilution series is made in tubes filled with a homologous medium containing 0.6% w/v melted Bacto agar (Difco) (ca. 40°C). The dilutions are simply made by pouring 1-2 ml from one tube into the next and mixing the contents by inverting the tubes once or twice. As soon as a mixture is made and the next tube inoculated, the contents are poured into a Petri dish and incubated at 20"-30°C. Details about enrichment media for chemo-organotrophic spirilla are given by Jannasch (1965a). A growth medium consisting of the usual minerals, 0.001% w/v yeast extract and 0.2% w/v lactate as carbon and energy source allows growth of a great many spirilla.

2. Separation of spirochetes in agar As described above, colonies of spirilla in soft agar rapidly extend, and use can be made of this property in isolation procedures. A similar approach may be applied with small spirochetes. Since the width of their cells is only 0.19-0.35 pm, these organisms can migrate through agar of 1-2% concentration. Since the diameter of many spirilla is much larger, these organisms do not show migration at these higher agar concentrations. I t should be emphasized, however, that migration through agar by spirochetes is markedly influenced by the nutrient concentrations of the growth medium. Saprophytic anaerobic spirochetes only show spreading movement at very low nutrient concentrations (e.g. mineral medium with O . O l ~ o w/v yeast extract); inclusion of 0.2% w/v of glucose in the growth medium reduces migration considerably. Further details are given by Veldkamp (1960,1965d).

3. Separation of Proteus The swarming abilities of Proteus can be used for its rapid isolation as follows. At the bottom of a freshly prepared agar slant, whose typical composition might beAgar slant Peptone

Bacto agar (Difco) Distilled water

5g 15-20 g 1 litre

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H. VELDKAMP

a tiny amount of manure is placed. After incubation overnight at 30"-37"C, Proteus will be found at the top of the slant as a thin film of cells. Less fast moving organisms like Serratia are never further up than half-way by then.

4. Isolation of Cellvibrio In many cases enrichment and isolation of cellulose-decomposing vibrios does not present difficulties. They can readily be obtained as follows (Harmsen, 1946). A piece of filter paper is placed in a Petri dish on a layer of soil of ca. 3 cm thickness and subsequently covered with 1-2 cm of soil. The plates are incubated at 2Oo-30"C, care being taken that the soil does not dry out. After 10-14 days' incubation the soil is removed and patches of decomposing paper are suspended in sterile tap water. Samples then are seeded in cellulose agar (prepared according to Skerman, 1967); the agar concentration should not exceed 0.7-0-80/, since growth of Cellvibrio is often inhibited by higher agar concentrations. T h e growth requirements of Cellvibrio are variable; some strains need vitamins and amino-acids for growth, whereas others grow profusely on cellulose suspended in a mineral medium. The more versatile organisms that grow, for instance, on peptone generally show weak cellulose-decomposing activities. Vigorous cellulose decomposition is often shown by those organisms which do not grow on peptone or glucose. Celluibrio forms characteristic colonies on as well as in the soft agar. I n the latter case the dense centre of the colonies is surrounded by a zone of low density (1-3 nun dia.) formed by cells which move into the agar. When Cellvibrio forms a minority population on the decomposing filter paper, it may be necessary to introduce a second enrichment procedure before inoculating cellulose agar. This can be achieved by using patches of partly decomposed paper as inoculum for a liquid enrichment culture. This consists of a shallow layer of a mineral base, including a piece of sterile filter paper. Another way to purify Cellvibrio is to make use of its mobility, according to a procedure introduced by Chalvignac (1952). A silica gel plate containing a basal mineral salts medium is prepared (Skerman, 1967). Across the dry and sterile surface a strip of filter paper, moistened with chlortetracycline solution (50 pglml), is placed. T h e inoculum is subsequently deposited on one end of the strip. Growth of Cytophaga and other organisms in the mixed population is inhibited by the antibiotic, which does not affect Cellvibrio. After 4-6 days the rapid spreading growth of this cellulolytic organism reaches the other end of the strip. Some strains show pale-yellow growth, but the extending growth of others may not be visible macroscopically, necessitating regular microscopic control. Two or three transfers are subsequently made, and the cells that reach the end of the last


353

strip are nearly exclusively those of Cellvibrio (Chalvignac, 1952). Finally, the organism can be purified in cellulose agar as described above. 5. Separation of spirilla in glass capillaries For the isolation of aerobic chemo-organotrophic spirilla occurring in enrichments containing various eubacteria, Giesberger (1936, 1947) introduced the following method. A small sample of the culture is taken up in a glass capillary filled with sterile liquid medium. Fast moving spirilla soon leave other organisms far behind as can be easilyestablished by microscopic examination. By cutting the capillary in the right place, it is possible to separate several spirilla from other organisms. Jannasch (1965a), who gave an extensive description of this method, recommends the use of flat capillaries; these can be obtained by flattening a heated glass tube with a pair of forceps before drawing the capillary, which is subsequently filled with sterile liquid medium and briefly dipped into the culture surface. T h e tip of the capillary is then immediately closed by dipping it in melted paraffin. Entrance of air should be avoided. Aerotaxis makes the spirilla move away from the closed tip and they can then be trapped as described above. In some cases it has been shown useful to coat the surface of the capillary with a silicone compound in order to prevent the liquid from becoming alkaline (S. C. Rittenberg, personal communication).

C. Microbial morphology as an aid in isolation procedures 1. The direct isolation of Arthrobacter There are many organisms for which no specific enrichment methods are known. Direct plating of a soil suspension on soil extract agar, for instance, reveals the existence of organisms that are rarely found among the dominant populations of enrichment cultures. Colony features of such organisms generally do not allow immediate identification. Exceptions are for instance the colonies of Cytophaga, Streptomyces and Bacillus nzycoides that can generally be recognized macroscopically. Among organisms that commonly occur in natural environments (e.g., soil, coastal waters) are those belonging to the genus Arthrobacter. Representatives of this genus occasionally form dominant populations in enrichments for aerobic chemo-organotrophic organisms. This may occur for instance when aromatic compounds, such as toluene, or the herbicide 2,4-dichlorophenoxyacetic acid (Alexander, 1967) are used as carbon and energy source. Stevenson (1967) found the ability to decompose aromatic hydrocarbons to be common among soil arthrobacters. The organisms commonly

354

H. VELDKAMP

occur on “non-selective” agar media seeded with samples from natural environments. Even though their colonies on such media are not very distinctive, the arthrobacters can rapidly be detected and isolated by making use of their characteristic life cycle. I n the stationary phase of growth the cells of Arthrobacter are coccoid, whereas pleomorphic rods are formed during the logarithmic growth phase. On nutritionally poor media, this life cycle is not very pronounced, and the cells remain very short throughout all stages of growth. However, when more complex media are used, the rods become much lcnger and very pleomorphic during the logarithmic growth phase. T h e life cycle of Arthrobacter forms the basis of an isolation method devised by Mulder and Antheunisse (1963). Their procedure is as follows. A soil suspension is plated out on a relatively poor growth medium, whose typical composition might bePoor growth medium K2HP04 (NH4)zS04 MgS04.7HzO Glucose Yeast extract (Difco) Isoelectric casein (Difco) Agar (Difco) Tap water pH 7 . 0

16 0.5 6

0.25 g 1rx 0.7g 16

15 6 1 litre

After 7 days’ incubation at 25°C colonies are examined microscopically and those consisting of coccoid cells are purified. T h e strains thus collected are then transferred to a rich agar medium as for instanceRich growth medium

Yeast extract (Difco) Glucose Agar (Difco) Distilled water

76

10 rx

15 g 1 litre

As soon as colonies become visible they are examined microscopically; those showing rods (generally of irregular shape) are Arthrobacter colonies. It is essential that colonies are examined when still in the early logarithmic phase of growth; the incubation time should preferably be 24 h or less. Further details concerning Arthrobacter are given by Mulder and Antheunisse (1963) and Veldkamp (1965~). Fragmentation of cells may also occur in organisms belonging to related genera, e.g., Nocardia and Corynebacterium. However, these organisms generally do not form spherical cells upon fragmentation.


355

ACKNOWLEDGMENTS

The author is very grateful to the following authors and copyright owners for their permission to reproduce Tables and Figures: Prof. R. Y. Stanier and Prentice Hall, Inc., Englewood Cliffs, N.J., U.S.A. (Tables I-IV), Prof. H. A. Barker and Springer Verlag, Heidelberg, Germany (Fig. l), Dr. E. G. Jefferys and Society for Applied Bacteriology (Fig. 2), and Prof. E. G. Mulder and Gustav Fischer Verlag, Stuttgart, Germany (Fig. 4). REFERENCES Alexander, M. (1967). In “The Ecology of Soil Bacteria” (Ed. T. R. G. Gray and D. Parkinson), pp. 270-284. Liverpool University Press, Liverpool. Allen, M. B. (1952). Arch. Mikrobiol., 17, 34-53. Allen, M. M., and Stanier, R. Y. (1968). J . gen. Microbiol., 51, 203-209. Anderson, J. I. W., and Hefferman, W. P. (1965). J. Bact., 90, 1713-1718. Baas Becking, L. G. M. (1934). “Geobiologie”. W. P. van Stockum en Zoon N.V., Den Haag. Babenzien, H. D. (1965). In “Anreicherungskultur und Mutantenauslese” (Ed. H. G. Schlegel), pp. 111-116. Gustav Fischer Verlag, Stuttgart. Barker, H. A. (1936). Arch. Mikrobiol., 7, 420-438. Barker, H. A. (1940). Antonie van Leeuwenhoek, 6 , 201-220. Barker, H. A. (1956). “Bacterial Fermentations”. Wiley, New York. Barnes, E. M. (1956). J. appl. Bact., 19, 193-203. Becking, J. H. (1961) PI. Soil, 14, 49-81. Beijerinck, M. W. (1911). PYOC. K. Akad. W e t . Amst., 13, 1237-1240. Beijerinck, M. W. (1913). Juarb. K . Akad. W e t . Amst. Beijerinck, M. W. (1921-1940). Verzamelde Geschriften, Vols. 1-6. Nijhoff, Den Haag. Borek, E., and Waelsch, H. (1951). J . biol. Chem., 190, 191-196. Bock, E. (1965). In “Anreicherungskultur und Mutantenauslese” (Ed. H. G . Schlegel), pp. 148-1 54. Gustav Fischer Verlag, Stuttgart. Brock, T. D. (1966). Lininol. Oceunogu., 11, 303-307. Brock, T. D. (1968). J . appl. Bact., 31, 54-58. Brown, M. E., Burlingham, S. K., and Jackson, R. M. (1962). PI. Soil, 17, 309. Bryant, M. P., Wolin, E. A., Wolin, M. J., and Wolfe, R. S. (1967). Arch. Mikrobiol., 59,20-31. Bukatsch, F. (1965). In “Anreicherungskultur und Mutantenauslese” (Ed. H. G. Schlegel), pp. 399406. Gustav Fischer Verlag, Stuttgart. Buyze, G. (1955). Ph.D. Thesis, University of Utrecht. Canale-Parola, E., and Wolfe, R. S. (1960). J . Bact., 79, 857-862. Canale-Parola, E., Rosenthal, S. L., and Kupfer, D. G. (1966). Antonie van Leeuwenhoek, 32,113-1 24. Canale-Parola, E., Holt, S. C., and Udris, 2. (1967). Arch. Mikrobiol., 59, 41-48. Casida, L. E. (1967). In “The Ecology of Soil Bacteria” (Ed. T. R. G. Gray and D. Parkinson), pp. 97-1 22. Liverpool University Press, Liverpool. Chalvignac, M. A. (1952). Annls. Inst. Pusteur, Paris, 83, 417419. Claus, D. (1965). In “Anreicherungskultur und Mutantenauslese” (Ed. IH. G. Schlegel), pp. 337-362, Gustav Fischer Verlag, Stuttgart. Conover, S. A. M. (1956). Bull. Bingham Oceanographic Collection, 15, 62-1 12. Davis, J. B. (1967). “Petroleum Microbiology”. Elsevier, Amsterdam.

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CHAPTER VI

The Isolation of Mutants DAVID A. HOPWOOD* Institute of Genetics, University of Glasgow, Scotland I.

Introduction

11. Clonal Considerations . A. Obtaining pure clones B. Pick each clone once only

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111. General Remarks on Mutagenesis . A. Spontaneousor induced mutants? B. Whichmutagen? C. How much mutagenesis? D. Takecare! .

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IV. Mutagens and Mutagenic Treatments A. Radiations . B. Chemical mutagens . C. Other mutagenictreatments V.

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Methods of Mutant Isolation A. The design of screening procedures . B. Selectivemethods . C. Replica techniques D. Screeningby visual examination of colonies E. Screeningfor diffusibleproducts .

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CharacterizationofMutants A. Auxotrophs . B. Temperature-sensitivemutants Conclusion

References

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I. INTRODUCTION This Chapter is aimed primarily at the microbiologist, biochemist, molecular biologist, or pathologist, who, in the course of an investigation, needs to obtain mutant organisms to further some aspect of his research. It may

* Present address: John Innes Institute, Colney Lane, Norwich, England.

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also aid a geneticist wishing to develop the experimental genetics of a hitherto neglected organism, in which a stock of mutants, or well-tried methods of obtaining them, are lacking. The remarks that follow are not intended for those engaged in studies of well-known genetical systems, nor in studies of mutagenesis itself, where much more specializedinformation is available than is summarized here. The objective is to obtain the desired mutants by the most economical route, and to consider the precise mechanisms involved only in so far as they bear on the choice of experimental procedures. Since two organisms will rarely respond in precisely the same way to a mutagenic treatment, or a mutant screening programme, it will often happen that a published procedure will have to be modified to yield optimal results in a new situation. This Chapter will have served its main purpose if it helps to reduce the time spent in the initial stages of an investigation, during which pilot experiments define the right mutagen, the right mutagenic conditions and the right screening technique for the job in hand. A second objective of this Chapter is to mention someof the kinds of mutants that have been isolated in various micro-organisms, and sometimes to make brief reference to the purposes for which they have been used. This information may aid those who, rather than needing a recipe for isolating a particular defined class of mutant, are at first undecided as to what kinds of mutant might serve their requirements best, or what classes of mutants it might be feasible to seek in the particular organisms with which they are dealing.

11. CLONAL CONSIDERATIONS A. Obtaining pure clones The isolation of mutants is normally greatly facilitated when a uninucleate haploid stage in the life-cycle of a micro-organism is available for mutagenic treatment and subsequent cloning. This follows from the fact that the great majority of mutant genes are recessive to the wild-type allele. When a mutation occurs in a uninucleate haploid cell, a pure mutant clone or colony can result. (It might be expected, since DNA is a duplex, that all mutation would produce mosaic colonies, part mutant-from the altered strand of the duplex-and part wild-type-from the unaltered strand. In fact all mutagens, for reasons which are not entirely clear, but which almost certainly involve, at least in part, the operation of repair systems (Holliday, 1962; Witkin and Sicurella, 1964; Haefner, 1967), produce a proportion of pure mutant clones ;the proportion varies between different mutagens, and can be high.) In contrast, if a mutation occurs in a diploid, the mutant allele finds itself in heterozygous condition and escapes detection, unless and until an opportunity for segregation in haploid or homozygous condition occurs. Mutation

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in a multinucleate haploid cell will give rise to a heterokaryon; again a recessive mutant gene is masked until an opportunity occurs, during subsequent nuclear and cell divisions, for segregation of cells homokaryotic for the mutant gene. Such opportunities for segregation can often be built into a mutant screening programme, by judiciously exploiting the life cycle of the organism, if one is forced to utilize diploid or multinucleate cells for mutagenic treatments. For example, Beadle and Tatum (1945), in their classic isolation of auxotrophic mutants of Neurospora crassu, irradiated the multinucleate (macro) conidia, then used these conidia to fertilize protoperithecia of an untreated culture of opposite mating type. Single nuclei from the conidia were involved in the nuclear fusion and segregation events in the asci, and the resulting ascospores contained, in single dose, chromosomes descended from the nuclei of the irradiated macroconidia ; a small fraction of them developed into pure mutant colonies of the required types. Such a roundabout cloning procedure was rendered unnecessary by the subsequent discovery of strains of N . crussa producing exclusively uninucleate (micro) conidia (Barratt and Garnjobst, 1949), which could be subjected to mutagenesis and then immediately plated out to develop into pure clones. The technique of using a meiotic sieve to achieve segregation was applied by Chen (1965) to isolating ascospore colour mutants of Sorduriu brewicollis, even though uninucleate conidia were available. These were irradiated and used to fertilize protoperithecia of a strain of opposite mating type, and asci from the cross were examined microscopically in a search for those containing 4 mutant spores and 4 of wild-type colour, indicating the presence of a mutant gene in the conidium that sired the asci. In this way, Mendelian segregation had already been established when the mutant was isolated. A similar procedure, but without mutagenic treatment, was used by Lissouba et ul. (1962) in Ascobolus immerm. In Paramecium, which is a diploid or dikaryotic organism, segregation of mutant alleles in homozygous condition is achieved by forcing the animals to undergo autogamy. In isolating mutants (e.g., Igarashi, 1966) specimens can be irradiated, isolated separately and starved to induce autogamy; a number of homozygous progeny from each culture is then screened for the presence of mutants. Bacterial cells, under most cultural conditions, rarely contain a single genome, so that cloning after mutagenesis would be expected to present problems. However they are usually rather easily overcome. For example growth in broth for a few generations after mutagenesis before plating for single colonies, results in the segregation of pure clones. This may even be unnecessary if mutagenesis is associated with appreciable killing, since any “viable” but mutant genome is unlikely to find itself in the same bacterium

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as a “viable” but non-mutant genome. Probably this is the explanation for the successful isolation of auxotrophic mutants by filtration enrichment (see later) after ultraviolet irradiation in strains of Neurospora bearing macroconidia (e.g., Catcheside and Overton, 1958).

B. Pick each clone once only Particularly when dealing with spontaneous mutants, or with mutants obtained by a procedure which includes provision for segregation of pure mutant cells before cloning, one has to be constantly on one’s guard against isolating strains carrying descendants of the same mutant gene. A satisfactory isolation programme has built-in safeguards against such an occurrence. For example, suppose we wish to isolate ten independent microbial mutants lacking a particular enzyme. It would be unwise to isolate our ten mutants from amongst the survivors of a single culture subjected to a mutagenic treatment; if we did so, we would have no way of knowing whether we had, in a proportion of cases, picked members of the same mutant clone. Instead we would first clone the culture, for example by plating it out for single colonies, isolate ten separate (non-mutant) colonies, treat each resulting culture with the mutagen, and isolate one mutant only from each culture; in this way we would ensure that each mutant gene in our sample had an independent origin from the wild-type allele. Of course, if two mutants from the same culture are demonstrably different in some aspect of their phenotype (provided that the difference is not due to additional mutations differentiating the two strains), the mutant genes they carry can be deduced to be of independent origin, or alternatively two mutant genes may be shown to be independent by being mapped at different positions on the genome. However, since mapping is usually much more laborious than repeating the mutagenic treatment on a separate culture, it is usually best to take no risks and stick to the “one culture-one mutant” rule when looking for a particular phenotypic class of mutants. The only situation in which this rule may safely be relaxed is when the yield of mutants after mutagenesis is much greater than in control cultures, and when no opportunity for clonal reproduction has occurred after mutagenesis and before cloning the survivors as single colonies. Under these circumstances it is much more likely that two mutant colonies stem from two independent mutational events suffered immediately before plating than that they belong to a single clone derived from an earlier, spontaneous, mutation. 111. GENERAL REMARKS ON MUTAGENESIS A. Spontaneous or induced mutants? Sometimes it is possible to design a selectiwe screening procedure for particular types of mutant (see below): for example non-mutant cells are


367

killed before plating (as in penicillin selection) or only mutant cells can form colonies on the plating medium (as in the isolationof drug-resistant mutants). Under these circumstances mutants can be isolated with a minimum of effort even from populations in which the proportion of mutant cells is much less than 1 x 10-6. It may then be possible to avoid using a mutagen and rely on the spontaneous origin of mutants. More often, the screening procedure is more or less inefficient, so that any increase, as a result of mutagenic treatment, in the proportion of mutants in the population to be screened results in an appreciable saving of effort.

B. Which mutagen? It is an empirical fact that the use of different mutagens usually results in isolating different “spectra” of mutants. For example mixed bags of auxotrophs isolated from the same strain on the same medium after different mutagenic treatments contain different proportions of mutants with the various classes of requirements (e.g., Zetterberg, 1962). Probably most of the differences are due to factors affecting the likelihood of recovery, in viable colonies, of mutations in particular genes, rather than to real differences in their frequency of occurrence (see for example Auerbach, 1966). Current notions on gene structure and the mechanism of mutagenesis by different agents lead to the prediction that any mutagen should be capable of inducing mutations at some sites in any gene, although particular sites (depending on the base-pair in the DNA) will be mutable by some, but not by all, mutagens. Furthermore, only certain mutagens lead to particular classes of mutational event, such as the deletion or addition of single bases (“frame-shift mutation”) by acridines and their nitrogen mustard derivatives (ICRcompounds), but not by base-analogues. It follows that, except in sophisticated studies of particular genes or particular mutagens, with which we are not concerned here, the choice of mutagen depends largely on considerations of practical convenience or empirical success. However, because of the vagaries of mutant recovery referred to above, choice of a single mutagen may be undesirable; in a search for a particular mutant, success may come more quickly with the use of more than one mutagen, each on a limited scale, than with a massive use of only one. Similarly, if the object is, for example, to isolate a wide variety of nutritional mutants for genetic markers, it may be most efficient to spread the net as widely as possible by using several mutagens. In order to find a mutagen effective in a little known organism, an “auxanographic” test for mutagenesis (Iyer and Szybalski, 1958) may be useful; this test uses reverse mutation as a criterion of mutagenesis, but the likelihood is that a mutagen effective in this test will also be suitable for the

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induction of the forward mutations that are usually required in isolating desired mutants. Large numbers of cells of an auxotrophic mutant are spread on minimal medium, and a small amount of the substance to be tested as a mutagen, possibly applied as a solution on a filter paper disc, or even as a crystal of the pure substance, is placed on the plate. After incubation, colonies of prototrophic revertants in a zone centred on the point of addition of the test substance indicate mutagenesis. Unfortunately a negative result means little; it may occur because reversion of the chosen auxotroph requires a base-change that is outside the scope of the mutagen, in which case use of other auxotrophs may yield a positive result. On the other hand a potentially useful mutagen may fail in this plate-test because conditions are too far removed from those optimal for mutagenesis.

C. How much mutagenesis ? It is not surprising that all mutagens, as well as leading to the production of mutant, but viable, individuals, also kill a proportion of the treated cells. Some of this killing is due to the induction of “lethal’,’mutations (a relative term, since a mutation may be lethal under some conditions-a nutritional mutant on a minimal medium-but not others-the same mutant on a complete medium) but this is certainly not the whole story. Otherwise it would be difficult to account for the enormously different proportions of a given mutant type in populations treated, to the same level of survival, with different mutagens. Thus some agents cause much “non-genetic” killing and others relatively little. The absolute number of viable mutant individuals obtainable in a mutagentreated population will vary inversely according to the ability of the mutagen to cause “non-genetic” killing; thus for high absolute numbers of mutants, mutagens causing little “non-genetic” killing are needed (N-methy1-N’nitro-N-nitrosoguanidine [NTG] is an example). For any mutagen, the number of viable mutants in the population will increase with dose up to a certain level, and then fall as killing overtakes the induction of new mutants. When a selective method of mutant isolation is available it may be best to aim for a high absolute number of viable mutants in the culture to be screened. However, in most screening programmes, the proportion of mutants amongst the sumivors is the important thing, since only the survivors have to be screened. This proportion in general increases with dose, often in an approximately linear fashion, though sometimes proportionally to a higher power of the dose, up to a dose level well above the optimum for absolute numbers. Eventually, with increasing dose, the proportion of mutants often begins to fall; at least in some instances this is due to genetical or physiological heterogeneity of the original population in respect of sensi-

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tivity to the killing and mutagenic effects of the mutagen so that, at high doses, the more resistant component comes to make up an increasing proportion of the survivors. Thus for each mutagen and each organism, there is usually an optimum “dose” (a combination of mutagen concentration, time and conditions of treatment), which leaves the highest proportion of a particular mutant class amongst the survivors. (The optimum may not be the same for different classes of mutants.) Do we always want to aim for the highest proportion of mutants obtainable? Perhaps not. To give a specific example, as we see below, N T G is such a potent mutagen that auxotrophic mutants may constitute up to 50% of the survivors of mutagenesis under optimal conditions. Assuming that mutations to auxotrophy occur at random in the population, if 50% of the survivors are auxotrophic mutants, about half of them will in fact harbour more than one auxotrophic mutation, a quarter of them more than two etc. In addition, the likelihood that other classes of non-lethal mutation will also be carried by the auxotrophs will be exceedingly high. Thus a high proportion of the isolated mutants will be troublesome to characterize nutritionally, and their genetic analysis will be complex. Probably mutagenesis should be moderated to yield not more than loo/, auxotrophs; even then one-tenth of them would be expected to be multiple auxotrophic mutants. As we see later, it is often possible to enrich a population in respect of rare auxotrophic or other mutants by a suitable procedure interposed between mutagenesis and plating. Thus auxotrophs may come to represent perhaps 50% of the survivors of the combined mutagenic and enrichment procedure, when they constituted perhaps less than O.lyo of the survivors of mutagenesis alone. Under these circumstances, the chance of recovering multiple mutants is very much reduced. We see that the mutagenic treatment best suited for a particular mutant isolation depends on many factors, including the nature of the test designed to reveal the desired mutants (whether selective or not), the availability or otherwise of enrichment procedures, the extent to which multiple mutants will give trouble, and others.

D. Takecare! Except with ionizing radiations, there is little likelihood of the experimenter accidentally inducing mutations in his own gonads. However, most, and possibly all, mutagens can also be carcinogenic under the right conditions, as well as being potentially harmful in other ways, so that more than usual care should always be taken in their handling, and disposal after use. 15

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IV. MUTAGENS AND MUTAGENIC TREATMENTS A. Radiations 1. Ionizing radiations Ionizing radiations (X-rays, y-rays, neutrons and other particles) may occasionally have applications in the routine induction of mutations when other mutagens are ineffective (for example because the only available stage in the life cycle of a microbe is not transparent to ultraviolet light or susceptible to the action of chemical mutagens), but should probably be regarded as a last resort ; this is because of the comparatively high likelihood that chromosomal breakage will occur, leading to structural changes like translocations and inversions, which will complicate subsequent genetic analysis of the mutants (e.g., Kafer, 1965, who analyses the origin and dissemination of structural changes, many induced by radiation, in genetic stock cultures of Aspergillus nidulans since its inception as a genetical organism). In the present state of knowledge, no mutagen can be completely exonerated from breaking chromosomes, but ultraviolet light and the chemical mutagens mentioned below are certainly less likely than ionizing radiations to do so. 2. Ultraviolet l$ht (UV) (a) General. This is an extremely convenient mutagen, provided that the cells to be treated are appreciably transparent to it (some strongly pigmented fungal spores, for example, are not). Mutagenic wavelengths lie between about 200 and 300 nm, the peak of absorption by nucleic acids. It is thus an extremely fortunate coincidence that a convenient source of UV, a low-pressure mercury vapour (“germicidal”) lamp, emits a very high proportion of its energy at 254 nm, close to the most effective wavelength. (High-pressure mercury vapour lamps spread their energy more uniformly over a wide range of wavelengths.) A good concise account of the practical essentials of UV-irradiation is given by Meynell and Meynell (1965, pp. 235-237) and only a few points will be amplified here. (b) Dose-response relations. Killing by UV is in many (haploid) organisms, exponential, often with a marked initial lag, probably at least in part due to the operation of repair mechanisms. T h e proportions of particular mutant classes amongst the survivors in general increase approximately linearly with dose, until, at doses giving very low levels of survival, the proportions may fall. A guide to determining the dose of UV that will leave the highest proportion of mutant survivors is to plot a survival curve and to use the highest

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dose that results in exponential killing; that is before any slowing down in the rate of kill with increasing dose is apparent. (c) Measurement of dose. In routine mutant isolation it is unnecessary to measure the UV dose in physical units (ergs/mm2); it is enough to plot a survival curve in terms of minutes of exposure to a given UV source under standard conditions, and choose for the induction of mutations an irradiation time giving a suitably low survival, say 0.1 to 1%. The physical measurement of dose-rates can be laborious, but a simple and inexpensive set-up was described by Jagger (1961) who also discussed the merits and de-merits of more complicated systems. (Photovoltaic cells of the type he describes, with good UV sensitivity, are available from Electrocell-Gesellschaft, Kalkenthal and Presser, Berlin-Dahlem, Germany, in addition to the source quoted by Jagger.) Physical measurement has the advantage of being applicable to day-to-day monitoring of a UV source to take account of changes in output due to voltage fluctuations, ageing of the tube, variations in reflectivity of lamp housings, and other causes. For onceand-for-all calibration of a single source, the biological assay based on bacteriophage T 2 inactivation (Latarjet et al., 1953) is probably the simplest technique; since killing of T 2 was found to be strictly exponential, after a shoulder extending to about 50 ergs/mm2, a calibration line of surviving fraction against dose in a semi-logarithmic plot may be drawn using the two reference points: 10-2 survival at 200 ergs/mm2 and 10-4 survival at 360 ergs/mm2. (d) Photo-reactivation. For reproducibility of experiments, and to prevent increases in viable count of stored suspensions, it may be necessary to avoid exposure to long wavelength UV or the shorter wavelengths of the visible spectrum (sunlight and light from fluorescent tubes are effective sources) during the period immediately following irradiation in order to avoid photo-reactivation (reviews ; Rupert, 1964; Rupert and Harm, 1966): the enzymic reversal of a proportion (often up to a maximum of about 60%) of the mutagenic and killing effect of a given UV dose. The action spectrum for photo-reactivation is not the same in all organisms (peaks at 375 nm and 435 nm for Escherichia coli and Streptomycesgrisws respectively : Kelner, 1951) but it can probably be assumed that wavelengths longer than about 525 nm will have negligible effect. Thus post-irradiation manipulations may be carried out in yellow light. An ordinary yellow-coated light bulb may be a good enough source for this purpose, or alternatively a darkroom safelight with Wratten Number OB filter. If a higher intensity of illumination is needed, or a large room has to be illuminated, a convenient and inexpensive source is a sodium vapour lamp (emitting at 589 nm) of the kind used in

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street lighting (e.g., Phillips SOX, nominal 40W, with appropriate ballast and capacitor). Photo-reactivation occurs unpredictably amongst micro-organisms ; for example Harold and Hopwood (1969) found that a certain wild-type isolate, K673, of Streptomyces coelicolor was strongly photo-reactivable, while another wild-type of the same “species”, A3(2), was not. I t is therefore worth testing an organism that is to be irradiated extensively to see whether the extra complications designed to avoid photo-reactivation are in fact required.

B. Chemical mutagens Innumerable compounds are mutagenic, but few are convenient for the routine isolation of mutants. Some of the most widely used are mentioned in what follows. The best mutagen and set of conditions for its use are not identical for all organisms (see for example the discussion of the use of N-methyl-N‘-nitro-N-nitrosoguanidine below) and have often to be arrived at rather empirically, but the notes and references below should aid the choice of suitable conditions for preliminary experiments. In general, chemical mutagens can be grouped into three classes according to their mode of action: those that cause a chemical change in one or more of the nucleic acid bases while they remain in situ in the molecule, and therefore can operate on non-replicating nucleic acids (nitrous acid, alkylating agents, NTG); those that mimic a natural base closely enough to be mistaken for it and inserted into a newly synthesized strand during nucleic acid replication (base analogues); and those that cause loss or addition of one or two bases in DNA, again during its replication or repair (frame-shift mutagens). If the object is, as it often is, to obtain a stock of non-leaky mutants (that is with as little as possible of the activity associated with the wild-type allele remaining), any of these classes of mutagen will serve. Frame-shift mutagens may result in the biggest proportion of non-leaky mutants, because mutation results in a shift in the reading frame during translation of the genetic message from the site of the deleted or added base onwards to the end of the message. Thus mutant protein with residual function is most unlikely to result. However the other classes of mutagen can give rise to nonsense mutations (that is to codons which cannot be translated, so that polypeptide synthesis is terminated), and these, like the frame-shift mutations, are likely to be non-leaky. A high proportion of non-leaky auxotrophs, isolated at random, does in fact turn out to carry frame-shift or nonsense mutations, rather than the missense mutations resulting in substitution of one aminoacid for another, and often giving rise to a protein with some, or even full, function. As a further complication, frame-shift and nonsense mutations


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are usually polar; that is they can abolish or severely reduce the activity of adjacent genes in the same operon, so that several functions (usually enzymes) may be lacking in the mutant; on the other hand missense mutants are normally not polar. The ability to act on “resting” DNA with mutagens (for example nitrous acid) allows extracted, transforming DNA to be treated and subsequently used to transform a recipient strain in respect of a particular selected marker; among the transformants new mutants involving loci linked to the selected marker can be found (Anagnostopoulos and Crawford, 1961).

1 . Nitrous acid This mutagen has the advantage that mutagenesis is very easily controlled, and the mutagen is a rather harmless substance. The cells to be treated are usually suspended in acidic buffer (for example 0.1 M acetate, p H 4.5), and nitrous acid is generated by adding a freshly prepared aqueous solution of sodium nitrite to a final concentration of, say, 0-1 or 0.2 M. After treatment for a suitable length of time, the reaction may be stopped simply by dilution during plating, by neutralization with a suitable volume of NaOH solution, or by pipetting samples of the reaction mixture into neutral buffer. The amount of mutagenesis is most easily controlled by varying the length of treatment. In bacteria (Kaudewitz, 1959), Aspergillus (Siddiqi, 1962) and Schizosaccharomyces (Loprieno and Clarke, 1965), survival was exponential after a marked initial lag, and the proportion of mutants among survivors increased more than linearly with time for a considerable period. Useful general references to nitrous acid mutagenesis are Kaudewitz (1959); Siddiqi (1962); Clarke (1963).

2. Ethyl methane sulphonate ( E M S ) , ethyl ethane sulphonate ( E E S ) and diethyl sulphate ( D E S ) Of the large class of alkylating agents, these compounds seem to have achieved the greatest use as routine mutagens. They are rather easily handled, and give relatively high mutant yields at high levels of survival. (Many commercial samples of DES are contaminated by a substance causing killing but no mutagenesis; Freese (1963) gives Fluka Chemical Co., Zurich, Switzerland, as a source of pure DES.) They are liquids, and are usually pipetted (use a bulb!) into a suspension, in water or a suitable neutral buffer (e.g., 0.1 M phosphate), of the cells to be treated, to give a concentration in the region of 1% v/v. After treatment (for say 15 to 30 min) it is usually sufficient to stop the reaction by dilution during plating; alternatively, samples of the reaction mixture may be pipetted into 10 volumes of buffer containing 2% sodium thiosulphate to inactivate the mutagen.

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Useful references to mutagenesis using these alkylating agents are: Loveless and Howarth (1959); Freese (1963).

3. N-methyl-N '-nitro-N-nitrosoguanidine(NTG) This substance (usually obtained from Aldrich Chemical Co., Milwaukee, Wisconsin, U.S.A. ; British agents R. N. Emmanuel Ltd., London S.E.l. or from Koch-Light Laboratories Ltd., Colnbrook) is one of the most potent mutagens known, producing, under the right conditions, extremely high mutant yields with comparatively little killing. (Up to 8-10% auxotrophs among survivors in Salmonella typhimurium (Eisenstark et al., 1965); Schizosaccharomyces pombe (Megnet, 1965) and Streptomyces coelicolor (DeliC, Hopwood and Friend, 1970), and even 50% in E. coli! (Adelberg et al., 1965).)Its minor disadvantages are the extreme care required in handling and the likelihood that the desired mutants carry additional mutations which may complicate their analysis ; however the latter difficulty, which is an inevitable corollary to high mutant yields, can easily be avoided by moderating the mutagenic treatment to any desired level. Adelberg et al. (1965) made the first really systematic study of conditions influencing mutagenesis and killing in E. coli, and their schedule has been followed by most users who, in general, have found it very effective. Their most important finding was that survival was low and variable if growth occurred during treatment, but that high mutant yields with constant, high survival were obtained when cells were treated under conditions of no growth. The relationship between N T G concentration and the proportion of mutant survivors in a given time of treatment was complex, different concentrations between 0.1 and 1.0 mg/ml giving maximum yields of different classes of mutants (for example auxotrophs or valine-resistant mutants). The time of treatment was relatively unimportant, perhaps owing to rapid binding of N T G to the cells: mutagenesis reached a maximum (at 37") by 15 min. p H 6 was optimal; they used a 0-05 M Tris-maleic acid buffer (6.1 g Tris, 5.8 g maleic acid per litre, adjusted to p H 6.0 with NaOH). After treatment, the cells may be harvested by centrifugation or on Millipore filters, washed with water, saline or buffer and plated. In a study of optimal conditions for N T G mutagenesis in Streptomyces coelicolor spores Delif, Hopwood and Friend (1970) found that nearly all factors operated differently compared with E. coli. I n particular mutagenesis proceeded much more rapidly under conditions in which the N T G was decomposing rapidly (at p H 8 or 9), mutant yields increased for periods of several hours, and with the concentration of the mutagen almost up to the limit of its solubility (about 4 mglml). These findings underline the pitfalls


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of applying a mutagenesis schedule that has proved successful for one organism to another without modification. For routine mutagenesis in Streptomyces, spores were incubated at 30" in 0-05 M Tris-maleic acid buffer at pH 9, containing 3 mg N T G per ml for 90-120 min after which some 10% of the survivors were auxotrophic mutants. There appear to be few cases in which N T G is not a convenient mutagen; in Chlamydomonas reinhardi some people have found the undesirable sideeffect of extensive meiotic lethality (P. J. Hastings, personal communication), but others have had no difficulty (D. R. Davies, personal communication).

4. Base analogues Base analogues, such as 5-bromouracil (BU) and 2-aminopurine (AP) (references in Freese, 1963 and Orgel, 1965) are less likely to be useful as routine mutagens, particularly in a little-known organism, than the other compounds mentioned here, because suitable conditions of treatment will probably be more laborious to establish. For example 5-bromouracil is normally incorporated into DNA, and therefore mutagenic, only when the organism is starved of (both exogenous and endogenous) thymine, usually by being auxotrophic for thymine; however Kammen (1967) recently described a method for increasing BU incorporation by non-thyminerequiring strains by supplying the bacteria with deoxyribonucleosides. Since mutagenesis only occurs when the organism grows in the presence of the base analogue, mutant clones are likely to arise within the culture during treatment ; consequently precautions to exclude the picking of members of the same clone are more important than in the case of other mutagens (seep. 366). 5. Frame-shift mutagens (a) Acridines. Owing to the singularities of acridine mutagenesis (see below) it is unlikely that these compounds will be found convenient for the routine isolation of mutants in most micro-organisms, although they may have applications in particular situations. It has been found that certain acridines (Orgel and Brenner, 1961, studied systematically the relationship between molecular structure and mutagenicity), although strongly mutagenic for bacteriophages T 2 and T4, are very weakly or non-mutagenic in other systems, for example bacteria. The T2-T4 system is singular in that, as well as DNA replication, recombination also occurs during the mutagenic treatment, since it is invariably associated with replication in these phages; this fact suggested an association between mutagenesis and recombination and led Lerman (1963) to propose unequal crossing-over as a mechanism leading to insertion or deletion of

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single DNA nucleotides (Brenner et al., 1961; Crick et al., 1961) and so to mutagenesis. Unequal crossing-over would be stimulated, on this model, by intercalation of acridine molecules between adjacent DNA bases, thus forcing them further apart than normal ; physico-chemical data on the interaction of acridine with DNA were in agreement with such a notion (Lerman, 1963). Lerman’s theory led to the prediction that acridines would be mutagenic in other organisms if treatment occurred at the time of recombination, and some evidence for this has been obtained (Magni et al. 1964; Sesnowitz and Adelberg, 1969). However, the situation may well be more complicated than these simple statements would imply (Streisinger et al., 1966). It should be mentioned that what has been said above about acridine mutagenesis applies to treatment carried out in the dark; in a number of systems, acridines combined with visible light have been shown to be mutagenic, and such mutations are probably not of the frame-shift type (e.g., Ritchie, 1965). An additional genetic effect of sub-lethal concentrations of acridines is their ability to eliminate certain episomes from growing bacteria, presumably by slowing the rate of reproduction of the episome relative to that of the cell. Hirota (1960) converted F + cells of E. coli K-12 into F - cells in this way and resistance-transfer factors can also be eliminated from Escherichia and Shigella strains (Mitsuhashi et al., 1961 ; Watanabe and Fukasawa, 1961). There is a tendency to assume that all episomes, in the non-integrated state, may be eliminable by acridines, and to use a negative result as evidence against the existence of such a factor. Such a conclusion is not justified: it is probable that the F factor, and its relatives, are peculiar in a chance high sensitivity of replication to acridines, which may not be a property shared by all episome “replicons” (Jacob and Brenner, 1963). Another well-known genetic effect of acridines, such as acriflavine, is their ability to give rise to cytoplasmic variants (e.g., Ephrussi, 1953). Present indications are that such effects, in involving cytoplasmic DNA’s, may be analogous to those involving bacterial episomes. (b) ICR Compounds. Whatever the precise explanation turns out to be, it is a fact that the ordinary acridines fail to yield mutants in most systems. Recently a new class of monofunctional nitrogen mustard derivatives of acridines, known as ICR compounds (after the Institute for Cancer Research, Philadelphia, where they were developed), has become available : at present only on a very limited scale, but perhaps commercially by the time this Chapter is published. These compounds cause appreciable numbers of mutations of the frame-shift type, at rather high survival levels, in Salmonella (Ames and Whitfield, 1966) and Neurospora (Malling, 1967); they are


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also known to be mutagenic for Drosophila (Refs. in Malling, 1967), so it looks as if they will become mutagens of general applicability.

C. Other mutagenic treatments Few mutagenic treatments except those involving radiations or chemical mutagens have found applications in routine mutant-induction, because they are either too laborious or too specialized. For example the introduction of one of the known mutator genes into a particular strain of E. coli, resulting in a greatly increased “spontaneous” mutation rate (for example up to 10% auxotrophs for the mutator known as astasia: Zamenhof, 1966), is not likely to provide a convenient routine procedure of mutagenesis. One treatment is worthy of mention because of its simplicity, even though its applicability is limited to bacteria with heat-resistant spores. Chiasson and Zamenhof (1965) heated spores of Bacillus subtilis in vaczm at a temperature of 115°C for short periods and found up to 23% of auxotrophs amongst the colonies that developed from the surviving spores.

V. METHODS O F M U T A N T ISOLATION A. The design of screening procedures 1. The basic idea The most important factor contributing to the successful isolation of a particular kind of mutant is an efficient screening procedure: that is a method for distinguishing the mutant strain with a minimum of observation or manipulation from its non-mutant brethren, which may, in the case of a particular well-defined kind of mutant, outnumber it by many millions to one. Time and effort are usually better spent in devising and perfecting a screening procedure which will result in easy, or even automatic, recognition of the rare mutant class, than in the laborious isolation and testing of random survivors of a mutagenic treatment until the desired mutant is happened upon by chance. As we see in the remaining Sections of this Chapter, the ease with which different mutants can be recognized varies enormously, but it is almost always possible to increase the efficiency of screening, above the basal level of random testing of survivors, by considering the expected properties of the mutant and how to render one of them economically manifest, even if the ultimate goal of “automatic” selection cannot be reached.

2. Plate tests (a) Resolution of the test. One of the basic props of the microbiologist is the Petri dish containing agar medium, on which the final step of most cloning procedures is performed. Since cloning, as we saw above, is indispensable

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in mutant isolation, it follows that the most efficient screening procedures operate directly on the plates used for cloning (or on replicas of these, when the plate test is destructive: see below). Thus an often profitable approach to a new problem of mutant isolation is to try to devise a plate test that will render the mutant colonies directly recognizable on the cloning plates. The number of colonies that can be screened on a single plate (which determines the “resolution” of the test) limits the efficiency of the whole procedure, and varies widely with the growth habit of the organism and with the nature of the plate test: a “histochemical” reaction, which stains the cells of the colony, has a much higher resolution than a test depending on the production, or lack of it, of a zone of colour, or clearing, around the colonies, because such zones coalesce, and may therefore obscure the presence of mutant colonies if their density is too high. T h e resolution can be improved by manipulating the plate test itself to reduce the fusing of reaction zones of adjacent colonies, as we see when discussing particular examples. Something can also be done to improve the resolution by modifying the growth habit of the organism. (b) Compact colony procedures. Some microbes, such as yeasts, Chlamydomonas, streptomycetes and many other bacteria, grow as small compact colonies on agar ; the colonies reach a few millimetres in diameter after a few days’ incubation and stop growing before they lose their identity by fusion with neighbouring colonies. It is then easy to count many hundreds or even thousands of colonies on the surface of a plate and possibly to recognize and isolate a mutant among such a large number of its brethren. Highly motile organisms may give trouble by moving about on the medium, but this can usually be overcome by the elementary precaution of drying the plates before use. Otherwise it may be desirable first to obtain a non-motile mutant and use this in subsequent mutant hunts. The colonies of filamentous fungi are usually less amenable to plate tests, because they spread so rapidly over the agar surface (Neurospora is a notorious example) that few colonies may be screened at a time. Environmental manipulation can often greatly improve the resolution. Early in the study of Neurospora genetics, sorbose added to the medium was found to restrict the rate of colony expansion, so that colony coalescence was hampered. More recently, Mackintosh and Pritchard (1963) found, following up an earlier observation of others, that anionic detergents, like sodium dodecyl sulphate and sodium deoxycholate, caused Aspergillus to grow in very compact colonies, so that up to 300 separate colonies could easily be handled on a plate (Fig. l), and moreover they were replicated easily by means of velvet (Lederberg and Lederberg, 1952: see later). Genetic control of colony expansion may also be harnessed to improve the


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FIG.1. Compact colony procedures. Environmental control of colony size. Equal volumes of a conidial suspension of Aspergillus nidulans were spread on these two plates; (a) normal medium; (b) the same medium plus 0.08% deoxycholate. (From Mackintoshand Pritchard,1963 ;reproducedby permission of Cambridge University Press.)

resolution of a plate test. Again in Neurospora, “colonial” mutants were early used to keep the organism in its place (Barratt and Garnjobst, 1949; Jensen et al., 1951), so that many discretely separated colonies could easily be scored on a plate. An extreme example is the colonial mutant strain K3/17 of Neurospora developed by K ~ l m a r kand used in many experiments on mutagenesis (e.g., Kolmark, 1956), whose linear growth rate is about one millimetre per week, under standard conditions, whereas the wild type covers this distance in about 15 min! (H. G. Kdmark, personal communication). Fig. 2 shows another example : the difference between the wild-type Ophiostoma multiannulatum (Fig. 2a) and a compact-colony mutant (Fig. 2b) : amycelial-14 (Kdmark, 1965). Probably some environmental or genetic modification can always be found to produce compact colonial morphology if the needs of the experimenter are sufficiently pressing to make its development worthwhile. (c) Determination of viable counts. It is very rarely possible to control a mutagenic treatment sufficiently precisely to predict the viable count accurately enough for plating at the optimal density for a particular platetest. Consequently it is usually best to carry out a pilot plating, designed merely to determine the viable count, after each mutagenic treatment, and to store the mutated organisms meanwhile under conditions which

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FIG.2. Compact colony procedures. Genetic control of colony size. (a) Wild-type Ophiostoma multiannulatum; (b) colonial mutant amycelial-14 (photographs kindly supplied by Dr. H. G . Kelmark).

cause no change in the viable count, until a sufficiently large-scale plating for the job in hand can be carried out at a precisely determined colony density; if for some reason storage is impracticable, platings at a range of dilutions must be made, some of which will result in unusable plates. Several factors must be controlled if viable counts are to remain constant on storage; for example the action of the mutagen must be stopped completely before storage, subsequent reactivation must be avoided, and the same medium must be used for the pilot and main platings, since survival rates may vary by a large factor on different media after mutagenesis. A well-known example of the effect of the plating medium is the much greater kill and mutagenesis effected by a given dose of UV if plating is carried out on a rich medium, instead of a “minimal” medium (Witkin, 1956).

B. Selective methods An article by Schlegel and Jannasch (1967), part of which covers some of the same ground as this section, appeared after the completion of this Chapter; it may be consulted for some further examples of selective methods of mutant isolation. 1, The need for delayed selection The ideally efficient screening procedure results in automatic recovery of the desired class of mutant, usually because it grows on a medium on which the parent strain did not (for example drug-resistant mutants), or because the non-mutant individuals die (as in penicillin selection or


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starvation enrichment) or are removed (as in filtration enrichment) during a procedure interposed between the mutagenic treatment and cloning. In this type of procedure it is more important than in the case of nonselective procedures to allow for delays in the establishment of pure clones, because of hybridity of the DNA or heterokaryosis of the cell, as well as the possible occurrence of phenotypic lag;that is a delay between the establishment of a homozygous mutation in the genome and complete manifestation of the new character (or complete loss of the original character) by the cell. T o give a specific example, resistance to T1 phage in E. coli occurs by a loss of specific phage-receptor sites on the bacterial cell-wall ; the resistant cell fails to adsorb the phage and escapes infection. When the allele responsible for phage susceptibility is replaced by the allele conferring resistance, no further receptor sites are synthesized, but pre-formed sites are not destroyed. Therefore the cell remains susceptible to phage infection, and so do its descendants, until the receptor sites are diluted out sufficiently by new cell-wall synthesis; this may take several cell-divisions (Hayes, 1964: p. 201). It follows that time for these cell-divisions must be allowed after mutagenesis and before exposure to selective conditions (in this case the phage) if the mutants are not to be killed. In the case of unicellular organisms, such as many bacteria, yeasts, or algae, this can be arranged by growth in liquid medium for a few generations before selective conditions are applied, and such an opportunity for mutant expression is always provided in mutant screening procedures with these organisms. I n the case of filamentous organisms, such as moulds and actinomycetes, the problem is not so easily overcome, although something can be done, at least when isolating resistant mutants. For example it may be possible (assuming that we are not dealing with an inhibitor like a virus which kills by a one-hit process) to operate on the differential growth rates of mutant and starting strains by making point inoculations (stabs) with large numbers of mutagen-treated organisms on a medium containing a concentration of the inhibitor sufficiently low for the parental cells to grow, but slowly, long enough for the segregation and full manifestation of resistance; resistant clones then emerge as fastgrowing sectors on the margin of the advancing front of growth. Alternatively cells can be plated immediately after mutagenesis and the inhibitor applied as a spray or overlay, or by transfer of the micro-colonies on a membrane to a fresh plate of medium containing inhibitor, after an appropriate number of cell divisions have occurred. Apirion (1965) isolated mutants whose resistance was only slightly greater than that of the starting strain by inoculating Aspergillus on the surface of inhibitor-containing agar and, after some hours’ incubation, overlaying with a further layer of the same medium. Resistant mutants were recognized because they were first to reach the surface of the overlay and develop there into colonies.

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Once the necessity for delayed selection is realized, it is usually not difficult to provide an opportunity for it to occur in any particular situation.

2. Direct selective methods (a) Resistant mutants. T h e normal first step in isolating mutants resistant to a chemical agent (antibiotic, metabolite analogue, metal ion) is to determine, by plating on a graded series of inhibitor levels, the minimum concentration that prevents growth of the starting strain. This should be determined on heavily inoculated plates, such as will subsequently be employed in mutant isolation; the cells in a massive inoculation may be able to grow synergistically at a concentration of inhibitor that prevents growth of isolated plating units. Resistant mutants can then be sought by plating large numbers of cells on a higher concentration of the substance. Any colonies that appear are sub-cultured and tested over a range of inhibitor concentrations to determine the upper limit of their tolerance. It is worth bearing in mind that the level of sensitivity to an inhibitor may vary markedly at different stages of the life cycle; for example ascospores and conidia of some strains of Neurospora differed greatly in acriflavine resistance (C. Auerbach, personal communication). The choice of inhibitor concentration in the plates used for the selection of the mutants depends on several considerations. If there is reason to believe that mutants tolerating an inhibitor level many times (perhaps hundreds) higher than that inhibiting the starting strain are obtainable, and these are the only mutants sought, then such a high level can be used in order to narrow the search. On the other hand if mutants with modest levels of resistance are also required, or may be the only ones obtainable, obviously a lower inhibitor level is needed. In general, antibiotics fall into two classes according to the range of resistance-levels conferred by individual gene mutations : the famous penicillin and streptomycin classes (summarized by Braun, 1965, pp. 117-1 19). In the former, no single mutation confers a high level of resistance, but a multiple-mutant strain can be built up in which the cumulative effects of several mutations result in a high level of resistance. I n the latter, different individual gene mutations confer very different levels of resistance, including some which result in highly resistant strains. There is no theoretical reason why all inhibitors should fall neatly into one or other of these classes, and almost certainly there is a complete range of behaviour between the two extremes. What matters is that a high level of resistance can be obtained as a result of a single mutation only to certain inhibitors. These are the ones to be preferred, other considerations apart, if the mutations are simply to be used as genetic markers, since there is then a minimum of


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ambiguity in classifying the resistant and sensitive segregants from a cross, and a minimum likelihood of new mutants arising during a cross and confusing the results. Lower levels of resistance can, of course, be handled if need be, but not always as selected markers in crosses when rare resistant recombinants are to be selected in the presence of large numbers of sensitive organisms. However Eriksson-Grennberg et al. (1965) successfully used low-level ampicillin-resistance (some ten times the wild-type level) as a selected marker in E . coli crosses; Bani; (1959) was also able to select lowlevel penicillin and chloramphenicol-resistant recombinants (this time transductants) in Salmonella, but control platings of the recipient strain yielded appreciable numbers of resistant colonies originating from new mutations. If the sole object is to obtain highly resistant strains, which are not to be subjected to genetic analysis, then there may be no objection to the stepwise build-up of resistance by successive mutation. Several short-cuts in isolating such strains have been described; for example the use of a chemostat with gradually increasing inhibitor concentration (Bryson, 1952), or the gradient-plate technique (Szybalski and Bryson, 1952; summarized by Braun, 1965, p. 120). In this method, medium containing an inhibitory concentration of antibiotic is poured into an inclined Petri dish and allowed to set to form a wedge; the dish is then placed horizontally and antibiotic-free medium poured to form a complementary wedge. Vertical diffusion of antibiotic now establishes a gradient of concentration across the plate, so that when inoculated with a sensitive organism, growth occurs on one side only of the plate, except for isolated colonies of resistant mutants within the zone of inhibition. These can now be streaked in the direction of increasing antibiotic concentration to select further mutants of increased resistance, and so on. The difficulty of using such strains for genetic analysis in crosses with sensitive strains is, of course, that high level resistance does not segregate as a simple alternative to sensitivity because its expression depends on the simultaneous presence of several mutant alleles ; thus recombinants with all degrees of resistance between those of the two parents are produced (Cavalli and Maccacaro, 1952). T h e only way of analysing the results is by the techniques of quantitative genetics, and the formal genetics of few microbes is at present well-enough advanced for this to be a profitable undertaking. The isolation of mutants resistant to agents like bacteriophages and bacteriocins is even simpler than in the case of resistance to simple chemical inhibitors, since resistance is usually all or none (often depending on the loss of specific surface receptors) ; therefore the precise concentration of the killing agent is unimportant provided that there is enough to kill all the sensitive cells plated.

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The isolation of radiation-resistant mutants can present special problems because a dose of radiation cannot be chosen that wipes out all organisms of a sensitive strain but leaves all resistant mutant organisms unscathed ; instead the two sub-populations, starting strain and mutants, will differ in the proportion of organisms killed by a particular dose of radiation. If the mutants have a sufficiently enhanced resistance compared with the original strain, it may be possible to isolate resistant mutants simply by irradiating a culture with a dose that kills a very high proportion of all the organisms: Witkin (1947) in her isolation of the famous B/r strain of E. coli (a radiation-resistant mutant of strain B) obtained 4 survivors out of a population of 5 xlO4 organisms, and all turned out to be of the B/r type. If the relative resistance of the mutant compared with the wild-type, or the proportion of mutants in the culture to be screened, had been lower, this direct selection might not have been successful. I n such a situation the alternate irradiation method outlined by Braun (1965, pp. 126-127) may be useful; this consists of irradiating a culture to a moderate survival level, growing the survivors to form a new culture, irradiating this in turn, and so on until resistant mutants are sufficiently preponderant for isolation. It is worth bearing in mind that Hill (1958) used precisely the same technique to isolate one of the first radiation-sensitive mutants of E. coli; 2 . 2 106 ~ bacteria irradiated with UV yielded 22 survivors, and one of the 12 of these that were tested turned out to be of the desired UV-sensitive type ! Most resistant mutants currently used in genetic analysis of the betterknown genetical microbes appear to have been obtained by the very simplest method: by spreading large numbers of sensitive cells or spores on the surface of plates containing the inhibitor, and isolating any colonies that grew up. Often, particularly in the case of filamentous fungi, the organisms had been subjected to mutagenesis immediately before, or even after, plating, with no opportunity for overcoming segregational delay or phenotypic lag. Almost certainly the maximum yield of mutants was therefore not obtained, but this was of little moment because selection was automatic. An indication of some of the inhibitors likely to yield useful genetic markers in a new organism can be obtained by considering the known resistance markers of organisms of the same group. For example, antibiotics like streptomycin, kanamycin, spectinomycin (Davies et al., 1965), azaserine (Siege1 and Bryson, 1967), cycloserine (Curtiss et al., 1965), erythromycin and lincomycin (Aplrion, 1967) have been useful in enteric bacteria, while most of these and several others-bryamycin, micrococcin, neomycin, oleandomycin (Dubnau et al., 1967) and mitomycin (Iyer, 1966)-have yielded resistant mutants in Bacillus subtilis; actidione (Hsu et al., 1965; Wilkie and Lee, 1965), acriflavine (Roper and Kafer, 1957; Hsu, 1962), purine analogues (Morpurgo, 1962; Arlett, 1966) and chlorinated nitro-


385

benzenes (Threlfall, 1968) in fungi; anti-amoebics such as emetine in myxomycetes (Dee, 1962); and acriflavine in bacteriophages (Edgar and Epstein, 1961). I n Staphylococcus (Novick and Roth, 1967) and yeast (Middlekauf et al., 1956; Laskowski, 1956) resistances to a number of metal ions have been obtained. Particularly interesting examples of resistant mutants are those of eukaryotes resistant to antibiotics normally regarded as acting specifically against bacteria; in some cases, at least, the sensitive target of the eukaryote cell turns out to be an organelle such as a mitochondrion having macromolecular similarities with bacteria. For example Wilkie et al. (1967) made use of the fact that yeast cells are sensitive to erythromycin and chloramphenicol, provided that the cells are forced to depend on the mitochondria by growth on a non-fermentable carbon source like glycerol; they were then able to isolate mutants resistant to these antibiotics, some of which arose by mutation in a non-chromosomal determinant, possibly the mitochondrial DNA (Thomas and Wilkie, 1968). In Chlamydomonas reinhardi a number of non-chromosomal, as well as chromosomal, mutants conferring resistance to streptomycin and the related antibiotic neamine have been obtained (see Gillham, 1965, who summarizes the pioneer work of Sager). A peculiarity of this system is mutagenesis by streptomycin for non-chromosomal markers, though not for chromosomal; NTG was mutagenic for both. Although antibiotic resistances were initially used simply as genetic markers, their interest has been enhanced more recently with the discovery that they provide useful tools in the analysis of particular cellular processes, particularly ribosomal function. Altered ribosomes have been implicated in resistance to streptomycin (Flaks et al., 1966), spectinomycin (Davies et al., 1965), erythromycin and lincomycin (Apirion, 1967), and actidione (Cooperetal., 1967). Metabolite (usually amino-acid) analogues (review: Fowden et al., 1967) have recently greatly widened the range of inhibitors for which resistant mutants are obtainable. Even more than in the case of antibiotics, such mutants have usually been sought as tools in the analysis of cellular processes, rather than as general genetic markers, although there is no reason why some of them should not be useful as such. Considering only amino-acid analogues, resistant mutants so far isolated have been shown to owe their resistance to mutation in at least five, and probably more, types of gene: (1) mutation in a regulatory gene or (2) in an operator, has resulted in de-repression of biosynthetic enzymes, thereby over-producing the antagonized amino-acid and so overcoming competitive inhibition by the analogue; (3) mutation in the structural gene of a permease for the antagonized amino-acid has resulted in reduced ability to take up the analogue; (4) 16

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mutation in the structural gene of an amino-acyl transfer-RNA synthetase (activating enzyme) has resulted in better discrimination between the antagonized amino-acid and the analogue during protein synthesis; (5) mutation in a structural gene of a biosynthetic enzyme subject to endproduct inhibition has resulted in loss of such feedback control (review: Cohen, 1965); mutation in a transfer-RNA producing gene may also have been involved. Examples of such mutants are summarized in Table I ; the characterization of the mutants listed has not always been adequate to implicate conclusively one of the mechanisms just mentioned. The table may serve as a source of references to conditions for mutant isolation, sources and concentrations of analogues etc. Although the mutants he isolated were not characterized in detail, Adelberg (1958) was among the first to seek over-producing mutants on the basis of analogue-resistance. Large populations of cells were spread on analogue-containing agar medium, and when resistant colonies appeared they were recognized as over-producers if they were surrounded by halos of growth of the analogue-sensitive background population as a result of excretion of the antagonized metabolite by the resistant colony. Some conceptually rather more complicated selective techniques have been used to obtain mutants modified in regulation of carbohydrate metabolism in E. coli or transport of its substrates, and a few admittedly specialized, but illustrative, examples follow. Buttin (1963) selected mutants on a medium containing galactose as sole carbon source, but in the presence of thiomethyl /3-D-galactoside, a non-metabolized inhibitor of induction of the enzymes of galactose metabolism by galactose; the colonies that developed were mutants constitutive for the galactose enzymes. In a rather similar way, Englesberg et al. (1965) selected constitutive mutants for L-arabinose metabolism on L-arabinose as sole carbon source in the presence of D-fucose. Isaacson and Englesberg (1965) obtained L-arabinose permease-less mutants by using the fact that, in a mutant defective in arabinose utilization because of mutation in the structural gene for one of the metabolic enzymes, L-arabinose concentrated by the wild-type permease inside the cell results in an inhibitory salt imbalance; thus secondary mutants resistant to L-arabinose were selected in this primary mutant strain. Loomis and Magasanik (1967) obtained mutants insensitive to catabolite repression essentially on the basis of resistance to glucose. In wild-type cells, glucose prevents fulI induction of the lac operon by P-galactosides; cells were plated on a selective medium containing glucose as inhibitor, isopropyl thio /3-galactoside as inducer, and N-acetyl lactonate as sole source of nitrogen (this compound can be utilized only when the lac operon is induced), and after some 12 days’ incubation, several of the colonies that had grown turned out to be of the desired type!


387

Such selective techniques are not limited to the isolation of mutants altered in carbohydrate metabolism. For example Torriani and Rothman (1961) selected mutants constitutive for alkaline phosphatase in E. coli on a medium containing inorganic phosphate, which represses alkaline phosphatase synthesis by wild-type cells, and p-glycerol phosphate as sole carbon source, which can be utilized only when first hydrolysed by alkaline phosphatase. Resistance to bacteriophages is usually conferred by loss of specific receptors by the sensitive cells, and this fact can sometimes be employed in the selection of mutants lacking particular cellular appendages when these are the sites of phage adsorption. For example Stocker et a1 (1953), and others since them (Joys and Stocker, 1965; Iino and Enomoto, 1966), obtained collections of non-motile mutants of Salmonella typhimurium, either lacking flagella or bearing only non-functional ones, by selection of mutants resistant to phage x which kills only motile cells (Sertic and Boulgakov, 1936; Meynell, 1961). I t is often useful to obtain female (F-) derivatives of male strains of E. coli K12, and these can be selected on the basis of resistance to the male-specific phage MS2 (or others) whose site of attachment to sensitive cells is the sex-pilus, carried exclusively by male bacteria (Crawford and Gesteland, 1964). Another specialized application of selection for phage-resistant mutants is in the isolation of mutants defective in repair of radiation-induced damage to DNA. Howard-Flanders and Theriot (1962) isolated such mutants in E. coli by exposure of populations of mutagen-treated cells to T1 phage that had received a heavy dose of UV; wild-type bacteria reactivated the phage and were killed, while the mutants did not, and survived. Selection for resistance to other agents besides chemicals and viruses can yield mutants with structural modification. For example Brenner and Barnett (1959) obtained mutants of phage T 2 resistant to the osmotic shock caused by rapid dilution from suspension in concentrated sodium chloride solutions; the mutants owed their resistance to an altered head protein resulting in a more robust phage head. (b) Reverse mutations of auxotrophs; suppressors. These are often the most straightforward classes of mutant to isolate, since not only is selection automatic, by plating on a medium lacking the required metabolite, but the selected allele is often (although by no means always in the case of suppressors) dominant to the original allele; thus segregational delay and phenotypic lag are less likely than in the case of resistance mutants to result in loss of mutants by premature exposure to selective conditions. However, more than in the case of resistant mutants, precautions may have to be taken against the possible inhibition of the mutants by very large numbers of

TABLE I

Some isolations of mutants resistant to amino-acidanalogues

Analogue

Antagonized amino-acid

Symbol of gene Mutant? ormutant Type of

Arginine Arginine Arginine Histidine Histidine

DR P AE FB

P

argP mgs TAR hisP

0 TR AE DR DR DR

his0 hisR hiss hisT hisU hisW

Valine

Histidine Histidine Histidine Histidine Histidine Histidine Isoleucine

0

oprA

Valine S,S,S-Trifluoroleucine 5,S,S-Trifluoroleucine 5,5,5-Trifluoroleucine

Isoleucine Leucine Leucine Leucine

FB DR FB FB

Canavanine Canavanine Canavanine 2-Thiazolealanine a-Hydrazino analogue of histidine 1,2,4-Triazolealanine 1,2,4-Triazolealanine 1,2,4-Triazolealanine 1,2,4-Triazolealanine 1,2,4-Triazolealanine 1,2,4Triazolealanine

argR

VR 19 PI-191 FLR92

$1-

Organism

Reference

Escherichia Escherichia Escherichia Salmonella Salmonella

Maas, 1961 Maas, 1965 Hirshfieldetal., 1968 Sheppard, 1964 Shifinetd., 1966

Salmonella Salmonella Salmonella Salmonella Salmonella Salmonella Escherichia

Roth et d.,1966 Roth et aZ., 1966 Roth et al.,1966 Rothetal., 1966 Finketal., 1967 Fink et al., 1967 Ramakrishnan and Adelberg, 1965 Leavitt and Umbarger, 1962 Calvo and Calvo, 1967 Calvo and Calvo, 1967 Websterand Gross,1965

Escherichia Salmonella Salmonella Neurospora

Ethionine Ethionine Ethionine Ethionine a-Methyl methionine Ethionine Nor leucine 3,4-Dehydroproline Azetidine-2-carboxylic acid 3,4-Dehydroproline p-Fluorophenylalanine p-Fluorophenylalanine p-Fluorophenylalanine B-H ydroxynorvaline

Methionine Methionine Methionine Methionine Methionine Methionine Methionine

AE DR P P FB DR DR

Proline

P

Proline Phenylalanine Phenylalanine Phenylalanine Threonine

FB AE DR DR FB

fPA Gif36

Escherichia Escherichia Aspergillus Aspergillus Escherichia

S-Methyltryptophan

Tryptophan

DR

trpR

Escherichia

5-Methyltryptophan 5-Methyltryptophan

Tryptophan Tryptophan Tryptophan Valine

FB

5-MT-R6 mtr

Eschenchia Neurospora Neurospora Escherichia

5-Methyltryptophan a-Aminobutyric acid

P

P 0

methAE r-eth-1 55701 etr metI metK

WP1

PhS fPE

4-MT OPrB

Coprinus Neurospora Neurospora Saccharomyces Salmonella Salmonella Salmonella

Lewis, 1963 Kappy and Metzenberg, 1965 Kappy and Metzenberg, 1965 Sorsoli et al., 1964 Lawrenceetal., 1968, Lawrence etal., 1968 Lawrence etal., 1968

Escherichia

Tristram and Neale, 1968 Baich and Pierson, 1965 Fangman and Neidhardt, 1964 Sinha, 1967 Sinha, 1967 Cohen and Patte, 1963 Cohenand Jacob, 1959; Itoand Crawford. 1965 L Moyed, 1960 Stadler, 1966 Lester, 1966 Ramaluishnan and Adelberg, 1965

s c (

8

3

=! z

0

{

t AE = activating enzyme; DR = de-repressed (molecular basis variable; sometimes a classical regulator gene) ;FB = feedback; 0 = operator; P = permease; T R = tRNA.

2

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D. A. HOPWOOD

individuals of the starting strain (Grigg, 1952); the remedy is to plate cells under selective conditions at a series of different densities to make sure that failure to recover mutants is not due to their suppression by too large a background population of non-growing cells. Some of the pitfalls in this kind of work, particularly if quantitative conclusions are to be drawn, are discussed by Auerbach (1962, pp. 95-110). (Even in the case of resistance, mutants may fail to grow on heavily inoculated plates: Saz and Eagle (1953) found penicillin-resistant cells of pneumococci and streptococci to be suppressed at very high plating densities, and Shaw (1965) found streptomycin-resistant Phytophthora cactorum to be inhibited even at rather low plating densities.) True reverse mutations are not often sought, except in studies of specific mutagenesis and coding which are outside the scope of this chapter, unless to rid a strain of a mutant allele which would interfere with a particular analysis. It is worth remembering that, since true reversal of a mutation demands a spec@ change in the DNA to restore it to the wild-type sequence (a particular substitution, addition or deletion of a base), the change will be mediated only by a certain limited range of mutagens. Strains carrying suppressors of auxotrophic mutants (review: Gorini and Beckwith, 1966) turn up on the same selective plates as true reverse mutants, and have considerably more applications than they. Even if completely uncharacterized functionally, they can provide useful genetic markers. When recessive, they have applications in the selection of homozygous or haploid segregants from heterozygous diploids (Pontecorvo and Kafer, 1958) ; the diploids, homozygous for an auxotrophic mutant and heterozygous for its suppressor, are auxotrophic, whereas segregants homozygous or haploid in respect of the suppressor are prototrophic. Suppressors act in the most diverse ways (often so far unknown); for example a suppressor of a tryptophan auxotroph in Neurospora appeared to act by lowering the intracellular concentration of zinc to a level at which it no longer interfered with the functioning of the mutant enzyme (Suskind and Kurek, 1959). Therefore it is usually a matter of trial and error to determine which of a range of auxotrophic mutants will revert by suppressors and which will not. The suppressors with the most theoretical implications and probably also the most practical applications are the “super-suppressors”. These suppress certain mutations in any of a very large number of genes; often those mutations which, in the best characterized systems (e.g., Stretton et al., 1966), turn out to result in substitution, in the RNA message, of an untranslatable “nonsense” codon (amber, UAG, or ochre, UAA) for the normal amino-acid-specifying triplet. Strains carrying the suppressors, at least in some cases, contain a new species of transfer-RNA capable of inserting an


391

amino-acid in the growing polypeptide in response to the nonsense codon (Smith et al., 1966). A similar explanation accounts for the action of at least some super-suppressors of missense mutations (Carbon et al., 1966). Amber and ochre suppressors have provided a good harvest of widely distributed markers in E. coli (Eggertsson and Adelberg, 1965; Gorini and Beckwith, 1966) and in yeast (Hawthorne and Mortimer, 1963). Still another class of well-characterized suppressors are additional frame-shift mutations in the same gene as the suppressed mutation (Crick et al., 1961; Brammar et al., 1967). Like that of true reverse mutations, from which they cannot be distinguished except by fine genetic or polypeptide analysis, their study is too specialized for the purposes of this Chapter. (c) Selection for new metabolic capabilities. In general, in a search for biochemical mutants, one starts from a wild-type strain and isolates mutants which have lost particular metabolic capabilities, for example the ability to synthesize a required metabolite or to ferment or grow on a carbon source. However it is sometimes possible to obtain mutants, by direct selection, which have acquired a new capability. For example, starting from wild-type E . coli strains which did not grow on glutamate, or a-oxoglutarate, as sole carbon source, because these substances failed to enter the cells, Halpern and Umbarger (1961) selected mutants able to grow on one or other substance, probably because they had acquired permeases for them. Schaefler (1967) obtained mutants of E. coli which, unlike the wild-type, were able to ferment P-glucosides such as salicin or arbutin, by selection on these substances as sole carbon source. In several instances, constitutive mutants have been isolated by direct selection. For example neolactose is a substrate for /3-galactosidase, but a poor inducer of it; this enabled Lederberg (1951) to select constitutive mutants on neolactose as sole carbon source. In a similar way, Loomis and Magasanik (1967) selected mutants constitutive for the lac operon on melibiose, which enters the cell through the lac permease, but induces it poorly. (d) Two-way selection. An ingenious system, potentially of wide application, for selectingforwardand reversemutations in the same gene has been devised by Apirion (1962,1965). The notion was that, when a substance is inhibitory by virtue of a “lethal synthesis”, a mutant may acquire resistance by impairment of such synthesis, and this in turn may result in a nutritional requirement not possessed by the wild-type. In the example provided by Apirion mutants of Aspergillus nidulans resistant to the analogue fluoroacetate were selected by plating on a suitable concentration of the inhibitor. One class of these had simultaneously lost the ability to utilize acetate as sole carbon

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D. A. HOPWOOD

source, so reverse mutations from them could be selected by plating on acetate-containingagar with no other carbon source. Various other selections based on this system were also discussed (Apirion, 1965). Of course there is no guarantee, apriori, that, in such a system, the forward and reverse mutations will in fact occur in the same gene, rather than involve suppressors. Another example of the principle of two-way selection is provided by certain mutants of A . nidulans resistant to p-fluorophenylalanine, which had become auxotrophic for tyrosine (Calvori and Morpurgo, 1966 ; Sinha, 1967). Another system of two-way selection was described by Apirion and Schlessinger (1968) who selected mutants (called nek) of E. coli on the basis of resistance to either neomycin or kanamycin and found them invariably to be cross-resistant to the other antibiotic. The reverse selection, to nek+, was provided by plating on chloramphenicol, since the nek mutants were more sensitive to this antibiotic than nek+ strains.

3. Enrichment methods for auxotrophs These methods do not achieve the ultimate goal of automatic recognitionof the desired mutant class realized by selective methods. However they can result in an enormous increase in the efficiency of a mutant screening programme by enriching, in respect of desired mutants, the population of organisms to be examined individually. The principle underlying the first two methods discussed below is essentially the same : non-mutant organisms in the culture are allowed to “grow” under conditions where synthesis of some essential cellular component is impossible, and the resulting unbalanced growth proves lethal. The desired auxotrophic mutants in the population are unable to grow, because of lack of their required growth factor, and they are thereby spared from unbalanced growth. In the first method, antibiotic enrichment, unbalanced growth is achieved by the action of a drug that specifically interferes with the synthesis of an essential cellular component; in the second method, starwation enrichment, the same end result is achieved by deprivation of a required metabolite specifically needed for this synthesis. It goes without saying that the choice of metabolite for which starvation is practised is critical : starvation for the great majority of metabolites does not prove lethal, otherwise these methods would not enrich the population in respect of auxotrophs! (a) Antibiotic enrichment. Probably the most fruitful single method for isolating mutants has been the penicillin enrichment method applicable to isolating nutritional mutants of bacteria, independently devised by Davis (1948) and by Lederberg and Zinder (1948). This method depends on the fact that penicillin, unlike many antibiotics, kills bacterial cells only if they are growing, because it interferes with the synthesis of the mucopeptide


393

component of the bacterial cell-wall. Thus cell-wall synthesis in sensitive cells growing in the presence of penicillin is unable to keep pace with cell expansion, and the bacteria “burst at the seams” (McQuillan, 1958); if for some reason, notably because it is auxotrophic for a metabolite lacking from the medium, a cell fails to grow, it is spared from the lethal effect of the penicillin. Thus if a population of bacteria, a small proportion of which are auxotrophic mutants, is grown in a minimal medium for a time in the presence of penicillin, a high proportion of the prototrophic organisms are killed, and most of the auxotrophs survive. If the culture is then cloned on a medium lacking penicillin, but containing the growth factors required by the auxotrophic organisms, a very high proportion of the resulting colonies turn out to be auxotrophs. If a particular class of auxotrophs is the only one sought, then the penicillin-containing growth medium can, of course, be supplemented with as many as possible of the growth factors required by other classes of mutants, so that as far as possible, the desired class is the only one protected from the lethal action of the penicillin. Certain precautions have to be taken if the penicillin method is to work effectively in practice. (i) Auxotrophic organisms in the culture of bacteria to be enriched must be depleted of any internal pool of the metabolite in question, for instance by incubation of the culture in buffer, before exposure to penicillin; otherwise the cells will be able to grow for a time in the presence of the penicillin and be killed. (ii) The cell density of the culture must not be too high, or else metabolites released from lysing prototrophic cells will reach a high enough concentration in the medium to cause growth of auxotrophs. (iii) The concentration of penicillin carried over on to the supplemented cloning medium must be reduced to a sub-lethal level; this can usually be ensured by centrifuging the culture and re-suspending in penicillin-free medium, or merely by diluting the culture by an appropriate factor before plating; if these procedures are not practicable, penicillinase may be added to the culture (e.g., Nester et al., 1963). Various improvements in the penicillin method were suggested by Gorini and Kaufman (1960), and their schedule appears to have been followed by most people using the method recently. They were able to treat populations of high cell density by using a much briefer exposure to penicillin than in previous versions of the technique. This was made possible by growing the culture to log phase in minimal medium before adding the penicillin. A second, less critical, modification was to add 20 % sucrose (to render the medium isotonic) and extra magnesium ions at the same time as the penicillin. The prototrophic cells then did not lyse, but were converted to spheroplasts, which were lysed only at the end of the period of exposure to penicillin by returning the cells to hypotonic medium. Thus cross-feeding of auxotrophs by lysing prototrophs was reduced.

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D. A. HOPWOOD

Adelberg and Myers (1953) described a modified penicillin technique in which the various steps in the procedure were performed by adding successive layers of agar medium to Petri dishes; one advantage of the method was that each mutant isolated represented a separate mutational event. Even when all possible precautions are taken, one should not expect, as with any enrichment method, one hundred percent recovery of the auxotrophs present in the original population, nor, alternatively, a random loss of different mutant classes. The method will obviously tend to select against the leakier mutants, since they will often undergo enough growth in the presence of penicillin to succumb to its action. Liika (1963) found that good yields of auxotrophs could be obtained even at very low cell densities in the populations exposed to penicillin (104 cells/ml) and that under these conditions the total number of surviving auxotrophs was higher than at higher cell densities ; thus dzyerential loss of different classes of auxotrophs was probably reduced. Ampicillin, a chemically modified derivative of penicillin, has been used for antibiotic enrichment of amino-acid auxotrophs in E. coli by Molholt (1967). This antibiotic is much more active against Gram-negative bacteria than penicillin and so could be used at a lower concentration. It may find applications in organisms insufficiently sensitive to penicillin. Molholt's schedule, which is reproduced here because of its inaccessibility, used two cycles of ampicillin treatment. Cells were grown to 2 x 108 per ml in glucosesalts medium, then given a heavy dose of UV to survival), before incubation for 3 h at 37" in the same medium plus amino-acids to allow for segregation of recessive mutants. The cells were washed twice in the glucose-salts medium and re-suspended in it for 3 h at 37" for starvation, then 20,ug/ml ampicillin was added and lysis of prototrophs occurred during incubation for 1 h. The cells were centrifuged, re-suspended in glucose-salts medium plus amino-acids and again allowed to reach 108 per ml. This culture, enriched in respect of auxotrophs, was again starved and treated with ampicillin as before. When the cells were finally grown to 108 per ml, after removal of the ampicillin by centrifugation, as many as 90% were auxotrophs. At any point in the procedure, a culture reaching 2 x 108 cellslml was diluted by a factor of ten in the same medium. Cycloserine is another antibiotic which interferes with mucopeptide synthesis, and Curtiss et al. (1965) were able to use it in an analogous fashion to penicillin for auxotroph enrichment, using an essentiallysimilarprocedure. They suggested, but did not test, the idea of using it in combination with penicillin to harness the known synergistic action of the two antibiotics. Antibiotic enrichment has been used on a very limited scale in microbes other than penicillin-sensitivebacteria, but there is no reason why it should not find wide application. The antifungal antibiotic nystatin has been used


395

successfully to enrich for auxotrophs in Sacchiiromyces cerevisiae (Snow, 1966), and could probably be used in other yeast-like fungi such as Ustilago maydis (R. Holliday, personal communication). 2-Deoxyglucose, an inhibitor of yeast cell-wall synthesis, was used for the same purpose in Schizosaccharomyces pombe by Megnet (1965). (b) Starvation enrichment. This method has found sporadic use in bacteria; for example Bauman and Davis (1957) enriched for auxotrophs by starvation for diaminopimelic acid, an amino-acid specifically required for cellwall synthesis, or for thymine, making use of the well-known “thyminelessdeath” of bacteria deprived of thymine. Starvation enrichment has achieved widespread use in the fungi, where general methods of antibiotic enrichment have not been available; there is no reason to believe that it could not be applied successfully to other groups of micro-organisms. Fries (1948) first noticed that cells of wild-type Ophiostoma, which required two vitamins (thiamine and pyridoxin), died at a rapid rate when incubated in a medium adequate for growth except for lack of the essential vitamins. On the other hand, if the strain had an additional unsatisfied growth requirement, it was usually protected from this rapid die-off, presumably because the lethally unbalanced growth of the culture starved of the essential vitamins was prevented by the additional nutritional requirement. Starvation enrichment was used extensively by Pontecorvo et al. (1953) for isolating auxotrophic mutants of Aspergillus nidulans. T h e initial auxotrophic strain, which was used as a starting point for the isolation of additional mutants, required biotin ; strains requiring other growth factors were not nearly as effective. It was not clear why an unsatisfied biotinrequirement led to such rapid death. On the other hand, in Neurospora crassa (Lester and Gross, 1959), Ustilago maydis (Holliday, 1962) and Schizosaccharomyces pombe (Megnet, 1964), the starting strains required inositol, and in this case their rapid death during inositol starvation probably resulted from a failure of cell membrane synthesis to keep pace with cell expansion (Shatkin and Tatum, 1961). T h e practical details of starvation enrichment are very simple. Most conveniently, cells of the population to be enriched are incubated on agar medium lacking the growth factor required by the starting strain and those required by the mutant class(es) to be isolated, for a period long enough to ensure a low survival of the starting strain. (This period has to be determined empirically in a new situation.) Then a layer of agar medium able to support the growth of the desired double mutant class(es) is poured over the plates, or growth factor solutions are pipetted under the layer of agar in the dishes

396

D. A. HOPWOOD

(Megnet, 1964); the population of colonies that grows up should now be rich in the desired mutants. (c) Enrichment by lethalsynthesis. There have been a few successful attempts to enrich for auxotrophic mutants by allowing prototrophic organisms in the population to incorporate an analogue, which subsequently kills them, leaving auxotrophs unharmed. Thus Lubin (1959) incubated cultures in the presence of tritiated thymidine, then stored them in the cold for a week or two. During this time, radioactive decay of the tritium, which had been incorporated preferentially by the prototrophic organisms, killed a high proportion of them so that, on subsequent growth, the cultures contained a greatly increased proportion of auxotrophs. Bonhoeffer and Schaller (1965) grew cultures for several cell generations in minimal medium containing 5-bromouracil, which was incorporated into the DNA of the growing prototrophs, but not of non-growing auxotrophs. Subsequent irradiation with UV of 313 nm wavelength preferentially killed the cells whose DNA had been labelled and resulted in a substantial enrichment in respect of auxotrophs on subsequent plating. Wachsman and Mangalo (1962) grew Bacillus megaterium in the presence of 8-azaguanine, which kills growing cells preferentially, and achieved a striking enrichment in respect of particular classes of auxotrophs; Wachsman and Hogg (1964) used 5-fluorouracil plus uridine for the same purpose. There are doubtless innumerable possibilities for developing enrichment procedures based on lethal synthesis in other groups of micro-organisms. (d) Filtration enrichment. This method, like that of starvation enrichment, appears to have been largely confined to fungi such as Ophiostoma, Neurospora, Coprinus and Aspergillus, but it should be applicableto any filamentous micro-organism. The principle is extremely simple : on incubation of a spore suspension in a liquid minimal medium, prototrophs develop into microcolonies which are filtered off by passage of the suspension through a suitable filter, leaving the filtrate enriched in respect of auxotrophs, which are then recovered by cloning on complete medium. A detailed account of the method is given by Fincham and Day (1963, pp. 54-56); its use in Aspergillus is described by Roberts (1967). Perhaps it is worth mentioning a filtration enrichment of a quite different kind, used by Van de Putte et al. (1964) to isolate mutants of E. coli defective in cell division at high temperature, so that they grew as long filaments. In this case the cellsfailing to pass through the filter (a membrane-filter) were saved and used to seed a new culture, which became enriched in respect of the desired morphological mutants. (e) Enrichment by heat inuctiwation. Iyer (1960) made use of the fact that the high heat tolerance of bacterial spores is lost on germination. Sporulated


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cultures of Bacillus subtilis were incubated in minimal medium until the majority of the spores had germinated, and then heated at boiling point. This heat treatment killed the majority of the prototrophs but not those auxotrophs that had remained ungerminated because of lack of a required growth factor. (f) The arninopterin method for thymineless mutants. This highly specialized method is mentioned here as an example of a very ingenious harnessing of some physiological observations to yield a very efficient enrichment method for a particular desired class of mutant. Aminopterin, by interfering in methylation and hydroxymethylation reactions, inhibits the synthesis of a number of metabolites. It was found empirically that, in certain strains of E. coli, provided 13 substances whose synthesisis likely to be blocked by aminopterin (glycine, leucine, methionine, serine, valine, adenosine, guanosine, cytosine, thymidine, uracil, pantothenic acid, pyridoxin and thiamine) are present in the culture medium, the growth of thymine-requiring mutants was inhibited to a lesser extent by aminopterin than that of prototrophic cells, and this fact was used by Okada et al. (1960) as the basis for a very effective enrichment procedure which sometimes resulted in up to 50% of the survivors of the treatment being thymine auxotrophs. Unfortunately the method was not universally applicable even among enteric bacteria: it did not work for certain substrains of E. coli K12, nor for Salmonella typhimurium. This limitation was overcome by Okada et al. (1962), by omitting the 12 substances previously added, in addition to thymidine and aminopterin, to the selective growth medium. Stacey and Simson (1965) successfully used trimethoprim instead of aminopterin for certain strains of E. coli in which aminopterin was not very effective. In the original version of the method, cells were grown in broth, first without thymidine, then in the presence of aminopterin but without thymidine, and finally in the presence of both substances, before cloning on thymidine-containing agar. Recently a simplified version of the method has been described (Caster, 1967), in which cells are incubated directly on agar containing thymidine and aminopterin (as well as the other substances mentioned above), when a majority of the colonies that develop are thymine auxotrophs, usually of independent origin. 4. Enrichment methods based on discontinuous selection In the situations described in the preceding section, a set of conditions could be established which resulted in a continued selective advantage for the desired mutants, so that selection could be continued until they constituted an acceptable proportion of the surviving population. In other

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situations, like that in the example about to be considered, selective conditions have to be discontinuous, because a permanent selective advantage for the mutants cannot be established. In such situations, methods based on an alternation of selective and non-selective conditions can be successful, each cycle of selection stepping up the proportion of mutants until it reaches a useful level. (See also alternate irradiation: p. 384.) (a) Alternate subculture for constitutive mutants. This method, devised by Cohen-Bazire and Jolit (1953))depends on the fact that mutants constitutive for an enzyme or enzymes necessary to metabolize a substance required for growth, for example because it furnishes the sole carbon, nitrogen or energy source of a culture, have an initial advantage over the original inducible population because they can start growing optimally without delay ; however this advantage lasts only until the cells of the original strain become induced. In the alternate subculture method, the population of cells to be enriched is incubated for a period in a medium which ensures that the metabolite in question has to be utilized for growth to occur; during this period, constitutive mutants are favoured and increase their proportion of the total population. When induction of the non-mutant cells has occurred, the culture is diluted into a medium in which the metabolite is replaced by one not requiring the induced enzyme(s) for its utilization, and grown long enough for de-induction of the inducible cells to occur. The cycle is then repeated, as often as necessary, each period of gr'owth under selective conditions giving an opportunity for an enrichment of the total population in respect of constitutive mutants, until, when the culture is finally plated out, they are numerous enough to be recognized with a minimum of effort. In practice, samples of the culture are assayed periodically for the relevant enzyme(s), under conditions of de-induction, and the culture is plated only when a significant rise in enzyme activity above the wild-type level is observed. The originators of the method used it to isolate mutants constitutive for amylomaltase and P-galactosidase (Cohen-Bazire and Jolit, 1953)) and it has also been used by Buttin (1963) to obtain mutants constitutive for the enzymes of galactose metabolism and by McFall (1964) to obtain constitutivity for D-serine deaminase. In the latter case the constitutive mutants, under selective conditions, had a double advantage over the inducible population because they not only obtained nitrogen for growth by metabolism of the D-serine which constituted the only nitrogen source, but also overcame its inhibitory effect as a competitive inhibitor of p-alanine in pantothenate synthesis. The technique has been discussed in terms of inducible enzymes, but could conceivablybe adapted tothe isolation of constitutivemutants incertain repressible systems.


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5. Miscellaneous enrichment methods (a) Repeated enticement for “behaviour” mutants. Armstrong et al. (1967), in a search for non-chemotactic mutants of E. coli, inoculated a dense suspension of motile cells in the centre of moist Petri dishes of soft agar containing a complex mixture of metabolites. The bacteria, responding chemotactically, spread out from the centre of the dish, the outermost ring of the expanding population of cells following a concentration gradient of the enticing substance, in this case oxygen, and subsidiary rings of smaller diameter following anaerobically utilizable metabolites. Only motile chemotactic organisms moved from the centre in this “directed” fashion, so that the central area of the plate was enriched in respect of non-motile, and motile but non-chemotactic, mutants. Had incubation been continued indefinitely, the population over the entire plate would presumably have become randomized, at least in respect of the two classes of motile bacteria. Instead, suitably early in the procedure, a sample of the cells from the centre of the plate was transferred to a fresh plate of the same medium and the bacteria allowed to race again. After several repetitions of the procedure, motile but non-chemotactic mutants were readily isolated from the enriched population, as well as a variety of non-motile types. (b) Enrichment for mutants with impaired motility. Non-motile mutants of Chlamydomonas moewusii were obtained by Lewin (1954), who made use of the fact that most of the motile cells keep near the surface of a stationary liquid culture, while non-motile cells sink to the bottom. By taking samples of cells from the bottom of a culture and using them to inoculate fresh medium, a considerable enrichment in respect of non-motile cells was obtained, so that on cloning the culture on soft agar medium, colonies of non-motile mutants were easily recognized by their compact nature, as distinct from the diffuse colonies of motile organisms (see p. 409). Lewin (1960) found this simple procedure inadequate for isolating nonmotile mutants of Euglena gracilis and invented an ingenious device in which mutagenesis and selection were combined. T h e upper layers of a liquid culture were irradiated continuously by UV light at 260 nm during a 3-week period of growth, and after this time samples of cells from the bottom of the culture yielded a high proportion of non-motile mutant colonies. During irradiation, wild-type cells, congregating in the upper layers of the culture, had constantly been subjected to the lethal and mutagenic effects of UV, so that the culture had become enormously enriched in respect of non-motile mutants, which fell down out of range of the UV. (c) Enrichment for phage host-range mutants by adsorption. Considerable use has been made of mutants of bacteriophages differing in the range of bacterial hosts to which they can adsorb, the best characterized system being

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the h+-h transition in T2. h mutants, which adsorb to E. coZi B/2 as well as B, can readily be obtained from h+, which adsorb only to B, by direct selection on strain B/2; selection in the opposite direction cannot be made directly, but a population of h phages could be enriched in respect of h+ mutants by allowing the phages to adsorb to cells of strain B/2, which were then removed by centrifugation, taking 90% of the h phages out of circulation and leaving the unadsorbed h+ mutants in the supernatant (Streisinger and Franklin, 1956). (d) Enrichment fw lysozyme-defective mutants of bacteriophage. Streisinger (1963) devised an ingenious enrichment procedure for these mutants in T4. Bacteria were infected at low multiplicity, to ensure that very few bacteria were mixedly infected, and after a few minutes non-adsorbed phages were inactivated by antiserum. Later, chloroform was added, which resulted in lysis, by phage-produced lysozyme, of bacteria infected by wild-type phages, most of which were inactivated by the antiserum. The unlysed bacteria were washed free of antiserum by centrifugation, then lysed by added lysozyme; many of the phages liberated by this procedure were defectivein lysozyme. (e) Enrichmentfor density mutants. It might be expected that many morphological and physiological changes resulting from mutation would be associated with a change in the buoyant density of microbial cells, and that such a density change might be used as the basis of isolation methods for such mutants. Such reasoning was applied by MacDonald et aZ(l967) to isolating mutants of E . coli in which the normal strict relationship between RNA and protein content had been modified; mutant cells with excess RNA were obtained by centrifuging a culture of mutagen-treated cells to equilibrium in gradients of potassium tartrate or caesium sulphate and isolating the small minority of cells that took up a position below the main band.

C. Replica techniques Replica techniques do not differ in principle from the least sophisticated mutant isolation procedure of all: individual testing of the survivors of a mutagenic treatment in respect of particular characteristics until the desired mutants are happened upon by chance. The only, and fundamental, difference is that hundreds of survivors are tested at a blow, thereby enormously increasing the efficiency of the procedure. Probably no microbiological manipulation comes near to the replica plating technique of Lederberg and Lederberg (1952) in terms of the saving of labour that has been achieved since its inception at so little cost. In principle the method uses a multiple inoculating “wire” instead of a single one


40 1

to transfer a sample of cells from each colony in a random (or regular) array on one Petri dish to one or many subsequent dishes, in such a way that the two-dimensional arrangement of the patches of cells deposited on each new dish corresponds faithfully to that of the colonies on the original plate. The genius of the method was to use velvet as the inoculator, the individual fibres of the pile functioning as innumerable inoculating wires, thereby achieving very accurate correspondence between “prints” and “master copy”. Other multiple inoculators have been described, usually based on a regular array of pins or wires (Szybalski, 1956; Roberts, 1959), and these may have applications in special situations, for example in inoculating organisms whose growth habit precludes the use of velvet (for example fungi with lax sporophores or spores that are very easily blown about), but if by environmental or genetic manipulation (see Figs. 1 and 2) the organism can be made amenable to replica plating by velvet, the effort is likely to be well worth while. The practical details of replica plating with velvet are very simple. T h e velvet should have a dense, short pile, to minimize lateral displacement of the individual fibres when pressure is applied, which would lead to poor resolution of the replicas. Discs of velvet rather larger than the Petri dishes in use (about 14 cm diameter for standard 9 cm dishes) are sterilized, either dry (for most bacteria) or damp (for certain fungi like Aspergillus) by autoclaving. Many discs can be stacked together in a suitable container, provided autoclaving lasts long enough for the heat to penetrate the whole stack (say 120°C for 30 min). T o use them, the discs are laid over a wooden, metal or plastic block, about 8 cm in diameter for 9 cm Petri dishes, and held in place by means of a ring rather larger than the block. The block and ring, of course, are not sterilized since they do not come into contact with the inside of the Petri dishes. The plate to be replicated is then placed on the velvet in a particular orientation determined by a reference mark on the dish and enough pressure applied to force the fibres of the pile into the colonies to be sampled; with plastic dishes, which are somewhat flexible, pressure should be applied uniformly over the base of the dish because it may not be distributed satisfactorily if applied only at the edges. The plate is then carefully removed, with no lateral movement, and replaced by the plate to be imprinted, or by several such plates in succession, orientated to correspond to the original plate by means of reference marks. T h e velvet discs are, of course, re-used repeatedly, usually being washed between successive uses; however some people merely brush them at infrequent intervals to prevent any gross accumulationof oldcellsormedium. I t has even been found possible to keep a single velvet pad permanently in position on the replicator and sterilize it for each use by wiping it with a chloroform-soaked rag (Kemp, 1967). 17

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1. Conditional lethal mutants In many situations, a wild-type strain of a micro-organism will grow under tw-o particular sets of environmental conditions, whereas mutants of this strain will fail to grow, or even to survive, under one of these sets of conditions. Such mutants may be described as “conditional lethals”, because lethality is conditional on the operation of a particular environmeptal factor. Examples are provided by auxotrophic mutants, which grow more or less normally provided their nutritional requirements are satisfied by the environment, but fail to do so if these are not satisfied. Two other broad classes of conditional lethals have achieved considerable renown in microbial genetics: sensitive mutants in a wide variety of microbes, and the amber mutants of certain viruses. Replica techniques have played a large part in the isolation of sensitive mutants. The largest class of sensitive mutants are the so-called temperaturesensitives, which grow at one temperature tolerated by the wild-type, but not at another (usually higher). Their usefulness is due to the fact that they arise by mutation in any of a high proportion of the genes of an organism (Horowitz and Leupold, 1951;Epstein et al., 1963); current theory would predict that missense mutations at particular sites in almost any structural gene would, by altering the amino-acid sequence of the corresponding protein, make its correct functioning more sensitive to elevated temperature than that of the wild-type protein. Such mutants not only provide a rich harvest of genetic markers; more important, they allow the recognition and study of genes whose products are indispensable to the cell and cannot be provided from outside, for example the structural genes for the amino-acyl transfer-RNA synthetases (activating enzymes) (Yaniv et al., 1965; Neidhardt, 1966; Bock et al., 1966), ribosomal components (Flaks et al., 1966), or nucleic acid polymerases (De Waard et al., 1965; Mendelson and Gross, 1967). They also make possible experiments in which a gene product is allowed to function during certain periods of time, but not during others, by appropriate temperature shifts: for example Bonhoeffer (1966) investigated, by means of temperature-sensitive DNA polymerase mutants, the effect on recombinant production of shutting off DNA synthesis in male or female E. coli during chromosome transfer, and Bock et al., (1966) used a mutant with a temperature-sensitive valyl tRNA synthetase to study the relationship between amino-acid activation and RNA synthesis. Other, more specific, classes of sensitive mutants are those less tolerant than the wild-type of a wide variety of agents such as radiation, antibiotics, etc. Enrichment methods for one class of conditional lethals in some organisms, namely auxotrophs, have already been discussed. T h e same methods are


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also applicable to isolating temperature-sensitive mutants in the same organisms; many such mutants in bacteria have in fact been obtained by penicillin selection at high temperature. In this section, we look at methods based on replica plating for isolating conditional lethals, where enrichment methods are inapplicable. (a) Auxotrophs. In a search for auxotrophs by replica plating, survivors of a mutagenic treatment are plated on a “complete” medium, if a mixed bag of auxotrophs is sought, or on a defined medium containing only one or a few growth factors, if the search is to be confined to auxotrophs of one or a few classes. Replica plates are made on media lacking the growth factor(s) for which auxotrophs are sought and compared with the originals. T h e amount of effort saved by replica plating in auxotroph isolation can be judged by the following example. In Streptomyces coelicolor, total auxotrophs rarely exceed 0.5% of the survivors of ultraviolet irradiation (Hopwood and Sermonti, 1962). This means that 200 separate inoculations of random survivors would be needed from “complete” medium to “minimal” medium for every auxotroph identified, whereas, since 200 colonies can comfortably be grown on a single plate, a single inoculation by replica plating is all that is required. (b) Sensitive mutants. In isolating such mutants, survivors of mutagenesis are plated under non-stringent conditions and replica plates exposed to the agent to which sensitive mutants are sought. For example, temperaturesensitive mutants are recognized by their failure to grow on replicas from cultures grown at a low temperature and incubated at a higher temperature, at which the wild-type can still grow (Kohiyama et al., 1966: E. coli at 30“ and 40°C; Hopwood, 1966: Streptomyces at 30” and 38°C; Hartwell, 1967: yeast at 23” and 36°C). Radiation-sensitive mutants are identified by failure to grow on replicas subjected to a dose of radiation killing perhaps 50% of wild-type cells, so that no wild-type colony fails to give rise to a patch of growth on the replica plate (Van de Putte et al., 1965: E. coli; Holliday, 1965: Ustilago; Davies, 1967: Chlamydomonas; Harold and Hopwood, 1969: Streptomyces). Most mutants concerned with antibiotic tolerance have decreased sensitivity compared with the wild-type and have been obtained by direct selection as described in a preceding section. However mutants with increased sensitivity have sometimes been isolated, by replica plating on a medium containing a concentration of the antibiotic tolerated by the wild-type, when some sensitive mutants have failed to grow (Sugino, 1966: methylene blue and acridine ; Apirion, 1967: erythromycin and lincomycin). In all these procedures, as in a search for auxotrophs, a proportion of colonies may fail to grow on the replica exposed to stringent conditions

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merely because of a trivial failure to be replicated satisfactorily. Such colonies can be largely eliminated from the hunt by the simple precaution of comparing two replicas from the original plate, the first exposed to stringent conditions (minimal medium, high temperature, radiation, antibiotic, etc.) and the second to non-stringent conditions (complete medium, low temperature, no radiation, no antibiotic, etc.).

2. Mutant salvage from destructive testing As we see in a subsequent section, certain plate tests based on “histochemical” reactions, or other visual procedures for recognizing mutants, kill the organisms. In these situations, replica plating is invaluable because a replica can be prepared before subjecting the original plate to destructive testing, and any mutants subsequently recognized can be rescued from the corresponding colonies on the replica.

3 . Fertility mutants A particularly interesting application of replica plating is in isolating bacterial variants of changed potentiality for sexual reproduction. Jacob and Wollman were the first to employ such a procedure, in their isolation of Hfr variants from cultures of F+ E. coli (see Jacob and Wollman, 1956). A densely inoculated plate of an F+strain growing on a non-selective medium was replicated to a lawn of F- cells plated on a selective medium on which neither they nor the F+ cells were able to grow. After incubation of the replica, a few recombinant colonies appeared. Areas of the original F+ plate corresponding in position to the recombinants were identified, cells from these areas were re-spread on fresh plates, thus enriching in respect of Hfr cells, and the operation repeated. After several such cycles, pure Hfr clones were often obtained. Clark and Margulies (1964) used a replica technique in their isolation of the first recombination-deficient (rec-) mutants of E. coli. Colonies of an F- culture surviving mutagenesis were replicated on to a lawn of Hfr bacteria spread on the surface of a plate of medium selective for one marker from each parent. Corresponding to each wild-type F- colony, a patch of recombinants grew on the replica; two out of 2000 colonies failed to give rise to such patches of recombinants and were subsequently characterized as rec- mutants. (The strategy was successful, of course, because of the normally incomplete transfer of the male chromosome into the female; the zygotes produced from the rec- mutant colonies, therefore, because of the fortunate choice of Hfr strain, normally lacked the reef allele of the male and so were phenotypically Rec-, although the rec- allele is usually recessive.) In Strejhtomyces coelicolor, spores of two parental strains mixed directly


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FIG.3. Recognition of “ultra-fertile” variants in Streptomyces coelicolor A3(2). Colonies of a starting strain on non-selective medium (top left) were replicated to a lawn of spores of a tester-strain on non-selective medium (top right). After incubation, this plate was replicated to selective medium (lower plate). An “ultra-fertile” colony gave rise to a denser patch of recombinants (arrowed). From Hopwood, Harold, Vivian and Ferguson (1969).

on a plate of selective medium normally give rise to recombinant colonies with negligible frequency (Hopwood and Sermonti, 1962); a period of mixed growth is required on non-selective medium before plating. However, by replica plating colonies of one strain on to a lawn of spores of the other strain spread on a plate of selective medium, it was possible to identify variants that were able to undergo “plate-mating” with high efficiency (Hopwood, Harold, Vivian and Ferguson, 1969). A better way of isolating the same class of mutants, which have been called

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“ultra-fertile”, was to replicate the colonies of the first strain to a lawn of spores of the tester strain on non-selectiwe medium and, after a period of incubation, to replicate this plate to the selective medium. Each normal colony gave rise to a small number of recombinants on the selective plate, whereas ultra-fertile mutants gave many more than usual (Fig. 3).

D. Screening by visual examination of colonies In this section we consider the isolation of mutants whose colonies differ, or can be made to differ, in some visually distinct way-in colour, morphology, size-from those of the starting strain. Sometimes use is made of a spontaneous difference in colour, texture or shape of the mutant colonies, but more often such a difference is enhanced or generated by some procedure carried out on the plates, for example incubation on a particular medium, or under specific environmental conditions, or staining of the colonies with a reagent diagnostic for a particular enzyme.

1. Colonial colour variations (a) Mutants with altered pigmentation. Many microbial colonies have a characteristic colour, either because the cells making up the colony are pigmented, as in bacteria like Serratia marcescens (red) or Sarcina lutea (yellow), or algae such as Chlamydomonas in which chlorophyll colours the cells green, or else because certain structures borne on more differentiated colonies are pigmented, as in Aspergillus nidulans (green conidia), Aspergillus niger (black conidia), Neurospora crassa (orange conidia), or Streptomyces coelicolor (fawn aerial spores). Whenever such a distinctive colonial colour is present, mutants showing departures from normal pigmentation can be isolated by visual inspection. In the case of an organism with a compact colonial morphology, rare colour mutants may be recognizable in the presence of very large numbers of non-mutant colonies. For example in Aspergillus or Streptomyces a Petri dish containing a confluent culture arising from many thousands of plated spores may be scanned systematically by means of a stereoscopic microscope at a magnification of about 25 and any patches of non-wild-type colour sub-cultured to fresh medium by means of a fine needle. When colonial morphology is less compact, fewer colonies can be screened on a single plate, but the isolation of colour mutants is nevertheless usually efficient. Amongst the better known genetical microbes, a variety of colour mutants have been obtained in A . nidulans (summary: Dorn, 1967), Aspergillus niger (Lhoas, 1967), N . crassa (summary: Barratt and Ogata, 1966), S t . coelicolor (D. A. Hopwood and H. M. Ferguson, unpublished results), and S. marcescens (Kaplan, 1959).


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(b) Colour as an indicator of morphological change. A possibly more important application of mutant recognition on the basis of colonial colour variation is in the isolation of morphological mutants. For example in B. subtilis (Schaeffer and Ionesco, 1960; Rouyard et al., 1967)) A. nidulans (A. J. Clutterbuck, personal communication) and St. coelicolor (Hopwood, 1967; Hopwood, Wildermuth, and Palmer, 1969), colonies recognized because they lack the colour characteristic of sporulation often turn out, not merely to lack the pigment of the wild-type, but to differ from it in morphology. In all three of these organisms, and doubtless in others, a rich harvest of mutants has been obtained in which the development of the spores or spore-bearing apparatus has been interrupted at different stages up to the point at which pigment deposition normally takes place. The advantage of recognizing the mutant colonies on the basis of colour is, of course, that screening can be carried out at low magnification, or even with the naked eye, whereas if a magnification high enough to reveal the morphological features of the mutants were to be employed, examination of large numbers of colonies would be laborious.

2. Morphological colony variations In some micro-organisms, mutants with altered colonial morphology have been isolated in abundance ;for example in Neurospora, whose mutants, following the traditional practice handed down from Drosophila genetics, have been given such picturesque names as crisp, caulapower, juflyoid and snowjake (see Barratt and Ogata, 1966). Such mutants may be useful as genetic markers even if the basis of the changed morphology remains entirely unknown ; apart from Neurospora, morphological markers have been used on a limited scale in most genetically investigated filamentous fungi: Aspergillus, Coprinus, Schizophyllum, Sordaria. Often, however, it may be possible to associate change in a character of interest with an altered colonial morphology, which can be used to recognize the desired mutant by inspection. There are many examples of changes in the morphology of bacterial colonies associated with changes in characters such as resistance or virulence (see Braun, 1965, pp. 155-163); a particularly rich harvest of mutants, altered in the polysaccharide component of the cell surface, has been obtained in Salmonella by picking colonies departing from the normal smooth colonial texture (see for example: Stocker et al., 1966). Such correlations are often first noticed by chance. For example, Roth, Ant6n and Hartman (1966) found that, among triazolealanine-resistant mutants, colonies of the desired sub-class consisting of strains with de-repressed levels of histidine biosynthetic enzymes had a characteristic wrinkled appearance when growing

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on a rather high concentration of a fermentable sugar. Another example is the distinction between the colonies of true revertants and (partially) suppressed strains which can often be made, usually on the basis of slow growth of the strains carrying suppressors. Probably such findings as these, even if entirely empirical, can be exploited in a wide variety of situations, once the correlation between colonial morphology and the desired mutant character is noticed. Let us now discuss some examples in which a deliberate attempt has been made to reveal a mutant character as a change in colonial morphology. (a) Auxotrophs. In several bacteria, auxotrophic mutants are routinely recognized by plating the suspension to be screened on a minimal medium, enriched with sub-optimal concentrations of growth factors, when the colonies of auxotrophic mutants are recognizable by their minute size (e.g., Eisenstark et al., 1965). The concentrations of growth factors in the medium have to be chosen so that the mutants undergo enough cell divisions to give rise to recognizable colonies, but stop growing long before these colonies reach the size achieved by those of wild-type organisms. Minimal agar medium supplemented with 2% by volume of nutrient broth may serve routinely, but special levels of supplementation may have to be chosen for the recognition of particular classes of auxotrophs. Goldberg et al., (1965) used an ingenious modification of this technique, combining mutagenesis and screening, to isolate auxotrophs of Bacillus cereus by plating on partially-enriched minimal medium containing diethylsulphate and picking the slow-growing sectors induced in a high proportion of the colonies by the action of the mutagen. In filamentous fungi, starvation for a required nutrient sometimes does not result in a slowing in the linear growth-rate, but rather in a restriction of branching, so that the colonies reach normal size but are thin, or “spidery”. Herman and Clutterbuck (1966) found that auxotrophs of A. nidulans fell into two classes according to whether starvation caused the formation of small compact colonies or large “spidery” ones and utilized this observation as the basis of a procedure for identifying auxotrophs. A strain requiring putrescine, starvation for which results in compact colonies, was grown on a limiting concentration of this substance, and “spidery” colonies were picked. These usually turned out to have a second requirement,inadditionto putrescine ; on the normal minimal medium, which contains nitrate as nitrogen source, the great majority lacked the ability to utilize nitrate and grew slowly at the expense of traces of reduced nitrogen in the agar. T h e technique could doubtless be adjusted to yield other desired classes of mutants.


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(b) Plaque-type mutants. The majority of bacteriophage mutants are distinguishable from their wild-type by changes in plaque size or morphology under conditions specific for particular mutant types. For example r (“rapid lysis”) mutants of T 2 and T 4 produce larger plaques than the wildtype, and with sharper margins, when plated on E. coli B; m (“minute”) mutants give small plaques; and tu (“turbid halo”) mutants give plaques with a broad diffuse margin (Doermann, 1953). Temperate bacteriophages produce turbid plaques on hosts they can lysogenize, owing to the growth of lysogenic bacteria within the plaque; mutants defective in lysogenization (“virulent” mutants) can be recognized because they produce clear plaques (Kaiser, 1957). Bresch (1953) used mutants of phage T1 whose plaques differed in colour from those of the wild-type when plated on bacteria growing on a medium containing certain dyes. Certain host-range mutants can be recognized on the basis of plaque morphology by plating on mixed indicator bacteria. For example h mutants of T2, which, unlike h+, can infect E. coli B/2 as well as B, give clear plaques on a mixture of B and B/2 bacteria, while h f phages give turbid plaques because they lyse only the B component of the mixed lawn of cells (Streisinger and Franklin, 1956). Edgar and Lielausis (1964) found that plaques of temperature-sensitive T 4 mutants (ts), which form plaques at 25” but not at 42”, could be recognized visually by plating at 25°C and, after a few hours’ incubation, transferring the plates to 42”. Plaques produced by wildtype phages continued to enlarge, but those of t s mutants were recognized by their being small and sharp-edged; however the efficiency was rather low in that only 5-20% of plaques with this morphology turned out to be tsmutants. An interesting extension of the plaque-type concept has recently been made to the cellular slime-mould Dictyostelium discoideum. This organism is normally cultured on lawns of bacteria growing on agar plates ; the slimemould consumes the bacteria, leaving a “plaque”, and it has been found that mutants producing altered plaque-types can be isolated (Loomis and Ashworth, 1968). (c) Motility mutants. By plating in semi-solid agar medium, in which wildtype colonies have a diffuse appearance owing to migration of the motile cells, it is often possible to distinguish colonies of non-motile mutants by their smaller size and increased density; this technique is applicable to many motile micro-organisms, including bacteria (Leifson and Palen, 1955; Iino and Mitani, 1966) and green algae (Lewin, 1954, 1960). (d) Mating-type mutants. Certain basidiomycetes (Coprinus lagopus and Schizophyllum commune are the best-known genetically) have a complex system of heterothallism such that fruit-bodies develop only in mixed

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cultures of two mycelia which differ in mating-type allele at each of two loci, A and B. If the mycelia have the same allele at one of the loci (say A), but different alleles at the other (say B), a heterokaryotic mycelium is produced, but this normally bears no fruit-bodies. Suppose, however, that a mutation occurs in the A gene in a nucleus of the heterokaryon, to produce a new A allele; this may form a compatible combination with the original A allele and, since two different B alleles are already present in the mycelium, a fruit-body may result, from which nuclei bearing the mutant A allele can be isolated. Thus a morphological change, production of a fruit-body, can be used as an indicator of the presence of a mating-type mutant (see Fincham and Day, 1963, pp. 202-203).

3. Recognition of metabolic defects by staining (a) Indicator media for fermentation. One of the oldest methods for the visual identification of mutants of a particular biochemical type is by using media which indicate the production of acid during carbohydrate metabolism : “fermentation” (Lederberg, 1947). Both fermenting and non-fermenting colonies are able to grow on the media, because a non-fermented carbon source (peptone) is included, but only the fermenters produce acid. Two such media in common use, Eosin Methylene Blue Agar (EMB), due to Levine, and MacConkey Agar, were originally devised for the routine distinction of lactose-positive coliforms from lactose-negative bacteria, but when the lactose is replaced by other single carbohydrates they serve as indicators for the fermentation of these. On EMB agar, fermenting colonies turn dark purple, with a metallic sheen, while on MacConkey Agar they become brick red ; on both media non-fermenting colonies remain transparent and show only the background colour of the medium. Since the colonies themselves are stained, the resolution of the plate test can be high, and mixed colonies are readily detected by being sectored. Levine’s EMB agar has the following compositionEosine methylene blue agar Peptone

K2HP04 Eosine Y Methylene Blue Agar Test carbohydrate Distilled water

The medium is shaken gently before pouring in order to oxidize the methylene blue. MacConkey agar usually has the following composition-


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MacConkey Agar Peptone Bile salts NaCl Neutral Red Crystal Violet Agar Test carbohydrate Distilled water

Full details of these media can be found in the manuals of the suppliers of culture media (Difco, Oxoid, etc.); these firms market the media ready prepared, but with lactose included, so that they are unsuitable for testing the fermentation, by lactose-positive bacteria, of the several other carbohydrates of interest to microbial geneticists. EMB medium can also be made up in a defined (minimal) form, with a non-fermentable carbon source such as succinate to support the growth of the colonies unable to ferment the test carbohydrate (see Meynell and Meynell, 1965, pp. 56-57). Other indicator media for fermentation have been used on occasion. Lederberg (1948) devised a medium containing a tetrazolium salt on which non-fermenting colonies became red owing to the deposition of insoluble formazan as a result of dehydrogenase activity, while fermenting colonies remained uncoloured, because of interference with dehydrogenase activity at acid pH. Schaefler (1967) used a medium containing bromthymol blue as an indicator of acid production during fermentation. (b) Histochemical staining for specijic enzymes. There are many examples of the detection of particular enzyme activities, or lack of them, by reactions carried out directly on plates of colonies, usually by spraying or pouring on a suitable reaction mixture. Examples are provided by tests for alkaline phosphatase and P-galactosidase in E. coli. For alkaline phosphatase, colonies can be sprayed with suitably buffered p-nitrophenylphosphate, a colourless substance, which is attacked by alkaline phosphatase to liberate yellow p-nitrophenol (Garen, 1960). In an analogous fashion nitrophenylgalactoside can be used as an indicator of p-galactosidase activity (Cohen-Bazire and Jolit, 1953, applied it to colonies, previously treated with toluene to liberate the enzyme, on filter paper and observed yellow spots on the paper corresponding to the colonies), and Schaefler and Maas (1967) used p-nitrophenyl P-glucoside for P-glucosidases. The resolution of such tests was increased by Messer and Vielmetter (1965). They eliminated diffusion of colour from the colonies by using a-naphthol phosphate and naphthol P-galactoside (later bromo-naphthyl P-galactoside was found to give better results: W. Messer, personal com-

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munication) and detected the a-naphthol released by the action of alkaline phosphatase and /I-galactosidase respectively by coupling with diazonium salts to form insoluble azo-dyes which were precipitated directly on the cell-surface of the bacteria; in this way negative mutants were detected on plates carrying up to 105 colonies. Novick and Richmond (1965) devised a plate-test of high resolution for detecting penicillinase production in Staphylococcus in which the acid produced by hydrolysis of penicillin changed the colour of an indicator. Such techniques are, of course, applicable to other microbes besides eubacteria. Dorn (1965) used a naphthol-azo-dye technique, of the kind independently employed by Messer and Vielmetter (1965) (see above), for the recognition of both acid and alkaline phosphatase activities in colonies of a filamentous fungus, A. nidulans. Lewis (1968) used a diazo technique to recognize colonies of U . maydis lacking nitrate or nitrite reductase activities. Urease was recognized in colonies of St. coelicolor by detecting, with an indicator, the p H change caused by the production of ammonia when a solution of urea was poured over the colonies (Hopwood, 1965). In this case excessively rapid diffusion of the ammonia during the time required to recognize urease-less colonies was prevented by pouring the reaction mixture (urea and bromthymol blue) over the colonies as a buffered agar overlay (Fig. 4).Kdmark (1965) overcame this problem in a different way, using an amycelial strain of Ophiostoma multiannulatum, by growing colonies on the mesh of a nylon fabric over agar medium, removing the fabric and placing it in a solution of urea and indicator at a suitable pH, when the colonies of urease-less mutants failed to change colour. All these techniques are applicable to the detection of mutants constitutive for the enzyme in question, when it is not already constitutive, as well as those lacking it. For this purpose the organism is grown and stained on a medium on which wild-type colonies are not induced, or are repressed, when colonies that give a positive reaction may be constitutive mutants. There is enormous scope for devising further plate tests for enzyme activities based on known, or new, histochemical staining procedures. Study of a standard work on histochemistry (Pearce, 1961; Barka and Anderson, 1963; Lillie, 1965) may give clues to the choice of suitable procedures. A slightly different “histochemical” procedure was used by Durwald and Hoffmann-Berling (1968) to identify mutants of E. coli defective in nuclease activity. They stained the colonies with a nucleic acid specific dye (for example Giemsa) after incubating them under toluene ; wild-type colonies were almost unstained because nucleases had degraded the nucleic acids during toluene treatment, but colonies lacking a potent nuclease were stained, and such mutants could be isolated from replica plates that had not been exposed to toluene.


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FIG. 4. Agar-civsrlay techniques. Recognition of urease-negative mutants of Streptomyces coelicolor A3(2). Colonies growing on a plate were overlaid with buffered agar containing urea and bromthymol blue. Ammonia produced by the action of urease in wild-type (urease-positive) colonies has turned the granules of indicator blue as a diffuse zone over each colony. A urease-negative colony near the margin of the plate at about 2 o’clock has no halo and remains sharply visible. (Nearby, a minute urease-positive colony is seen.)

(c) Recognition of auxotrophic colonies by staining. Messer and Vielmetter (1965) devised an ingenious method for the visual identification of the colonies of E . coli and B. subtilis auxotrophs, making use of the fact that cells are unable to synthesize an inducible enzyme under conditions of no growth. Colonies were grown on membranes (filters or Cellophane discs) so that they could be transferred without disruption from one medium to another. The first medium (“growth medium”) contained growth factors for the desired mutants and led to no synthesis of the test enzyme, either because it contained high levels of inorganic phosphate (if alkaline phos-

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FIG. 5 . Colonies of Escherichiu coli with auxotrophic sectors (white) stained by the method of Messer and Vielmetter (1965) for alkaline phosphatase using naphthyl-AS-MX-phosphate and Fast Blue R.R. (Photograph kindly supplied by Dr. W. Messer.)

phatase was being used) or lacked P-galactosides (for /3-galactosidase). When the colonies were of a suitable size, they were transferred to a “starvation” medium, of the same composition as the first except for lacking growth factors for the desired mutants, to allow exhaustion of intracellular pools. After a few hours, the colonies were transferred to plates of “induction” medium, still lacking the growth factors, but allowing synthesis of alkaline phosphatase (because it lacked inorganic phosphate) or /3-galactosidase (because it contained lactose). When induction of wild-type colonies had taken place, the plates were stained for enzyme activity (see a previous Section) and auxotrophic colonies, or sectors, were identified by being colourless (Fig. 5). Since the colonies themselves were stained, the resolution of the test was very high; it was possible to isolate auxotrophs from plates carrying 100,000 colonies, examined with a stereoscopic microscope. A method for yeast and yeast-like fungi involves the incorporation of the dye Magdala Red into the growth medium, when the colonies of auxotrophic mutants stain intensely red and are easily distinguishable from the pale prototrophic colonies (Horn and Wilkie, 1966). Included amongst the auxotrophs is a high proportion of respiratory-deficient (“petite”) mutants, which are also revealed by plating on other dyes (Nagai, 1963).


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(d) Accumulation of coloured products. Sometimes a metabolic block caused by mutation results in the accumulation of a coloured material which stains the colonies. For example certain adenine-requiring mutants (“adeninepurple” strains) of fungi accumulate a purple compound, and this can be used to recognize such mutants visually. De Serres and Kpllmark (1958) used this system for quantitative studies of “forward” mutation rates in Neurospora. In yeast, Roman (1956) used a modification of the system for the same purpose; he started with an adenine-purple strain and recognized white mutant colonies, which usually arose by mutations in other genes controlling steps in the synthesis of adenine, before the one blocked in the adenine-purple strain. The same system was used by Nasim and Clarke (1965) in Schizosaccharomyces pombe. In Streptomyces scabies. tyrosinase produced by wild-type colonies causes the production of brown pigment, possibly melanin, in the colonies when they are growing on a medium rich in tyrosine. Tyrosinase-negative colonies could be recognized by lacking brown pigment (Gregory and Vaisey, 1956). Alderson and Scazzochio (1967) used a somewhat different approach in A . nidulans where mutants lacking xanthine dehydrogenase were recognized visually because their colonies remained green on plates containing 2-thioxanthine, whereas wild-type colonies were yellow because 2-thiouric acid, produced by the action of xanthine dehydrogenase, inhibited conversion of yellow to green conidial pigment.

4. Fluorescence techniques In several instances, fluorescent materials, accumulating as a result of a metabolic block, impart a visually recognizable fluorescence to microbial colonies. The accumulation of anthranilic acid, which fluoresces bright blue in long ultraviolet, by certain tryptophan-requiring mutants is a well-known example. Such accumulations are not confined to auxotrophs : Giles and Partridge (1954) found that about 2% of prototrophic colonies of N . crassa surviving irradiation gave a recognizable long UV fluorescence. In at least two instances, specific classes of mutant have been isolated by a colony screening procedure based on fluorescence. Bennoun and Levine (1967) used an ingenious technique to identify mutants of Chlamydomonas reinhardi impaired in photosynthetic ability. They exposed colonies to visible light, filtered to exclude the red part of the spectrum (above 610 nm), when the wild-type colonies showed a faint red fluorescence. Mutants of the desired type, owing to impairment in photosynthetic electron transport, exhibited an increased fluorcscence and could be distinguished visually from the wild-type colonies, or better by photography on red-sensitive film, with a filter passing only light of wavelength longer than 640 nm.

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Eigner and Block (1968) isolated mutants of E. coli defective in nuclease activity by making use of the fact that colonies grown on a medium containing Acridine Orange fluoresced in long UV because of interaction between the acridine and nucleic acids. If the plates of colonies were treated with toluene for 24 h at 37"C, fluorescence was lost because of degradation of nucleic acids by intracellular nucleases. Colonies of mutants lacking a potent nuclease, in their case endonuclease-I (a DNase) continued to fluoresce and could be isolated from a replica plate prepared from the original before toluene treatment; this procedure is a modification of that devised for the same purpose by Durwald and Hoffmann-Berling (1968) described above.

5. Screening by autoradiography Levine (1960) was able to recognize photosynthetic mutants of C.reinhardiwith fairly high efficiency (150 colonies/plate) by illuminating the plates in an inverted position over a source of 14C-labelled carbon dioxide for a few minutes, then replicating the colonies to a sheet of filter paper. This was treated with hydrochloric acid fumes to drive off unfixed carbon dioxide, then an autoradiograph was prepared. Wild-type colonies caused blackening of the emulsion, while the colonies of photosynthetic mutants which had fixed no 1*C, caused no blackening. This method was later superseded by a fluorescence method (see above), but may still find application in particular situations.

E. Screening for diffusible products 1. Plate tests based on zones of extracellular activity There are several instances in which the production, or lack, of an extracellular enzyme, inhibitor, or other material by microbial colonies has been used as the basis of a plate test for mutant recognition. I n these procedures, the colonies may be grown on a diagnostic medium on which a zone of extracellular activity around each colony is recognizable visually, or can be rendered visible by some subsequent treatment carried out on the plate. The resolution of the test is related to the size of the halo around each colony, and this depends on several factors, including the depth of the layer of agar medium. It may happen that, if the medium is deep enough to support satisfactory growth of the colonies, halos are poorly defined because extracellular activity has not spread through the full depth of the medium; or if time is allowed for full penetration, the diameter of the halos becomes so great that few colonies can be screened on a single plate. In these situations, resolution can often be improved by growing the colonies on a basal layer of normal medium and, when they have reached the right size, pouring on a


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thin overlay, as a layer of normal or semi-soft agar, of diagnostic medium. Activity spreads rapidly through this thin layer, and in this way a small, sharply-defined halo may be obtained. If the halos are still too large, a “barrier layer” of agar, possibly containing an adsorbing material such as activated charcoal, may be interposed between the colonies and the diagnostic layer to reduce the halo size (Kelner, 1948). (a) Detection of extracellular enzymes. Extracellular enzymes for which plate tests are readily devised are largely those degrading macromoleculesproteases, amylases, celluloses, chitinases, lipases, pectinases, nucleases; the function of the enzymes may often be nutritional. In these situations it is rare for a single enzyme to degrade the native highly polymerized substrate to its constituent monomers; usually a number of enzymes with differing specificities work in conjunction to achieve this result. When degradation is accomplished only in the presence of the complete set of enzymes, loss of any one results in an interruption of the process and mutants lacking degradative ability should be revealed. More often, there is partial or complete overlap in the roles of certain of the enzymes, so that lack of a single enzyme, as a result of a single-step mutation, may have little or even no effect on overall degradation. This may account for the fact that, although plate tests for amylases and lipases, for example, are available, based on zones of digestion of soluble starch (revealed by iodine-staining) or emulsified tributyrin (revealed by clearing) incorporated in the medium, there appears to have been no report of isolating mutants lacking these enzymes. In less extreme cases one may have to be content to pick single-step mutants with reduced activity, due to the lack of one enzyme, rather than with no activity, and use these as a starting point for the successive isolation of further mutants lacking other enzymes.

Proteases. The commonest indicator medium for the visual recognition of protease production contains milk (for example 1% skim milk) ; proteases diffusing from the colonies solubilize the casein, which accounts for the opacity of the milk, and so give rise to a clear halo (Gorini and Fromageot, 1949). Mutants of A. nidulans (A. W. J. Bufton, personal communication) and B . megaterium (Millet and Aubert, 1964) lacking extracellular protease were obtained by this procedure. Agar containing elastin has been used for detecting microbial colonies secreting elastase (Sbarra et al., 1963). Nucleases. Extracellular ribonuclease and deoxyribonuclease activities can be detected by incorporating crude RNA or DNA in the growth medium (at 0.3-3% depending on the degree of polymerization of the material, etc.) and later precipitating the unhydrolysed nucleic acid as a visible opacity in the medium by flooding the plates with normal hydrochloric acid or

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exposing them to the fumes of concentrated hydrochloric acid ; this acid treatment kills the cells, so a replica is first taken for subsequent mutant isolation. In this way Gesteland (1966) obtained mutants of E. coli lacking ribonuclease-I and R. Holliday (personal communication) isolated mutants of U . maydis lacking the main excreted deoxyribonuclease. Lanyi and Lederberg (1966) devised a non-destructive plate-test for ribonuclease in which the colonies grew on a medium containing RNA and acridine orange, which fluoresced green under ultraviolet illumination, except where extracellular ribonuclease had acted.

FIG.6. Agar-overlay techniques. Recognition of an antibiotic non-producing mutant of Streptomyces coelicolor K673. Colonies growing on a plate were replicated to a fresh plate and, after a few hours’ incubation, were overlaid with agar containing spores of S t . coelicolor A3(2). Haloes of inhibition of A3(2) are seen around each wild-type K673 ; an antibiotic non-producer is seen halfway between the centre and the edge of the dish at 6 o’clock.


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Pectinase. Mutants of Pectobacterium carotovorum with severely reduced ability to degrade pectin were recognized by J. M. Duncan (personal communication) on plates of 1.5 % sodium polypectate gel containing 1yo yeast extract, poured over a basal layer of agar, when wild-type colonies were surrounded by a zone of liquefaction of the pectate gel, while the mutants were not. (b) Detection of antibiotic activity. Kelner (1948) appears to have been the first to use overlays of medium containing a sensitive test organism to detect antibiotic activity of colonies as zones of inhibition of the test organism. The method is applicable to isolating mutants with enhanced, as well as reduced, antibiotic activity. It may therefore have been used in the recognition of high-yielding mutants of some industrially important micro-organisms ; however, in view of the notoriously poor correlations between the size of inhibition zones on test plates and antibiotic yields under industrial conditions, most improved strains have been obtained by assaying antibiotic production of random survivors of mutagenesis under conditions more nearly approximating those in an industrial fermenter. A difficulty in isolating mutants lacking antibiotic activity, especially when dealing with an industrial strain, is that the halos of inhibition surrounding non-mutant colonies may be so large that very few colonies can be screened on a single plate. It may be possible to overcome this difficulty by replica plating a set of colonies to a fresh plate, which is overlaid with the test organism either immediately, or after a suitable time lag. Fig. 6 illustrates the recognition, by this technique, of a mutant of strain K673 of S t . coelicolor which fails to produce the antibiotic, active against strain A3(2) of S t . coelicolor, that is produced by the wild-type K673 strain. VI. CHARACTERIZATION OF MUTANTS The characterization of mutants, once identified, is largely outside the scope of this Chapter. If the mutants are to be used simply as genetic markers, there may in fact be no compelling reason to characterize them in any detail. More often, the studies for which they are to be used will result in their rather precise characterization. For example a stock of non-motile mutants might be isolated (see p. 387) as the raw material for an analysis of the normal structure and function of flagella; different mutants might then be characterized in great detail morphologically, chemically, functionally, and indeed according to any criteria that could be devised, in order to discover in what ways they departed from normality. Often a mutant isolation procedure is so specific that the very process of isolation serves to characterize the mutants very precisely ; for example in the case of many “histochemical” tests designed to reveal specific enzyme

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losses. However at least two commonly isolated mutant classes are so broad that some preliminary characterization of the mutants is essential and may be regarded merely as a slight extension of the process of isolation itself: these classes are auxotrophs and temperature-sensitive mutants. A. Awotrophs The complete characterization of an auxotrophic mutant, arising by a mutation in a structural gene, which has so far been achieved in extremely few, if any, systems, might involve: identification of the enzyme implicated in the auxotrophy; mapping the mutation to its site within the gene; determination of the nature of the change in the DNA at that site (particular base substitution, addition, deletion), the nature of the corresponding change in the polypeptide (amino-acid substitution, premature chaintermination), the effect of this change on the function of the mutant enzyme (lack of or reduced function, altered specificity), and of the changed function on the physiology of the cell (lack of or reduced synthesis of the end-product of the pathway, accumulation of precursors, changed end-product inhibition); evaluating the consequences of such changes for the fitness of the mutant organism compared with the wild-type in mixed populations under varying environmental conditions. . . We are here concerned merely with the preliminaries to the first step of this analysis.

1. Determination of requirements Auxotrophic mutants, unless mutants of a particular restricted class were sought, are often first recognized by their growth on a complex “complete” medium and their failure to grow on a “minimal” medium. I n order to determine which of the many compounds that distinguish the two media, usually amino-acids, water-soluble vitamins and nucleic acid bases, with a smaller number of more complex substances, are actually required by the mutants, at least two approaches are possible : successive approximation and one-step characterization. I n each, the usual first step is to pick the mutant colonies from the original cloning plates on which they arose, possibly purify them by streaking in case they are heterogeneous in respect of mutant and wild-type cells, and then inoculate them in a defined pattern on a “master plate” of complete medium, This plate is then replicated to a series of diagnostic plates of variously supplemented minimal medium. In successive approximation (for example Lederberg, 1950; Pontecorvo et al., 1953), such plates usually contain: a pool of amino-acids (possibly supplied as acid-hydrolysed casein plus tryptophan); a pool of watersoluble vitamins (p-aminobenzoic acid, biotin, choline, inositol, nicotinamide, pantothenic acid, pyridoxin, thiamine) and a pool of nucleic acid bases (either hydrolysed nucleic acid or an artificial mixture of bases).


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Growth on one only of these plates indicates requirement for one or more of the substances in that pool. At this point it is usually best to switch to auxanography (see Pontecorvo, 1949, for a discussion of this technique) to determine which substance is implicated, using one dish to test up to about eight substances from the pool. Mutants which failed to grow on one of the pools may have a multiple requirement or may require a substance present in the complete medium but absent from the pools, and necessitate further testing. In one-step characterization (Holliday, 1956), the master-plate of mutants is replicated to a number of dishes, each containing a pool of substances, arranged in such a way that each substance is present in two different pools; 12 plates are needed to test 36 substances, each plate containing six substances, but the number may be reduced (say to 11 plates for 30 substances, or 10 plates for 25 substances) if experience has shown that certain classes of requirement are negligibly rare. A mutant requiring one substance grows on two plates, which serve to identify it. An advantage of the method is that mutants having multiple requirements are rather easily characterized, by growing on one plate only, provided their requirements are anticipated and incorporated together into one of the pools : well-known examples include requirements for isoleucine plus valine, purines plus thiamine, methionine plus threonine, arginine plus uracil, phenylalanine plus tyrosine plus tryptophan, etc. Apart from this advantage, one-step characterization would seem to be easily superior on the basis of economy : supposing that 25 mutants can be handled on one master plate, only 12 plates are needed for their characterization in respect of up to 36 substances, whereas by the method of successive approximation, at least 3 plus 55 plates are required, assuming that 15 of the mutants need an amino-acid, necessitating the use of three plates for auxanographic testing, while 10 need a vitamin or base, testable on a single plate. However, in practice, a rather large proportion of the growth-reactions on the pools of substances in the one-step method may need confirmation by auxanography, so that many more plates than the minimum number are used. Auxanography also yields semi-quantitative information on growth requirements and information on inhibitions of auxotrophs by other metabolites (Pontecorvo, 1949). Thus, in practice, both methods are usually found to have particular applications, and it is rare to find one used to the exclusion of the other. In general, pools of substances used in the preliminary characterization of auxotrophs, in order to keep the numbers of substances within reasonable limits, consist largely of the end-products of biosynthetic pathways. Once such an end-product is found to satisfy the needs of an auxotroph, the next step is often to discover what known or possible precursors of the endproduct can substitute for it in this role, in order to begin to identify the

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particular biosynthetic step defective in the mutant. T h e choice of substances to test can often be made by referring to previous publications dealing with cognate auxotrophs in other organisms, or by studying one of those invaluable charts of metabolic pathways, in conjunction with several catalogues of the suppliers of fine chemicals. Sources of frustration in such an approach are many, and include: incomplete knowledge of the pathway, lack of availability of known precursors, their extreme lability or their inability to pass permeability barriers of the cell-for example the only precursor of histidine biosynthesis in Salmonella that can substitute for histidine is histidinol, so that mutants of the ten genes specifying the enzymes specifically concerned with histidine synthesis can be grouped into only two classes on the basis of growth on histidine precursors (Ames and Hartman,

1963).

2. Syntrophism Two mutants requiring the same end-product of a pathway can be deduced to be biochemically different, even if nothing is known about the pathway of biosynthesis, or possible growth on precursors, if one mutant is able to “cross-feed” the other; the converse is not true. T h e basis of cross-feeding (syntrophism) is the accumulation of a precursor immediately before a metabolic block by one mutant, its excretion into the medium, uptake by the other mutant, whose metabolic block must be earlier in the pathway of synthesis, and its conversion to the end-product. Reasons for a failure to cross-feed, even though the biochemical blocks in the two mutants bear the right relationship to one another, are many and include failure of the precursor to pass the permeability barriers of the cells, extreme lability of the precursor, or its conversion by the first mutant to a product not on the direct pathway of synthesis of the required nutrient. A test for syntrophism normally consists of streaking the two mutants in close proximity on a medium containing a low concentration of the required growth factor, so that they are able to grow to a limited extent, and looking for a stimulation of growth of one mutant by the other (e.g., Smith, 1961). The principle of syntrophism has recently been applied, in another context, by Deli6, Pigac and Sermonti (1969)to the biochemical grouping of non-antibiotic-producing mutants of tetracycline-synthesizing streptomycetes. Two mutants are grown in close proximity and production of antibiotic by one of the mutants, presumably by conversion to the antibiotic of an intermediate secreted by the other mutant, with a block at a later point in the biosynthetic pathway, is detected by cutting a strip from the culture and laying it over agar containing an antibiotic-sensitive test organism.


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B. Temperature-sensitive mutants Classifying a mixed bag of temperature-sensitive mutants isolated merely on the basis of their normal growth at, say, 30°C but a failure to grow normally at, say, 40°C is a much less straightforward proposition than the characterization of a group of auxotrophs, chiefly because the range of cellular functions that may be implicated is so much wider. Any attempt to classify a group of temperature-sensitive mutants must proceed by subjecting them to a series of tests; the series described by Kohiyama et al. (1966) does not claim to be definitive but serves to illustrate the problems involved. These authors isolated a large number of temperature-sensitive mutants of E . coli by replica plating and studied the following properties of the cultures at 40"C, the temperature at which the mutant phenotype was expressed : change in optical density (whether it increased, though more slowly than in a wild-type culture, remained constant, or decreased due to lysis); survival (whether the viable count increased, remained constant, or decreased) ; incorporation of radioactive leucine (as an indicator of protein synthesis), uracil (for RNA synthesis) or thymine (for DNA synthesis); ability to act as a host to various bacteriophages; morphology and fine structure of the cells. Most of these characters are complex; therefore not surprisingly classification in respect of them did not result in precise identification of the function implicated in temperature-sensitivity. In two cases, however, a more precise identification was possible : when incorporation of labelled thymine ceased preferentially on transfer to N"C, an essential factor in DNA replication was implicated, although not identified with certainty; when incorporation of labelled leucine ceased preferentially at 40"C, protein synthesis was specifically interrupted, and the cause was found to be, in several mutants, a temperature-sensitive valyl-tRNA synthetase (Yaniv et al., 1965). Later a mutant in which synthesis of all three classes of macromolecule, protein, RNA and DNA, ceased within a short time of transfer to 40°C was found to be mutant in a gene concerned with the synthesis of adenylate kinase, which is required for energy production (Cousin, 1967).

VII. CONCLUSION We have seen in this Chapter that, although a suitable mutagenic treatment is an essential factor contributing to an efficient mutant isolation procedure, unless the procedure is selective, the test used to identify the desired class of mutant is of even greater importance. Great ingenuity has been applied by many people in devising efficient tests. Some of the examples

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quoted in this Chapter are so specialized that they are unlikely to be of direct application in new situations, but the reasoning which led to the successful devising of the tests will hopefully help to set the reader’s mind working towards devising suitable tricks for his own use. Microbial genetics is expanding at an exponential rate, and the range of mutants that it is now feasible to isolate is likewise increasing (compare the previously standard linkage map of E. coli of Taylor and Thoman (1964) with its 100 loci with the current map of Taylor and Trotter (1967), which embraces some 220 loci). It follows that many informative and useful procedures of mutant isolation will have been overlooked in writing this Chapter. I apologize to those whose papers I unknowingly ignored, and should be very grateful for information that would help to make a revised version of this Chapter more complete. ACKNOWLEDGMENTS

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allele. A particular form of a gene, that is a unique sequence of nucleotides, occupying a defined position, or locus, on a chromosome. autogamy. The process of internal self-fertilization in Paramecium whereby a heterozygous animal gives rise to completely homozygous individuals. (For cytological details of this nuclear reorganization see Beale, 1954.) awotroph. A strain having a nutritional requirement (for example an aminoacid or vitamin) not possessed by another strain with which it is compared, for example the strain from which the auxotroph arose by mutation. base-analogue.A purine or pyrimidine not naturally found in nucleic acids but capable of being incorporated into it in viwo by virtue of its resemblance to one of the naturally occurring bases. chain-termination.The phenomenon whereby the synthesis of a growing polypeptide chain ceases because of the presence in the messenger RNA of a codon that is not translated (nonsense codon). clone. The descendants of a single cell or individual by processes of nuclear and cell division not involving genetic recombination between cells. cloning. A procedure designed to give rise to recognizably distinct clones. “complete” medium. A complex medium containing the nutritional requirements of as many as possible of the usually encountered auxotrophic mutants, usually by containing such things as amino-acids, water-soluble vitamins, nucleic acid bases, etc. episome. A unit of DNA that can exist and replicate independently of the chromosome of a bacterial cell (autonomously) or alternatively integrated into the chromosome : examples are transfer factors and the genomes of temperate bacteriophages. eukaryotes. All organisms other than bacteria, blue-green algae and viruses (protokaryotes), distinguished by possessing a nuclear membrane. Correlated with this feature are many other differences involving the organization of DNA, chromosome divisions ribosomal structure, etc. forward mutation. A mutation from the wild-type allele to a mutant allele; this usually involves a change in the D N A nucleotide sequence at any one of a considerable number of possible positions (each giving a different mutant allele)compare reverse mutation. frame-shift mutation. An addition or deletion of a number of contiguous nucleotide pairs, not a multiple of three, in a gene such that the reading frame of translation of the corresponding messenger RNA is put out of the normal register. F+, F-, Hfr. Different fertility types of Escherichia coliand related enteric bacteria. F+ and Hfr possess the fertility factor (F) and are male; F- bacteria lack F and are female. Hfr (high frequency recombination) males have F integrated in the chromosome and are very efficient donors of genes during conjugation with F- bacteria; Ff males have F autonomously in the cytoplasm and are inefficient gene donors. genetic markers. The genes that differentiate the parents in a cross, which allow the inheritance of particular chromosomal regions from one or other parent by the progeny to be experimentally determined. genome. In haploid microbes, the DNA representing the total set of chromosomal genes in an organism. heterokaryosis.The presence in the same cytoplasmic unit (cell, coenocyte, etc.) of nuclei of more than one genetic constitution. heterothallism.The existence of two or more mating-types in a microbial species, usually morphologically identical, such that sexual reproduction occurs only between appropriate, different, mating-types.

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inversion. The reversal of the sequence within a segment of a chromosome, relative to a standard or original sequence, as a result of breakage of the chromosome in two positions and re-joining with the included segment in reverse orientation. leaky mutant. A strain not completely lacking the function that distinguishes it from the wild-type; for example a leaky auxotroph does not fail completely to grow on a medium lacking the nutrient required for optimal growth. lysogeny. The condition in which the DNA of a temperate bacteriophage (prophage) forms part of the make-up of a host-bacterium such that prophages are inherited by the descendants of the originally infected bacterium; in the beststudied systems, this usually occurs by integration of the prophage into the host chromosome. metabolic block. An interruption of a biosynthetic pathway as a result of a mutation causing lack or inactivity of the enzyme catalysing a particular reaction step in the pathway. minimal medium. A usually chemically defined medium which is nutritionally the simplest on which the wild-type strain of a micro-organism can grow; auxotrophic mutants, by definition, grow on a minimal medium only when it is supplemented additionally with one or more nutrients. missense mutation.A nucleotide substitution in a gene which results in insertion in its polypeptide product of an amino-acid different from the one present at the corresponding position in the wild-type polypeptide. mutant. An individual or strain differing from the wild-type by one or more mutations. mutator gene. A gene which leads to a greatly increased frequency of mutation of some or all genes of the strain bearing it. non-selected markers. Genetic markers that are not selected by culture on a particular medium so that progeny from a cross are free to vary in respect of allelic pairs of such markers. nonsense mutation. A nucleotide substitution in a gene which results in the presence in the corresponding messenger RNA of a triplet of bases (amber UAG ; ochre UAA; or umber UGA) which cannot be translated into an amino-acid; the result is chain-termination at this position in polypeptide synthesis. operator. A sequence of nucleotides at the beginning of an operon (the end at which transcription is initiated) which is the site of interaction of regulator gene product(s). operon. A group of contiguous genes whose function is co-ordinately regulated. phenotype. The characters expressed by an organism. polar mutation. A mutation that reduces or abolishes the function, not only of the gene in which it occurs, but also of the genes in the same operon on the side away from the operator. protokaryotes. Bacteria and blue-green algae, organisms lacking a nuclear membrane. petite mutant. A mutant of yeast lacking functional mitochondria and therefore growing more slowly than the normal type (that is as small colonies) under aerobic conditions. recombination. The sequence of processes resulting in the combination in the same chromosome or organism of nucleotide sequences initially present in different chromosomes or organisms. regulator gene. A gene whose product, in conjuction with a small molecular


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weight compound (co-repressor or inducer), influences the activity of one or more structural genes. replicon. Any smallest unit of nucleic acid capable of initiating and controlling its own replication. reverse mutation. A mutation from a mutant allele to the wild-type allele; this usuully involves a return of the nucleotide sequence of the DNA to that in the wildtype gene and therefore requires a specific base substitution, addition or deletion (compare forward mutation). segregation. The separation to different cells or organisms of members of a pair of alleles initially present in the same cell or organism. selected marker. A genetic marker whose presence in a growing recombinant clone derived from a cross is ensured by some feature of the medium; for example a gene conferring antibiotic resistance is selected on a medium containing the antibiotic. sex-pilus. A specialized surface appendage borne by male cells (F+and Hfr) of Escherichiu coli which is concerned in conjugation between male and female cells and also bears receptors for certain male-specific bacteriophages. spontaneous mutant. A mutant that arises as a result of an event unknown to or uncontrolled by the experimenter. structural gene. A gene, other than a regulator gene, for which the information encoded in its nucleotide sequence is translated into a polypeptide consisting of a corresponding sequence of amino-acids according to the rules of the genetic code. suppressor mutation. A mutation causing the partial or complete reversal to the wild-type phenotype of a mutant phenotype conferred by a mutation at a different position in the DNA. temperate bacteriophage. A bacteriophage capable of establishing a lysogenic relationship with a bacterium that it infects. transduction. The transfer of bacterial DNA from one organism (donor) to another (recipient) by the agency of a bacteriophage, resulting in the production from the recipient of recombinant progeny. transformation. The transfer of bacterial DNA from one organism (donor) to another (recipient) without the agency of a specialized vector (contrast transduction), resulting in the production from the recipient of recombinant progeny. translocation. The re-arrangement of chromosome segments in new sequences as a result of breakage and re-joining. unequal crossing-over. An exchange of material between homologous chromosomes such that one recombinant chromosome gains and the other loses genetic material (contrast normal crossing-over in which there is no net change in content of the participating chromosomes). virulent bacteriophage. A bacteriophage incapable of establishing a lysogenic relationship with a bacterium that it infects and therefore infection obligately leads to lysis. wild-type. Usually the starting strain of an organism, isolated from nature, from which mutant individuals or strains are subsequently derived.

18


CHAPTER V I I

Improvement of Micro- organisms by Mutation, Hybridization and Selection C. T.CALAM Imperial Chemical Industries Ltd, Pharmaceuticals Division, Alderley Park, Macclesjield, Cheshire, England I. Introduction

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11. Basic Problems in Planning and Initiating Strain Improvement Work

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Selectionof Cultures for Increased Productivity

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I. INTRODUCTION One of the most important, if not the most important, methods of improving the efficiency of fermentation processes is the production of improved strains by mutation and selection. It is hard to get precise information about a subject which is so much tied up with commercial factors, but a long series of mutation and selection steps carried out mainly at the University of Wisconsin by Backus and Stauffer (1955) increased the output of penicillin by Penicillium chrysogenum from 250 to 2500 ,um/ml between 1945 and 1953. Further advances to 8000 or 10,000 ,um/ml have been mentioned in the literature and these were achieved by the same procedure. Alikhanian (1962) gives a number of illustrative examples. Although success cannot be guaranteed, advances of two to four times have often been obtained in a few year’s work, and of ten to twenty times over longer periods, With large

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increases in production, medium modifications are often essential. Improved performance is also often obtained at the same time, as fewer unwanted by-products are produced, resulting in easier extraction and purification of the product, Thus whenever a fermentation process is being used for production purposes the search for improved strains should be regarded as an important aspect of the development programme. The methods used for this type of work are basically similar to those used in research work in microbial genetics. There is therefore no need to attempt to give details of the techniques used since these will be found in other Chapters in this book (see Hopwood, this Volume, p. 363). There are some differences in emphasis, however, and it is these that it is proposed to deal with here, together with a discussion of some of the main problems involved. These subjects have been treated by Calam (1964), by Alikhanian (1962) in a detailed article and a further review has been provided by Bradley (1966). Papers on mutation and selection occur rather widely scattered in the literature. They are conveniently abstracted in “Plant Breeding Abstracts”, under the heading “Economic lower plants”. A book by Esser and Kuenen (1967) reviews fungal genetics from the academic standpoint, and includes interesting material on mutagens and methods of mutation. Great interest was aroused in 195 1when Pontecorvo et al. published details of the parasexual process for the hybridization of fungi. Later several workers have described similar processes in actinomycetes, as well as transformations induced by a phage. It was hoped by Pontecorvo et al. (1951) that the parasexual process would be adapted to industrial strain improvement. Although many interesting results have been recorded, on the whole the method has not proved outstandingly successful and it is proposed to discuss here some of the possible reasons for this. Briefly, it can be said that hybridization of micro-organisms and mutation-selection work are analogous to plant breeding rather than genetics, and although microbial genetics have been studied extensively, virtually nothing has been done on methods of breeding. The background on which to base the choice of mutagens is discussed by Hopwood, this Volume, p. 367. The handling and selection of isolates are based on scientific principles, and these will beexplained in the course of the description of the methods used. This Chapter first discusses the basic factors which are important in planning mutation-selection work. This is followed by an outline of the methods used for mutation including a discussion of the choice of mutagens. After this is an account of parasexual methods for hybridization. The Chapter will be concluded with a description of methods of screening and a discussion of the main problems of mutation and selection and of hybridization.

VII. MUTATION, HYBRIDIZATION AND SELECTION

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11. BASIC PROBLEMS I N PLANNING AND INITIATING STRAIN IMPROVEMENT WORK Strain improvement by mutation and selection depends on the alternate processes of diversification, selection and rediversification, so that better and better strains are successfully picked out and further improved. The method used for diversification is mutation. This process involves changes in the nucleus of the organism which lead to increased productivity. While in the early stages of mutation work considerable advances are usually possible, a time comes when the advances are relatively small and it is at this point that the greatest need arises for careful planning and development of the programme. The search for new and improved commercial strains differs mainly from the selection of mutants for genetic work in that small quantitative changes are sought, rather than clear-cut positive or negative effects. Thus the first task in genetic work is the production of a stock of clearly marked mutant strains with stable well-defined properties. In genetic work an important factor is the initial choice of the species of organism to be used ; for instance, Aspergillus niger was selected because of its well-marked characteristics, rapid growth and other features which have made it so valuable for research purposes. I t proved well worthwhile to spend a long time in achieving this very satisfactory starting point for genetical work. The position is entirely different in commercial strain improvement work. Very often only a single culture is available as a starting point and it is chosen not for its suitability for development but because it produces a particular product. Success in commercial development work usually requires an early start to mutation selection with as rapid a turnover of strains as possible. It is rarely convenient to stop the programme for a general review. Since the criteria in selection are quantitative rather than qualitative not only is the work more laborious and more limited in scale but it is also less clear cut owing to the natural variations which always arise in biological work. For these reasons the whole approach to mutation selection is quite different to that of genetic research, the data available are less clear cut and decisions often have to be based on intuition and experience rather than on the painstaking accuracy which is a feature of genetic research. The two subjects have, however, one thing in common: the need for clear and well-thought-out objectives. Mutation and selection offer so many possibilities that too many requirements may be brought in, for instance not only greater production but also faster growth, lack of various cultural disabilities or the production of a purer product. Too great a broadening of the programme must be strenuously resisted as it can so overload the physical possibilities of selection that only failure will result.

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Thus the first principle is the careful definition of the objective. After this it is necessary to consider the possible rate of advance and the time at which certain targets should be reached, for instance a certain increase in yield. To produce a significant effect usually requires the screening of about 1000 isolates, followed by checks and repeat tests, and a decision should be taken as to whether this should be achieved in 3 say or 6 months or longer. A fast rate of working is usually desirable but undue haste leading to confusion and unreliability is naturally undesirable. A balance should be struck between the requirements of process development and rapid but reliable working. As a result of this it is possible to consider the effort and apparatus required to bring about the desired results. In addition a target should be set in advance against which progress can be assessed. This could well be an advance in yield of so many percent in 12 months, or the screening of so many isolates in that time. This makes it easier to assess the practical difficulties to be overcome and find the main features which are likely to impede progress. In the first place it is advisable to set a relatively easy target. The next question is to determine the method of testing the mutants produced. This usually involves two stages, a production stage in which the organism is grown, followed by an assay of the product. If the process has already been established for some time no difficulty need arise, but when a new product is involved most of these stages may still be relatively unreliable. It is essential before screening starts that both stages are brought to an adequate degree of reliability. The culture stage should be adjusted to give reliable growth of the organism, giving the highest possible yield at the time of harvesting. Shaken cultures are nearly always used, and it is better to use 500 ml flasks and get good results, than small flasks or test tubes which give less reliable results, even though more can be handled. Frequently the product is a mixture of substances, only one of which is desired, and the assay must be carefully worked out so that its significance is understood and the result given is closely related to the concentration of the desired product (for a discussion of the principles of bioassay see Boyce and Roberts, this Series, Vol. 7). Often a really sound method is laborious and unsuitable for mass use, and an inferior but easy method has to be used. This is quite tolerable provided the position is fully understood. Both these questions can present appreciable difficulties, and it may be difficult to arrive at a satisfactory compromise, It is, however, hardly worth starting screening until a soundly based position has been reached. So many variations can arise during mutation work that an unreliable selection method can be fatal to success. On the other hand to put off starting too long can also be detrimental. Once screening has started it is undesirable to change the process frequently. Having established the culture conditions and assay, the weekly


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routine should also be laid down so as to provide an adequate rate of progress. A further discussion of these points will be given later in the appropriate section. T h e final choice to be made is the organism to be mutated. If only one organism is available then no problem arises. Quite frequently several strains are available and the choice is more difficult. Where one gives a higher yield than the others it is better to use it, even if it has some apparent weaknesses. When several strains are available, giving equal results, two or three may be mutated and screened alongside each other, each ieceiving one third of the effort available. One or other will soon reveal an advantageous response to mutation and will provide a basis of further work. It is, however, inadvisable to mutate too many cultures or effort may be spread too thinly over the field to yield useful results. Following the choice of strain and screening methods, it is desirable also to consider the question of the storage and maintainence of strains during the selection programme. This has been discussed by Calam (1964)and is illustrated in the article by Backus and Stauffer (1955).I n selection work several months may elapse between the isolation of a mutant and its final selection for plant trials. It is not safe to rely on keeping a slope and making occasional sub-cultures, and a preservation system involving slopes preserved under mineral oil, soil cultures or lyophilization should be established. If this is not done a variety of difficulties may arise due to real or apparent changes in the culture which prejudice the final trials and frequently involve a considerable amount of unnecessary work. When these questions have been dealt with the programme of work can be laid out on a suitable scale, and the necessary facilities provided. As a rule at least a year’s work should be allowed before a critical assessment of results is attempted.

111. METHODS O F MUTATION AND CHOICE OF MUTAGEN The mutation process involves two steps, the treatment of the organism with the mutagen and then the isolation of the mutants prior to testing and selection. Mutagens commonly used fall into four main groups, ultraviolet light, radiation such as X-rays and gamma-rays, fast neutrons and chemical mutagens. T h e methods used for mutation and for plating and isolating colonies are similar to those described elsewhere in detail in this book (Hopwood, this Volume, p. 370); they will be outlined briefly here in order to provide continuity. The choice of mutagens is by no means easy and an account will be given of those commonly used for industrial selection work.

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It is generally accepted that dose and degree of kill have an effect on the efficiency of the mutation, but optimal conditions can only be found by trial and error.

A. Methods of mutation 1. Ultraviolet light The suspension of spores or cells is placed in a Petri dish situated a few inches beneath an ultraviolet lamp. The suspension should be stirred during the exposure, for instance with a magnetic stirrer or by rocking the dish, in order to ensure that all the spores are subjected to the action of the rays, as these only penetrate a short distance into the suspension. A 15 W bactericidal lamp giving light of wavelength 253 - 7 nm is suitable. Exposures are in the range 4to 20 min depending on the sensitivity of the organism, and kills of 90-99.9 % are usually employed. In order to provide a treated suspension suitable for plating it is advisable to use an initial concentration of cells of the order of lO7/ml. Considerable differences of opinion exist as to the most suitable degree of killing; some workers consider that the greatest number of mutants is obtained with a relatively small kill in the range of 30-70 %. However, the general experience among industrial workers appears to suggest that a higher kill in the order of 90-99% or more is more effective in producing cultures of increased productivity. With long exposures and certain types of ultraviolet lamp heating of the suspension may occur resulting in accidental death of cells from a cause other than that primarily used for mutation. A well-known phenomenon is that of photoreactivation, whereby the spores or cells when exposed to daylight after treatment are reactivated so that it may appear that little or no killing has taken place. It has been suggested (Alikhanian, 1958) that the photoreactivation effect could be used to allow repeated mutation treatments to be applied, thereby giving an increased mutation effect. I n fact experiments on these lines have given some indication that this is a possibility; but in spite of this photoreactivation is usually avoided by keeping the suspensions in the dark or placing them in a refrigerator. The ultraviolet lamp provides a considerable personnel hazard. It should be shielded and dark goggles should be worn by the operator during the mutation process.

2. X-rays and gamma-rays X-rays and gamma-rays have been used to a considerable extent in mutation work although at the present time they do not appear to be regarded as particularly effective. The culture may be exposed to the rays in a small thin-walled test tube or in a plastic container. The culture may be in the


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form of a spore suspension but is more usually exposed as an agar colony. A sporing colony approximately 5 mm in dia., if sporing well, will provide sufficient cells to form the basis of a mutation and selection programme. The action of these radiations is increased by the presence of oxygen and this is usually available when a colony is irradiated in a small tube. If, however, a suspension is being irradiated arrangements should be q a d e for it to be aerated during the process. Commercial X-ray machines may be used for the irradiation, usually with doses of 20,000-50,000 R. Gamma-rays are available from cobalt-60 sources and facilities for irradiation with gammarays can be obtained from the United Kingdom Atomic Energy Authority, Harwell, Berks. The doses for gamma-rays are similar to those used for X-rays. Provided suitable apparatus is available these methods of mutation are extremely convenient; with the doses mentioned kills of the order of 99% may be expected.

3. Fast-neutrons The value of fast-neutrons has been stressed by a number of workers. Mutation is obtained by exposing a colony or cell suspension in a plastic container in an atomic pile. In this country arrangements can be made for fast-neutron irradiation through the United Kingdom Atomic Energy Authority at Harwell. Exposures used are usually 20,000-150,000 rad. Samples are introduced into the pile by means of a probe or pneumatic “rabbit”. To hold the sample itself we have used plastic containers approximately 1.5 cm in dia. and 3-5 cm long, which are readily available commercially. A small colony of the organism on agar is placed in these containers prior to exposure. During transport, before and after exposure, the containers are kept in ice in a vacuum flask.

4. Chemicals Chemical mutagens are extensively used for the induction of mutation. The normal practice is to produce a suspension of spores or cells in buffer, pH 6-7, and then add a small quantity of the mutagen, which may be added as a solid which will dissolve in the buffer or as a concentrated solution prepared beforehand. Mutagens vary considerably in their solubility and stability in solution, and the best way to use them must be determined in each case. Chemicals are often used in conjunction with physical factors such as ultraviolet light. A great variety of chemicals has been used for the induction of mutations. In the early days nitrogen mustard was commonly used (cf., Morpurgo and Sermonti, 1959). In a recent book Kihlman (1966) has described a number of mutagenic chemicals and their action on plant cells. This provides a convenient summary of the position, since although there are many

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differences between plants and micro-organisms, the principles involved are not dissimilar. The following are some of the more important mutagens used at present, with references to papers describing their use. Ethyleneimine, especially in combinationwith ultraviolet light, is a powerful mutagen which is valuable for general use (Alikhanian, 1962). The ethyleneimine is applied for 1 4 h at dilutions of 0.5-1 g/litre, followed by exposure to ultraviolet light after addition of a few crystals of sodium thiosulphate. Diethyl sulphate (1-2 g/lOO ml) may be used in the same way. Diethyl sulphate may also be used as a vapour (Alikhanian and Shishkina, 1967). The use of methyl ethane sulphonate and beta-propiolactone is described by Kihlman (1966), who also mentions the use of azaserine, streptonigrin, 6-mercaptopurine, 8-ethoxycaffeine and other chemicals. N-Methyl-"nitro-N-nitrosoguanidine (MNNG) is a powerful mutagen, described by Adelburg et al. (1965) and by Lingens and Oltmanns (1966) with Saccharomycescerevisiae. Goldat et al. (1967) have described the use of nitrosomethyl urea and other mutagens in work with Actinomyces aureofacim. The use of a variety of mutagens for selection work with FElsarium monileforme has been described by Erohina and Sokolova (1966). Esser and Kuenen (1967) provide a valuable review of the chemicals used for mutation work at present, and give examples of methods of using them and results obtained with fungi. Many of the chemicals are relatively non-toxic so that the degree of kill cannot be used as a guide to efficiency. The chemicals are usually used at concentrationsof about 0.05 M with exposures of 0.5-12 h. It is only possible by experiment to find out what treatments are most productive, but the papers quoted provide a basis for experimentation. It is very difficult to discover the optimal dose for mutation. The production of morphological mutations or of biochemical deficiencies can be used to show that mutation is occurring. However these are not necessarily a good guide to the production of mutations affecting productivity. The latter can only be assessed by studying the distribution of productivity of the isolates which can be expressed in statistical scatter diagrams (cf. Alikhanian, 1962, and other references given in the previous paragraph). Although the interpretation of results is not easy, there is no doubt that the chemical mutagens can give very useful results. Treatment is carried out by adding the chemical to a suspension of cells, which is incubated (preferably with agitation) at a constant temperature (25°C)for various times (5 min to several hours). Sodium thiosulphate crystals are added and the suspension diluted and plated; this should be done as soon as possible after the exposure. Many of the chemical mutagens used at the present time are capable of producing blisters and other harmful effects on the skin. During weighing and other operations precautions


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should be taken to avoid any risk of contact between the mutagens and the skin either directly due to spillages or by flying particles of dust. Rubber gloves should be worn and inhalation of mutagens avoided.

B. Plating and isolating cultures Following mutation the suspension of cells should be diluted appropriately and plated on agar to yield individual colonies. Normally 50-100 colonies should be allowed per plate. The correct dilution can be calculated if the kill is known, but as a rule only an approximate idea of the kill can be obtained and several dilutions are therefore necessary. I n order to avoid unnecessary dilution it is convenient to assume that one of the doses of mutagen will produce a kill of say 99% and to dilute all the samples from each of the exposures as if a kill of 99 % had been achieved. Then it will be found that one of the exposures has given the desired concentration of spores and a satisfactory set of plates. If a different kill is preferred the dilution system can be adjusted accordingly. The medium and conditions for plating and isolating the colonies should have been prepared beforehand. The plates after spreading are incubated for a suitable length of time and the colonies can then be picked off onto slopes and incubated. The colonies on the plates after exposure frequently show great variations in appearance. This, however, is not a true guide to the extent of mutation. When sub-cultured onto slopes the colonies show much less variation and the appearance on the slopes is a much better guide to the effect of mutation. A considerable number of studies have been published, especially by the Russian workers (cf. Alikhanian, 1958), suggesting that optimal conditions for the production of morphological variation are not usually the best for the production of variation in productivity. When mutating moulds or actinomycetes it is normal to plate the spores directly after mutation, If further genetic changes occur during growth these will be within the mycelial threads. With bacteria and yeasts it is the practice of some workers to let the cells subdivide before plating to give any further changes a chance to express themselves and thus avoid the production of unstable forms. In industrial mutation work the occurrence of unstable forms is not usually too harmful, since owing to their instability they soon drop out of the screening procedure. As the circumstances differ with each type of organism, and indeed with each individual organism, this is a matter best dealt with as it arises. If a programme is arranged to avoid all possibility of a mistake, it is likely to become so cumbersome that no progress is made. The slopes which have been obtained from the isolation plates form the basis of the screening programme. The number of isolates to be used will

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depend on the arrangements for screening which have been made and may be from 50 to 200 or more from each mutation. Some extra isolates should be kept so as to ensure that an adequate number is available when the final selections for screening are made. Once the slopes have grown they may be used to produce slopes which are actually used for the screening purposes. The precise number of slopes to be made will depend on the system which is employed, but it is convenient to make several slopes from each culture. These can be used both for the first screen and for a second screen and also for the preparation if necessary of a master culture. Clearly many of these slopes will be discarded after the first screen but considerable time is saved in this way since it is unnecessary to wait while further slopes are prepared for further screening tests to be carried out. Before starting screening the slopes should be carefully examined for morphological effects and also to ensure that good growth has been obtained. The difficulty of producing good slopes at this stage will depend on the properties of the culture. In some cases, especially when spores are formed, this is easy but in other cases special precautions should be taken. For instance, in preparing the first slope from the isolation plates it may be advantageous to remove the whole colony to crush it in a small quantity of water and use the suspension for the inoculation of the preliminary slope of the isolate. Alternatively the suspension could be used to prepare several slopes which will cover the entire screening and selection programme, provided that variability and culture purity are satisfactory.

C. Problems involved in the choice of mutagens for selection work Mutagens vary in their action on the cells. The nucleus may undergo fragmentation, exchanges among chromatids or minor changes in parts of the chromosomes. On the whole mutation effects are exceedingly complex, and when a mutation occurs at one point in the nucleus it frequently appears to have a widespread effect on other parts. Thus when biochemical requirements are induced productivity is usually altered, and strains having the same biochemical requirements often differ in other respects. Mutagens affect the cells in different ways, thus powerful radiations tend to cause extensive chromosomal damage followed by reassembly, while chemicals have more limited effects. For these reasons it is often advisable to use a variety of mutagens in turn so as to enable different effects to be brought into play. This has been discussed by Calam (1964) who gives references to work in this field, while Kihlman (1966) gives a valuable discussion of the effects of chemicals on cells. A feature of selection work is a progressive increase in resistance to mutation as a programme develops. Not only are greater doses required to induce

TABLE I Examples of mutagenic specificity in t h e production of mutants (based on Auerbach, 1966) Significant factor Organism Effect observed Physiological background Ephestiu pupae Ratio of two types of somatic mutations varied greatly depending on dose of X-rays and preliminary storage temperature Sexual isolates bearing the same methionine allele divide Genetic background Yeasts into two classes: (1) diepoxybutane (DEB) increases reversions, (2) DEB does not. UV gives reversions with both classes Escherichia coli Tryptophane allele stable to DEB but mutable after introduction of arginine requirement Nutritional regime Yeast with adenine Methionine in the isolation medium influences the DroDor. I auxotrophy tion of adenine reversions. The effect is weak with HNOz as mutagen, strong with UV Yeast with ad. meth. Methionhe is essential for obtaining adenine reversions auxotrophy Addition of MnClz to the medium selectively suppresses Penicillium mutations to resistance to azaguanine when induced by nitrogen mustard but not by X-rays, UV or diethylsulphate with isolation on complete medium With isolation on complete medium (CM), N-nitrosoOphiostonza methylurethane gave many more his mutants than did UV-light. However numbers were equal on minimal medium +histidine due to suppression of UV-his-mutants by constituents of CM Nature of mutagen Neurospora with Ratio of adenine/inositol reversions increases from 0.6 to 3.9 with DEB as mutagen, when dose is increased and doubleauxotrophy survival falls from 49 to 12 %. The curves response/dose (ad-, inos-) differed widely also when other mutagens were used (UV light, X-rays). Formation of ad+ reversions increased if DEB treated mores were washed and keDt in warm water.

E

f wl

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the same degree of mutation, but the effect on productivity becomes less. This can sometimes be offset by a change in mutagen, but the possibilities become more limited after a time. Alikhanian (1962), has stressed the importance of “great mutations” which radically alter the cells so that after being resistant to mutational changes they once more become plastic and other effects can be induced. Many workers (e.g., Gregory, 1961) have stressed that mutation responses depend more on the organism treated than on the mutagen used. For these reasons a mutation-selection programme should be designed to run with several lines in parallel so that advantage can be taken of favourable responses as they occur in one line or another. This is discussed in Section V. The specificity of the action of mutagens has been discussed by Auerbach (1966). A number of the examples she quotes are summarized in Table I, which also indicates the factor which was significant in the expression of specificity. In mutation work some chemical mutagens often give rise to large numbers of particular biochemical mutants, suggesting that a specific type of mutagenic action is involved, in contrast to this, when improved producers are being sought a change in mutagen is often followed by success. A study of the information given by Auerbach shows how complex the situation can be and stresses the empirical nature of mutation and selection work. A typical mutation-selection programme involves several mutation steps, perhaps twenty or more in number, succeeding each other quite rapidly over a course of time. As will be discussed below, success usually depends more on carrying out successful mutations than on the extent of screening. As many mutations produce little effect it is advisable to provide numerous mutation steps with only a small degree of selection between each. This means that it is necessary to provide a variety of mutagenic procedures so as to avoid the production of resistant cultures and provide opportunities for the appearance of new types of mutation. It is of course very difficult to compare mutations in industrial work. In genetic research mutagens can be assessed by noting the frequency of specific mutations which they produce. In quantitative work the actual mutations involved cannot be identified, assessment is therefore largely a matter of experience. However, some guidance can be obtained by noting the mutagens which continue in use over the years, as these are the ones which are felt to be most reliable and effective. Since mutations are frequently complex, in genetic work agents such as ultraviolet light which produce mutations with a minimum of side effects are frequently used. The object is partly to avoid complications in the interpretation of results, but also to make it easier to introduce further successful mutations later. Powerful mutagens are avoided so as to avoid excessive nuclear damage. These

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problems are not important in strain improvement work where the only objective is to obtain strains giving improved productivity. However, it would be advantageous if a series of mutations could be chosen which would avoid an excessive disturbance to the nucleus and thus allow successful mutations to continue as far as possible. Unfortunately there appears to be no ready method of doing this, though it is probably advisable to alternate mutagens of different types. The nucleus is visualized as a complex structure which can be affected in different ways by mutagens. When several million cells are treated with a mutagen the majority are destroyed. Among the survivors, in some cases the nuclei may have missed the mutagenic action. Where an effect has been produced, weak mutagens will have brought about small changes, while powerful mutagens such as energetic radiations will have completely broken up the chromosomes, which have then reorganized and repaired themselves so that deep-seated changes have been brought about in the structure, The populations resulting will contain mutants showing changes in production and morphology. Changes in productivity may be positive or negative, and are usually of the latter kind. Positive changes may be small (5 % or less) or large (25 yoor more). These are immediately obvious effects. The more fundamental effects induced in the nucleus may mean (a) that further treatment with a particularly weak mutagen can have little or no chance of a further favourable effect, or (b) that big changes produced by the powerful mutagen have again stabilized the nucleus so that further treatment is useless. Alternatively mutation may have left the nucleus in a condition in which considerable further favourable changes are possible. It it, however, a matter of judgement whether subsequent treatment should be mild or strong. It is often considered better to follow one or two mild treatments by strong ones which should throw up a few new strains again receptive to small changes, while a strong mutation should be followed by weaker treatments capable of inducing minor but productive changes. Of the radiations used as mutagens, ultraviolet light is a useful agent which is both convenient and effective. It has a relatively mild effect on the nucleus. Ultraviolet lamps are cheap and easily installed and reproducible results can be obtained without difficulty. Effective results have been obtained with ultraviolet light often over a series of treatments. X-rays and gamma-rays have been used to a considerable extent for increasing productivity. They are not as effective as might be hoped, and for most workers they are not very convenient as the special apparatus required is not always readily available. They are best kept in reserve for occasional use. Fast neutrons are highly recommended by the Russian workers (Zhdanov and Alikhanian, 1964). In a mutation programme with a strain of Streptomyces erythreus (producing erythromycin) they compared the

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effects of fast neutrons, ultraviolet light, diethyl sulphate and a combination of diethyl sulphate and ultraviolet light. Fast neutrons proved the most effective; by comparison the other treatments had relatively little effect. The fast neutrons were particularly effective in producing “plus-variants”, having increased productivity. Fast neutrons have a powerful effect on the nucleus, and it would seem that they could usefully be introduced into mutation programmes at not infrequent intervals. Although it may be troublesome to have to take material to an atomic pile for treatment, this is a relatively easy matter when compared to the generally laborious nature of mutation and screening work. The other treatment which the Russians have found particularly effective is ethyleneimine followed by ultraviolet light (Alikhanian, 1958; 1962). The papers cited above contain much information on screening results, distribution of productivity and the like, and are therefore useful for general reading. When a mutation programme is being planned it is recommended that ultraviolet mutations should be tried in the first place, with kills of 90-99 yo or 99.9 yo.With wild strains this type of mutation usually gives considerable progress in the first instance (cf. Backus and Stauffer, 1955). After two or three treatments in this way a change to ethyleneimine followed by ultraviolet light will often be found effective. At this point it may be advisable to change to a programme of mutations involving fast neutrons, or ultraviolet light with ethyleneimine with occasional use of the chemicals which have been mentioned in the previous paragraph. By the time this work has been done some indications as to the most successful mutagens should have been obtained and at this point judgement can be based on experience and the programme continued in the most productive way.

IV. METHODS OF HYBRIDIZATION

A hybridization method which has aroused a great deal of attention during the last ten years or so is the parasexual process, which allows crossing to take place between asexual organisms such as the fungi imperfecti and actinomycetes. In addition to this a large number of other possibilities exist, such as processes involving the intervention of phages. Sexual crossing is also used, expecially in the case of yeasts.

A. The parasexual process The parasexual process was originally described, in fungi, by Pontecorvo al. (1951). The original patents give a clear account of the methods used and the results obtained. Work of industrial interest with penicillinproducing organisms has been described by Sermonti (1957; 1961) and by Macdonald et al. (1964). This work has been reviewed by Calam (1964);

et


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it contains many valuable details of the methods used and problems involved. Hybridization of actinomycetes by similar methods has been described by Alikhanian (1962), in whose laboratory transformation and induction have also been used in hybridization work. Later Hopwood (1967) has given details of the methods used in crossing strains of Streptomyces coelicolor. Vladimirov and Mindlin (1967) have discussed the problems involved in strain improvement work with actinomycetes. See also Bradley (1966). The following is a description of an experiment on the crossing of two penicillin-producing strains of P. chrysogenum in which penicillin production and sporulation were measured. The method of hybridization was based on that used by Sermonti (1954). Improved methods of working have been described by Macdonald, Hutchinson and Gillet (1963a)b, c). 1. Two media were used, Minimal Medium (MM) (Czapek-Dox) and Complete Medium (CM) [lactose 30 g/litre, corn-steep solids 2.5 g/litre ; pH 6.1-6.3, trace elements solution (MgS04.7HzO 2.5 g/litre, FeS04.7HzO 0.6 g/litre, MnS04.4HzO 0.2 g/litre, CuS04.5HzO 0.2 g/litre) 20 ml/litre, agar 20g/litre]. For subcultures the complete medium was used with corn-steep solids increased to 20 g/litre. Productivity tests were carried out in shaken culture in the usual way. 2. The parental strains used were the Wisconsin mutant Wis. 49-133 and another mutant 301. I n shaken flasks these gave 2000 and 3300 ,um/ml respectively. Deficient parental strains were obtained using ultraviolet light and X-rays respectively. These were non-leaky and gave 1500 ,um/ml (green) and 1925 ,um/ml (white) respectively. They were not further characterized. 3. Mixed spore suspension from the two deficients was plated on M M when a number of colonies developed. A raised colony was transferred to an M M slope when confluent white growth developed, showing sporulation on the raised folds after 17 days. The slope was used to inoculate an 8-02 medical flat bottle containing MM, when again confluent growth occurred from which raised “beehive”-shaped green colonies arose, sporulating freely (18 days); these were regarded as heterokaryotic. I n later work the methods described by Macdonald et al. (1963a, b, c) gave similar results and were more convenient. 4. The spores from the medical flat were plated on M M and CM. At high dilutions on M M only tiny colonies appeared, on CM green and white colonies arose (proportion 64 green : 43 white). These colonies represented the deficient parents whose spores were produced by the heterokaryon. When plated at high concentration (e.g., 104-105 spores/plate) onMM colonies obtained from heterokaryotic spores appeared, and a small number of dark green diploid colonies (twenty-two on sixty plates). These grew very fast

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and sporulated after only 3 days. A typical diploid was chosen as giving homogeneous dark green growth (H61). It had enlarged spores (5.9 x 4.9 pm: parents averaged 4.95 x 4.1 pm) and gave 1580 pm/ml of penicillin in shaken flasks. 5. In order to obtain recombinants the diploid (H61) was mutated via two successive steps. This was done because it seemed to be extremely stable, giving little chance of recombination. After treatment with ethyleneimine (2+7 h at l/SOOO) kills of 99-99.9 % were obtained and on plating 40-70% of small colonies were obtained, the remainder being of the H61 type. Out of 372 colonies fifty-four were biochemically deficient. When tested in shaken flasks the best two gave 3131 and 3051 pm/ml, a dozen others exceeded 2500 pm/ml. These results nearly equalled the best of the parents(301). The best mutant (H61129) was remutated using ethyleneimine (1/500, l+ h) followed by ultraviolet light to give kills up to 99-99.6 %. Two hundred colonies from the plates were tested in shaken flasks, the best penicillin titres reaching 40301155 pm/ml. The sporulation of some of the best strains was tested. Under standard conditions some of the best mutants were equal to or better than the high-yielding parent. Although the hybridization experiment gave an advance in penicillin production, parallel work by mutation alone gave equally good results. The methods used were of the simplest type and it is impossible to draw any conclusions as to the genetical changes involved in the hybridization achieved. This experiment was chosen as an example because it was a simple one which illustrated the various steps involved and the type of results obtained when the parasexual process is applied in strain development. With other types of organism or even with other strains of P. chrysogenum the effects observed may be different. Thus diploid formation may be less clear cut: it does not appear as a separate stage with actinomycetes. In other cases recombination is less clearly developed and it may be confused by reversion of one or other of the biochemically deficient parents. Clearer results are obtained with doubly marked strains, but this increases the preliminary work which has to be done before hybridization can be started. It will be noticed that the example involved four mutation steps, two for the production of biochemically deficient parents, and two to produce high-yielding recombinants. A considerable amount of time was required for the production of heterokaryotic spores for plating to yield diploids. If the parent 301 had been submitted to straightforward mutation and screening procedures, at least four or five successive steps could have been done in this time, probably with a substantial increase in productivity. Thus hybridization is often at a disadvantage when increased production is the


45 1

only objective. This and other problems and possibilities in hybridization will be discussed below.

B. Other methods of hybridization As the writer has no practical experience in these fields, only a brief review will be attempted. The hybridization of yeasts has been of interest for many years, both as a basis for genetical studies and as a means of strain improvement. Lindegren (1949) has given a general account, while Bunker (1961) has reviewed industrial aspects. See also Rose and Harrison (1969). Extensive work on the hybridization of the ascomycete Neurospora crassa played an important role in the development of our knowledge of biosynthetic pathways. In the case of bacteria, hybridization, or transfer of properties, is possible by three routes; conjugation by a sexual process, transduction via a phage and transformation by means of DNA which carries genetic material from one cell to another. Alikhanian and Iljina (1957) reported the mutagenic effect of an actinophage on Actinomyces olivaceous, up to 99% of the isolates representing new forms. The ability to transfer production of streptomycin to a non-producing mutant has also been demonstrated (Alikhanian and Teteryatnik, 1962). Further studies with these strains led to the production of high-yielding variants (Teteryatnik et al., 1962). The performance of different phages and the cross-resistance of strains was also studied. A high frequency of variation has also been obtained in Streptomyces erythreus by this method (Retinskaya and Alikhanian, 1962). The relation between viruses and bacteria and the participation of viruses in the transfer of genetic material is attracting much interest at the present time. Apart from transduction and transformation, parts of viruses can become permanently incorporated into the chromosomal system of bacteria, bringing with them new metabolic activities. Lysogeny is a particularly well-known example of this, while the so-called “transfer factors’’ carry determinants for drug resistance, especially to antibiotics. Production of diphtheria toxin is known to be associated with lysogeny (e.g., Anderson and Cowles, 1958), while penicillinase production is associated with certain plasmids in Staphylococcus aureus (Novick and Richmond, 1965). The existence of all these mechanisms suggests the possibility that they could be used for strain improvement. However, a considerable amount of development work would be necessary for this to be achieved.

C. Problems of hybridization in strain improvement work Having described methods which can be used for hybridization it is now necessary to discuss some of the problems involved in their application.

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This will be mainly centred around parasexual cross-breeding, but the problems involved will be similar in other cases. Technically the work is not particularly difficult, once the various procedures have been mastered. The most serious difficulties arise during the cross, where, even if all goes well, it may not be easy to decide precisely what has taken place, and interpretation of results depends on the complete reliability of the work done. Thus prototrophs may arise on plates because of reversions, or because of infection with a related culture arising from parallel mutation and screening work. It is therefore essential to establish thoroughly sound techniques at all stages of the work. It may be noted that in genetic work well-marked strains are used throughout so as to facilitate checking. In industrial work there is rarely sufficient time for this to be possible. The first stage is the production of suitable biochemically deficient parents. These should come from different biological lines and should preferably be distinguishable from one another. A clearly marked deficiency should be inserted; the deficient strains should be stable and non-leaky. For some reason it is usually difficult to achieve this for some time, but progress seems to become easier with experience. As productivity is the object, the deficients should show, in the case of moulds, no loss of productivity alongside the deficiency. In most cases the deficiency causes a reduction in productivity, and it is necessary to continue the search for deficient strains until this problem is overcome. If moulds with reduced productivity are crossed, there is little prospect of reaching the original level. With actinomycetes this difficulty is not so serious. In genetic work colour-marked strains are used, but this may not be possible in development work and the formation of diploids and recombinants is less easy to detect with certainty. The formation of heterokaryons and diploids is, however, usually fairly easy to observe, since new types of colony commonly arise, diploids giving vigorous growth and spores of increased size. Actinomycetes do not form diploids but here also the various changes involved are not difficult to recognize. The final breakdown of diploids to form recombinants is usually more difficult than the early literature suggests and mutation is nearly always necessary. Ideally ultraviolet light is recommended for producing recombinants, but stronger mutagens may be necessary. Isolates are finally tested by screening. Observations may also be made of the occurrence of biochemical deficiencies and colour changes. Throughout a hybridization programme a series of choices has to be made, which are by no means easy. Thus, it may be hard to decide which pairs of organisms to start from. Ideally these should be from different lines and give roughly equal degrees of production. If they are too far apart genetically it may be difficult to secure crossing. If one is much inferior to


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the other, all that may be achieved is a slight improvement of the inferior partner. Unfortunately in industrial work there is usually only a single mutant line to start from, so that the crosses should lead back to a hypothetical mutual parent. Often, however, two or three mutational lines are available. Basically the crossing of these lines should lead to some degree of improvement. Ideally, the recombination process should give rise to new biochemical deficients which could be used to start new crosses, combining the best features of the various lines available. Unfortunately recombinants tend to be very stable diploids which are in their nature unsuitable for further crossing. As was found by Sermonti (1957) and by Macdonald et al. (1963a, b, c; 1964) the usual result is a small improvement in productivity, no more than would be obtained by mutation. One advantageous result may be the production of a more vigorous culture. This can be valuable since after a long period of mutation, a culture may become weak in growth and sporulation. The reasons for the difficulties which occur in using hybridization for strain improvement have been discussed by Calam (1964) and Bradley (1966), and by Mindlin (1967), and were anticipated by Sermonti (1957). They are in part the microbiological problem of securing the right kinds of diploidization and recombination. I n this connection Daglish and Calam (1968) have described difficulties in crossing strains of Penicillium patulum. In an extensive series of experiments carried out by MacDonald on the hybridization of P.chrysogenum it became clear that diploidization processes are more complex than might be expected; in particular Macdonald (1966) found that diploids of the same two strains but prepared in different ways showed unexpectedly different properties. More particularly, problems arise from the difficulty of obtaining suitable and genuinely different parental lines and in the handling of the large numbers of isolates required when multi-gene crosses are involved. Thus productivity is controlled by several genes rather than by one, and all would have to be altered in the favourable direction in order to obtain an improved result. Although published data are not very specific on this point it appears that hybridization expresses the maximum potential activity of the parents used, but this is rarely significantly higher than that which is available by mutation alone. The increased plasticity towards mutation which was hoped for in the early days does not seem to have materialized. A subject of the utmost importance is the time factor. Hybridization work is complex and it is not easy always to make the right choice. Often programmes to investigate alternative routes and procedures are valuable. Techniques based on phages or on transfer factors could well be useful if the necessary development work was done. As has already been pointed out,

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the preparation and selection of parental strains may well cause a delay of six months before breeding work can actually start. In industrial strain improvement work progress depends on the steady operation of the screening system. It is obvious that if this stops there is no possibility of an improved culture being discovered. The decision to stop screening while hybridization is started is therefore bound to bring progress to a halt. If, however, mutation and selection continues while hybridization is set in motion, it is likely that the mutation work will get so far ahead that the hybridization programme can never catch up. The more elaborate the hybridization programme the more it is at a disadvantage. These paragraphs make clear the points at which hybridization can be used advantageously; that is when mutation work is no longer making progress, or when the mutants have become such that it is worth stopping attempts to increase productivity while new properties are introduced such as better sporulation or improved growth or other similar features. It will already have been observed that there have been few references to the latest theories of genetic control and their application to strain improvement work. Strain improvement involves fine changes in genetic structure which contrast with the on-off type of phenomena which are the usual basis of genetic studies. No doubt our knowledge of genetic processes will eventually reach the stage when a more rational approach can be made.

V. SELECTION OF CULTURES FOR INCREASED PRODUCTIVITY After the mutation or hybridization stage has been carried out, the isolates should be tested for productivity. The best strains can then be remutated and a further selection of mutants can be made. Methods of selection have been described by Calam (1964) and by Alikhanian (1962) and are detailed by Hopwood, this Volume, p. 377, while Davies (1964) has described the statistical background to the optimization of selection methods ; this paper was based on a study of the populations arising in mutation programmes (see below). Robson et al. (1967) have also discussed the distribution of deviates in segregating populations. In industrial work selection is nearly always based on shaken culture, as this enables large numbers of isolates to be tested under standard conditions. Whenever batches of flasks are set up it is advisable to incorporate a few controls using an established strain, since batch to batch variation in production occurs, and this can be misleading if controls are not available. Mutation and selection programmes pass through several phases. At first relatively large jumps in productivity occur, which are easy to detect.


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The selection procedure is therefore not very critical. At this stage the fermentation process will probably be not too well established and therefore more variable than is desirable. The impression may be gained that selection is more difficult than is really the case. There may be a desire to increase replication so as to improve accuracy and screening. Unless variation is very high (and if this is so it would be better to try to reduce it rather than persist with screening) it is always better to have single flasks in the first screen. The fact that the test is a rough one is compensated by retesting a fairly high proportion (25-50 %) of the strains by more accurate tests later on. The principles involved are (u) that the isolates being screened are part of a population and (b) that the more strains screened the better the chance of encountering one with maximal productivity. T o test fewer strains with a high degree of accuracy reduces the chance of encountering a high-yielding strain. As mutation and selection proceeds the advances in titre become smaller, and the selection procedure becomes more critical. Another factor which must be taken into account, as yields become higher, is that some change in the medium may be necessary to exploit to the full the efficiency of the new mutants. Thus when productivity reaches several g/litre or even 10-15 g/litre, an increase in production of 25 % may call for a considerable increase in the amount of intermediates or raw materials available for the production of an antibiotic. This emphasizes the need for constant attention to the composition of the medium employed. T h e discussion of methods of screening will be based on the work of Davies (1964). This involved the simulation of screening programmes, taking as a basis the results of mutation work on penicillin production. At the time of this investigation the mutation programme had been in progress for a considerable time and only relatively small advances were being made. A full summary of this work is given by Calam (1964). As a basis for calculation the following data were used: (a) 200 shaken flasks were available for continuous use, each fermentation taking 7-8 days, including growth of inoculum, when a two-stage fermentation was used ; (b) a standard error per flask of lo%, in estimating the amount of penicillin produced; and (c) a distribution of improved mutants among the mutated populations which provided in a “poor” population 1 : 2000 giving an extra 5 % and 1 : 200,000 giving an extra lO%,in a “good” population 1 : 50 giving an extra 5% and 1 : 300 giving an extra 10%. With the facilities described, it was possible to complete a mutation and a two-stage selection every month, including all the sub-culturing involved. It is evident from the distributions of improved mutants, which were based on the study of actual populations, that there were many more cultures giving a 5 yo increase, than cultures giving a 10% increase. This was so much so that it was evidently more economical to aim at two successive

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steps of 5 % , than at a single step of 10%. When mutation and selection work was first begun it was customary to mutate and then screen hundreds or even thousands of isolates in the hope of obtaining a strain with the greatest possible improvement in titre. This proves not to be the best procedure when high-yielding strains are present only in low proportions. Successive mutations and the screening of a lower number of isolates each time (e.g., 200) should be more profitable. A second point is that with the standard error which exists in the test, it would be difficult to pick out the best strain without a great deal of testing. Under the circumstances it would be better to reduce the 200 original isolates by two steps to about five strains, one or two of which would be likely to be better than the original. This is best done by testing the 200 isolates with one flask each, and then the best fifty with four flasks each. Calculation showed that a two-stage screen, along the lines proposed, was nearly always the most economical method. After reducing the isolates to five, the next stage is to mutate all five strains, plate and pick off forty isolates from each, and then repeat the screen of the 200 isolates obtained and reduce to five in the same way. The cycle is then repeated several times. As this process is carried out slow changes will become apparent, each of the five strains selected becoming gradually replaced by superior mutants. It also becomes apparent which line of mutants is most successful. With the “poor” population mentioned above, the advance after ten months would be expected to be about 15 %, with the “good” population about 100%. An obvious advantage of this system is that since several isolates are mutated at each stage, there is a possibility of finding strains which, though not necessarily the best, are the most sensitive to mutation. When a system of this type is mentioned, the criticism is sometimes raised that the numbers of isolates from each mutation which are proposed are too small to offer a chance of success. It will be seen, however, that after several months large numbers are in fact screened. The difference is that this number involves numerous mutations. I t is considered better to test 1000 isolates from many mutations than from only one mutation. What the programme does, apart from allowing more mutations to be involved, is to avoid the problem of picking out a single mutant at too early a stage. T o pick out a mutant with an improvement of 10-20% is difficult. To pick a single isolate for mutation and then for a large-scale screening programme is either to risk choosing an inferior one, or to waste a lot of time checking and rechecking results. Multi-stage programmes of this type are used in crop-selection and are supported by Alikhanian (1962), who points out that several mutation steps are usually needed before an appreciable advance is obtained.

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When working in this way, with successive mutations constantly following one another, it is necessary to set up a programme which makes the intervals between mutations as short as possible. By careful organization the intervals between mutations can often be reduced to 4-6 weeks. In achieving this the rapid handling of assay samples and reporting of the results are important factors. Following mutation and screening work along the lines described, improved mutants should be obtained and these can be submitted to stirred culture trials with a view to use in the plant. It is important to make sure that a sound master culture is available. It is also advisable to consider the sort of advance that will be effective under plant conditions. Attempts to exploit mutants which give too small an advance are often disappointing and confusing. I n later mutation work, when improved isolates become even rarer, different screening systems may be appropriate. The ideas suggested by Davies (1964) may be used as a basis for these modifications. With programmes of the type suggested, in which mutation and screening are rapidly alternated, it may be difficult to keep staff and equipment constantly in use. T o avoid this difficulty, it may be decided to start a second overlapping mutation programme. If this is done it will nearly always be found that one of the lines is always behind the other, and that it does no more than keep the staff occupied. Another method is to divide the screening work into two overlapping parts, testing one group of mutants in one half, the other group in the other half. This can be successful but the best mutants should be constantly crossed over so as to make sure that the work is concentrated on the most profitable lines. It is often best to concentrate on a single line and use any free capacity for other types of development work or for finalizing the selection of the best mutants.

VI. GENERAL DISCUSSION OF MUTATION AND SELECTION PROBLEMS This Chapter has given a general outline of the possibilities of mutation and selection as a means of process improvement, and the methods used in this type of work. It is disappointing to report that at present hybridization is proving less useful than was hoped. The writer feels that under certain conditions it still has something to offer, but it is not recommended as a procedure for beginners. It is to be hoped that as time goes by improved methods of breeding will become available which will lead to hybridization becoming more generally useful.

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C.

T. CALAM

The importance of the individual strain being used as a factor in mutation and selection work has been stressed. The behaviour of the strain seems to depend on the type of product being produced rather than the species it belongs to. This individuality among strains stresses the need to choose methods of mutation and selection in a very flexible manner. The selection programmes outlined in this Chapter provide flexibility of approach. Considerable experimentation is usual before progress is achieved, and even when a successful routine has been established care must be taken both to avoid being misled and to seize any opportunities that appear. It has to be realized that mutation work depends very much on chance, and a balance must be struck between a too careful approach which reduces the scale of work to a level which is unlikely to produce new strains, and carelessness which is unable to find them if they are present. A good deal of patience and confidence are needed as often long periods occur when progress appears to be absent. It has been suggested that mutation work should be started as soon as possible. This is primarily to allow plenty of time to work out reliable methods of mutation, plating, isolation of cultures, testing and assay of products and in working out routines and methods of presenting the data obtained. It is essential to reduce this side of the work to a routine. This is not always easy since in development laboratories the emphasis is often on change. Yet unless a sound system of operation is set up and held reasonably constant it is difficult to tell what is going on. It is often necessary to compare results spread over several years, and this can only be done if the work is carefully planned. This necessity to continue for considerable periods between the assessment of results is important. There is a natural desire to know what is happening, and it must be realized that it may not be possible to reach any conclusions about this except at long intervals. Even then care may be necessary, small but useful advances of say 10-15 yo may easily be missed if the test experiments are incorrectly designed or assessed. Having discussed the start and operation of a mutation and selection programme, the final question is when it should be stopped. This is primarily an economic question. Hastings (1958) and others have suggested that above certain levels of production, increases in titre have little effect on economics. This depends to some extent on whether the plant is working at full capacity or not. It may well be that a mutation programme shows little progress over 6-9 months. Whether to continue will depend on the degree of advance needed to show a practical advantage on the manufacturing scale and the hope of achieving it in a reasonable time. Added to this is the question of the minimum advance that it will be possible to detect in plant work. A correct decision can only be made by taking each of these factors into consideration.


459

REFERENCES Adelburg, E. A., Mandel, M., and Chen, C. C. G. (1965). Biochem. biophys. Res. Commun., 18, 788-795. Alikhanian, S. I. (1958). Bjull. Mosk. Obsa. Ispyt. Prirod. Ordel. Biol., 63, 79-96. Alikhanian, S. I. (1962). Adv. appl. Microbiol., 4, 1-50. Alikhanian, S. I., and Iljina, T. S. (1957). Nature, Lond., 179,784. Alikhanian, S. I., and Shishkina, T. A. (1967). Genetiha (Moscow),No. 1 , 159. Alikhanian, S. I., and Teteryatnik, A. F. (1962) Mikrobiologiya,XXXI, 54-60. Anderson, P. S., and Cowles, P. B. (1958).J. Bact., 76, 272-280. Auerbach, C. (1966). Genetika (Moscow),No. 1, 3-11. Backus, M. P., and Stauffer, J. F. (1955). Mycologia, 47, 429-463. Bradley, S. G. (1966). Adv. appl. Microbiol., 8, 29-59. Bunker, H. J. (1961). Prog. ind. Microbiol., 3, 1-41, (Heywood, London). Calam, C. T. (1964). Prog. ind. Microbiol., 5 , 1-53. Daglish, L. B., and Calam, C. T. (1968). Biochem.J., 106, 37P. Davies, 0. L. (1 964). Biometrics, 20, 576-591. Erohina, L. I., and Sokolova, E. V. (1966). Genetika (Moscow),No. 1, 109-115. Esser, K., and Kuenen, R. (1967). “Genetics of Fungi” (English Ed.). Springer Verlag, Berlin, Heidelberg and New York. Goldat, S. Yu., Makarevich, W. G., and Laznikova, T. N., (1967). Genetika (Moscow) No. 3,130. Gregory, W. C., (1961). “Mutation and Plant Breeding.” Publ. No. 891, National Academy of Science, National Research Council, Washington, D.C., pp. 461-486. Hastings, J. J. H. (1958). “Biochemical Engineering” (Ed. R. Steel), p. 299-318. Heywood, London. Hopwood, D. A. (1967). Bact. Rev., 31, 373-403. Kihlman, B. A. (1966). “Actions of Chemicals on Dividing Cells.” Prentice-Hall, Englewood Cliffs, N.J., U.S.A. Lindegren, C. C. (1949). “The Yeast Cell, Its Genetics and Cytology.” Educational, St. Louis. Lingens, F., and Oltmans, O., (1966). 2. Naturj., Zlb,660-663. Macdonald, K. D. (1966). Antonie wan Leeuwenhoek, 32, 431-441. Macdonald, K. D., Hutchinson, J. M., and Gillett, W. A. (1963a, b, c). J. gen. Microbiol., 33, 365-374, 375-383, 385-394. Macdonald, I

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