METHODS IN MICROBIOLOGY,VOLUME 3B, Volume 3B (v. 3B)

METHODS in MICROBIOLOGY This Page Intentionally Left Blank METHODS in MICROBIOLOGY Edited by J. R. NORRIS Milstead ...

Author: Author Unknown

26 downloads 1815 Views 19MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

METHODS in MICROBIOLOGY

This Page Intentionally Left Blank

METHODS in MICROBIOLOGY Edited by J. R. NORRIS Milstead Laboratory of Cliem’cal Enzyiiiology, Sittingbourne, k‘ent, England

D. W.RIBBONS Department of Bioclieinistry, University of Miami School of iIIedicine, and Howard Hughes Medical Institute, Miami, Florida, C7.S.A.

Volume 3B

@

ACADEMIC PRESS, INC.

( I izirr.ourt Brare J o v o i i o v i c ~ h Puldishrrs) .

London Orlando Toronto

Montreal

San Diego Sydney

New York Tokyo

ACADEMIC PRESS INC. (LONDON) LTD 24-28 Oval Road London NW1

U.S.Edition published by ACADEMIC PRESS, INC.

Orlando. Florida 32887

Copyright 01969 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: 12-521543-6

PRINTED INTHE UNITEDSTATESOFAMERICA

85 86 87 88

9 8 7 6 5 4

LIST OF CONTRIBUTORS ELLAM. BARNES,Food Research Institute, Norwich, Norfolk, England EVEBILLING,East Mulling Research Station, Maidstone, Kent, England T. D. BROCK,Department of Microbiology, Indiana University, Btoomington, Indiana, U.S.A. N . G. CARR,Department of Biochemistry, University of Liverpool, England VERAG. COLLINS,Freshwater Biological Association, Ambleside, Westmorland, England M . R. DROOP, Scottish Marine Biological Association, Oban, Scotland R. J . FALLON,Ruchill Hospital, Glasgow, Scotland N . E. GIBBONS,Division of Bwsciences, National Research Council, Ottawa, Canada P. N. HOBSON,The Rowett Research Institute, Bucksburn, Aberdeen, Scotland R. E. HUNGATE,Department of Bacteriology, University of California, Davis, California, U.S.A. JOHN E. PETERSON, Department of Botany, University of Missouri, Columbia, Missouri, U S .A. A. H . ROSE,School of Biological Sciences, Bath University, Bath, England P. WHITTLESTONE, School of Veterinary Medicine, University of Cambridge, England A. T . WILLIS,Public Health Laboratory Service, Luton and Dunstable Hospital, Luton, Beds., England

V

ACKNOWLEDGMENTS For permission to reproduce, in whole or in part, certain figures and diagrams we are grateful to the following publishersMessrs Baird & Tatlock (London) Ltd; Council of the Marine Biological Association of the United Kingdom; Gustav Fischer, Stuttgart; H. K. Lewis & Company Ltd; Masson et Cie, Paris; Royal Society of Sciences of Uppsala, Sweden. 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 laboratory, 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 October, 1969

vii


CONTENTS LISTOF CONTRIBUTORS.

V

.ACKNOWLEDGMENTS.

vi

PREFACE

.

vii

Chapter I. Isolation, Cultivation and Maintenance of AutotrophsVERAG. COLLINS .

1

Chapter 11. Growth of Phototrophic Bacteria and Blue-Green Algae-N. G. CARR .

53

Chapter 111. Techniques for the Study of Anaerobic, Spore-forming Bacteria-A. T. WILLIS .

79

Chapter IV. A Roll Tube Method for Cultivation of Strict Anaerobes-R. E. HUNGATE .

117

Chapter V. Rumen Bacteria-P.

N. HOBSON

.

Chapter VI. Methods for the Gram-negative Anaerobes-ELLA M. BARNES . Chapter VII. Psychrophiles and Thermophiles--T. A. II. ROSE

133 Non-sporing

151

D. BROCKAND 161

Chapter VIII. Isolation, Growth and Requirements of Halophilic Bacteria-N. E. GIBBONS .

169

Chapter IX. Isolation, Cultivation and Maintenance of the Myxobacteria-Jol%N E. PETERSON .

185

Chapter X. Isolation, Cultivation and Maintenance of Mycoplasmas -R. J. FALLON AND P. WHITTLESTONE .

211

Chapter XI. Algae-hl.

K. DROOP .

Chaptcr M I . Isolation, Growth and Preservation of Bacteriophages -EVE BILLING . ,

269 315

AUTHORINDEX

.

331

SVBJLCT INDEX

.

345

ix


Isolation, Cultivation and Maintenance of Autotrophs VERAG. COLLINS Freshwater Biological Association, Anibleside, Westmorland, England I. The Environmental Site in Nature

.

1

.

4

111. Isolation and Cultivation of Some Representatives of the Various Groups of Organisms . .

12

IV. Discussion on the Autotrophic Way of Life in the Natural Environment

25

11. Groups of Autotrophic and Facultatively Autotrophic Organisms

V. Media and Methods References

.

.

. .

26 49

I. T H E ENVIRONMENTAL SITE I N NATURE A. Stratified freshwater lake A stratified freshwater lake offers an ideal natural site for the isolation of facultatively autotrophic bacteria. T h e period of stratification of a lake starts in the late spring and early summer, when the temperature of the surface water rises owing to increased solar radiation. T h e resulting difference in density of the water gives rise to the formation of two distinct layers of water, the upper layer or “epilimnion” and the lower layer or “hypolimnion”. Under some conditions the temperature is approximately the same at all depths in the epilimnion; below this layer, in the transitional zone known as the “thermocline”, the temperature falls rapidly with increasing depth. The nature of the thermocline and the dcpth at which it occurs vary greatly, but in general the depth tends to increase as the summer advances and, therefore, the ratio of the volume of epilimnion to volume of hypolimnion increases. When the layering has begun, the amount of dissolved oxygen becomes gradually less in thc hypolimnion owing to the stagnation of the water and the increased activity of the aerobic bacterial flora of thc mud surface (Collins, 1963). l’his incrcascd activity of thc hcterotrophic facultatively 2

2

V. G . COLLINS

anaerobic bacteria and the mud's chemical oxygen demand results in an oxygen deficit in the bottom waters of a stratified lake. When this situation prevails, the first group of autotrophic bacteria to become active are the sulphate reducers. This results in the production of hydrogen sulphide and the formation of ferrous sulphide on the mud surface and in the overlying Bacteria, 1000/ml

!32527 29 31

3 (C)

i I

3-

1

A

I

I

..

4-

,

.

5-

I E

6-

'.

f a 0) 7 n

C

Temperoture, "C 17 18 19 2 0 21 ?I 22 2? 2 3

10

20

jb

I

30 40 I 60 6b Oxygen, % saturation

I

I

70

I

I

80

FIG.1. A typical environmental situation for a stratified lake. Bfelham Tarn, Boathouse Buoy, August 20th, 1968. Curves: (a) bacteria; (b) temperature; ( c ) oxygen. A. epilimnion; B thermocline ; (c) hypolimnion.

water, creating ti zone of anaerobiosis. At the interface between this zone and the upper zone containing oxygen, members of the coloured sulphur bacteria develop where the light penetration is sufficient to support their photosynthetic mechanisms. Above this zone in the lake and therefore in the thermocline system, where there is sufficient oxygen and organic matter in the form of decayed plank-

I . AUTOTROPHS

3

tonic organisms, members of the photosynthetic purple non-sulphur bacteria develop. Within this system, during stratification, the activities of the filamentous iron organisms must be superimposed. They are not recovered from the oxygenated water immediately above the mud surface, but they are present in very large numbers in the thermocline and above it, and offer, by means of their filamentous growth, a matrix of colloidal sheaths impregnated with iron compounds-an ideal site for the attachment of other micro-organisms. A typical environmental situation for a stratified lake is shown in Fig. 1, where the temperature and dissolved-oxygen determinations were measured by means of a Mackereth oxygen electrode (Mackereth, 1964). This instrument can be obtained from The Lakes Instrument Co., Ltd., Oakland, Windermere, Westmorland. T h e results for bacteria per ml were obtained from aerobic plate counts on a standard medium (Collins and Willoughby, 1962).

B. Sampling methods 1. Water samples For the purpose of obtaining water samples from a profile depth series in any lake system, a piece of apparatus known as a Friedinger water bottle is invaluable. These bottles can be purchased from Messrs Hans Buchi, Berne, Switzerland, and are made in two capacities, 1 litre and 2 litre. They can be sterilized, if necessary, between different depth samples by alcohol swabbing. If it is essential to obtain samples of water at a given depth in the sampling profile without any risk of contaminating water from any other depth entering the sampler, then the bacteriological sampler designed by Mortimer (1940)is recommended.

2. Mud samples Samples of mud can be obtained by means of a Jenkin surface-mud sampler, Fig. 2. ‘I’his apparatus can be obtained from The Lakes Instrument Co., Ltd., Oakland, Windermere, Westmorland. The sampler takes a mud core approximately 30 cm in depth, with the ovcrlying water in situ ovcr the mud. l’here is some slight disturbance of the surface mud during its enclosure in the Perspcx sampler tube, but not enough to make any significant difference to the contents of the sample. 1he samples, collcctcd by means of this apparatus, are excellent for the purpose of making Winogradsky cylinders (Winogradsky, 1887) for enrichment culture techniques, for measuring redox potentials of mud, and for respiration studies using pcrfuscr methods. r ,

4

V. G . COLLINS

11. GROUPS OF AU'L'O'rROI'HIC AND FACULTATIVELY AUTOTROPHIC ORGANISMS

A. Group a: Ammonia-oxidizing bacteria ORDER I I-'SElJDOMONADALES Rergey (1957) SUBORDER I I €'SEIJI)OMONAI)INEAl~ Family I NITl~OiIA(:TIiIlh~'fiAE Genus I Nitrosomonas

FIG. 2. Jenkin surface-mud sampler. (a) The sampler; (b) empty core tube mounted on the sampler; (c) mud-core sample. Photograph by A. E. Ramsbottom.

5

I. AUTOTHOPHS

B. Group b: nitrite-oxidizing bacteria ORDER I PSEUDOMONADALES Bergey (1957) SUBORDER I1 PSEUDOMONADINEAE Family I NITROBACTERACEAE Genus VI Nitrobacter

C. Group c : sulphur-oxidizing bacteria, that oxidize inorganic sulphur compounds and deposit sulphur globules internally ORDER Family Genus Genus Genus ORDER Family Genus

VII I I 111 IV VII IV I

BEGGIATOALES BEGCIATOACEAE Beggiatoa Thioploca Thiothrix REGGIATOALES ACHROMATIACEAE Achromatium

Bergey (1957)

Bergey (1957)

For illustration of representative species of Group c, see Fig. 3. D. Group d : sulphur-oxidizing bacteria that oxidize inorganic sulphur compounds and deposit sulphur both internally and externally ORDER SUBORDER Family Genus Genus Genus Genus

I I1 I11 I1 I11 IV V

PSEUDOMONADALES Bergey (1957) PSEUDOMONADINEAE

THIOBACTERIACEAE Macromonas Thiovulum Thiospira Thiobacillus

For illustrations of representative species of Group d, see Fig. 4. E. Group e: photosynthetic sulphur bacteria, producing red to

purple pigments, and able to use hydrogen sulphide as a hydrogen donor ORDER ' I SUBORDER I Family I Genus II Genus III Genus IV Genus V Genus VI Genus VII Genus VI I I Genus IX Genus X Genus XI Genus XI I Genus XI11

PSEUDOMONADALES Bergey (1957) RH~DOBACTERIINEAE

THIORHODACEAE Thiopedia Thiocapsa Thiodictyon Thiothece Thiocystis Lamprocystis Amoebobacter Thiopolycoccits Tiiiospirilluni Rhabdomonas Rhodothrce Chrowatittm

For illustrations of some rcprescntativc species of Group e, see Figs 5 and 6.

FIG.3. Group C : illustrations of some representative members of this microbial group. 1. Beggiatoa mirabilis (from Bavendamm, 1924), x 115 ; 2. Beggiatoa arachnoidea (from Skuja, 1956), x 670; 3. Beggiatoa minima (from Bavendamm, 1924), x 450; 4. Beggiatoa alba (from Bavendamm, 1924) x 450; 5. Thiothrix nivea (from ; Thiothrix niveu, growing attached to an algal cell Bavendamm, 1924), ~ 4 5 0 6. (from Bavendamm, 1924), x 67; 7. Thiothrix annulata (from Bavendamm, 1924), x 125; 8. Thiothrix tenuis (from Winogradsky, 1949), x 50; 9. Thioploca ingrica (from Bavendamm, 1924), x 225; 10. Achromatium oxuliferum, cells about to divide, original; 11. Achromatium oxalz’ferum, internal granules, original ; 12. Achromatium mobile (from Bavendamm, 1924), ~ 3 5 5 .T h e diagrams from Ravendamm are reproduced by courtesy of Gustav Fischer, Stuttgart; that from Skuja b y courtesy of the Royal Society of Sciences of Uppsala, Sweden; that from Winogradsky by courtesy of Masson et Cie, Paris.

I. AIJTOTHOPNS

7

Fig. 4. Group d : illustrations of some.representative members of this microbial group. 1 . Macronionas mobilis (from Skuja, 1956), x 840; 2. Macromonas hipiinctata (from Skuja, 1956), x 804; 3. Marvomonas frtstyormis (from Skuja, 19ih), x 804; 4. Mucromonas minutissiwta (from Skuja, 1956), x 804; 5 . Tltin7:zthtm ntinrrs (from Skuja, 1956), x 513; 6. Thiovitlum nrajiis (from Skuja, 1956), x 513; 7. Thinzw/rim Mttlleri (from Skuja, 1956) x 5 1 3 ; 8. Thiospira agilis (from Skuja, 19.56), x 804; 9. Thiospira Winogradskzi (from Bavendamm, 1924), x 420; 10. ‘I%io.ipira rfcstogyru (from Skuja, 1956), x 804; 11. 7hiospira tanrii.r (from Skuja, 1956), x 804; 12. Tlliospira Winogradshy (from Skuja, 1056), x 804. T h e diagrams frcim Skuja arc reproduced b y courtcsy of the Iioyal Society I J f Sciences of Upps;rl;i, Swedcn ; that from naveridamm by courtcsy of (iustav I:ischcr, Stuttgatt.

FIG.5 . Group e : illustrations of some representative members of this microbial group. 1. Thiopedia rosea (from Bavendamm, 1924), x 560; 2 . Thiocapsa Y O S ~ O pevsicina (from Bavendamm, 1924), x 486 ; 3. Thiodictyon elegans (from Ravendamm, 1924), x 560; 4. Thiothece gehtinosa (from Bavendamm, 1924), x 5 6 0 ; 5. Thiocystis violarea (from Bavendamm, 1924), x 560; 6. Lamprocystis roseo-persicina (from Bavendamm, 1924), ( a ) x 135 ; (b) x 486; 7. Lamprocystis roseo-persicina (from Winogradsky, 1949), schematic ; 8. Amoebobacter roseum (from Winogradsky, 1949), (a) colony of cells, (b) cells prior to colony formation; schematic; 9. Amoebobacter bacillosiis (from Winogradsky, 1949), schematic ; 10. Thiopolycoccus rubm (from Ravendamm, l924), x 56; 11. lihodotliccr ntrda (from Skuja, 1956), x 804; 12. Rhodothece conspicira (from Skuja, 19-50), x 804. The diagrams from Bavendamm are reproduced by courtesy of Gustav Fischer, Stuttgart; those from Winogradsky by courtesy of Masson e t Cie, Paris; those from Skuja by courtesy of the Royal Society of Sciences of Uppsala, Sweden.

Fla. 6. Group c : illustrations o f some representative mcrnbers of this microbial group. 1 . Thiospiuillrcm Hosenberfiii (from Skuja, 1950), x 804 ; 2. 7*l~io.spi~ill1c~~~ jeizcnsc (fn,m Skuja, 1056), x 804; 3. H / r u / ) ~ f ~ / c . / / / . c t i i(R/rnhcfoirronu.s) /u~;~/i/~ ro.sfw/i (from Skiija), x 804; 4. R/rabdoc.lrrowrutiriiir Sp. (from I%;iventlarnm,1924), involution forms, x 67.5 ; 5 . Rlrahdoiiroirus S'p., original ; 6. I~l/cihtfoc.lcri)iiintirc?ir (Rlrabdomonas) frrsifornre (from Ih\-cmtlamm, 1024) ; 7. ~ ' / ~ r o i i r o t ; r c.Sp., ~ u original ; 8. Clruoviatirem Sp., intcrml glol~tilc~s, original. 'I'hc diiigranis f r o m Skuja arc rcproduccd by courtesy of thc R ( J ~ : Socicty II o f Scicnccs i)t' Lrpps:il:i, S\vedcn ; those from I h \ endamm by courtcsy o f (; u s t : i \ , I;ischcr, Stuttgart.

10

v.

G . COLLISS

FIG.7. Group f : illustrations of some representative members of this microbial group. 1. Clathrochloris hypolimnica (from Skuja, 1956), x 804; 2. Chlorochromatium glebultcm (from Skuja, 1956), x 804; 3. Pelodictyon Sp., original; 4. Cylindrogloea bacterifera (from Skuja, 1956), x 804; 5. Chlorobiztm Sp., enrichment culture, original. T h e diagrams from Skuja are reproduced by courtesy of the Royal Society of Sciences of Uppsala, Sweden.

11

I . AUTOTHOPHS

F. Group f: photosynthetic sulphur bacteria, producing green pigments and able to use hydrogen sulphide as a hydrogen donor ORDER SUBORDER Family Genus Genus Genus Genus Genus

I I 111 I II III V VI

PSEUDOMONADALES Bergey (1957) RHODOBACTERIINEAE C'IfLORODACTERIACEAE

Chlorobirim Pelodictyon Clathrochloris Cl~lorochrornatirini Cylindrogloea

For illustrations of some representative species of Group f, see Fig. 7 .

G. Group g : photosynthetic, non-sulphur bacteria, producing red to brown pigments ORDER SUBORDER Family Genus Genus

I II I II

PSEUDOMONADALES Bergey (1957) RHODOBACTERIINEAE ATZIIORHODACEAE

Hhodopseudomonas Rhodospirillum

H. Group h: bacteria capable of reducing sulphates, and able to obtain their energy from atoms of hydrogen or the labile hydrogen atoms of low molecular organic substances ORDER I SUBORDER I I VII Family Genus II

PSEUDOMONADALES Bergey (1957) PSEIJDOMONADINEAE SPIRILLACEAE Desulfovibrio

I. Group i :methane-oxidizing bacteria ORDER I SUBORDER I1 Family I1 G~I~LIS I

PSIXJDOMONADALIIS Uergey (1957) PSEUDOMON~DINEAE METHANOMONADACEAE Alethanomonas

J. Groupj :hydrogen-oxidizing bacteria ORDER SUBORDI:R Family Genus

I1 I1 II II

PSEUDOMONADAIXS I3ergey (1957) PSEUDOMONADINEAE METHANOMONAIMCEAE Hydrogenomonas

K. Group k: carbon monoxide-oxidizing bacteria ORDER I SUBORDICR I I Family II Genus III

PSEUD0MONADALI:S PSEUDOM0NAT)INEAE R.~ET€1ANOMONAI)A('I:i\E

Carbosyc-iomonas

Uergey (I 9.57)

v.

12

G . COLI.INS

L. Group 1: bacteria capable of oxidizing iron and manganese compounds ORDER

I1 CIHLAMYDOBACTERIALES Bergey (1957)

Family Genus Genus

I Splraerotihrs I1 Leptotkrix

Family Genus Genus

I1 PELOPLOCACEAE I Pcloploca I I I’cloncma

Family (;enus (;cnus

ORDER SUBORDER Family Genus Famiiy Genus Genus Genus Genus Genus Genus Genus

1 CI-ILAMYDODACTERIACEAE

I I I CI3ENOT RICI3ACI:AE I C‘renotlirix I 1 I CIonotlirix I PSEUDOMONADALES I1 PSEUDOMONADINEAE

Bergey (1957)

V CAULOBACTEHACEAE

II Gallionella

VI SIDEROCAPSACEAE I IV V VI VI I IX

x

Siderocupsa Ferribacterium Sideromonas Naumanniella Ochrobium Siderobacter Ferrobacillus

For illustrations of some representative species of group 1, see Figs 8, 9 and 10. 111. ISOLATION AND CULTIVATION OF SOME REPRESENTATIVES OF THE VARIOUS GROUPS 01; ORGANISMS

A. Group a ; ammonia-oxidizing bacteria, Nitrosotnonus species 1. Site These organisms can be isolated from the surface muds of stratified lakes, where the mud is covered with shallow layers of water within the oxygenated zone. An ideal recovery site in a freshwater lake is the surface mud where a sewage bearing inflow first flows into the lake.

2. Methods Samples of the surface mud are obtained by the use of the Jenkin surfacemud sampler. T h e surface layer of the mud core thus obtained is removed aseptically and transferred to a sterile, covered 500 ml beaker. Sterilized CaC03 is added in sufficient quantity to cover the layer of mud, the beaker

I . AUTOTROPHS

13

and contents then being incphatcd overnight at 20°C. Enrichment cultures are then made, kiftcr miling thc contents of the beaker, by inoculation of 5 g amounts of the mud with l’opc and Skcrman’s liquid mineral salts medium with additions (Slterinan, 1967). Alternatively inoculations can

FIG.8. Group c : illustrations ( I f some represcntati\,e membcrs of this microbial group, also shown in Figs 9 and 10.1. Spiiaerotilits natans, original; 2. Leptotlirix Sp., original; 3 . Leptothrix ochracea, a chain of rods gliding out of a sheath impregnated with iron, original; 4.Leptotlirix ochracea, a chain of rods completely emerged from an iron impregnated sheath, original ; 5 . I,eptot/irix sidevopozrs, Filamcnts growing out of an iron impregnated attachment disc, original ; 6. Crrnothrix polyspora (from Dorff, 1934), x 750; 7 . Clonothrixfitsca (from Uorff, 1934), x 500. ‘I’he diagrams from Dorff are reproduccd by courtesy o f Gustav I:ischcr, Stuttgart.

14

FIG.9. G r o u p 1 : 1. Pelonenia tenur (from Skuja, 1956), (a) x 600, ( t i ) x 402; 2 . Peloplocaferruginea (from Skuja, 1956), (a) x 810, (1)) x 600, ( c ) x 2 8 2 ; 3. Gallionella ferruginea (from Do&, 1934), (a) x 600, (b) x 1440; 4. (;ulliondla tenuicaulis (from Skuja, 1956), x 804; 5. Gallionella tcniiicaulzs, showing the “kidney cells” a t the ribbon terminals (from Skuja, 1956), x 804. T h e diagrams from Skuja arc reproduced by courtesy of the Royal Society of Sciences of Uppala, Sweden; that from Dorff by courtesy of Gustav Fischer, Stuttgart.

I. AUTOTROPIIS

15

FIG.10. Group 1 : 1. Sicferorapsa Treitbii (from Dorff, 1934). schematic; 2. Siderocupsa major (from Dorff, 1934), schematic; 3. Siderorapsa coronata (from Dorff, 1934), schematic; 4. Siderocupsa geniinata (from Skuja, 1956), x 670; 5 . Sideyoderma (Ferribacterium)dubiicm (from Skuja, 1956), x 670; 6. Naunianniella neustonica. (from Dorff, 19341, schematic; 7. Sideyocystis (Sideromonas) conjercurum (from Dorff, 1934), x 500; 8 Sidcrobacter (from Ihrff, 1934), (a) Sidmhacter lineare, (b) Siderobucter di+/ex, both schematic; 9. Ochrobiun/ tecfitnt (from I h r f f , 1934), x 1000; 10. Ochrobium techtni (from Skuja, 1956), x 670. T h e diagrams from I>orf€ are reproduced by courtesy of Gustav E’ischer, Stuttgart; those from Sltuja b y courtesy of the Royal Society of Sciences of Uppsala, Sweden.

16

v. c. COLLINS

bc niaJc into the medium of Meililcjohn (Skerman, 1967, p. 218). Subsequent transfcrs are inadc to silicd-gel plates, with nutrient additions, using the method of Sonimcrs and IIarris (1968) for preparation of the silica gel. When colonies develop on the siliea-gel plates, they are then transferred back to the above liquid media for A’itrosomonas, with the addition of a shallow layer of sterile fine sand on the bottom of the culture flask. Purity checks on the flask contents can bc performed by the inoculation of ordinary nutrient agar plates. As growth of the pure culture proceeds, estimations of the accumulation of nitrite (Skerman, 1967, pp. 218-220) and the absence of growth on repeated purity checks on nutrient agar give clear evidence of the presence of Nitrosonionas species. Re-plating on silica-gel plates, and subsequent transfer of pure colonies back to liquid media, with sand, should give pure cultures. T h e medium and methods of Soriano and Walker (1968) are greatly to be recommended.

B. Group b: nitrite-oxidizing bacteria, Nitrobacter species 1. Site The environmental site in a freshwater lakc is the same as for Group (a), as are the sampling operations.

2. Method T h e mud for enrichment cultures of Croup (b) is inoculated into Skerman’s medium for Nitrobacter (Skerman, 1967, pp. 215, 216) and into Meiklejohn’s medium (Skerman, 1967, p. 218). Again purity checks can be made on nutrient agar plates. Cultures of the nitrite-oxidizing bacteria are maintained in the liquid medium. As growth proceeds, determinations of the disappearance of nitrite and the formation of nitrate (Skerman, 1967, p. 218) in the culture flask, combined with the absence of growth on the nutrient agar purity check plates, give positive evidence of the presence of Nitrobacter species. For pure culture studies and literature reviews relating to the organisms of Groups (a) and (b), reference should be made to the follouing pubiications; hleiklejohn (1950; 1952; 1953a; 1953b; 1954), I,ws (19S4), and Smith and Iloare (1968).

C. Group c :sulphur-oxidizingbacteria that are capable of oxidizing inorganic sulphur compounds, depositing sulphur as globules within their cells-Beggiatoa, Thioploca, Thiothrix and Achromatium 1. Site T h e representatives of this group are found on the surface muds of stratified lakes, just prior to the complete de-oxygenation of the hypolimnion

I. AUTOTROPI-IS

17

water when the dissolved oxygen concentration is in the range of 0*15-0*30 mg &/litre in the deepest part of the lake. I n the English Lake District, this oxygen depletion of the hypolimnion occurs in five lakes where the depth ranges from 13 to 21 m.

2. Method The samples of mud are obtained from Jenkin surface-mud cores, taken at the maximum depth of the lake sampling profile. This enables the actual environmental conditions which prevail at the time of sampling to be maintained under laboratory conditions. That is, until the facultative heterotrophicbacteria increase their respiration rate by the increased temperature of the laboratory, and remove the remaining traces of oxygen. Therefore, it is essential for the successful recovery of this group of organisms to make enrichment cultures from the surface mud before the onset of complete de-oxygenation within the Jenkin core-sampling tube. As a procedure to check for the presence or absence of these organisms in the surface mud of the sample, small drops of the surface mud, removed by pipette, can be mounted on microscope slides under cover slips and viewed directly under the microscope, using darkground illumination and a lox objective (Leitz Heine system phase-contrast) or a 25 x phasecontrast objective with bright field phase lighting. T h e unique morphology and size of the organisms of this group makes rapid identification possible. An essential feature of successful enrichment cultures is a slow generation of hydrogen sulphide and a low concentration of dissolved oxygen. One enrichment method consists of removing the top layer of mud from a Jenkin core sample, and adding it directly to a Winogradsky Cylinder (Winogradsky, 1887; Larsen, 1952; Collins, 1963). I t is advantageous to siphon off the overlying water of the original mud core and use this as the liquid for the medium of the Winogradsky cylinders. Air is then slowly bubbled into the top layer of the water in the cylinder of the enrichment culture. Jenkin core tubes act as excellent cylinders. T h e entire unit is then stored in a cool incubator (Gallenkamp) at a temperature of ll-l2”C, this being the temperature of the natural environment of the surface mud at the time of sampling. The majority of the organisms in this group will develop on the surface of the mud and in the zone of water immediately abbe the mud. The slow generation of hydrogen sulphide from the bottom of the cylinder and the slow diffusion of oxygen from the top of the water column in the cylinder enables an artificial “poising” of the oxygen concentration. Insertion of an oxygen probe into the cylinder during incubation enables measurement of the dissolved-oxygen content to be carried out. Control of the rate of oxygenation can then be maintained between 0.15-0*50 mg Oz/litre in the over3

18

V. G. COLLINS

lying water of the cylinder. This type of enrichment culture will yield viable cells of all the organisms of this group, over a period of six to eight weeks. T h e next stage of obtaining pure cultures of these organisms is more difficult. T h e author, so far, has only succeeded in isolating species of Beggiatoa in pure culture using the methods of Faust and Wolfe (1961), and the methods of Cataldi (1940). However, for taxonomic purposes it is possible, from the enrichment culture technique, to harvest large concentrations of the cells of Thioploca, Thiothrix and Achromatium for microscopic examination. For reference purposes the descriptive work of the following authors should be consulted: Keil(l912); West and Griffiths (1913); Bavendamm (1924); Ellis (1932); Winogradsky (1949); Scotten (1953); Bissett and Grace (1954); Pochmann (1959); Lackey (1961); and Burton and Morita (1964).

D. Group d: organisms capable of oxidizing inorganic sulphur and usually depositing sulphur externally-Mucromonus, Thiovulum, Thiospira and Thiobaciilus 1. Site The main source of material for this group is from Jenkin surface-mud cores taken in the oxygenated zone of a stratified lake, that is mud from above the thermocline regime. I n this case the overlying water of the mud would have an oxygen concentration from 70 to 80% saturation, but the surface mud would have avery low oxygen value, of the order of 1-0-2.0 mg Oz/litre. An excellent time to take samples for this group is on the occasions when mass blooms of algae occur in the surface waters of a lake. When the algal cells die and sink onto the surface mud above the thermocline, large populations of Macromonas, Thiovulum, and Thiospira develop on the surface mud.

2. Method The simple expedient of storing the Jenkin mud cores with the top of the tube uncovered, at a temperature of 15"-20"C, usually results in large numbers of the afore-mentioned species developing on the surface of the mud. I n the case of Thiovulum, dense zones of growth develop in the overlying water about half-way between the surface of the tube and the mud. Pure cultures of these three species have as yet not been procured by the author, microscopic studies only have been possible for taxonomic purposes. For enrichment cultures of thiobacilli direct from the natural environment of the surface mud of a freshwater lake, 1 g amounts of mud are inoculated into the medium of Lieske (1912), Waksman and Starkey (1922); Starkey (1953) using tk serial-dilution technique. T h e liquid media in this instance are dispensed in shallow layers in 100 ml Pyrex conical flasks, and sterilized by steaming for one hour on three consecutive days. Two other

I. AUTOTROPHS

19

useful media are those described by Skerman (1967, p. 216), using the PopeSkerrnan basal mineral salts medium with additions. Enrichment cultures are best incubated at 30"C, when turbidity and pellicle formation are evident in the flask contents. A transfer to medium of the same composition with the addition of agar usually results in pure colonies of Thiobacillus species from the higher dilutions of the mud. Subsequent transfer of colony material back to liquid media, accompanied by determinations to assess the disappearance of thiosulphate gives positive evidence of the presence of thiobacilli. For the methods of estimation involved in determining the reactions of these organisms during active growth, see Skerman (1967, pp. 234-238). For studies on the species of Thiobacillus concerned in the oxidation of thiocyanate the work of Happold et al. (1954) should be consulted. The following publications should be used for reference to the ironoxidizing species of Thiobacillus: Lyalikova (1958) ; Colmer (1962); Lazaroff (1963); Razzell and Trussell (1963); and Duncan et al. (1964). The anaerobic thiobacilli have been extensively studied by the following workers: Woolley et al. (1962); London (1963); Hutchinson et al. (1965; 1966; 1967); and Jackson et al. (1968). For general reviews on the organisms of Group (d) the following work should be consulted; Bunker (1951); Baalsrud (1954); Lees (1955); Vishniac and Santer (1957); and Sokolova and Karavaiko (1964).

E. Group e : photosynthetic, sulphur bacteria, producing red to purple pigments-Thiopedia, Thiocapsa, Thiodictyon, Thiothece, Thiocystis, Lamprocystis, A moebobacter, Thiopolycoccus, Thio spirillum, Rhabdomonas, Rhodothece, Chromatium 1. Site The source of material is Jenkin surface-mud cores obtained from the de-oxygenated zone 'underneath the thermocline of a shallow, stratified lake, when the temperature' at the bottom of the lake is in the range of 11"-12°C and the dissolved-oxygen concentration 0.09 mg Ozflitre.

2. Method The original mud-core sample can be used as a Winogradsky cylinder, either stored, closed to laboratory atmosphere while exposed to diffuse daylight, or incubated untouched, at 3OoC, with a series of 25 W light bulbs positioned at a distance of 20-30 cm. The natural generation of hydrogen sulphide proceeds in the mud core, and any remaining oxygen is taken up by the heterotrophic bacteria present on the surface mud under the completely closed conditions of the core tube. When pink pigmented patches appear on the inside walls of the tube,

20

v.

G. COLLINS

enrichment cultures can be made by siphoning off the overlying water of the mud core, the entire core being removed from its tube by means of a piston. Slices of the mud are then placed in Petri dishes, mixed with a glass rod and 5 g amounts transferred to empty sterile 4 oz glass-stoppered bottles. T h e bottles are then completely filled with the medium devised by Pfennig (1961; 1962), and incubated at 30°C with 25 W light bulbs placed at a distance of 20-30 cm from the bottles. As growth of the enrichment cultures proceeds, the contents of the bottles turn a bright pink to deep red in colour. At this stage, pure cultures can be achieved by inoculating 5-10 ml of the enrichment culture into deep-culture tubes of van Niei’s medium (van Niel, 1931). These tubes are constructed from pieces of glass tubing 8 in. in length, and having an internal diameter of $ in. They are plugged at both ends with cotton wool and sterilized. After sterilization a sterile rubber bung is inserted at one end of the tube, the enrichment culture inoculum is added, the cooled molten agar medium is poured in, a sterile rubber bung is inserted in the other end of the tube, and the contents are gently mixed by repeated inversion of the tube. When mixing is complete, the tube is immersed in cold water to allow quick setting of the agar. When the agar has set, the tubes are removed from thc water, dried, and a transparent Viscap sealing cap (Baird and Tatlock, Ltd.) placed over the rubber bung at either end of the tube. For incubation at 30°C the tubes are mounted in a rack to allow maximum length of the tubes to be exposed to the 25 W light source. Brightly pigmented colonies soon develop in the agar, which can be removed by opening the tube and, using a sterile glass piston slightly smaller than the diameter of the tube, pushing the long plug of agar out and slicing it into sterile Petri dishes. Separate colonies are then cut out of the agar slices and transferred back to the medium of Pfennig, as liquid cultures in glass stoppered bottles, for maintenance and sub-culture purposes. For descriptive works with many illustrations see Winogradsky (1 888; 1949); Bavendamm (1924); Skuja (1956). For culture studies see the work of van Niel (1931), and Pfennig (1961; 1962).

F. Group f: photosynthetic, green pigment producing sulphur bacteria-Chlorobium, Pelodictyon, Clathrochloris, Chlorochromatium, Cylindrogloea 1. Site T h e source is the same as that for Group (e). 2. Method For this group enrichment cultures are obtained by the Winogradsky (1887) and Larsen (1952) cylinder method, and exposed to the same conditions as those described for Group (e). Pure cultures are obtained using the

I. AUTOTROPHS

21

methods and medium of Larsen (1952)) or the methods for green sulphur bacteria described by Pfennig (1961 ; 1962). For reference purposes the review by Larsen (1954) covers both groups of photosynthetic sulphur bacteria. T h e isolation by Pfennig (1968) of new species of green sulphur bacteria is proof that the green sulphur group of organisms is far from complete.

G. Group g : photosynthetic, non-sulphur bacteria, producing red to brown pigments-Rhodopseudomonas, Rhodospirillum 1. Site , The source is the same as that for Group (e). T h e bacteria can also be obtained from water samples taken in the thermocline zone of a stratified lake towards the end of the period of stratification in August and September of each season, when the temperature within this zone would be in the range of 17'-19"C, and the dissolved oxygen concentration within the range of 55-90% saturation.

2. Method Enrichment cultures can be obtained from Winogradsky cylinders using the surface mud, and from direct inoculation of the water samples using the methods and medium of van Niel(l94-4). For maintenance of pure cultures of these organisms the methods and media of Hutner (1944; 1946; 1950) give excellent results.

H. Group h: organisms capable of reducing sulphates and able to obtain their energy from atoms of hydrogen or the labile hydrogen atoms of low molecular organic substances. Also able to use low organic substances and C 0 2 as C-sourcesfor growth-Desulfovibrio 1. Site The source is Jenkin surface-mud cores taken from the deepest part of a stratified lake, when the bottom waters of the lake have remained stagnant for the maximum period under the thermocline. This period usually extends from the end of May to the end of September, just before the overturn and complete mixing of the lake.

2. Method The overlying water of the mud-core sample is siphoned off, and the entire core of mud is sliced into Petri dishes and placed directly into an anaerobic jar and incubated at 30°C. After 2 days incubation, sub-samples from the mud in the Petri dishes are inoculated into freshly autoclaved medium that has not been exposed to laboratory atmosphere. The methods and media recommended and used by Butlin et al. (1949) are excellent. These authors modified the original medium of Baars (1930) and Starkey

22

V. G. COLLINS

(1938); their media and methods are described by Skerman (1967, pp. 267268). These media are excellent for enrichment cultures. For serial dilution counts direct from the natural environment, and for pure culture maintenance and experimental work, the medium first described by Miller (1950) and later used by Crossman and Postgate (1953), is to be recommended; this medium is quoted by Skerman (1957, pp. 268-269). It is quite acceptable to omit the incubation of the mud in an anaerobic jar, and to inoculate the slices from the mud core directly into any of the above-mentioned liquid media in glass-stoppered 2 oz bottles. Serial dilutions can be used for enumeration purposes, blackening of the bottles after 7 days incubation at 30°C is a good indication that active sulphate reduction is taking place. Transfer of enrichment culture material to agar media of the same composition incubated in an anaerobic jar usually results in pure colonies of sulphate-reducing bacteria. For pure culture studies see Postgate (1965). T h e culture vessel system designed by Pankhurst (1967) serves as an excellent method of maintaining pure cultures of these organisms for a considerable time period, before sub-culturing is necessary. For studies on the spore-forming sulphate-reducing bacteria reference should be made to the publications of Campbell et al. (1957), Coleman (1960), and Postgate and Campbell (1963). A very good general study of sulphate reducing bacteria is that of Miller et al. (1968). J. Groupj :hydrogen-oxidizingbacteria-Hydrogen0 rnonas 1. Site T h e source is mud cores obtained from the littoral regions of freshwater lakes, especially lakes that receive their drainage from agricultural land which has been fairly heavily manured and also treated with inorganic fertilizers. T h e surface mud, obtained from such an environmental site as this, is always exposed to fully oxygenated overlying water.

2. Method T h e author has tried a number of different methods for the isolation of this group of organisms from the natural environment. For maintenance of autotrophic growth conditions the method of Atkinson and McFadden (1954) should be used. Cohen and Rurris (1955) also describe a very effective method of culturing these organisms in liquid media, which serves as an ideal basis for obtaining enrichment cultures from direct inoculation of the surface mud to the culturing system. Transfer of material to either silica-gel plates or agar, as described by Schatz and Bovell (1952), and Wilson et al. (1953) usually results in the growth of pure colonies of these organisms. For maintenance purposes, cultures survive for a longer period of time under the conditions described by Cohen and Burris (1954).

I. AUTOTROPNS

23

Enrichment cultures of related strains of these organisms have also been recovered from surface mud from the decpcr parts of a stratified lake, when the oxygen saturation has been in the range of 6-15%. Below 6% oxygen saturation of the overlying water of the mud, attempts at isolating this group of organisms have been negative. For information on the autotrophic and heterotrophic growth of this group of organisms, reference should be made to De Cicco and Stukus (1968). For the isolation of pure cultures of this group, under reduced oxygen tension conditions, the medium of Schatz and Bovell(l952) is ideal; this is described by Skerman (1967, p. 220).

K. Group k: carbon monoxide-oxidizing bacteria-Carboxydomonas The author has no personal experience with this group of organisms; repeated attempts to obtain enrichment cultures from the surface mud of a freshwater lake have been negative. On several occasions, using the medium of Kistner (1953), several isolates have been studied and were found to oxidize CO to CO2. However, without exception these isolates also oxidized hydrogen when grown under the conditions described by Cohen and Burris (1954). Kistner’s medium is described by Skerman (1967, p. 221), and on the following pages Skerman describes a good technique for the observation of the oxidation of carbon monoxide in actively growing cultures of this group. Since some organic matter in some form or another is nearly always present in the natural environment of a freshwater lake, this may not be an ideal site for the initial isolation of this group of organisms. It is a matter for speculation that some of the other facultative autotrophs may be capable of oxidizing CO to C02 under certain conditions of laboratory culture. Reference should be made to Bergey (1957, p. 77) for a discussion on the one species of this group so far described.

I. Group i :methane-oxidizing bacteria-Methanomonas 1 . Site The source is Jenkin surface-mud cores obtained from the de-oxygenated zone of a stratified lake, and stored, covered, in the laboratory, with some of the overlying water removed to leave an “airgap” in the closed-core tube. After a period of 2-3 weeks, when the mud has warmed to laboratory temperature (i.e., 14”-18”C), the top 6-8 cm of the mud core breaks away, and rises to the top of the Fube, This is due to the evolution of gases proceeding in the lower depths of the column of mud. !

$

2. Method Samples, taken from the bottom of the 6-8 cm “plug” of risen mud, and inoculated into the autotrophic medium for Methanomonas, described by Skerman (1967, p. 221), and used under an atmosphere of 50% methane

24

V. G . COLLINS

and 50% air, yields enrichment cuItures of this group. For pure cultures the use of washed agar is essential. T h e inorganic salts of the aforementioned medium are incorporated and the cultures maintained in an atmosphere of one part methane and two parts air. Skerman (1967, p. 221) gives a very useful description of methods for observing the oxidation of methane, and also methods for the preparation of gas mixtures. For alternative cultivation methods see Overbeck (1969, Anagnostidis and Overbeck (1966).

L. Group 1: organisms capable of oxidizing compounds of iron and manganese-Sphaerotilus, Leptothrix, Crenothrix, Clonothrix, Gallionella, Siderocapsa, Naumanniella, Ochrobium, Siderobacter, Ferribacterium, Sideromonas, Peloploca, Pelonema, Ferrobacillus 1. Site Sources are water samples and mud cores obtained from a stratified lake during the period of summer stagnation. 2. Method Enrichment cultures for most of the organisms in this group can be obtained directly from water samples and mud cores, stored at laboratory temperatures. I n both cases the samples should be stored with access to atmospheric oxygen, which allows the reduced iron compounds, in samples from the de-oxygenated zone of the lake, to slowly oxidize. As re-oxygenation of the sample proceeds, large flocs of iron “complex” material are formed, and with the flocs copious growths of the iron bacteria develop in the stored samples of water and mud. For organisms belonging to the Sphaerotilus-Leptothrix group, the methods and media of Mulder and van Veen (1963) are ideal for the purpose of obtaining both enrichment and pure cultures. For species of Gallionellu, the methods and media of Nunley and Krieg (1967) are the only ones that the author has found successful for the pure culture isolation of Gallionella. See also Hanert (1968). T h e iron flocs that form on the surface of stored mud cores provide a good source of material for the isolation of Ferrobacillus species, using the medium and culture growth methods of Silverman and Lundgren (1959a). For pure culture studies concerned with the chemical activities of these organisms, reference should be made to the work of Dugan and Lundgren (1964), and Temple and Colmer (1951). T h e author has not been able, so far, to isolate any of the species of Siderocapsaceae, in pure culture. Enrichment cultures for these organisms can be obtained from the surface of submerged plants in the littoral regions of a stratified lake. It is possible to sediment large accumulations of the growth of these organisms from water samples taken at the period of over-

I. AUTOTROPHS

25

turnof astratified lake. This is the period when the temperature and dissolved oxygen concentration are the same from the surface of the lake to its greatest depth, the temperature range is ll0-12"C, and oxygen usually 90-100% saturation, A useful technique for obtaining material representative of the organisms in the Siderocapsaceae for taxonomic purposes is to submerge squares or strips of P.V.C. sheeting in the lake, at depth intervals during the stratified phase and also on the overturn of the lake. These organisms very quickly colonize the P.V.C. sheeting and, on removal to the laboratory, small sections of the sheeting can be cut out and mounted on microscope slides for direct observation. Since most of the species in this family form some kind of attachment extrusion, surrounded by a "torus" of iron compounds, the entire assemblage can be viewed microscopically on the pieces of P.V.C. sheeting. Descriptive studies on these organisms have been published by Cholodny (1926); Dorff (1934); Hardman and Henrici (1939); and Beger (1941). Pringsheim (1949) gives a most useful review of the iron bacteria with 199references. A most useful study on the bacteria associated with manganese nodules from the aquatic environment is reported by Trimble and Ehrlich (1968). Concerning the filamentous bacterium Sphaerotilus natans a great deal of published work now exists. Particularly because of its association with paper mill effluents, stream pollution and sewage treatment plants. Some of the industrial implications of the biological activities of this organism are described in the following publications; Waitz and Lackey (1958); Mulder (1964); Phaup and Gannon (1967); Muellar and Litsky (1968); and Phaup (1968). IV. DISCUSSION O N T H E AUTOTROPHIC WAY OF L I F E I N T H E NATURAL ENVIRONMENT A stratified freshwater lake has been chosen by the author as a natural environmental site for the isolation of the major groups of organisms thought to be autotrophic. From the evidence presented by detailed ecological and microbiological studies, covering twenty-one years of research on the freshwater environment, it would appear that there are very few strictly autotrophic bacteria existing and functioning in their natural environments. Many of the so-called autotrophic bacteria can be recovered from the natural situation of a stratified lake when it is known that organic matter is present in both the waters of the lake and the surface mud at the bottom of the lake. The gap existing between a laboratory culture of an apparently autotrophic bacterium and the natural site from whence the culture was derived is a very large one indeed. In many cases, the ability of thc cultured

26

V. G . COLLINS

organism to grow without organic matter in vitro may simply be the result of our inability to measure the micro amounts of organic matter derived from “contaminating” substances in the ingredients used for making the culture media in which the organism is growing. T h e author therefore prefers the use of the term “facultative autotroph” when referring to organisms known to be actively participating in the various chemical cycles of the natural environment, such as the nitrogen cycle, the sulphur cycle, and the iron cycle, T h e reader, the author hopes, will appreciate that there are many more natural sites where these organisms can be recovered, and reference to the “habitat” descriptions in Bergey (1957) give adequate proof of this fact. It should also be noted that the author has omitted certain genera of the various groups under consideration, having had no personal experience with the particular genera omitted.

V. MEDIA AND METHODS A. Organisms of groups a, b, d, i, j, and k For these organisms Pope and Skerman mineral salts media are used (Skerman, 1967). For the preparation of various mineral salts media the solutions described in Tables I and I1 are required. They should all be prepared with glassdistilled water and acid-cleaned glassware. TABLE I Solutions required for the preparation of various mineral salts media, Group A (from Skerman, 1967) Solution __---

__

Amount

.

-

__

2. 0.074 M 3. Solution from 2 , above

1 litre 1 litre 200 ml

4. Solution from 3, above

1 litre

1.

N

NaOH

5. NaHC03 in 100ml water 6. CaClz in 100ml water 7. NaNOz in 100 ml water 8. Glucose in 100 ml water 9. Mannitol in 100 ml water 10. Sucrose in 100 ml water 11. Sodium citrate in 100 ml water 12. Phenol in 100 ml water 13. Na&03 in 100 ml water

Procedure

_.

8.333 g 5 .O g 5 .O g 10.0 g 10.0 g 10 .O g

Sterilize at 121°C for 20 min Sterilize at 121°C for 20 min Dilute to 2000 ml(O.0074 M); sterilize as for Solution 1 Neutralize with use of N NaOH ; sterilize as for Solution 1 Sterilize as for Solution 1 Sterilize as for Solution 1 Sterilize as for Solution 1 Sterilize at 110°Cfor 25 min Sterilize at 110°Cfor 25 min Sterilize at 110°Cfor 25 min Sterilize at 121“C for 20 min

2.0 g an hydrous (or 2.77g hydrated) Sterilize at 121“Cfor 20 min 10 g Sterilize at 121°C for 20 min 10 g

27

I. AUTOTROPHS

TABLE I-continued Solution

Amount . .

-

100 ml 1 litre

14. 0.5 M He1 15. 0.0167 M H3P04

16. Monoethylamine hydrochloride in 100 ml water

5 ml

___

__

Procedure ~

___

Sterilize at 121"Cfor 20 min Neutralize 500 ml with N NaOH; sterilize as for Solution 11 Sterilize by filtration

TABLE I1 Solutions required for the preparationof various mineral salts media, Group B (from Skerman, 1967) Amount per Solution

100 ml solvent

Solvent required

3.0 g 6.6 g 21 .O mg 80.0 mg 106.0 mg 600.0 mg 123.0 mg 110.0 mg 109.0 mg 60.0 mg 30.0 mg 30.0 mg 629.0 mg 1.4g 36 .O mg 300.0 mg

0.0074 M H3P04 0.074 M H3P04 0.0074 M 0.0074 M 0.0074 M H 8 0 4 0'0074 M H3P04 0.0074 M 0 -0074 M 0.0074 M 0.074 M Water Water 0.074 M &PO4 Water Water Water

Final concentration (pgllitre medium) 300,000 660,000 21 80 106 600 123 110 109 60 30 30 629 140,000 36 300

Preparation of the basal mineral salts medium is carried out as folIowsStep 1. Pipette into a 1 litre standard flask the following amounts of solutions from Group B : 10.0 ml of Solutions 1 and 2 and 0.1 ml of each of Solutions3-10. Step 2. Add approximately 600ml of 0.0074~H3PO4(Solution 3, Group A) and 210 ml of water. Step 3. Adjust the p H to 7.0 with N NaOH (Solution 1, Group A). Step. 4. Add 0.1 ml of Solutions 11 and 12 from Group B. Step 5 . Take 0.1 ml of the MnClz solution (Solution 13, Group B), add 9.9 ml of 0,074 M &Po4 (Solution 2, Group A), and adjust the p H to 7.0. Autoclave and filter. Add the filtrate to the medium.

28

V. G . COLLINS

Step 6. Add 10 nil of Solution 14, Group B, and 0.1 ml of 15 and 16, Group B. Step 7. Using the neutralized 0.0074 M &PO4 (Solution 4, Group A), make the final volume to 1 litre. Step 8. Sterilize at 121°C for 20 min. This solution is crystal clear and will remain so for long periods if kept in acid-washed glassware. It provides a complex mineral salts base with ammonium-N, which has been found suitable, after addition of specific components, for a wide range of autotrophic and exacting heterotrophic bacteria. The use of Pope and Skerman mineral salts media for some of the groups of organisms covered in the preceding Sections is described below.

1. Group a :ammonia-oxidizing bacteria, Nitrosomonas species The medium for Nitrosomonas (Skerman, 1967) is used. (a) Liquid medium. Adjust the pH of the basal mineral salts medium to 8-2 before sterilizing. Add aseptically 20 ml of the sterile NaHC03 solution (Solution 5, Group A) per litre. (b) Silica Gel Plates. In the preparation of the silica-gel plates the medium is diluted 1 : 2. T o allow for this, prepare a double strength basal mineral salts medium (D.S.B.M.S.M.) as followsFollow the instructions for the single strength medium to Step 2. Add 105 ml of distilled water and make the volume up to approximately 400 ml with the use of the 0.0167 M H3P04 (Solution 15, Group A). Neutralize with N NaOH (Step 3). Then follow Steps 4, 5, and 6 as indicated. Make the final volume to 500 ml with the use of the neutralized 0.0167 M H3P04 (Solution 15, Group A) and sterilize at 121°C for 15 min. Prepare the silicic acid by the method of Pramer (see preparation of silica gel below). Before gels can be prepared it is necessary to determine the quantity of N NaOH (Solution 1, Group A) required to adjust the pH to the desired level after the addition of NaHC03 and CaClz solutions (Solutions 5 and 6, Group A). As a trial, mix 10 ml of silicic acid and 10 ml of D.S.B.M.S.M. Neutralize with N NaOH and note the amount added (x). Add 0.4 ml of the NaHC03 solution (Solution 5, Group A), and then adjust the pH to 8.2 with N NaOH (Solution 1, Group A). Divide the sample (approximately 20 ml) into four aliquots and add varying amounts of the CaClz solution to each. Allow 2 to 3 h to gel, and then determine the most suitable amount of CaClz solution (y) required for the whole 20.0 ml sample (approximately 0.6-0.8 ml is required),

29

I . AUTOTROPIIS

Before procecding to pour the plates, prepare another test plate as follows. MixSilicic acid D.S.R.M.S.M. N NaOH (Solution 1, Group A) CaClz solution (Solution 6, Group A) Inoculum NaHC03 solution (Solution 5 , Group A)

10 ml 10 ml x ml 0.6-0.8 ml 1 ml 0.4 ml

Immediately determine the p H and add N NaOH (Solution 1, Group A) until the pH rises to 8.2. Note this amount (XI). To prepare the plates substitute the value of (x + XI) for x in the previous mix. Allow 2 h to gel and then bcubate in a humid chamber at 28°C. (c) Preparation of silica gel. Pramer’s method is used as described by Skerman (1967). (d) Preparation of silicic acid sols. To prepare an ion-exchange column take approximately a 70 cm length of 25 mm glass tubing and fit it at one end with a glass tap and mount it vertically in a stand. Place some glass beads in the base and cover with a layer of glass wool. Pack 120 g (wet weight) of IR-120 Amberlite resin in the tube and add sufficient 2~ HCI to cover the resin. Remove any air bubbles from the column with a glass rod. Open the tap and pass 1000 ml of 2 M HCI through the column. Drain and then flush with distilled water until the effluent no longer gives a test for chloride with silver nitrate. Leave the column full of water. Immediately after using the column for the preparation of the silicic acid, wash it again with water and regenerate with 2 M HCl. Prepare 500 ml of a solution of sodium silicate containing 1.5% of SiOz and allow it to flow through the column at 5 ml/min. Check the p H of the effluent and collect for use when the p H falls below 3.4. Adjust the pH of the solution to 2.0 with HCI. T h e solution should be stable at this pIi. Sterilize at 110°C for 25 min.

2. Group b: nitrite-oxidizing bacteria, Nitrobacter species The medium for Nitrobacter (Skerman, 1967) is used. (a) Liquid medium. Prepare the basal mineral salts medium with omission of the ammonium sulphate (Solution 2, Group B) and sterilize. Add aseptically 8 ml of the NaNOz solution (Solution 7, Group A) and 20ml of the sterile NaHC03 solution (Solution 5 , Group A) per litre of medium. Adjust the p H aseptically to 8.8 with sterile N NaOH (Solution 1, Group A).

30

V. G . COLLINS

(b) Silica gels. Proceed as for Nitrosotnonas with omission of the ammonium sulphate (Solution 2, Group B) in the preparation of the double strength basal mineral salts medium (D.S.B.M.S.M.). The mixSilicic acid D.S.B.M.S.M. NaNOz solution (7, Group A)

10 ml 10 ml 0.16 ml

Neutralize with N NaOH (Solution 1, Group A), noting the amount used (x). Add 0.4 ml of the NaHC03 solution (Solution 5 , Group A), and then adjust the p H to 8.8. Divide the sample into four aliquots; add varying amounts of CaCl2 solution (Solution 7, Group A) to each and allow to stand for 2-3 h to gel. Determine the amount of CaCl2 solution (y) that will give the optimal gel in the total (approximately 20 ml) sample. Prepare another mix as followsSilicic acid D.S.B.M.S.M. NaNOz solution (Solution 7, Group A) Inoculum N NaOH (Solution 1, Group A) CaClz solution (Solution 6, Group A) NaHC03 solution (Solution 5, Group A)

10 ml 10 ml 0.16 ml 1 ml x ml Y ml 0 - 4 ml

Determine the p H immediately; adjust to p H 8.8 with N NaOH (Solution 1, Group A) and note the amount (XI). To prepare plates substitute the value of (x+xl) for x in the previous mix. Allow 2 h to set and then incubate at 28°C in a humid chamber.

3. Group a and Group b (a) Autotrophic media for Nitrosomonas and Nitrobacter (J. Meiklejohn personal communication to Skerman 1967). NaCl MgS04. 7Hz0 FeS04.7HzO H2O 0.1 M KH2P04

+ (NH4)zS04 (for Nitrosomonas)

+ NaNOz (for Nitrobacter)

0.3 g 0.14 g 0.03 g 90 ml 10 ml (Previously boiled for 30 min, cooled, and made up to volume). 0.66 g 0.5 g

Dilute to 1000 ml and add 10 g of powdered CaC03 and 0.4 ml of a trace element solution supplying Mn, 22 pg; €3, 21 pg; Cu, 17 pg; Zn, 16 pg; and CO, 14pg. Dispense in layers not more than 1 cm deep in Erlenmeyer flasks. Sterilize at 121°C for 15 min.

I. AUTOTROPHS

31

(b) Quantitative determination of nitrite. T h e method of Lees and Quastel described by Skerman (1967) is used. Dilute a sample containing approximately 0.5-5.0 pg of nitrite-N to 11 ml with distilled water. Add 2 ml of Griess-Llosvay reagent. After 30 min read the colour density in a photoelectric colorimeter with the use of a suitable filter and determine the nitrite-N concentration from a curve prepared from a series of standard nitrite solutions. The Griess-Llosvay reagents are prepared as follows(i) Sulphanilic acid solution: Dissolve 0.5 g in 30 ml of glacial acetic acid. Add 100 ml of distilled water and filter. T h e reagent is stable for onemonth. (ii) a-Naphthylamine solution: This should not be more than one week old. Dissolve 0.1 g of a-naphthylamine in 100 ml of boiling distilled water. Cool and add 30 ml of glacial acetic acid. Filter. For use mix Solutions 1 and 2 in equal quantities. The standard nitrite solution is prepared as followsDissolve 0-493 g of pure sodium nitrite in water. Make up to 1 litre. Dilute 10 ml of this solution to 1 litre. This final solution contains 0.001 mg of N as nitrite. T h e standard curve should be prepared over a range of 0.0005-0.001 mg of nitrite nitrogen.

4. Group a : ammonia-oxidizing autotrophic bacteria, additional medium The medium of Skinner and Walker (1961), as described by Soriano and Walker (1968), is used. The medium contains: (NH4)$304, 0.5 g; KHzP04, 0.2 g; CaC12.2HzO 0.04 g; MgS04.71-120, 0.04 g; Fe (as ferric citrate or Fe-EDTA chelate), 0.5 mg; phenol red, 0.5 mg; distilled water, 1. litre. Plates are prepared by adding 1-1.5% of special Noble (Difco) or purified (Merck) agar to the above solution. After sterilizing at 120°C for 15 min, the p H of the medium is adjusted to 7.5-8.0 by adding a sterile aqueous 5% sodium carbonate solution. The author has used the isolation techniques described by Soriano and Walker (1968). These are excellent and, therefore, are included here for reference. Dilution. Serial dilutions in sterile medium were made from an enrichment medium culture so that the final dilution contained an average of 1or 2 organisms/ml. Tubes (usually forty to fifty) containing 8 ml of medium were inoculated with 1 ml of the final dilution and incubated at 25" or 30°C for some weeks. At intervals, tests were made for the presence of acid and nitrite, and positive tubes were subcultured on peptone agar to check for heterotrophicorganisms,

32

V. G. COLLINS

Plating. An inoculum consisting of a drop of undiluted or diluted enrichment culture was spread over the surface of a dried agar plate by means of a sterile bent platinum wire (the microspatula of Beijerinck). T h e plates were incubated at 25” or 30°C in a closed jar containing dilute aqueous ammonia until nitrite was detected in the agar and presumptive colonies of nitrifiers could be seen under the microscope by transmitted light at x 80 or x 100 magnification. Contaminant colonies were mostly visible without magnification. Isolated microcolonies were sucked into capillary pipettes and transferred to tubes of sterile liquid medium. B. Organisms of groups c, e, f, and g Winogradsky cylinder technique for Group (c) non-photosynthetic sulphur bacteria, Groups (e) and (f) photosynthetic sulphur bacteria and Group (g) photosynthetic non-sulphur bacteriaT h e mud is mixed with CaS04 and some insoluble organic matter, e.g. cellulose, poured into a tall glass cylinder, whereupon the cylinder is filled completely with water containing O.lyoNH4C1 and 0.1% phosphate buffer at pH 7.3 and incubated with continuous illumination. T h e enriched mud serves as a continuous H2S generator (bacterial sulphate reduction), and the photosynthetic sulphur bacteria soon appear as coloured spots at the mudglass interphase. T h e superiority of the Winogradsky method is not only due to the fact that it permits the use of large amounts of mud as inoculum; additional advantages are the continued formation of H2S and the localization in space of the photosynthetic bacteria, thus facilitating their detection in the form of “colonies”.

1. Group c (a) Enrichment culture techniquefor Beggiatoa species. This is based on a modification of Cataldi’s (1940) technique, and described by Faust and Wolfe (1961). Dried roadside grass, which has been exposed to the weather for the autumn months is cut into small pieces and added to boiling tap water in a ratio of about 100 g of grass to each litre of water. After boiling for about 10 min, the water, which then contains soluble components from the grass, is decanted. Additional water is added and boiling is repeated. After five cycles of boiling and decanting, the extracted hay is allowed to stand overnight in water, and on the following day the extraction procedure is repeated three additional times before the hay is spread out and dried. About 0.5 g of extracted hay and 60 ml of stream water are added to each 125 ml Erlenmeykr flask. Each flask is inoculated with a small piece of decaying leaf or other debris from a polluted stream and is plugged with

33

I. AUTOTROPHS

cotton to retard evaporation. Enrichment flasks are incubated at room temperature (about 28°C). (b) Methods for obtainingpure cultures of Beggiatoa species (Faust and Wolfe, 1961). By means of a capillary pipette, a tuft of trichomes from a crude enrichment flask is transferred through several drops of steril tap water to remove most of the contaminating bacteria. T h e tuft is then placed on the dry surface of a solid medium (medium A of Faust and Wolfe) which consists of yeast extract, 0.2 g; agar, 1.5 g ; tap water, 10 ml; glass distilled water, 90 ml; and is adjusted to p H 7.0. Standard sterilization techniques are used in all media preparation. A dry surface is obtained by allowing excess water to evaporate from the surface of the medium in a 30°C incubator for about 2 h with the lid of each dish propped open slightly. Between 4 and 6 h after inoculation each plate is examined under a dissecting microscope at 30 x magnification. A small agar block containing a well isolated trichome is cut and transferred aseptically to 2 ml of sterile liquid medium A. Pure cultures are then obtained, after repeated plating and washing procedures where necessary, and the pure trichomes of the organism are finally transferred to a semi-solid medium (medium D of Faust and Wolfe). This is adjusted to p H 7.0 and contains yeast extract, 0.2 g; agar, 0.2 g ; CaC12,O.Ol g; sodium acetate, 0.1 g; and distilled water, 100 ml. T h e medium is filled out into test tubes (15 by 150 mm) with screw caps, and growth occurs in a narrow disc about 0.5 cm beiow the surface of the medium. This characteristic is typical of gradient organisms which require a specific oxidation-reduction potential for growth. Incubation should be at 17"C, on this medium; for maintenance of stock cultures repeat transfers must be made every seven to ten days.

2. Group d: isolation media (a) Themedium of Lieske (1912). This medium, described by Collins (1963), is particularly useful for the isolation of Thiobacillus dentrificans, and consists of the following ingredientsNazSz03.5H20 KNOB NaHC03 K2HP04 MgCh

0.2g 0.1 g

CaCIz

traces

FeC13 distilled water

traces 1 litre

5.og

5.Og 1.og

(b) The medium of Waksman and Starkey (1922). This medium, described by Collins (1963), is useful for the isolation of Thiobacillus thio-oxidans, and

34

V. G . COLLINS

is composed of the following ingredients per litre of distilled water0.2 g 0.1-0.5 g 0.01 g

0.25 g 3-5 g 10 g

(c) The medium of Starkey (1935). This medium, described by Collins (1963), is useful for the isolation of Thiobacillus thioparus, and is composed of the following ingredients per litre of distilled water5.0 g 0.4 g 4.0 g 0.25 g 0.5 g 0.01 g

(d) Neutral medium for nonaciduric species of Thiobacillus (Skerman, 1967). Use the Pope and Skerman Basic Mineral Salts Medium. Follow the preparation of the basal medium, except at Step 2 add 110 ml of water instead of 210, and at Step 7 make the final volume to 900ml instead of 1000. Sterilize at 121°C for 20 min. Cool. Then add aseptically 100 ml of sterile NazSz03 solution (Solution 5, Group A). The medium should be dispensed in a depth of not more than 1 cm in Erlenmeyer flasks.

3. Groups e and f : isolation media (a) Detailed description for the preparation of culture medium for red andgreen sulphur bacteria. This is described by Pfennig (1961 ; 1962), and in personal communication to the author. Solution 1 Distilled water CaClz anhydrous

2500 ml 2s

500 ml of this solution are autoclaved separately in an Erlenmeyer flask. 2000 ml are distributed in amounts of 75-80 ml into 127 ml (4 0 2 ) screwcapped bottles and autoclaved. Before adding sterile filtered sohtions 2 and 3 (below) the bottles are kept cold (4"-1OoC).

35

I. AUTOTROPHS

Solution 2

Distilled water Heavy metal solution Vitamin-Biz solution KH2P04 KCI NH4CI MgCI2.6H2O Na ascorbate

32 nil 50 ml 15 ml 1g 1g 0.8 g 0.8 g

2.4 g

(To be added only as a separate sterile solution to the ready prepared medium. Ascorbate can be omitted, if larger inocula are used.) Solution 3 900 ml 4.5 g

Distilled water NaHC03

Gaseous CO? is bubbled through for at least 30 min; p H about 6.1. Solution 4

Distilled water NazS .9Hz0

200 ml 3g

Prepared using a magnetic stirrer rod. This solution is autoclaved. After CO:! saturation of solution 3, solution 2 is added and the mixture immediately filtered through a Seitz filter using C 0 2 pressure to push the liquid through (no suction). T h e sterile filtered solution 2+ 3 is added to the 127 ml bottles containing cold 75-80 ml CaClz-solution 1. T h e sterilized cold solution 4 is partially neutralized by adding (on a magnetic stirrer) drop by drop 2 ml sterile 2 M HzS04. This partially neutralized solution is added to the bottles in 6 ml amounts for Chromatiurn and Chlorobium and 3 ml per bottle for Thiospirillum jenense. T h e bottles are nearly filled up with liquid by adding sterile solution 1 (from the 500 ml flask). A small air bubble is left. T h e bottles are immediately closed. ?'he final pH has to be 6.6-6.8, and this will be obtained, if these directions arc followed exactly. The freshly prepared medium becomes slightly turbid due to the oxidation of some HzS to sulphur by dissolved oxygen. After storage of the medium for 1-2 days the turbidity disappears and a slight sediment is formed which turns black after some days. If the tightly closed bottles are stored in the dark the culture medium keeps for several months. The freshly prepared cdture medium should be aged in the bottlcs for at least 24 h before inoculation., The cultures are incubated at room temperature (20°-25"C) at a distance of 30-40 cm from a 40 Wtbulb. Light periods of 16 h and dark periods of 8 hare favourable.

36

V.

G. COLLINS

If the growing organisms have used up both the HzS and the stored sulphur (disappearance’ of the chalky appearance) fresh neutralized NaZS solution has to be added. A suitable volume of solution 4 is added to a sterile Erlenmeyer flask with a magnetic stirrer rod and neutralized on a magnetic stirrer by adding drop by drop sterile 2 M HzS04 only until a slight sulphurturbidity appears, This turbidity will disappear if an excess of HzS04 is avoided. T h e solution will be slightly yellow but clear. 5-6 ml of the neutralized solution arc added to each 127 ml bottle of the Chromatium or Chlorobium strains; 3 ml are added to the Thiospirillum jenense cultures. After the addition of the sulphide solution the cultures are kept in the dark for some hours; thereafter they are put back into the light. Heavy metal solution Distilled water Ethylene diaminetetra acetate

Modified “Hoagland trace element solution” FeS04.7Hz0 ZnS04.7HzO MnClz .4Hz0

1000 ml 1* 5 g (EDTA has to be dissolved first) 6 mi 200 mg 100 mg 20 mg

Vitamin Bl2 solution Vitamin BIZ(Cyanocobalamin, Merck) 2 mg/100 ml distilled water Vitamin solution 100 ml Distilled water 0.2 mg Biotin Nicotinic acid 2 mg 1mg Thiamine p-Aminobenzoic acid 1 mg Pantothenic acid 0.5 mg 5 mg Pyridoxamin-HC1 Modified “Hoagland trace element solution” AIC13 IF! KI 0.5 g KBr 0.5g LiCl 0.5 g MnCk .4Hp0 7g H3B03 11 g ZnCla f g CUCI? Ig NiCls 1g COClZ Ig SnClz. 2H20 0.5 g BaCl2 0.5 g NazMo04 0.5 g NaV03. H20 0.1 g Se-salt 0.5 g

I. AUTOTROPHS

37

Each salt is dissolved separately in distilled water. Before mixing together, the pH of each solution is adjusted below pH 7.0. The total final volume is 3.6 litres (1/5 of the total final volume of Hoagland, 18 litres). The pH of the final solution is adjusted to pH 3-4. The flaky yellow precipitate which is formed after mixing transforms after a few days into a very fine white precipitate. Before use the solution is mixed thoroughly. (b) Group e: the medium of van Niel (1931). This medium is described by Collins (1963). Inoculate water or mud samples into medium of composition 1.0g NHdCI, 0.5 g K2HP04, 0.2 g MgC12, 1.0 g NaHC03, 1.0 g Na2S.9HzO per litre of distilled water (pH 8-8*5), contained in 4 oz glass-stoppered bottles. The NaHC03 is prepared as a 5’7’ solution, and sterilized by filtration through a Seitz filter; the Na2S.9H20 is prepared as a 10% solution and autoclaved separately, the remainder of the ingredients being autoclaved in bulk. After sterilizing, 10 ml/litre of the NazS. 9Hz0 are added to the bulk, along with 20 ml/litre of the NaHC03 solution to complete the medium. (c) Group f: the medium of Larsen (1952). This medium is described by Collins (1963). The medium consists of tap water with 0.1% each of NH4C1, KH2P04, and Na2S.9H20; 0.05% MgCl2; 0.2% NaHC03, and NaCl as required; the initial pH is adjusted to 7.3 (van Niel, 1931). See also the description of the Winogradsky (1887) cylinder enrichment method, as described by Laisen (1952). (d) Group g (photosynthetic non-sulphur bacteria): the medium of van Niel (1944). This medium is described by Collins (1963). The composition of the medium is 1.0 g (NH&S04 or NHdCI, 0.5 g KzHPO4, 0.2 g MgS04.7H20 or MgCl2, 2.0 g NaCI, 5.0 g NaHC03, 0.15-2% proteose peptone, or an alternative organic substance per litre of distilled water (pH 7.1-7-2 adjusted by means of sterile solutions of lisp04 and Na3C03 as required). Note, when chloride is substituted for MgS04 use 0.25 ml of a saturated solution of MgClz per Iitre, an approximate equivalent. (e) Group g: Hutner’s (1946) method and medium for agar slant cultures. Many different media proved suitable for the maintenance of cultures. The substratewas adequately supplied as lactate, 0*2-0-4%, or malic acid (natural or synthetic), 0.1-0.4~0;occasionally sodium acetate (hydrated), 0.05-0.1%, or sodium butyrate, 0*0470,was added to malate and lactate media. Media not containing malate contained sodium citrate (hydrated), 0.025-0.1 %, to ensure full availability of heavy metals and calcium. The unidentified requirements were adequately supplied as trypticase (Baltimore Biological

38

V.

G. COLLINS

Laboratories), 0.1--0*2y0,or thiopeptone (Wilson), 0.10/,. Yeast extract (Difco) was inhibitory to inany strains, while trypticase was non-inhibitory and permitted extremely good growth. T h e remainder of the medium consisted of agar, 1.5yo; small amounts of K2HP04, MgS04.7H20, (NH&HP04, and Fe, 0.1-0.4 mg%; and Mn, 0.05-0.2 mg%. T h e p H was adjusted to 6.5-6.8. These slants were made in screw-capped tubes (bottles would do equally well) with the rubber liners of the metal caps removed. Media were autoclaved for 10 min at 118°-121”C. Hutner states that a few isolates designated “Rhodovibrio”, which appeared to be microaerophilic, and a few isolates of Rhodospirillum rubrum, which although uninhibited by air, seemed unusually sensitive to inhibitory substances or were exacting for other reasons, were grown on the same media rendered semi-solid by decreasing the agar to 0.2-0*4%. T h e growth of slant cultures was usually heavy in 24-48 h. They were then stored in the dark at 6°C. They remained satisfactorily viable for at least a month. There was no obvious impairment of photosynthetic ability as a result of this treatment. (f) Group g: Hutner’s (1950) medium for growth-factor requirements under anaerobic conditions. KaHP04 MgS04.7HzO DL-Mak acid Sodium succinate (hydrated) L-Glutamic acid Glycerol Potassium acetate Sodium indigodisulphonate or benzylviologen(B.D.11.) Zn Ca

Mn Fe

cu Mo

co

0.05 g 0.025 g 0.3 g 0.4 g

0.2 g 0.2 g 0.1 g 1.0 g 0 . 5 rng 1 .O m g 0.4 g 0 . 2 rng 0.1 mg 0.1 rng

0.05 mg 0.05 mg

Distilled water was added to 100 ml, pH adjusted to 6.8-7.1 with KOH. Sodium formaIdehyde sulphoxylate 0.05 g/100 ml was added separately as a freshly autoclaved 2% solution (w/v) reducing agent. Growth factors supplied when necessary as follows: aneurin, 0.1 mg; nicotinic acid, 0.1 mg; p-aminobenzoic acid, 0.01 mg; and biotin, 0.4 pg. T h e concentrations of metals listed refer to the metal content of the salt used. These were usually sulphates.

39

I . AUTOTROPIIS

Hutner also lists a very useful “preservative” mixture for stock solutions as follows: a mixture (v/v) of part o-fluorotoluene, 2 parts n-butyl chloride, and 1 part 1,2-dichloroethane (Hutner and Bjerknes, 1948). This preservative volatilizes on autoclaving. C. Organisms of Group h-Desulfovibrio

1. Gvoup h: isolation media (takenfrom Skerman, 1967). The following modifications of media described by Baars (1930) and Starkey (1938) are recommended by Butlin, Adams, and Thomas (1949). Baars’ Medium K2HP04 NH4Cl CaS04 MgSO4.7HzO Sodium lactate, 70% solution Tap water

0.5 g 1.og 1.og 2-0 g

5.0 g 1000 ml

Dissolve the salts; adjust the p H to within the range of 7-0-7.5 and sterilize at 121°C for 20 min. Prepare separately a 1% solution of FeSO4. (NH4)2S04.6€1~0and sterilize by steaming for 1 h on three successive days. Add 5 ml of the supernatant per 100 ml of the above medium immediately before use. Note: This medium has a heavy precipitate but is quite suited for crude cultures. Starkey’s Medium K2HP04 NH4Cl Na&04 CaC19.2H20 h~gS04.71320 Sodium lactate, 70:/0 solution Distilled water

0.5 g 1.og 1.og 0.1 g 2.0 g 5.0 g

1000 ml

Dissolve the ingredients and adjust the p H to between 7.0 and 7.5. Sterilize at 121°C for 20 min. Note: This medium has a slight precipitate, which may be removed by filtration after sterilization, following which the medium may be resterilized. Prepare a 1% solution of FeS04. (NH&S04. 6 H Z 0as for Baars’ medium above, and add 5 m1/100 ml of medium just before use. For halophilic strains, add 1-3% NaCl to each of the above media before sterilization,or, alternatively, replace the tap or distilled water with seawater.

2. Group h: medium for the isolation and maintenance of pure cultures (from Skerman, 1967) The media of Baars and Starkey, described above, are not ideal for pure culture studies. Skerman recommends the following medium, described by

40

V. G . COLLINS

Butlin and his associates and modified by Postgate. T h e medium is similar in most respects to that published independently by Miller (1950). K2HP04 NH4Cl Na2S04 CaClz .6H2O MgS04.7Hz0 Na lactate, 70% solution (sterilize separately) Difco yeast extract FeS04.7HzO Distilled water

0.5 g 1.og 1.og

0.1 g 2-0g 3.5 g 1-Gg 0.002 g 1000 ml

Dissolve the ingredients, adjust the p1-I to 7.5, and autoclave at 121°C for 20 min. Filter off the sediment. dispense as required, and resterilize. Prepare separately a 0.6% solution of cysteine hydrochloride in distilled water, and sterilize by autoclaving at 121°C for 20 min. This acid solution has a p H of 1.8 and is relatively stable to oxidation, provided it is not neutralized. Add 1 ml to each 9 ml of medium immediately before use. T h e final concentration of cysteine is 5 pmoles/ml. Pick black colonies showing the correct morphological types on microscopic examination into the liquid medium. Incubate aerobically. If it is desired, the cultures may be plated on the same medium containing 2% agar and incubated anaerobically in an atmosphere of hydrogen and 5% C 0 2 in a McIntosh and Fildes Jar, with a dried pad of absorbent cotton wool, impregnated with lead acetate, between the cultures and the catalyst. See text for references to the authors mentioned in connection with this medium.

D. Organisms of Groupj-Hydrogenomonas A synopsis of a method for growing organisms of this group as described by Cohen and Burris (1955). The medium for growth of 11.facilis contains the following macro-nutrients per litre: NaHC03, 1-0g ; NHdCl, 1 4 g; KH2P04, 0.5 g; MgS04.7I180, 0.1 g; NaCl, 0-1 g ; CaC12, 0.1 g ; and Fe(NH&(S04)2. 6H20, 8 mg. T o this is added a mixture of micro-nutrients containing: H3B04, 228 pg; CoCl2.6H20, 80 pg; CuSO4. 5H20, 8 p g ; MnC12.4Ha0, 8 pg; ZnS04.7Hz0, 176 pg; and NazMoO4.2H20, 50 pg. The gas mixture made from commercial cylinder gases consists of 6 parts (by volume) of hydrogen, 2 parts oxygen, and 1 part carbon dioxide. The temperature for growth is maintained at 30°C by means of a thermostated bath. T h e pH of the medium as determined with a glass electrode after equilibration with the gas phase is initially 6.8-7-0. A small amount of Dow Corning “antifoam A” or silicon stopcock grease is added as an antifoam agent. The 8 litre of sterile medium are inoculated with 250-300 ml of a 36-48 h old culture grown in liquid medium in shaken flasks; each 500 ml

I. AUTOTROPHS

41

shaken flask contains approximately 125 ml of medium and the gas mixture described. In growing the hydrogen bacteria a gas mixture is circulated with a pump through the gas reservoir and thcn into the culture vessel; concomitantly the pressure of the system is maintained at essentially atmospheric pressure by displacing water into the gas reservoir as the gas mixture is used. T h e culture vessel is a narrow 10 litre Pyrex bottle (Corning No. GBYDI) fitted with a rubber stopper through which the gas inlet, gas outlet, and medium sampling outlet are attached. Both the gas inlet and outlet are connected externally to filter plugs (40 by 200 mm tubes packed with plugging cotton). A porous gas dispersion ball (“Marc0 Ball O’Mist Releaser”, obtained from the J. B. Maris Co., Bloomfield, N. J.) is connected to the end of the gas inlet, which extends nearly to the bottom of the vessel. In practice, 8 litre of medium are placed within the vessel and autoclaved. After cooling it is inoculated, and the sterile rubber-stopper assembly is put into position. T o avoid the hazard of evacuating this large vessel, C02 is blown through it for 5 min to displace air; the leads into and out of the vessel then are clamped off, and the vessel is set into the thermostated bath and connected to the gas reservoir and pump. After evacuating and filling the reservoir system with the gas mixture, the leads are freed, and the pump is turned on. Then the displacing water reservoir is connected. Gas reservoir bottles are 9 litre Pyrex serum bottles with concave bottoms. These bottles are suitable for evacuation. Connections in the system are made with rubber stoppers, butyl rubber tubing, and glass tubing; and these are made gas tight by sealing with either a mixture of 1 part beeswax to 1 part rosin or with ordinary finger-nail polish. T h e system is checked for leaks by partial evacuation. A gas inlet and evacuation port are connected through a three-way T stopcock to the apparatus. A manometer for the system serves in making up gas mixtures as well as in checking the system for leaks. The authors state that they employ four serum bottles to give a gas reservoir volume of 36 litre because it is convenient to fill the system with gas in the late afternoon and to harvest the next morning without having to refill the system. Any type of bottle which holds slightly less than the total gas volume of the system may be used as a water reservoir. Sulphuric acid sufficient to lower the pH of thc displacing fluid to p€I 4 is added to prevent binding of COa as bicarbonate in the displacing fluid. A “Ccnco Pressovac-4 Pump” is used to circulate the gas mixture; cotton gauze filters and a simple trap formed by using a 500 ml filter flask serve to filter the oil spray from the,gas train. A bypass around the culture vcssel permits control of the volume of gas passing through the culture vessel. In summary, the authors state that rapid growth of H . facilis was achieved by growing the organism in liquid culture, with vigorous aeration, main-

42

V. G . COLLINS

tenance of a reasonably constant gas pressure, and addition of micronutrient elements to thc medium were important. The medium of Schatz and Bovcll(1952), as described by Skerman (1967), 1s-

KHzP04 NH4N03 MgS04.7Ha0 FeS04.7H.O CaClz .2Hz0 Distilled water

0.1 g 0.1 g 0.02 g 0.001 g 0.001 g

to 100 ml

Adjust the pH to between 6.8 and 7.2. Where desired, incorporate 1.5% washed agar. For autotrophic growth, supplement the base with 0.05% NaHC03. Autoclave stock solutions of the NaHC03 separately, flush with COz, and add to the sterile medium before inoculation. Incubate under an atmosphere of 10% C 0 ~ , 3 air, 0 ~and ~ 60% hydrogen. 1. Group j : autotrophic medium using the Pope and Skerman mineral salts solutions (Skerman, 1967) Prepare the same medium as that employed for Nitrosomonas but adjust the p H to 7.0. Incubate under an atmosphere of 10% COz, 30% air, and 60% hydrogen,

E. Organisms of Group k-Carboxydornonas 1. Group k : autotrophic medium using the Pope and Skerman mineral salts solutions (Skerman, 1967) Use the same medium as for Hydrogenomonas but incubate under an atmosphere of 20% oxygen and SOYo carbon monoxide. 2. Group K : the medium of Kistner (1953) (Skerman, 1967) Kh'03 KzHP04 MgS04.7HzO Peptone HzO

2.0 g 1.og 0.1 g 0.2 g 1OOOmI

Dissolve the ingredients and adjust the p H to 7.2. Sterilize at 121°C for 20 min. For an agar medium use only sufficient agar to make a moderately firm gel. An agar that is too hard inhibits growth. Incubate under an atmosphere of 80% CO and 20% 0 2 . 3. Group k : Parkes and Mellor's method for producing carbon monoxide fm thegrowth of organisms (Skerman, 1967) Close a round-bottomed 500 ml flask with a rubber stopper fitted with a gas outlet tube just penetrating the stopper and a dropping funnel with the

I. AUTOTROPHS

43

lower end reaching almost to the bottom of the flask. Add concentrated sulphuric acid to the flask so that the tip of the dropping funnel is immersed. Place concentrated formic acid in approximately half this volume in the dropping funnel. Place the flask over a steam bath and connect the gas outlet tube via a concentrated NaOH wash bottle of soda-lime tube to the gascollecting apparatus. Heat to 100°Cand then admit the concentrated formic acid drop by drop until the required amount of gas has been collected. Allow for the exclusion of air before collecting the gas.

HCOOH~I-I~SO~-~I~~SO~E-~~O+CO 4. Groups i, j and k : Skerman’s (1967) recommended method and description ofan apparatusfor the collection and mixing of gases In culturing autotrophic gas-utilizing organisms, it is necessary to prepare gas mixtures of various types. This can be done simply with the apparatus shown in Fig. 11. This consists of a series of 500 ml gas-collecting

H

Frc. 1 1 . Apparatus for the collection and mixing of gases.

burettes, A , h’,C, and I>, conncctcd at thc base via a manifold to a 3 litre rescrvoir, E,containing dilute sulphuric acid; and via taps at thc top to a second manifold closed at both cnds by taps F and G. ‘The tube from tap G

44

V. G . COLLINS

is connected to the gas generator and both G and F are opened. Gas is allowed to stream through G and F to expel air or any previous gas. Then with F still open, the tap to A is opened and then F is closed. T h e gas is diverted into A and dilute acid is expelled to E. If desired, E may be lowered to reduce the back pressure on the gas generator. With A nearly filled, open F and then close A immediately. Disconnect the gas generator and then close G and F. Repeat with other gas to B and C. Any number of gas burettes may be employed, but allowance must be made for mixing. To prepare a mixture of 200 ml of A, 100 ml of B, and 200 ml of C and D, open F and then A to flush out the manifold. Close F. Lower E so that its meniscus is level with the 200 ml mark in D. Open D and let the gas flow in, adjusting E so that its meniscus is finally level with the water in D at the 200 ml mark. Close D and A. Raise E and open B and F to flush the manifold. Close F. Lower E to the 300 ml level of D and open D. Allow gas from B to flow into D to the required level. Close D and B. Raise E and open C and and F to flush the manifold. Close F. Lower E to the 500 ml mark in D and open D.Allow gas from C to flow in to the required level. Close D and C and replace F. T o discharge D into the required containers, open G and D and flush the manifold. Close G. Connect F to the apparatus and open F.Allow some of the gas mixture to flow through the connection to the apparatus to expel any air before admitting the gas.

F. Organisms of Group i-Methanomonas 1. Group i: autotrophic medium using the Pope and Skerman mineral salts solutions (Skerman, 1967) Use the same medium as for Hydrogenomonas but incubate under an atmosphere of 50% methane and 50% air. For the production of methane use Weygand’s method as described by Skerman (1967), or purchase the highest grade of commercial methane. Pure methane may be prepared by reducing methyl iodide in alcohol with a copper-zinc couple. Add 100 g of zinc dust to 250 ml of a 4% aqueous solution of copper sulphate. Shake thoroughly and then allow the powder to settle. Wash several times with water by decantation and then dry. Place the powder in a 100 ml Erlenmeyer flask fitted with a stoppcrcd dropping funnel and a gas outlet tubc. Allow a mixture of cqual volumes of methyl iodide and absolute ethyl alcohol to drop slowly onto the zinccopper couple. Collect the methane over water aftcr allowing the air from the flask to escape.

45

I. AUTOTROPHS

2. Group :Foster's autotrophic medium (Skerman, 1967) NaN03 MgSO4.7Hz0 FeS04.7HzO NasHP04 NaHzP04 CuSo4.5Hz0 fIsnO3 MnS04. HzO ZnS04.7HsO MOO3 KCl CaClz HzO

2.0 g 0.2 g 0.001 g 0.21 g 0.09 g 200.0 pg 60.0 pg 30-0 pg 300.0 p g 15.0 pg 0.04 g 0.015 g 1000 ml

Dissolve the salts and sterilize. Incubate under an atmosphere of 50% methane and 50% air.

3. Groups i, j and k : Skerman's (1967), recommended method for obsercing the oxidation of carbon monoxide, hydrogen, or methane by growing cultures The apparatus is illustrated in Fig. 12. A consists of a 10 ml graduated

pipette sealed at the tip and joined above the 0.0 ml mark to a 12 ml tube fitted with a 12 mm side arm and pear-shaped bulb with a 16 mm outlet. The tube is plugged at the outlet with cotton wool and sterilized. B is a glass tube of 6 mm outside diameter; bent so that the external arm liesparallel to the main axis of the tube when the tube is inserted as illustrated. The external end is unconstricted and is plugged with cotton wool. T h e internal end is only slightly constricted and extends into the tube until it almost touches the wall when the rubber stopper D fits snugly into the base of the side arm. A small 1 mm aperture, E, is made in the tube exterior to the rubber stopper. A cotton wool plug is rolled around the stem of B between the rubber

46

V. G . COLLINS

stopper and the bend. The lower end (bearing the stopper) is inserted into a 150 by 16 mm tube; and the assembly is sterilized. A third tube, C, bent in the same manner as B but without the small hole E and with the external end extended to a length that brings its tip level with the top of the graduated tube, completes the apparatus. This tube is fitted with a rubber stopper similar to D. Method for Use: Pipette 40 ml of the sterile synthetic medium into the tube A and inoculate the medium. Replace the plug in A with tube B, leaving the stopper D only loosely seated in its base. Connect B to the gas-mixing burette and, holding the tube on its side, loosen the stopper D and allow a quantity of gas to escape into the pearshaped bulb to exclude the air in tube B. Then turn the tube upright and collect approximately 9 ml of gas. If too much is collected it can be released by inclining the tube. Disconnect the gas supply and seat D firmly into the side arm. Supporting the tubes by the outlet tube, immerse them for 20 min in a water bath at a temperature as near that of the room as possible. Holding the tubes by the outlet tubes, slightly tilt the tube until the menisci in the closed arm and pear-shaped bulb are level; read the gas volume (Vl) and note at the same time the temperature of the water bath. All subsequent gas volume readings must be taken at the same temperature. Incubate the tubes on their sides attached to a rocking arm in a water bath. This provides a maximal gas-liquid contact area during incubation. The small aperture E allows movement of liquid between the two bulbs, but the position of the internal end of B does not allow escape of gas. After incubation adjust to room temperature to eliminate errors due to gas expansion. Replace tube B with tube C. Seat the stopper well into the side arm and introduce water through C, while holding the tube in the vertical position by the outlet tube, until the levels in the closed arm and in C are equal. Read the gas volume (V2). Release the stopper in the side arm and insert 4 pellets of solid sodium hydroxide (approximately 0.4 g) into the closed tube. Seal the tube and slowly rock it on its side in a water bath at room temperature for 20 min to absorb CO2 and re-equilibrate the temperature. Place in a vertical position, readjust the levels through C, and again read the volume (V,). The differknce (V2 - V,) is C02. Release C and introduce 0-5 g of solid pyrogallic acid by allowing it to drop through into the closed arm. Reseal; rock the tubes to absorb the oxygen; equilibrate the temperature; adjust the levels; and read the volume (V4). The difference (V3-V4) is oxygen. The residual gas will be hydrogen, carbon monoxide, or methanc, depending upon the mixture uscd for culture,

I. AUTOTROPHS

47

If the final gas analysis is compared with the initial onc, the ratio of gas oxidized to oxygen utilized can bc dctermined.

G. Organisms of Group 1 1. Group I: the culture media of Mulder aiid van Veen (1963) Basal culture solution. 27 mg It. Similar ciirvcd org;inisms h;i\.c hcen follntl in rntaat-curiny hrincs(1 Icnryc~tol., 19.57; Iiutti;iilx, 1057).

D. Pathogenic halopkiles Ily definition, i t is doiil)tful i f iiny 1 i i i i n ; i n o r i i n i i i i ; i l ~xitli~igcn i s a true ha1ol)hilc. I Iowc\~cr,some h;ivc I~ecndcscriI)cti. A micrococcus thiit grcw poorly in ordinary media, but wcll in media containing 3--S(,’{)salt wits isolated from infected lesions on the fingers of seal hiintcrs (‘l’hj6tta anti Knittingcn, 1949). ‘l’his is probably a marine organism that can grow in human serum. In Japan, several outbreaks of food poisoning have been attributed to halophiles. Various salted products have been implicated, principally sea fish. Although the organism was first isolated on blood agar, its later isolasalt agar, under the assumption that staphylococci were involved, tion on 4(;{’ resulted in its “halophilic” character being stressed. How organisms meeting the definition of halophiles could multiply in the human intestine was not clear. Sakazaki et nl. (1963) have reviewed the whole question and, after testing numerous strains, identified the causal organism as Vibrio parahaemol3~ticz~s. ‘rhey statc that the organism grows well in brain heart infusion broth or on t h o c l agar without added salt, but poorly on peptone water or on plain agar with ordinary salt concentration (presumably 0.5%). This raises the whole question of halophilism in moderate halophiles, or at lcast in some organisms that h w e been placed in this category. In my opinion, sufficient is known to place the extreme halophilic rods in a separate category, quite apart from other bacteria. T h e position of the moderate halophiles is still in doubt. More information is needed on their nutrition and on thcir physiology in relation to their ionic requirements. Further information on their chemistry and biochemistry may reveal whether they have unique properties similar to the extreme halophiles. REFERENCES Abram, D., and Gibbons, N. E. (1960). Can. J . Microbid., 6, 536--543. Baas-Uecking, L. G. M.(1931). Scient. Mon., 32, 434-446. Baumgartner, J. C . (1937). Fd. Res., 2, 321-329. Block, M. R. (1963). Scient. Am., 209, 89-98. Boring, J., Kushner, D. J., and Gibbons, N. E. (1963). Can. J . Microbid., 9, 143154. Brown, PI. J., an d Gibbons, N. E. (1955). Cun.J. bi‘icrobiol., 1,486--494. Buttiaux, 11. (1 957). In “The Microbiology of Fish and hleat-Curing Brines”, pp. 137-148. Pi.,lI. Stationery Oflice., Idondon. Cho, K. Y . , Doy, C. H., and Mercer, 1:. 13. (1967).,T Uact., 94, 196-201. Christian, J . €3. 13. (1956). D.Phi1. Thesis, University of C‘amhridgc.. Dundas, I. D., and Larsen, 1-1. (1962). Arch. Microbird., 44, 233 -239. Dundas, I. I)., and Larsen, 11. (1963). Arch. Mic.robio/.,46, 19--22. Dundas, I . I)., Sribivasan, V. It., and Ilnl\orson, l I. 0 . (19hB). (:an. J. iWicvobio[., 9,619-624. I)ussault, 1 I . 1’. (19S3). /’MI,y. /

METHODS IN MICROBIOLOGY,VOLUME 3B, Volume 3B (v. 3B)

The Man in 3B

Harrison, Harry - Deathworld 3b

3b. Chez les brigands

Handbook of Monetary Economics, Volume 3B

Starting with English 3B (Book)

California Math Triumphs Vol 3B RATIOS (CALIFORNIA MATH TRIUMPHS VOL 3B)

Handbook Of Labor Economics, Vol. 3B

Cumitech 3B: Quality Systems in the Clinical Microbiology Laboratory

(novel) (ebook) - Perry Rhodan 0006 - (3b) The Mutant Corps

(novel) (ebook) - Perry Rhodan 0006 - (3b) The Mutant Corps

Lock On No. 23 - Lockheed S-3B Viking

Chemistry of Carbon Compounds: 2nd Supplement to 2r.e v.3B, C, D

(novel) (ebook) - Perry Rhodan 0006 - (3b) The Mutant Corps

Sequences and Power Series Guidelines for Solutions of Problems Calculus 3b

The Prosopography of the Later Roman Empire 2 Part Set: Volume 3B, AD 527-641 (Vol 3)

METHODS IN MICROBIOLOGY,VOLUME 6A, Volume 6A (v. 6A)

METHODS IN MICROBIOLOGY,VOLUME 4, Volume 4 (v. 4)

METHODS IN MICROBIOLOGY,VOLUME 8, Volume 8 (v. 8)

METHODS IN MICROBIOLOGY,VOLUME 5B, Volume 5B (v. 5B)

Methods in Microbiology (v. 5A)

Methods in Microbiology: v. 9

Volume V

Methods in Microbiology: Volume 18

Methods in Microbiology, Volume 2

Methods in Microbiology, Volume 7B

Methods in Microbiology: Volume 6A

Methods In Microbiology, Volume 12

Methods in Microbiology: Volume 13

Inorganic Syntheses. Volume V

METHODS IN MICROBIOLOGY,VOLUME 3B, Volume 3B (v. 3B)

The Man in 3B

Harrison, Harry - Deathworld 3b

3b. Chez les brigands

Handbook of Monetary Economics, Volume 3B

Starting with English 3B (Book)

California Math Triumphs Vol 3B RATIOS (CALIFORNIA MATH TRIUMPHS VOL 3B)

Handbook Of Labor Economics, Vol. 3B

Cumitech 3B: Quality Systems in the Clinical Microbiology Laboratory

(novel) (ebook) - Perry Rhodan 0006 - (3b) The Mutant Corps

(novel) (ebook) - Perry Rhodan 0006 - (3b) The Mutant Corps

Lock On No. 23 - Lockheed S-3B Viking

Chemistry of Carbon Compounds: 2nd Supplement to 2r.e v.3B, C, D

(novel) (ebook) - Perry Rhodan 0006 - (3b) The Mutant Corps

Sequences and Power Series Guidelines for Solutions of Problems Calculus 3b

The Prosopography of the Later Roman Empire 2 Part Set: Volume 3B, AD 527-641 (Vol 3)

METHODS IN MICROBIOLOGY,VOLUME 6A, Volume 6A (v. 6A)

METHODS IN MICROBIOLOGY,VOLUME 4, Volume 4 (v. 4)

METHODS IN MICROBIOLOGY,VOLUME 8, Volume 8 (v. 8)

METHODS IN MICROBIOLOGY,VOLUME 5B, Volume 5B (v. 5B)

Methods in Microbiology (v. 5A)

Methods in Microbiology: v. 9

Volume V

Methods in Microbiology: Volume 18

Methods in Microbiology, Volume 2

Methods in Microbiology, Volume 7B

Methods in Microbiology: Volume 6A

Methods In Microbiology, Volume 12

Methods in Microbiology: Volume 13

Inorganic Syntheses. Volume V

Recommend Documents