Microbiology and Biochemistry

Microbiology And Biochemistry r "This page is Intentionally Left Blank" MICROBIOLOGY ANn BIOCHEMISTRY Dr. Madan ...

Author: Madan Lal Bagdi

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Microbiology And Biochemistry

r

"This page is Intentionally Left Blank"

MICROBIOLOGY

ANn BIOCHEMISTRY

Dr. Madan Lal Bagdi

MANGLAM PUBLICATIONS DELHI-II0053 (INDIA)

Published by: MANGLAM PUBLICATIONS L-2111, Street No. 5, Shivaji Marg, Near Kali Mandir, J.P. Nagar, Kartar Nagar, West Ghonda, Delhi -110053 Phone: 9968367559,9868572512 Email: [email protected] manglam. [email protected]

Microbiology And Biochemistry

©Reserved First Edition: 2009 ISBN 978-81-906785-0-6

All rights reserved no part ofthis work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, phtocopying, recording or otherwise, without the prior permission in writting from publisher of this book.

PRINTED IN INDIA Published by D. P. Yadav for Manglam Publications, Delhi-110053, Printed at Sachin Printers, Moujpur Delhi-53

Preface The present title Microbiology and Biochemistry is an authoritative text book compilated for under-graduate and post-graduate students of various Indian Universities offering this subject. It would· be equally useful as a text in courses in molecular biolo,gy, pharmacology and certain other desciplines of biology. All kinds ofmicroorganisms have been touched to create an impression about the divl!rsity. The scope and practices of using different micro-orgabisms have been shown which may attract future generation. The enormous prospect of application of microbiology and biochemistry have been indicated The author expresses his thanks to all those friends, colleagues, and research scholars whose continuous inspirations have initiated him to bring this title. The author wishes to thank the Manglam Publications, printer and staff mambers for bringing out this book. Constructive criticisms and suggestions for iniprovement of the book will be thankfully acknowledged. Author

"This page is Intentionally Left Blank"

Contents 1.

Introduction ......................................................... 1-35 1.1 Host-Parasite Relations ........................................ 2 1.2 1.3 1.4

1.5

1.6

Diagnosis of Parasitic Infections ............................. 4 Laboratory Procedures ......................................... 5 Procedures for Intestinal Parasites ........................... 6 1.4.1 Collection and Handling of Fecal Specimens ... 6 1.4.2 Gross Examination of Feces ...................... 8 Procedures for Microscopic Examination ..................................... 8 1.5.1 Calibration and Use of an Ocular Micrometer. 9 1.5.3 Direct Wet Mount.. ............................... lG 1.5.4 Concentration Procedures ........................ 12 1.5.5 Permanent Stains .................................. 17 (ii) Unpreserved specimens with PV A fixative .................................. 18 (iii) PV A fixative-preserved specimens ......... 18 Egg Counts ......................................... 21 1.5.6 Duodenal Material ................................ 21 1.5.7 Sigmoidoscopic Material ......................... 12 1.5.8 Abscess Material ................................ , . 22 1.5.9 Cellophane Tape ................................... 23 1.5.10 Examination of Cellophane Tape ................ 23 1.5.11 Culture for Amoebae ............................ ,. 23 1.5.12 1.5.13 Larval Maturation ................................. 24 Adult Worms ....................................... 25 1.5.14 Blood and Tissue Parasites ................................... 25 (i)

CONTENTS

(ii)

1.7

1.8

1.9 1.10 1.11 1.12 1.13 1.14

Collection and Handling of Blood Specimens .............. 7fj 1.7.1 Tissue ............................................... Z7 1.7.2 Aspirates of Bone Marrow or Spleen ........... 28 1.7.3 Fluids ............................................... 28 1.7.4 Skin Snips .......................................... 28 l. 7.5 Concentration Procedures for Blood ............. 28 1.7.6 Membrane Filter Concentration for Filariae .. 19 1.7.7 Saponin Lysis Concentration for Filariae ...... 19 Staining Procedures ........................................... 30 l.8.1 Giemsa Stain Procedure .......................... 30 1.8.2 Gram-Weigert Stain Procedure .................. 30 1.8.3 Culture Procedures for Blood and Tissue Parasites ................................................. 31 Urine ............................................................ 32 Sputum .......................................................... 32 VAginal Material ............................................. 32 Referral of Materials ....................... ~ ................. 32 Safety ........................................................... 33 Quality Assurance ............................................. 35

2.

Origin of Microbiology .......................................... 36--51 2.1 Beginnings of Microscopy .................................... 37 2.2 The First Microscopes ........................................ 38 2.3 Microorganisms and the Origin of Life .............................................. 42 2.4 "Diseases" of Wines .......................................... 46 2.5 Pasteurization .................................................. 47 2.6 Pasteur on Specificity of Disease ........................... 48 2.7 Pasteur of Spontaneous Generation .......................... 48 2.8 Modem Style ................................................... 49 2.9 Chemical Evolution ........................................... 49

3.

Microbiology of Fungi ....••.......•.....••.......•....•........• 52--82 3.1 Characterization ............................................... 52 3.2 Collection and Storage of Specimens ....................... 53 3.3 Direct Exainination ........................................... 53 3.4 Culture and Isolation .......................................... 56 3.5. Identification ................................................... 63

(iii)

CONTENTS 3.5.1 3.5.2 3.5.3 3.5.4 3.5.5 3.5.6 3.5.7 3.5.8 3.5.9 3.5.10 3.5.11 3.5.12

4.

Aureobasidium Spp ................................ 70 Cladosporium Spp ................................. 70 Curvularia Spp ..................................... 71 Drechslera Spp ................... _................ 72 Exophiala Spp ...................................... 74 Fonsecaea spp ...................................... 76 Phaeococcomyces Spp ............................. 77 Phialophora Spp .................................... 78 Rhinocladiella Spp ................................ 79 Scedosporium Spp. . ............................... 79 Scytalidium Spp .................................... ~ Sporothrix Spp. . ................................... ~

Microbiology of Bacteria ...................•.................. 83-105 Shape of Bacteria ............................... " ...... '" .... S4 4.1.1 Size of Bacteria ................................... 85 4.2 The Bacterial Cell ............................................ 87 4.2.1 Cytoplasmic Membrane .......................... 87 4.2.2 Cell Wall ........................................... 88 4.2.3 Capsules ............................................ 89 4.2.4 Polysaccharide Structures ........................ 92 4.2.5 Nucleus ............................................. 92 4.2.6 Metachromatic Granules ......................... C)l 4.2.7 Fat Globules ....................................... 95 4.2.8 Motility ............................................. 95 4.2.9 Motion of Colonies ................................ 99

4.1

5.

Microbiology of Viruses .............................•........ 106-167 The Nature of the Virus Particle. ... ..... .... ....... ...... 108 The Classification of Viruses .............................. 115 The Virus Host .............................................. 116 Quantification of Viruses ................................... 117 General Features of Virus Reproduction ................. 120 Early Events of Virus Multiplication ..................... 123 Viral Genetics ............................................... 128 General Overview of Bacterial Viruses .................. 130 RNA Bacteriophages ........................................ 131

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9

(iv)

CONTENTS 5.10 5.11 5.12 5.13 5.14 5.15 5.16

Single-Stranded Icosahedral DNA Bacteriophages ....................................... Single-Stranded Filamentous DNA Bacteriophages ....................................... Double-Stranded DNA Bacteriophages................... Large Double-Stranded DNA Bacteriophages ....................................... Temperate Bacterial Viruses: Lysogeny .................. A Transposable Phage: Bacteriophage Mu ........................................... General Overview of Animal Viruses. . . . . . . . . . . . . . . . .. . ..

134 138 139 143 147 156 160

6.

General Metabolism .......................•.•..•.............. 168-178 6.1 Characterization of Metabolism.. . . . . . . . . . . . . . . . . . . . .. .. .. 168 6.2 Energy Cycles in Animate Nature ........................ 170 6.3 Energetics of Biochemical Reactions......... ............ 173 6.4 Hight-Enegry and Low-Energy Phosphates: General Considerations ............................................... 175 6.5 Energy Transfer in Biochemical Processes ............... 177

7.

Metabolism of Saccharides ................................... 179-193 7.1 Carbohydrage Catabolism in Tissues ...................... 179 7.1.1 Pentose Phosphate Cycle ....................... 180" 7.1. 2 Interrelation of the Pentose Phosphate Cycle and Glycolysis............... 183 7.1.3 The Biological Function of the Pentose Phosphate Cycle. . . . . . . . . . . . . . . . ... 184 7.2 Biosynthesis of Carbohydrates in Tissues. . . . . . . . . . . .. .. .. 186 7.2.1 Gluconeogenesis ................................. 186 7.2.2 Biosynthesis of Glycogen (Glycogenogenesis) 189 7.2.3 Biosynthesis of Other Oligosaccharides and Polysaccharides ........ 190 7.3 Carbohydrate Metabolism Control in the Organism .... 191

8.

Metabolism of Fats and Glycerides .......................... 194-214 8.1 Degradation of Lipids in Tissues .......................... 194 8.1.1 Intracellular Hydrolysis of Lipids ............. 194 8.1.2 Oxidation of Glycerol ........................... 195 8.1.3 Oxidation of Fatty Acids ....................... 195

CONTENTS

8.2

8.3 8.4 8.5 9.

(v)

Biosynthesis of Lipids in Tissues .......................... 8.2.1 Biosynthesis of Fatty Acids .................... 8.2.2 Biosynthesis of Triglycerides .................. 8.2.3 Phospholipid Biosynthesis ....................... 8.2.4 Biosynthesis of Ketone Bodies ................. 8.2.5 Biosynthesis of Cholesterol ..................... Regulation of Lipid Metabolism in the Organism ............................... Pathology of Lipid Metabolism ............................ Applications of Lipids and Their Components in Pharmacotherapy ..........................

200 200 203 204 206 200 2fJ)

211 213

Metabolism of Nucleic Acid ••..•...••••.••.••••...••........ 215-257 9.1 Functional Roles of DNA .................................. 215 9.1.1 DNA as the Genetic Material ................. 215 9.1.2 Cellular Location of DNA ..................... 217 9.1.3 Clinical Comment. . . . . . . . . .. .. . .. . ............. 220 Other Conformations of DNA ................. 221 9.1.4 The "Central Dogma" .......................... 222 9.1.5 Strand Separation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 223 9.1.6 DNA Polymerases ............................... 224 9.1.7 Bacterial DNA Polymerases ................... 224 9.1.8 Stages of DNA Synthesis ....................... 226 9.1.9 9.1.10 Bidirectional Synthesis ......................... 727 9.2 DNA Synthesis in Animal Cells ........................... 230 9.2.1 DNA Polymerase a ............................. 230 9.2.2 DNA Polymerase b ............................. 231 9.2.3 DNA Polymerase g ............................. 231 9.2.4 Reverse Transcriptase .......................... 231 9.2.5 Nucleosome Formation ......................... 234 9.2.6 Transposable Genetic Elements ............... 235 9.3 Molecular Basis of Mutation ............................... 236 9.3.1 Mutagens ......................................... 237 9.3.2 Physical Agents .................................. 239 9.3.3 Excision Repair .................................. 239 9.3.4 Postreplication Repair .......................... 240 9.4 Chemical Carcinogenesis ................................... 241

CONTENTS

(vi)

9.5 9.6

9.4.1 Initiating Agents ................................. 9.4.2 Promoting Agents ........................... , ... , 9.4.3 Oncogenes ........................................ DNA Sequence Analysis ................................... Recombinant DNA Technology in Medicine ............. 9.6.1 Restriction Endonucleases ...................... 9.6.2 Restriction Maps ................................ 9.6.3 Cloning of Recombinant DNA ................ , 9.6.4 Clones ............................................. 9.6.5 Libraries of Genomic DNA .................... Detection of Recombinant DNA .............. 9.6.6 Clinical Comment .......................... , ... , 9.6.7 Reverse Genetics ................................ 9.6.8 Polymerase Chain Reaction .................... 9.6.9 9.6.10 Cloning and Sequencing the Human Genome .............................

242 242 243 246 248 248 249 249 250 253 254 255 256 256 257

1

Introduction Parasitic diseases continue to cause significant morbidity and mortality in the world, particularly in lessdeveloped tropical and subtropical countries. In the United States, indigenous malaria was eradicated long ago, and indigenous nematode infections such as ascariasis, trichuriasis,· and hookworm infection have markedly decreased in both incidence and severity. Some other infections are increasing. Giardiasis is a frequent public health problem, with outbreaks related to water supplies and day care centers for children. Giardia, ameba, and Cryptosporidium infections are increasing in male homosexuals. Pneumocystis carinii, Cryptosporidium species, Strongyloides stercoralis, and Toxoplasma gondii are increasingly important causes of serious infections in immunoco-mpromised hosts, especially those with AIDS (acquired immune deficiency syndrome). In addition to infections which are indigenous to the United States, a wide variety of infections may be seen in U.S. citizens who have traveled or worked in foreign countries or in foreign nationals who are visiting or now residing in the United States. Many of these people, such as persons infected with malaria, may be asymptomatic for months or years before disease develops or relapses occur. Some people are recognized as having malaria only when a recipient of their blood develops transfusion-induced malaria or when a baby develops congenital malaria. Other diseases such as echinococcosis may require years before becoming clinically evident. Efforts to eradicate parasite infections have had variable success. Sanitary fecal disposal, improved water supplies, and improved hygiene in food production and preparation have aided in the control of intestinal parasites. However, much of the earlier enthusiasm for

2

MICROBIOLOGY AND BIOCHEMISTRY

the eradication of malaria has been tempered by the realization that malaria eradication is going to be difficult because parasites are becoming resistant to chemotherapeutic agents, mosquito vectors are becoming resistant to common insecticides, and the use of some insecticides may harm the environment. Human modifications of the environment, such as the building of dams and irrigation systems, have provided an appropriate environment for vectors such as snails and thus allowed diseases such as schistosomiasis to flourish in areas where these diseases had been uncommon. In addition, immunization programs for parasite infections have developed more slowly than was anticipated.

1.1 HOST-PARASITE RELATIONS A knowledge of parasite life cycles is crucial in the understanding of the ways infection is acquired and spread, the pathogenesis of disease, and the ways in which disease might be controlled. Some parasites which infect only humans, such as Enterobius vermicularis (pinworm), have a narrow host specificity, whereas others such as Trichinella spiralis infect numerous species. When oth~ animals harbor the same parasite stage as humans, these animal species may serve as reservoir hosts. Humans infected with a parasite stage usually seen in other animal species are referred to as accidental hosts. In the simplest life cycle, the parasite stage from humans is immediately infective for other humans, as in pinworm infection or giardiasis. In other infections such as ascariasis or trichuriasis, a maturation period outside the body is required before the parasite is infective. However, for many parasite infections, a second or even a third host is required for completion of the life cycle. Hosts are defmed as intermediate hosts if they do not contain the sexual stage and as definitive hosts if they do contain the sexual stage. Some protozoa, such as the amebae, flagellates and hemotlagellates, do not have a recognized sexual stage. In the intermediate host, there may be a massive proliferation of organisms, as occurs in -humans harboring malaria parasites or snails harboring schistosome intermediate stages, or there may be no proliferation, as in mosquitoes which harbor microfilaria undergoing maturation. There may be proliferation in definitive hosts, as in mosquitoes harboring the sexual stage of malaria in which thousands of sporozoites are produced, or there may be no proliferation, as in helminth infections in which one adult is developed from each infective larva. However, in the latter, the adult helminths do produce numerous eggs or larvae.

INTRODUCTION

3

TABLE 1.1 : INCIDENCE OF INTESTINAL PARASITES IN 322,735 FECAL SPECIMENS EXAMINED BY STATE HEALTH DEPARTMENT LABORATORIES No. of % of positive Parasite examinations specimens Protozoa 12,947 4.0 Giardia lamblia 2,409 Entamoeba histolytica 0.8 1,880 0.6 Dientamoeba fragilis 7 Balantidium coli 1 Isospora spp. 21,120 6.5 Nonpathogenic Nematodes 5,481 1.7 Trichuris trichiura 4,630 Ascaris lumbricoides 1.4 4,344 Enterobius vermicularis 1.4 2,035 Hookworm 0.6 Strongyloides stercoralis 602 0.2 Trichostrongylus spp. 14 Trematodes Clonorchis-Opisthorchis 205 0.06 Schistosoma mansoni 48 Fasciola hepatica Paragonimus westermani Cestodes Hymenolepis nana 1,068 0.3 Taenia spp. 251 0.08 Diphyllobothrium latum 20 Hymenolepis diminuta 12 Dipylidium caninum 7 In some helminth infections, a migration through various body tissues is essential for maturation, as in ascarasis or schistosomiasis, whereas in other infections, the larva leaves the egg and simply matures in the intestinal tract, as in trichuriasis and enterobiasis. Host tissues involved vary depending upon the parasite. In severely immunocompromised patients, sites may be involved that are not involved in normal hosts.

4


Parasites of humans proliferate tremendously at certain stages, with thousands or even millions of forms being produced for every one that survives to perpetuate the parasite. Parasites may be quite hardy. For example, certain stages, particularly eggs and cysts, may survive for weeks or months in the environment. Parasites have often developed unique ways of protection from the defense mechanisms of the host. The survey does not include laboratories in Guam, Puerto Rico, or Virgin Islands. One or more parasites were found in 14.7% of specimens. Percentages are not calculated for parasites identified less than 100 times. These mechanisms include the ability to change antigenic characteristics so that although the host forms antibody, the antibody does not react with the modified parasite, or the parasite may be coated with host immunoglobulins, as in schistosomiasis, so that the host does not recognize the parasite as foreign. Macrophages and both cell-mediated and humoral immunities appear to be important in the host response to infection. Eosinophils are particularly important in the defense against tissue-invading helminths.

1.2 DIAGNOSIS OF PARASITIC INFECTIONS The diagnosis of most parasitic infections is dependent upon the laboratory. For intestinal and blood parasites, morphologic demonstration of diagnostic stage(s) is the principal means of diagnosis, whereas for tissue infections, immunodiagnostic teChniques are generally more important. During the early stages before diagnostic forms are produced (prepatent period), patients may be symptomatic. For example, patients may have pulmonary symptoms and eosinophilia due to ascaris larva migration at a time when eggs are not produced. In such patients the physician may suspect parasite infection, but the actual diagnosis must be based on a clinical impression or immunodiagnostic tests, or diagnosis must await the production of diagnostic stages. In establishing a diagnosis, the clinician places a great deal of trust in the laboratory. This trust can be misplaced if laboratory personnel are not competent to identify or exclude parasites. The literature clearly documents instances in which outbreaks have been overlooked due to incompetent laboratory diagnosis or in which inflammatory cells or other objects have been identified as parasites and outbreaks have been diagnosed when none existed. The results of proficiency-testing programs also suggest that laboratories have

5

INTRODUCTION

difficulty with the identification of some parasites, especially intestinal protozoa. Identification may be by gross examination for adult helminths or, more commonly, by microscopic examination for protozoa, helminth eggs, and larvae. The diagnostic forms of some parasites, such as the eggs of Ascaris spp., are present on a regular basis. Other forms, such as malaria parasites, Taenia eggs, or Giardia cysts, vary from day to day. TABLE 1.2 : PARTICIPANT PERFORMANCE No. of Parasite

Avg correct specimens

identification

(%) Formalin-fixed fecal specimens Ascaris lumbricoides eggs Hookworm Strongyloides stercoralis larvae Trichuris trichiura eggs Diphyllobothrium latum eggs Hymenolepis diminuta eggs Taenia sp. eggs Paragonimus westermani eggs Giardia lamblia cysts Entamoeba coli cysts No parasite seen PVA-fixed specimens Entamoeba histolytica Entamoeba coli Endolimax nana Negative for parasites

6 6 4

6 6 5 6 5 8 9 6 5 4 4

3

90 92 88 93 81 91 87 83 65 88 92 73 52 51 77

Most immunodiagnostic tests used today for parasitic infections detect antibody. In recent years, the sensitivity and specificity of many such tests have improved. A number of antigen detection tests have recently been described and show promise, but none of these tests are currently available commercially.

1.3 LABORATORY PROCEDURES Many methods for diagnostic parasitology have been described.

6


'There are advantages and disadvantages to each method. Some are particularly valuable for epidemiologic studies or for evaluations of new therapeutic agents, whereas other methods are used primarily for laboratory diagnosis. In this chapter we emphasize the diagnostic procedures. From the numerous methods, we have selected those which are widely used in this country and which are sensitive and relatively easy to perform. These methods should prove adequate for most laboratories. For additional procedures, laboratory manuals or parasitology books should be consulted. When alternative methods or methods for specific parasites are indicated, references will be given, but the methods will not be described.

1.4 PROCEDURES FOR INTESTINAL PARASITES Intestinal and biliary parasites are generally diagnosed by finding diagnostic stages in feces or other intestinal material such as duodenal or sigmoidoscopic aspirates. Studies have shown that the eggs of most parasites are uniformly distributed in the fecal mass due to the mixing action of the colon, although some, such as schistosome eggs, which originate in the distal colon, may be more numerous on the surface of formed fecal specimens. The distribution of protozoan forms is more variable. There may be fewer protozoan trophozoites in the first part of an evacuation than in the last because they have deteriorated while in the lower colon. 1.4.1 Collection and Handling of Fecal Specimens The numbers and times of collection for fecal specimens depend somewhat on the diagnosis suspected. As a routine, because some organisms are shed in a variable pattern, it is advisable to examine multiple specimens before excluding parasites. The general recommendation is to collect a specimen every second or third day, for a total of three specimens. From a hospitalized patient, one specimen each day for three days may be more cost effective. A number of substances may interfere with stool examination. Particulate materials such as barium, antacids, kaolin, and bismuth compounds interfere with morphologic examination, and oily materials such as mineral oil create small, refractile droplets that make examination difficult. Antimicrobial agents, particularly broadspectrum antimicrobial agents, may suppress amebae. If any of these substances have been used, specimens should not be submitted until the substances have been cleared (generally 5 to 10 days). A fecal specimen may appear satisfactory by gross examination when there is still barium, etc., which can interfere with microscopic examination.

INTRODUCTION

7

Fecal specimens are best collected into widemouthed, water-tight containers with tight-fitting lids such as waxed, pint-sized ice cream cartons or plastic containers. Usually patients can defecate directly into such containers. Urine should not be allowed to contaminate specimens, as it is harmful to some parasites. If specimens are to be collected in a bed pan, the patient should micturate into a separate container before the specimen is collected. Toilet paper should not be included with the specimen. Stool should not be retrieved from toilet bowl water, as various freeliving protozoa in water might be confused with the parasites. In addition, water is harmful to some parasites such as schistosome eggs and amebic trophozoites. If the patient is producing formed specimens, stool may be collected by having the patient squat over waxed paper to defecate. Purgation with sodium sulfate or buffered phosphosoda may be helpful in the diagnosis of amebiasis in some patients. Purgation is usually done after a series of fecal specimens have been negative, and it requires the order of a physician. Prior arrangements must be made with the laboratory, and specimens must be collected during regular laboratory hours. The patient is given the appropriate salt solution orally. In approximately 1 to 1.5 h, the patient will begin to pass stool specimens, and each specimen should be promptly transported to the laboratory for examination. Clinical information such as the suspected diagnosis, travel history of the patient, and clinical findings should be included on the requisition. In addition, the time the specimen was passed and the time it was placed in fixative should be noted. If the specimen is in fixative, the consistency of the original specimen should be stated, or a portion of unfixed specimen should be included with the fixed specimen. A laboratory may have specimens placed in fixatives in the home or patient care area immediately after passage, may place portions of specimen in fixatives at the time they are received in the laboratory, or may examine the specimen unfixed. Many laboratories use a combination of these methods depending on the location of the patient, consistency of the specimen, time of day, and laboratory work load. Prompt examination or fixation is particularly important for soft, loose, or watery specimens, which are most likely to contain protozoan trophozoites. Formed specimens, which are likely to contain protozoan cysts or helminth eggs or larvae, can remain satisfactory for a number of

8


hours at room temperature or overnight in a refrigerator. Soft and liquid specimens should be examined or placed in fixatives promptly (within 1 h). Specimens which cannot be examined or fixed promptly should be either refrigerated or left at room temperature. They should not be incubated, as incubation speeds the deterioration of the organisms. Feces for parasite examination must not be frozen and thawed. The fixative system generally used is a two-vial technique with one vial containing 5 to 10% buffered Formalin and the other vial containing polyvinyl alcohol (PV A) fixative. A portion of the specimen is added to the fixative in a ratio of approximately 3 parts fixative to 1 part specimen and thoroughly mixed to ensure adequate fixation. An alternative to Formalin is Merthiolate-iodine-formaldehyde (MIF), which fixes and stains at the same time. If unfixed specimens are processed in the laboratory, fecal films may be prepared and immediately fixed in Schaudinn fixative.

1.4.2 Gross Examination of Feces Specimens should be examined grossly to determine the consistency (hard, formed,loose, or watery), color, and presence ofgross abnormalities such as worms, mucus, pus, or blood. It may be profitable to examine flecks of mucus, pus, or blood for parasites. If adult worms or portions of tapeworms are sought, the feces may be carefully washed through a screen. (Small worms may be difficult to see if gauze is used.) The identification characteristics of adult worms are not discussed in this chapter, so parasitology books should be consulted.

1.5 PROCEDURES FOR . MICROSCOPIC EXAMINATION The three principal microscopic examinations performed on stool specimens are direct wet mount, wet mount after concentration, and permanent stain. Although each examination can contribute to diagnosis, the yield of some methods is small with certain kinds of specimens. As a minimum, formed specimens should be examined by a concentration procedure. Soft specimens should be examined by concentration and permanent stain, and, if submitted fresh, by direct wet mount. Loose and watery specimens should be examined by wet mount and permanent stain. If specimens are received in fixative and the consistency is not known, concentration and permanent stain should be performed. Other examinations may be helpfuL Special procedures which may assist in the diagnosis of specific parasites are noted below in discussions of the parasites.

9

INTRODUCTION

1.5.1 'Calibration and Use of an Ocular Micrometer Size is important in the differentiation of parasites and is most accurately determined with a calibrated ocular micrometer, thus, each laboratory performing diagnostic parasitology must have such a micrometer. An ocular micrometer is a disk on which is etched a scale in units from 0 to 50 or 100. To determine the micrometer value of each unit in a particular eyepiece and at a specific magnification, the unit must be calibrated with a stage micrometer. A stage micrometer has a scale 2 mm long ruled in fine intervals of 0.01 mm (10 p.m). 1.5.2 Calibration of the Ocular Micrometer 1. Insert the micrometer in the eyepiece so that the micrometer rests on the diaphragm, with the etched scale facing the eye. In many new microscopes, the micrometer can be dropped in and secured with a ring retainer. (It is helpful to have an extra ocular in which the micrometer may be left.) 2. Place the stage micrometer on the microscope stage. TABLE 1.3: LABORATORY EXAMINATIONS FOR VARIOUS TYPES OF FECAL SPECIMENS Type of specimen

Direct wet mount

Unpreserved Formed + ++ Soft + + Loose and watery ++ Preserved Formalin Formed or soft + Loose or liquid PVA fixative Formed Soft, loose, or liquid Essential for basic examination;

Permanent stain

++ ++

±

±

++

+ +

++

+ +,

Method Concentration

+

± ++ +,

recommended for basic examination;

±, optional for basic examination. 3. Focus on the etched scale. Since the micrometer must be calibrated for each objective, begin with the lowest magnification

10


(e.g., x 10). 4. Align the two scales so that the zero points are superimposed. 5. Find a point far down the scales at which a line of the stage micrometer coincides with a line of the ocular micrometer. Count the number of ocular units and the number of stage units from zero to these coinciding lines. 6. Multiply the number of stage micrometer units by 1,000 to convert millimeters to micrometers. 7. Divide the. product of step 6 by the number of ocular units to determine the 'value of an ocular unit. Repeat the calibration for each objective. Keep a record of the unit value for each objective for each microscope used. Calibration must be done separately for each microscope and must be repeated if an ocular or objective is changed. Use of the micrometer. Insert the eyepiece containing the calibrated ocular micrometer in the microscope. Count the number of ocular units which equal the structure to be measured. Multiply the number by the micrometer value of the ocular unit for the objective being used. If an ocular micrometer is properly used, parasites which are similar in appearance but different in size can be readily differentiated. 1.5.3 Direct Wet Mount The direct wet mount made from unconcentrated fresh feces is most useful for the detection of the motile trophozoites of intestinal protozoa and the motile larvae of Strongyloides spp. It is also useful for the detection of protozoan cysts and helminth eggs. For fixed feces, the direct wet mount may allow the detection of parasites which do not concentrate well. This method is also useful for the examination of specific portions of feces, such as flecks of blood or mucus. Direct wet mounts are prepared by placing a small drop of 0.85% saline toward one end of a glass slide (2 by 3 in. [ca. 5 by 7.5 cm]) and a small drop of appropriate iodine solution (see below) toward the other end. With an applicator stick, a small portion of specimen (1 to 2 mg) is thoroughly mixed in each diluent, and a no. I cover slip (22 mm) is added. The density of fecal material should be such that newspaper print can be read with difficulty through the smear. The material should not overflow the edges of the cover slip. Grit or debris may prevent the cover slip from seating and may be

INTRODUCTION

11

removed with a comer of the cover slip or an applicator stick. Mounts may be sealed with Vaspar (50% petroleum jelly, 50% paraffin) which is melted on a hot plate (not over an open flame). A cotton applicator or small brush is used to apply small drops of Vaspar to opposite comers to attach the cover slip and then to seal it with even strokes. The amount of Vaspar on top of the cover slzip should be minimal. Sealing slows drying and allows oil immersion magnification to be used. Alternatively, drying can be slowed by placing wet gauze or paper toweling in a petri dish, laying portions of applicator sticks or glass rods on the moist material, laying the slide on the .sticks or rOds, and replacing the lid of the dish. The iodine solution should be that of Dobell and O'Connor (1 %) or a 1:5 dilution of Lugol iodine. Iodine solution, if too weak, will not stain organisms properly, and if too strong, it will cause clumping of fecal material. Stock iodine solution should be stored in a tightly stoppered brown bottle away from sunlight. Keep the iodine and saline solutions in small dropper bottles, and replace (don't replenish) the solutions weekly. Iodine solution keeps longer if it is refrigerated. Iodine stain solution can be quality controlled by the observation of appropriate staining in positive clinical specimens or Formalin-fixed specimens kept for that purpose. For the examination of wet mounts, the light of the microscope must be properly adjusted. To achieve optimal resolution, the condenser should be centered and focused for Kohler illumination (racked up). To achieve contrast of the objects in the field, light intensity is diminished with the iris diaphragm of the condenser rather than by lowering the condenser. The entire saline wet mount cover slip should be systematically scanned with x 100 to X 200 magnification. Suspicious objects are confirmed at higher magnification. In addition, the preparation should be scanned at higher power (X 400 to x 500) for a couple of passes across the cover slip to look for protozoan cysts which might be missed with lower power. Screening a slide should take an experienced microscopist about 10 min. If debris is covering a suspicious object, the debris may be removed by pressing or tapping on the cover slip with an applicator stick. This pressure may also help in reorienting an egg, as when one is looking for an operculum. The saline wet mount is best for the detection of helminth eggs and larvae, and it is especially good for protozoan cysts, which appear refractile. The principal usefulness of the iodine mount is to study

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the morphology of protozoan cysts, as this stain shows nuclear detail and glycogen masses (but does not stain chromatoid material). If suspicious objects are seen, they can be examined under oil immersion ( xl, (00). If definite or possible protozoan cysts or trophozoites are detected which cannot be identified in wet mounts, permanent stains are required. A solution of buffered methylene blue (pH 3.6) may be used as a vital stain for the examination of fresh specimens for protozoa. The wet mount is prepared as described above, with buffered methylene blue substituted as diluent and 5 to 10 min allowed for the dye to become incorporated in the organisms before examination. Organisms become overstained in 20 to 30 min.

1.5.4 Concentration Procedures Concentration procedures are used to separate parasites from fecal detritus. These procedures are based on differences in the specific gravity of parasite forms and fecal material. In sedimen-tation, the parasite forms are heavier than the solution and are found in the sediment, whereas in flotation, solutions of high specific gravity are used, and parasite forms float to the surface. An initial washing step removes some of the soluble or fmely particulate material, and straining removes larger portions of debris. A wide variety of sedimentation and flotation methods have been described. The Formalin-ethyl acetate modification of the Formalin-ether sedimentation technique and a zinc sulfate flotation technique are widely used and are the only methods described in this chapter. Both methods require that centrifugation be performed in centrifuges with free-swinging carriers. Squeeze bottles for Formalin, saline, or water simplify the processing of large numbers of specimens. 1.5.4.1 Formalin-ethyl acetate centrifugal sedimentation The original procedure from which the Formalinethyl acetate centrifugal sedimentation technique was adapted was the Formalinether concentration method of Ritchie. The Formalin-ethyl acetate procedure avoids problems with the flammability and storage of ether. This procedure can be performed on specimens which have been fixed in Formalin for a time or on specimens with Formalin added during the processing. The procedure can also be performed on material fixed in MIF. The procedure with Formalin-preserved specimens is as follows. 1. Thoroughly mix the formalinized specimens.

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2. Depending on the density of the specimen, strain a sufficient quantity through gauze into a 15-ml conical centrifuge tube to give the desired amount of sediment. (Wet gauze in a 4-oz [ca. 120-ml] conical paper cup with the tip cut off can be used for straining.) 3. Add tap water or saline, mix the solution thoroughly, and centrifuge it at 650 x g for 1 min. The amount of the resulting sediment should be about 1 ml. The amount of sediment may be adjusted by the addition of more feces and centrifugation again or by the addition of water, suspension again, the removal of an appropriate amount of material, and then recentrifugation. 4. Decant the supernatant, and wash it again with tap water, if desired. 5. To the sediment, add 10% Formalin to the 9-ml mark, and mix the solution thoroughly. 6. Add 4 ml of ethyl acetate, stopper the tube, and shake the tube vigorously in an inverted position for 30 s. Remove the stopper with care. 7. Centrifuge the solution at 450 to 500 x g for 1 min. Four layers should result: ethyl acetate, plug of debris, Formalin, and sediment. 8. Free the plug of debris from the sides of the tube by ringing the tube with an applicator stick, and carefully pour the top three layers into a discard container. With the tube still tipped, use a swab to remove debris from the sides of the tube. This step is very important, for lipid droplets which reach the sediment make examination difficult. 9. Mix the remaining sediment with the small amount of fluid that drains back down from the sides of the tube (or add a drop of saline or Formalin). If mounts are to be prepared later, a small amount of Formalin may be added to the sediment and the tube may be stoppered. 10. Prepare wet mounts as described above, and examine them. The procedure for Formalin-ethyl acetate centrifugal sedimentation with fresh specimens is as follows. 1. Comminute a portion of stool about 1.5 cm in diameter in 10 ml of saline or water. 2. Strain about 10 ml of the fecal suspension into a 15-ml conical centrifuge tube. 3. Centrifuge the suspension at 650 x g for 2 min. This step

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should provide about 1 ml of sediment. If not, adjust the amount of sediment as described above. 4. Wash the sediment again if desired. 5. To the sediment, add 10% buffered Formalin lo the 9-ml mark, mix thoroughly, and allow the mixture to stand for 5 min or longer. 6. Proceed as for step 6 of the procedure for fixed specimens. (Note that either saline or water can be used. Tap water will lyse Blastocystis hominis. If schistosomiasis is suspected, the specimen should be preserved in Formalin before concentration to prevent hatching.)

1.5.4.2 Zinc sUlfate centrifugal flotation The zinc sulfate concentration method originally described by Faust et al. may be performed on unfixed or Formalinfixed specimens, although the specific gravity of the zinc sulfate solution required differs. The disadvantages of the zinc sulfate concentration are: (i) dense schistosome eggs do not concentrate well; (ii) opercula often pop, and thus operculate eggs may be missed; and (iii) larvae and cysts may collapse. The modified procedure with Formalin-fixed feces slows the collapse of larvae and cysts and largely prevents the popping or opercula. The advantages are that it leaves a rather clean background, has less grit than the sedimentation procedure, and is better for the concentration of some parasites, such as Giardia cysts. The procedure for Formalin-preserved specimens is as follows. The specific gravity of the zinc suflate must be 1.20. Centrifugation must be performed in round-bottomed tubes such as 16- by l00-mm disposable tubes. 1. The Formalin-preserved fecal material is mixed, strained through one layer of cheesecloth into a conical paper cup, poured into the tube to a level about 1 cm from the top, and then centrifuged. 2. The tubes are centrifuged for 3 min at about 650 x g. There should be 1 to 1.5 cm of sediment. 3. Decant the supernatant from each tube, and drain the last drop against a clean section of paper towel. 4. To the packed sediment of each tube, add zinc sulfate to within 1 cm of the rim. 5. Insert two applicator sticks, and thoroughly mix the packed sediment. 6. Immediately centrifuge the suspension at 500 rpm for 1.5 min.

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7. Very carefully transfer the tubes to a rack of the proper size, so that the tubes remain vertical. Do not disturb the surface ftlms, which now contain the parasites. Allow the tubes to stand for 1 min to compensate for any movement. The countertop must be vibration free. 8. With a loop which is bent at a right angle, transfer to a slide (2 by 3 in.) two loops of surface material beside I drop of saline and two loops beside I drop of iodine. With the heel of the loop, mix first the saline and then the iodine with the surface material. Cover each mixture with a 22-mm no. I cover slip. The slide should be made within 20 min. 9. To retard drying, place each prepared slide on a bent glass rod or portions of applicator sticks in a petri dish containing a damp paper towel. Petri dishes may be placed in the refrigerator if examination will be delayed. Alternatively, cover slips may be sealed with Vaspar. The procedure with fresh specimens is as follows. / 1. Comminute a fecal specimen about 1 cm in diameter in a tube (16 by 100 mm) half filled with tap water. Add additional water to within 1 to 2 cm of the top. 2. Centrifuge the tube at 650 x g for 1 min. 3 .. Discard the supernatant, and add a zinc sulfate solution of specific gravity 1.18 to within 1 cm of the rim. 4. Proceed as from step 5 above. 1.5.4.3 Sheather sugar ./lotation She ather sugar flotation is recommended for the concentration of Cryptosporidium cysts. Although these oocysts will concentrate when the Formalin-ethyl acetate or zinc sulfate technique is used, they are more readily detected with the Sheather sugar flotation, for they stand out sharply from the background in this solution of high specific gravity. This procedure may be performed on unfixed or Formalin-fixed feces. The procedure for Sheather sugar flotation is outlined below. l. (a) Fonned stool. Place approximately 0.5 g of stool in a tube (16 by 100 mm) about half full of Sheather sugar solution. Mix the solution thoroughly, and then add more sugar solution to within 1 cm of the rim. (b) Watery stool. Centrifuge the fecal specimen and mix 0.5 to 1 ml of sediment with Sheather solution as described above.

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2. Centrifuge the solution at 400 x g for 5 to 10 min. 3. Remove the top portion of the sample with a wire loop bent at a right angle. Place severalloopfuls on a glass slide (2 by 3 in.). Cover the specimen with a 22-mm cover slip, and examine the slide with a x 40 objective. Oocysts are found just beneath the cover slip and are refractile. Saline or iodine is not used in the pr~paration of these mounts.

1.5.4.4 Baermann concentration The Baermann concentration technique has greater sensitivity for the detection of strongyloides larvae than do the standard concentration techniques described above. This technique is useful clinically for the diagnosis and monitoring of therapy of strongyloides infections, and it is useful epidemiologically for the examination of soil for the larvae of nematode parasites. A funnel with a clamped rubber tube on the stem is placed in a ring stand. A circular mesh screen is placed across the funnel approximately one-third from the top, a portion of coarse fabric such as muslin is placed on the screen, and feces is added. Tap water at 37°C is added so that the water just touches the feces. Let the specimen stand I h, remove 2 ml of fluid from the stem, and centrifuge the sample at 300 x g for 3 min. Prepare a wet mount of sediment, and examine it for larvae. 1.5.4.5 Hatching technique for viable schistosome eggs Place a large amount of feces (5 to 10 g) in a large flask (1 to 2 liters), and add water while mixing to break up the feces to a fine suspension. Bring the water level to 2 to 5 em from the top of the flask. Cover the sides of the tlask with foil or other material to shield all but the top of the liquid from light. Allow the tlask to stand at room temperature for several hours. With a hand lens, examine the material at the top of the tlask neck for swimming miracidia. Remove the miracidia with a Pasteur pipette for examination with a x 10 objective. It is not possible to determine the species of schistosome from the miracidia. 1.5.4.6 Other concentration procedures Concentration procedures have been described for feces preserved in MIF, sodium acetate-Formalin, or PYA fixative. MIF- or sodium acetate-Formalin-fixed feces may be used in place of Formalin-fixed feces in the Formalin-ethyl acetate concentration procedure. Some workers feel that organisms do not concentrate as well from material

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fixed in PV A fixative or from material which has been in MIF for extended periods. If large amounts of specimen are to be concentrated, as when specimens of C)ggs are prepared for teaching, gravity sedimentation is usualiy used. The feces is thoroughly mixed in liquid (water, saline, or 10% Formalin) and allowed to settle in a sedimentation jar or funnel for several hours ot overnight. Supernatant fluid is discarded, and the sediment is again suspended and allowed to settle. This procedure can be continued if desired until the supern-atant is clear.

1.5.5 Permanent Stains Permanent stains of fecal smears are most needed for the detection and identification of protozoan trophozoites, but they are also used for the identification of cysts. Wet mounts of fresh feces, even with stains such as methylene blue, are not as sensitive for trophozoites and therefore do not substitute for permanent stains. It is sometimes difficult to identify cysts which are detected in wet mounts; thus, for each specimen, regardless of consistency, it may be worthwhile to fix a portion in PV A fixative or to prepare two fecal films fixed in Schaudinn fixative so that permanent stains can be performed if needed. Permanent stains also provide a permanent record and are easily referred to consultants if there are questions on identification. A number of staining procedures have been described. Some stains, such as chlorazol black, require fresh specimens and are not widely used. A variety of stains for fecal smears preserved by Schaudinn or PV A fixative have been described, including various hematoxylin stains. The stain most widely used in the United States is the Wheatley trichrome stain, which is the only permanent stain described in this chapter. The trichrome staining procedure uses reagents with a relatively long shelf life and is easy to perform. Note that there are differences in staining times depending on whether the specimen is fixed in Schaudinn or PV A fixative, as penetration is slower in the latter. Preparation of smears. (i) Unpreserved specimens with Schaudinn fixative. 1. To prepare thin, uniform smears, place a drop of saline on a glass slide (l by 3 in. [ca. 2.5 by 7.5 cm]). With an applicator stick, transfer a small, representative portion of the specimen to the drop of saline, and mix the two. Spread the solution into a film by rolling the applicator stick along the surface. Remove any lumps. Before watery specimens are smeared, apply an adhesive such

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as serum or albumin to the slide. Liquid specimens may be centrifuged, and the sediment may be used for smear preparation. 2. Place fresh smears immediately into Schaudinn ftxative. Do not allow the smears to dry at any time before they are stained. Smears should ftx for at least I h at room temperature or for 5 min at 50°C; however, they can be left in ftxative for several days. After ftxation, slides may be kept in 70% alcohol indeftnitely before they are stained. (ii) Unpreserved specimens with PV A fixative. 1. On a slide (l by 3 in.), thoroughly mix I drop of unftxed

specimen ~ith I drop of PYA ftxative. 2. Spread the specimen as described below. 3. Allow the smear to dry, preferably overnight, before it is stained. (iii) PVA fixative-preserved specimens.

1. Preserve I part specimen in 3 parts PV A fixative. Mix thoroughly. Fix for at least I h. Specimens keep indefinitely. 2. Add I drop of PV A-fixed specimen to a slide. (a) If there is little sediment, remove a portion of the sediment with a Pasteur pipette. (b) If there is abundant sediment, mix the specimen thoroughly, and add I drop of specimen to a slide with applicator sticks or a Pasteur pipette. 3. Spread the material over the center third of the slide by rolling the specimen with an applicator stick. Remove any lumps. The film should extend to both the top and bottom edges of the slide, as this helps prevent peeling. 4. Allow the slide to dry overnight at room temperature or 35°C. In an urgent situation, the slide can be dried for 4 h at 35°C and then stained.

1.5.5.1 Trichrome staining procedure Table elsewhere outlines the steps in the trichrome staining procedure. Permanently stained slides may be mounted with a cover slip or may be air dried and examined after oil is added. Slides should be examined at a magniftcation of x 400 to X 500 or greater after they are scanned under lower power to fmd optimal areas. A x 50 oil immersion objective is particularly helpful, as it allows the easy use

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of aX 100 oil immersion objective for the detailed examination of organisms while allowing more rapid screening with a x50 objective. Oculars of x 5 or x 6 can provide the same result. A x20 dry objective may also assist in screening. Permanently stained slides should be kept for 2 years.

1.5.5.2 Stain reactions In an ideal stain, the cytoplasm of cysts and trophozoites is bluegreen tinged with purple. Entamoeba coli cyst cytoplasm is often more purple than that of other species. Nuclear chromatin, chromatoid bodies, erythrocytes, and bacteria stain red or purplish red. Other ingested particles such as yeasts often stain green. Parasite eggs and larvae usually stain red. Inflammatory cells and tissue cells stain in a fashion similar to that of protozoa. Color reactions may vary from the above. Incompletely fixed cysts may stain predominantly red, and organisms which have degenerated before fixation often stain pale green. Poor fixation due to an inadequate mixing of the specimen in fixative may result in both of these" appearances. In some specimens, degeneration has occurred before the specimen is placed in fixative, either in the patient before the specimen was evacuated or because of delay in fixing the specimen. 1.5.5.3 Troubleshooting the trichrome stain Except for problems with delayed or inadequate fixation as noted above, problems with the trichrome stain are usually related to reagents other than the stain. If crystalline material is apparent after the specimen is stained, the crystals are probably mercuric chloride in the fixative which was not adequately removed because the iodine in the alcohol-iodine solution was too weak or because the slide was in this solution too short a time. If crystals are present after treatment with proper-strength iodine-alcohol, they are present in the specimen, which is thus unsatisfactory, and another specimen should be requested. If the stain appears washed out, it is likely that the slide was destained too much. This washed-out appearance can be either because the specimen was left too long in the acid-alcohol destain or because the alcohol wash after the acid-alcohol destain had become acidic as a result of transfer by previous slides. The trichrome may become diluted by carry-over alcohol if more than 10 slides per day are stained in one Coplin jar. To restore the

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stain, the lid may be left off for several hours to allow alcohol to evaporate, and then the volume is replaced with new stain. Control slides should be used to monitor the staining. Specimens containing protozoa are best for controls; however, feces containing inflammatory cells or added buffy-coat leukocytes also are satisfactory.

1.5.5.4 Restaining Should the stain be unsatisfactory, the slide can be destained by placing it in xylene to remove the cover slip or immersion oil and then placing it in 50 % alcohol for 10 min to hydrate the slide. Destain the slide in 10% acetic acid in water for several hours, and then wash it thoroughly first in water and then in 50 and 70% alcohols. Place the slide in stain for 8 min, and then complete the stain procedures. It is helpful to e.Jiminate or shorten the destain step.

1.5.5.5 Acid-fast stain for Cryptosporidium sp. Acid-fast staining for Crypiosporidium. sp. has recently become important because this parasite is now recognized as a cause of severe diarrhea in immunodeficient patients such as those with AIDS, and it can cause transient diarrhea in immunocompetent individuals. The modified acid-fast stain recommended is similar to that used to stain Nocardia spp. in that it uses milder acid decolorization. A variety of acid-fast and fluorochrome staining procedures have been described for Cryptosporidium spp., and all the procedures appear to work. The following procedure is useful for staining Nocardia species as well as Cryptosporidium species. This procedure may be used on fresh or Formalinfixed material or on material from concentration procedures. If the specimen is liquid, centrifuge it, and use the sediment to prepare a smear. 1. Pick a portion of material with an applicator stick, mix the material in a drop of saline, spread it on a glass slide (1 by 3 in.), and allow it to dry. 2. Fix the dried film in absolute methanol for 1 min, and air dry the slide. 3. Flood the slide with Kinyon carbol-fuchsin, and stain the smear for 5 min. 4. Wash the slide with 50% ethyl alcohol in water, and immediately rinse it with water. 5. Destain the smear with 1 % sulfuric acid for 2 min or until no color runs from the slide. 6. Wash the slide with water.

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7; Counterstain the smear with Loeffler methylene blue for 1 min. 8. Rinse the slide with water, dry it, and examine the smear with oil inunersion. The results are that Cryptosporidium oocysts stain bright red, and background materials stain blue or pale red.

1.5.6 Egg Counts Egg-counting methods are used in clinical studies to assess the intensity of infections (especially infections by intestinal nematodes) and the efficacy of therapeutic agents, and these methods are commonly used for epidemiologic studies. Methods used for scientific studies, such as Kato thick smear or Stoll egg counting, require greater accuracy than methods used for patient care. The simplest, most practical method is to use a standard fecal suspension which contains approximately 2 mg of feces mixed in a drop of saline and covered with a cover slip. The entire cover slip is examined at a magnification of 100 x, field by field, and the number of eggs is counted. For research work, the density of the smear can be standardized with a light meter, but this standardization is not essential for patient care. The number of eggs per cover slip provides a rough index of the severity of the infection. 1.5.7 Duodenal Material The examination of duodenal fluid or duodenal biopsy material may be useful for the diagnosis of giardiasis, strongyloidiasis, or other upper intestinal parasite infections in patients in whom parasites cannot be detected in the feces. In addition, duodenal fluid occasionally can be useful in showing whether helminth eggs are originating in the biliary or intestinal tract. Duodenal material may be obtained by passing a tube through the nose and stomach into the upper small intestine and then aspirating enteric fluid. As an alternative, a string test may be used. A weighted gelatin capsule attached to a string is swallowed, and the proximal end of the string is taped to the face of the patient. Over a period of several hours, helped with small sips of water, the string reaches the upper small intestine. After 4 to 5 h, the string is retrieved, and the material on the bilestained portion is stripped from the string and examined for parasites with direct wet mounts or with permanent stains when wetmount fmdings are questionable. Aspirated duodenal fluid is examined in a similar fashion. The material for permanent stains can be fixed in Schaudinn or PV A fixative, although the latter may adhere better

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to the slide. If question able organisms are seen in the direct wet mount, the coverslip can be removed, the material can be mixed with a drop of PV A fixative, and a film can be made for later permanent staining. A duodenal biopsy can be used to demonstrate Giardia organisms. A biopsy is usually obtained by a swallowed biopsy capsule. In searching for Giardia spp., it is generally preferable to make both impression smears and sections of biopsy tissue. Giardia spp. are usually present in mucus or attached to epithelium rather than in tissue. Biopsies occasionally can confirm a diagnosis of strongyloidiasis or cryptosporidiosis. 1.5.8 Sigmoidoscopic Material Materials obtained by sigmoidoscopy may be helpful in the diagnosis or monitoring of amebiases, schistosomiasis, or cryptosporidiosis. Patients suspected of having amebiasis may have ulcerations of the l:olon which can be visualized by sigmoidoscopy or colonoscopy. Scrapings or aspirates of material from ulcers can be examined by direct wet mounts and permanent stain as described above. The rmding of typical, erythrophagocytic, motile trophozoites in direct wet mounts or in permanently stained preparations allows a diagnosis of amebiasis. Material is best aspirated with a pipette or scraped with an instrument. Swabs should not be used, as the parasites may be killed or trapped by swab material. Biopsy material for amebiasis should be processed for surgical pathology and then examined for ulcers containing amebae. The periodic acid-Schiff stain counterstained with hematoxylin is particularly helpful because amebae stain more positively with periodic acid-Schiff stain than do inflammatory cells, and amebae show typical amebic nuclei. Of course, there are no amebic cysts in tissue. Biopsy material for schistosomiasis is better examined in teased preparations than in sections, as the entire thickness can be examined at once, and the viability of eggs can be determined by observation of the movement of the larvae within the eggs. In cryptosporidiosis, biopsy material shows organisms at the luminal surface of the epithelial cells, but the organisms are small, and the study of structural detail requires electron microscopy. 1.5.9 Abscess Material Abscesses suspected of being caused by Entamoeba histolytica may be aspirated, and the material may be submitted to the laboratory.

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The last material aspirated is most likely to contain amebae. Material may be examined microscopically in wet mounts and permanent stains, and in addition, it can be cultured for amebae if bacteria are also added to the culture as described below. Abscess material is often thick and difficult to examine. It may be treated with streptokinase and streptodonase enzymes to liquefy the specimen. 1. Reconstitute streptokinase and streptodonase per the instructions of the manufacturer. 2. Add 1 part enzyme solution to 5 parts aspirated material. 3. Incubate the mixture at 35 to 37°C for I h. Shake the mixture at intervals. 4. Centrifuge the mixture at 300 to 400 x g for 5 min. 5. The sediment may be used for microscopic examinations for amebae (wet mounts and permanent stains) and for the culture of amebae.

1.5.10 Cellophane Tape Cellophane tape is used for finding the eggs of Enterobius vermicularis or Taenia species from the perianal area. The tape used must be clear cellophane and not slightly cloudy or opaque. Alternatively, a Vaspar swab may be used. Specimens from more than 1 day may be required to diagnose light infections.

1.5.11 Examination of Cellophane Tape 1. If the specimen is difficult to examine, raise the tape from the front of the glass slide, add a drop of toluene to the slide, and replace the tape smoothly with an applicator stick. (Remember. Enterobius spp. and Taenia solium eggs are infective!) 2. Examine the entire tape, including the edges, with x 100 magnification (x 10 objective). 3. Confirm suspicious objects with high dry objectives (x40 to x50).

1.5.12 Culture for Amoebae Cultures for amoebae have improved detection in some studies, but they are not widely used. Although Giardia spp. have been cultured in research laboratories, cultures are not useful for diagnosis. A variety of culture media for amebae have heen described, and some may be purchased from commercial meUlum manufacturen. The method described here uses the modified charcoal agar slant diphasic medium described by McQuay.

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1. Place 3 ml of sterile 0.5% phosphate-buffered saline on a charcoal agar slant. 2. Add approximately 30 mg of sterile rice starch to the tube. 3. Warm the medium to 35°C before it is inoculated. 4. (a) Inoculate the medium with fecal specimen (approximately 0.5 ml of liquid specimen or a 0.5-cm sphere of formed specimen) which is mixed with the saline overlay. (b) If abscess material is cultured, bacteria must be inoculated into the culture in addition to the inoculation with 0.5 ml of specimen. A heavy inoculum with Clostridium perjringens or Escherichia coli is satisfactory. 5. Incubate the culture at 35°C. 6. At 24 and 48 h, remove I drop of liquid from the lowest point of the overlay, and prepare a wet mount. 7. Examine the wet mount for amebae. 8. Permanent stains can be prepared by the fixation of sediment in PV A fixative, with the subsequent preparation of smears and staining. 1.5.13 Larval Maturation Larval maturation studies, sometimes referred to as cultures, can be performed on fecal specimens applied to wet filter paper. Nematode larvae such as Strongyloides spp. or hookworm mature to the filariform stages in the culture container and migrate from feces into water, where they are detected microscopically. The procedure can be performed in a petri dish with a square of filter paper or in a large test tube with a strip of filter paper. 1. Smear approximately 0.5 g of feces on the filter paper. 2. (a) For the tube ·method, insert the filter paper strip into the tube so that the bottom of the strip is in 3 ml of water. The fecessmeared portion of the strip need not be immersed in the water. (b) For the petri dish method, place feces on one half of a piece of filter paper. Lay the feces-bearing end of the filter paper on a glass rod or a portion of an applicator stick in the petri dish. Add approximately 3 ml or sufficient water so that the feces-free end of the filter paper is in the liquid. 3. Leave the tube or dish at room temperature in the dark. Add water as needed to ensure that the filter paper is in contact with the water. 4. Examine the liquid for larvae either by direct microscopic

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examination with an inverted microscope or by examination of a wet mount of sediment from the liquid. With the petri dish method, the surface of the feces also may be examined with a dissecting microscope. 5. Examine the specimen on days 3, 5, and 7. Strongyloides filariform larvae are found on days 2 and 3, and hookworm larvae are found on days 5 through 7. Larvae are identified by their morphological characteristics. 1.5.14 Adult Worms Adult worms, or objects suspected to be adult worms, may be submitted to the laboratory. The laboratory must determine if these are helminths and, if so, if they are parasites. Identification characteristics are described in standard references. Tapeworm proglottids, particularly those of the Taenia species, are difficult to differentiate grossly unless they are cleared so that the internal structure can be seen and the number of lateral uterine branches can be counted. One procedure for clearing the proglottids is outlined below.

1.5.14.1 Clearing Taenia proglottids and other helminths Proglottids are first relaxed by placing them in warm saline (56°C) for I h and then clearing them in carbolxylene while they are kept flat. They may be kept flat in a number of ways. One way is to press the proglottid between two glass slides held together with membrane clips or string. Clearing takes from several hours to overnight. The proglottid is examined under a dissecting microscope or with a hand lens, and the uterine branching is observed. Glycerine and beechwood creosote can also be used with good results. Cleared proglottids may be mounted or stained if desired. Small nematodes may also be cleared in carbolxylene or beechwood creosote and mounted in permount or balsam. This method is particularly good for hookworm adults.

1.6 BLOOD AND TISSUE PARASITES Blood and tissue parasites whose diagnostic forms circulate in the peripheral blood are generally diagnosed by the demonstration of parasites in Giemsastained thick or thin films of blood. Special concentration techniques may be helpful for the diagnosis of some diseases such as filarial or trypanosornal infection. Other tissue parasites which do not circulate in the blood may be diagnosed by the detection of parasites in skin snips, lesion scrapings, body fluids,

26


or biopsy material or by the detection of antibody or antigen in serum or other body fluids.

1. 7 COLLECTION AND HANDLING OF BLOOD SPECIMENS The timing of the collection of blood specimens depends on the parasite disease suspected. For example, for certain filarial infections, specimens are best obtained between 10:00 p.m. and midnight, whereas for other infections, specimens are best obtained during the day. In malaria, the numbers and stages of parasites in the peripheral blood vary with different parts of the cycle. Blood films are best made from blood which is not anticoagulated, such as that obtained from finger stick or ear lobe puncture. Anticoagulants may interfere with parasite morphology and staining. Care should be taken that the alcohol disinfectant is allowed to dry before the area is punctured, or there may be fixation of erythrocytes, which will interfere with the preparation and staining of thick films. Both thick and thin films can be prepared from blood obtained by venipuncture, although it is best if the blood remaining in the needle of the venipuncture device is used, because it is anticoagulant free. Thick and thin films can be prepared from blood that is anticoagulated, but the staining characteristics are not as good. EDTAanticoagulated blood is better for staining than citrate- or heparinanticoagulated blood. Both thick and thin blood films are useful. Thick films are more sensitive because the same amount of blood can be examined in a thick film in 5 min as can be examined in a thin film in 30 min. However, thin films allow the study of the effects of parasites on erythrocytes and provide better parasite morphology. Thick and thin films may be prepared on separate slides or on the same slide, with the thick film at one end and the thin film at the other end. The thick film is prepared by spreading 1 drop or puddling several small drops of blood into an area approximately 1.5 cm in diameter. A properly prepared thick film should be thin enough so that newspaper print can barely be read through it. If the film is too thick, it will fragment and peel, and if the film is too thin, the increased sensitivity will be lost. Thick films should be allowed to dry overnight and should be stained within 3 days. They must not be heated, and they should be protected from dust. If the erythrocytes are fixed, they will not dehemoglobinize. If prompt examination is

INTRODUCTION

27

required, prepare a slightly thinner thick film, dry it for I h, and stain it. The thin film is prepared in the same manner as a film for a differential leukocyte count. A small drop of blood is placed on one end of a microscope slide. A second slide held at an acute angle of 30 to 45 0 is backed into the drop of blood, which spreads along the junction of the slides. The spreader slide is then pushed along the slide, and it pulls the drop of blood along behind the angled edge of glass. A properly prepared thin film should have a significant area near the end which is only one erythrocyte thick and in which the erythrocytes show good morphology. The angle and speed of the spreader slide and the size of the drop of blood will influence the thickness and size of the film. Slides with only a thin film can be fixed by being immersed in absolute methyl or ethyl alcohol for I min and allowed to air dry. If the thick film is on' one end of the slide and the thin film is on the other end, the thin film is fixed by a brief flooding or by immersion in alcohol and allowed to air dry, while the thick film is protected from alcohol or alcohol fumes. In a well-ventilated area, the slide may be dried vertically with the thick film up or horizontally after the thick film is covered with a dry paper towel. Thick and thin films are best stained with Giemsa stain, as it provides the most detailed and intense staining of parasites. Wright stain can be used for thin films but not for thick films, as it contains alcohol, which will fix the erythrocytes. Wright stain does not stain parasites as well as Giemsa stain. The staining procedure is outlined below. 1. 7 .1 Tissue Biopsy or necropsy tissue may be examined by histology sections or impression smears. To prepare impression smears, tissue should be blotted to remove as much blood or other fluid as possible and then pressed against glass slides (1 by 3 in.) to make a series of impressions. Tissue should stick to the slide slightly and leave an irregular film on the slide. Similar impressions may be made on multiple slices from the same portion of tissue. Portions of biopsy tissue with different gross appearances can be used with the impressions from each portion placed in a longitudinal row. Impressions must be close together, preferably with slight overlapping to make slide scanning easier. Impressions from small fragments may be placed in a small area (1

28


cm in diameter). After being dried, the area with impressions is circled with a diamond marker to facilitate the location and scanning of the material. Fixatives and stains appropriate for the parasites suspected are used. If amebiasis is suspected, impression smears must be fixed promptly in Schaudinn fixative and not allowed to air dry. For most other parasites, the slides are allowed to dry before fixation in methyl alcohol. Giemsa is the usual stain, but other stains such as Gram-Weigert or hematoxylin may be used depending on the parasite suspected. 1.7.2 Aspirates of Bone Marrow or Spleen Aspirates of bone marrow or spleen may be useful in the diagnosis of infections such as leishmaniasis, trypanosomiasis, and occasionally malaria. In such instances, Giemsa stains of alcoholfixed bone marrow films are most useful. Splenic aspiration is r~ely performed in the United States because it is dangerous. 1.7.3 Fluids Fluids such as tissue aspirates, cyst fluid, bronchial washings, cerebrospinal fluid, pleural fluid, and peritoneal fluid can be examined directly, or they can be centrifuged and the sediment examined by wet mounts or stains (or both), depending on the parasite suspected, as described above for abscesses or tissue. 1.7.4 Skin Snips Skin snips may be useful in the diagnosis of microfilarial infections such as onchocerciasis in which the parasites circulate in the skin and not the blood. A small (2-mm) skin snip is taken with a needle and a knife. The needle point is stuck into the skin, and the skin is raised. With a sharp knife or razor blade, the skin is excised just below the needle. Alternatively, a scleral punch may be used. The skin snip is then placed in a small volume (0.2 ml) of saline in a tube or a microtiter well, teased, and allowed to stand for 30 min or more. The microfilariae migrate from the tissue into the saline, which is then examined microscopically to demonstrate the wiggling microfilariae. 1. 7.5 Concentration Procedures for Blood A number of procedures have been described for the concentration of blood specimens. Most of these procedures have been developed to diagnose filarial infections. The three most widely used methods are membrane filter, saponin lysis, and Knotts concentration. Procedures for the first two methods

INTRODUCTION

29

will be given here, as these methods are the most sensitive. Membrane filter techniques use 5- or 3-lLm filters. Both filters give satisfactory results, but the procedures with the Nuclepore filters do not require the lysis of erythrocytes. Parasites on filters are often not as suitable for morphologic study as are those in thick films.

1.7.6 Membrane Filter Concentration for Filariae 1. Collect approximately 7 ml of blood in EDTA. 2. With a syringe and firm pressure, pass 5 to 7 ml of blood through a 5-lLm Nuclepore filter held in a Swinney adapter. 3. Wash the membrane several times with a small amount of distilled water or physiologic saline. 4. The moist filter may be e}t'cUtlined directly or fixed and stained in the usual fashion for a thin blood film.

1. 7.7 Saponin Lysis Concentration for Filariae The saponin lysis method can be performed on either EDT'\- or citrate-anticoagulated blood. Saponin solution to lyse erythrocytes is available in most laboratories for use with automated hematology instruments. 1. Centrifuge up to 10 ml of blood at 150 x g for 10 min. 2. Remove and discard the plasma. 3. Mix the packed erythrocytes with 50 ml of 0.5% saponin solution in 0.85% saline. 4. Mix the solution at intervals for 15 min. 5. Centrifuge the solution at 650 x g for 10 min. 6. Decant and discard the supernatant (there should be about 1 ml of sediment). 7. Spread several drops of sediment on a glass slide (1 by 3 in.), and examine two such uncovered wet mounts for motile microfilariae. Allow the wet mounts to dry before they are fixed and stained. 8. Prepare four or five similar wet mounts and examine them as described above. To. each slide, immediately add 2 drops of 1 % acetic acid solution and mix it well (microfilariae will be killed and straightened). Allow the slide to air dry. 9. Dip the dried slides in buffered methylene bluephosphate solution. 10. Rinse the slides in distilled water, and let them air dry. 11. Stain the mounts for 10 min in a 1:20 dilution of Giemsa

30


stain in buffered water. 12. Examine the slides microscopically.

1.8 STAINING PROCEDURES 1.8.1 Giemsa Stain Procedure The procedures for staining thick. and thin fIlms differ. Staining is usually done in a Coplin jar. The ,&tain must be made fresh each day. Stain slides with only a thin fIlm as follows. 1. Fix and dry the blood film as described above. 2. Prepare a 1:40 dilution of stock Giemsa stain in neutral buffered water, pH 7.0 to 7.2 (generally, 2 ml of Giemsa stock plus 38 ml of buffered water with 0.01 % Triton X-l00). 3. Stain the film for approximately 60 min (the time, which will vary slightly with different lots of stock Giemsa stain, can be determined by the staining of leukocytes and erythrocytes). 4. Wash the slide brietly by dipping it in buffered water. 5. Air dry the slide in a vertical position. Note that, alternatively, a 1:20 dilution for 20 to 30 min may be used. Stain slides with only a thick fIlm as follows. .,1. Do not fix the slide . 2. Prepare a 1:50 dilution of stock Giemsa stain in neutral buffered water (pH 7.0 to 7.2). 3. Stain the film for approximately 50 min (the optimal time may vary with different lots of stain). 4. Wash the slide by placing it in buffered water for 3 to 4 min. 5. Air dry the slide in a vertical position. For combination thick and thin fIlms, the procedure is as follows. 1. Fix the thin film but not the thick film as described above. 2. Stain the film in a 1:50 dilution of Giemsa stain in neutral buffered water (pH 7.0 to 7.2) for approximately 50 min. 3. Rinse the thin film brietly by dipping it in buffered water. Wash the thick film by immersing it in buffered water for 3 to 5 min. 4. Dry the slide in a vertical position with the thick film down. 1.8.2 Gram-Weigert Stain Procedure The Gram-Weigert stain is used to stain the cyst walls of P. carinii cysts. It also stains fungi and many bacteria. Impression

INTRODUCTION

31

smears, sediment smears, or sections are fixed in methanol and air dried. For sections, when reagents are added, flood the slide gently from the end opposite the section, and rinse the slide carefully so that the tissue is not washed from the slide. The stain procedure is as follows. 1. Stain the slide with eosin Y for 5 min. 2. Wash the slide with water. 3. Stain the slide with crystal violet for 5 min. 4. Rinse the crystal violet from the slide with Gram iodine solution. 5. Leave the iodine solution on the slide for 5 min. 6. Rinse the slide with water. 7. Blot the smears carefully (do not blot the sections). 8. Wipe the reverse of the slide. 9. Air dry the slide completely. 10. Decolorize the smear in aniline-xylene, agitating the slide gently until no purple runs from it (the use of a second Coplin jar of aniline-xylene after the majority of blue stain has been removed aids the visual assessment of decolorization). 11. Rinse the slide in xylene. 12. Air dry the slide, add immersion oil to it, and examine it. P. carinii cysts and fungi stain dark blue and somewhat irregularly. Cell nuclei may stain blue if they are inadequately decolorized, but they are not as dark as P. carinii cysts. 1.8.3 Culture Procedures for Blood and Tissue Parasites Culture procedures have been developed for a number of blood and tissue parasites, but these procedures are used primarily in research. The culturing of Leishmania spp. and Trypanosoma cruz; may be helpful for diagnosis, and the procedures are easy to use. Biopsy or blood specimens may be cultured for Leishmania spp. or T. cruzi with Novy-MacNeal-Nicolie (NNN) medium. Biopsy specimens are ground in a small amount of saline. Biopsies from skin lesions or other tissues which may contain bacteria may have penicillin (0.1 ml of 1,000 U/ml) added to the medium with the inoculum. The inoculum is 1 drop of ground tissue or blood. Incubate the culture at room temperature (22°C), and at days 3 and 7, examine a direct mount of liquid from the bottom of the slant at x400 magnification. These cultured organisms are potentially infective for humans.

32


1.9 URINE Urine specimens usually are examined for the eggs of Schistosoma haemotobium or the trophozoites of Trichomonas vaginalis, although occasionally the larvae of Strongyloides stercoralis may be found in patients with hyperinfection syndrome. Urine is the usual specimen for the diagnosis of Trichomonas infection in males. See below (Vaginal Material) for culture method. Urine is centrifuged, and the sediment is examined microscopically.

1.10 SPUTUM Sputum may be examined to diagnose Paragonimus infection or hyperinfection due to Strongyloides stercoralis. Occasionally an amebic abscess or hydatid cyst may rupture, and amebic trophozoites or hydatid sand, respectively, may be found in sputum. Entamoeba histolytica must be differentiated from Entamoeba gingivalis, which may be found in the oral cavity of over 30% of people. Occasionally, the migrating larvae of ascarids, strongyloides, or hookworm can be found. Sputum may be examined directly by wet mount or treated with a mucolytic agent such as Nacetyl-cysteine and then concentrated by simple centrifugation, with subsequent examination of the sediment.

1.11 VAGINAL MATERIAL T. vagina lis frequently infects the vagina, and Enterobius vermicularis adults or eggs occasionally may be found. Direct wet mounts of vaginal material for typical, tumbling T. vagina lis organisms are widely used and generally allow the diagnosis of symptomatic infection, but wet mounts are not as sensitive as culture methods. Vaginal material is best submitted as liquid in a tube, although swabs submitted in a small amount of saline may be used. A drop of the material is covered with a cover slip and examined with reduced light. To culture, 1 or 2 drops of urine sediment or vaginal exudate are inoculated into tubes of warmed, modified Diamond medium. If vaginal swabs are submitted, the swab is immersed in the medium and pressed against the side of the tube to express material. Tubes are incubated at 35°C, and drops of culture are examined by wet mount at 48 and 72 h for motile trophozoites.

1.12 REFERRAL OF MATERIALS Few laboratories perform complete parasitological examination, whereas many perform limited studies, and some perform none. Referral laboratories may provide services not available in the individual

33

INTRODUCTION

laboratory and can provide consultation on specimens with questionable laboratory findings. Referral laboratories with a special interest and competence in parasitology may be found in major cities, university medical centers, and state public health laboratories. The major national resource is the Centers for Disease Control in Atlanta, Ga. Specimens for the Centers must be sent via the state health laboratory, and appropriate clinical information must be provided. Of course, the recommendations of the specific referral laboratory should supersede' these guidelines.

1.13 SAFETY The parasitology laboratory has infection hazards for personJil.el. Blood, feces, and other body materials as well as parasite cultures may be infective. Eggs of Ascaris spp. can survive and embryonate even in Formalin, and Cryptosporidium oocysts are hardy. In fresh fecal specimens, the cysts of Entamoeba histolytica and Giardia spp .. the oocysts of Cryptosporidium spp., the eggs of Enterobius vennicularis. Taenia solium, and Hymenolepis nana, and the lanae of Strongyloides stercoralis may be infective. In addition, feces may contain other infectious agents such as hepatitis A, rotavir~s, Salmonella spp .. Shigella spp., and Campylobacter spp. Blood and tissue specimens can be infectious for trypanosomes, Leishmania sp~., malaria, and Babesia spp., as well as for non-A, non-B hepatids, hepatitis B, and possibly AIDS.

TABLE 1.4 : HANDLING OF SPECIMENS FOR Specimen Feces, for Helminths Protozoa

Cryptosporidium spp.

Material from suspected amebic abscess

Duodenal aspirate

REFERRA~

Handling Fix in 10% buffered Formalin. Fix a portion in 10% buffered Formalin and either fix a portion in PVA fixative or prepare three Schaudinn-fixed fecal films. Fix a portion in 10% buffered Formalin. Place the last material aspirated in a sterile tube and send it on ice for culture (do not freeze). Prepare Schaudinn-fixed fecal films, or fix a portion in PVA fixative. Obtain serum for serology. Centrifuge, and remove the supernatant.

34

MICROBIOLOGY AND BIOCHEMISTRY Prepare two films from sediment. Fix in Schaudinn or PYA fixative. Preserve the remainder of sediment in 10% Formalin.

Urine, for Trichomoniasis

Schistosomiasis

Sputum, for Nematode larvae or Paragonimus eggs

Amebae

Blood Malaria and babesiasis

Filariasis

Trypanosomiasis

Centrifuge. Cover the sediment with sterile saline and send it on ice (not frozen) for direct mounts and culture. Centrifuge entire midday urine. Add an equal volume of 10% buffered Formalin to the sediment.

Break up mechanically or digest I part sputum plus 5 parts 3% NaOH for 1 h. Centrifuge, and preserve the sediment in an equal volume of 10% buffered Formalin. Prepare films fixed in Schaudinn fixative, or fix a portion in PYA fixative.

Send unstained and, if available, Giemsastained thick and thin films. Fix thin film (but not thick) in alcohol before it is sent. Send 5 ml of citrate- or EDTA-anticoagulated blood on ice (not frozen). Unfixed thick films may be sent in addition. Send serum for serologic tests. Send 5 ml of anticoagulated blood as for filariasis (above).

Send fixed thin films. Cerebrospinal fluid Trypanosomes, toxoplasma, leishmania, trichinella Free-living amebae Sigmoidoscopic material Tissue

Send on ice (not frozen). Send in a sterile container without refrigeration. Fix films in Schaudinn fixative or mix material with PYA fixative. For impression smears when E. histolytica

INTRODUCTION

35 is suspected, fix in Schaudinn or PVA fixative. When toxoplasma, leishmania, Pneumcoystis spp., or Trypanosoma cruzi is suspected, prepare multiple impression smears and fix in methyl alcohol. For surgical pathology, fix the tissue in buffered Formalin.

Whole worms or proglottids

Wash debris from the specimen and send it in saline. If there are mUltiple worms or proglottids, some may be fixed in Formalin.

~eagents such as mercury-containing fixatives may be toxic, and solvents such as ether may be flammable. These materials must be handled and discarded properly.

1.14 QUALITY ASSURANCE The parasitology laboratory must have an up-todate procedure manual and appropriate reference materials which might include color atlases or 35-mm slide collections permanently stained glass slides, wet fecal material containing parasites, and one or more standard reference books on laboratory methods or general medical parasitology. The persons performing parasitic examinations must be competent in the identification of parasites which might be found in patients from whom they receive specimens. Methods should allow the ready use of outside consultants, if there is a question of diagnosis. Personnel may maintain proficiency through participation in formal courses or workshops, review of self-study sets, and periodic review of known positive materials. Participation in external survey programs is particularly valuable, as the performance of the laboratory in the identification of unknown specimens can be compared with the performance of other laboratories. If a laboratory is unable to do accurate parasitology because of either the types of procedures offered or the quality of personnel available, it should arrange to have specimens appropriately prepared and submitted to a reference laboratory.

tI

2

Origin of Microbiology Before the dawn of civilization in the Mesopotamian regions and farther east some 7000 to 8000 years ago there was little exact knowledge of either the causes or nature of natural phenomena. However, scholarly thinkers and their works were not wholly lacking. By the time writing and written history had been "invented" 5000 to 6000 years ago 'in Sumeria, Egypt, Syria and adjacent regions, many keen and ambitious minds in the ancient priesthoods, secular upper classes and royal families had learned of the medicinal and poisonous properties of certain plants and of the venoms of certain snakes and insects. They knew how to exploit nature for political and other purposes. For thousands of years after the beginnings of civilization magic, incantation, abracadabra and witchcraft passed for science and usually also for religion. Even as recently as the Middle Ages (c. 500-1400 AD.) and later in the European Renaissance (c. 1400-1700 A.D.) astrology (aided by imaginative charlatans, with weird grimaces and impressive passes) passed for astronomy; alchemy (strongly flavoured with wizardry) masqueraded as chemistry; the most outrageous quackery was accepted, even by royalty, as medicine. As always, however, honest, imaginative and inquisitive men here and there were still capable of analytical and creative thought and the proposing of working hypotheses to be tested experimentally. They were sometimes reviled, persecuted and tortured for their supposed dealings with "The Evil One." Century after century these pioneer scientists (seekers after experimentally demonstrable truth) began to establish a system of knowledge based on accurate, purposeful 36

ORIGIN OF MICROBIOLOGY

37

observation; logical inference; imaginative hypothesis; and ingenious experiments designed to establish indisputable fact or destroy fallacy. Because of great difficulties in travel and communications, ancient scholars shared little of one another's learning. As the centuries passed, exploration began and travel became more common, populations increased, and vast interminglings of peoples occurred because of wars and trade. Scientific information thus began to spread from country to country and, more recently, from continent to continent. Instead of a few great scholars who were thought (even by themselves!) to know everything, men began to realize that there were boundless deserts and plains and illimitable dark forests of ignorance only awaiting the axe and plow of the devoted researcher to yield rich crops of wonderful, golden knowledge. Men also realized the awesome truth that knowledge is power-to create or to destroy utterly. Eventually scientific thought, experimentation and communications became permissible and even respectable. They also became incalculably profitable, and frightening. Scientists interested in the mysteries of life collected, over the centuries, a considerable mass of reasonably accurate information about such living things as could be seen with the naked eye, and even with "magnifying glasses" (magnifications of about 10 diameters). By 350 B.C. Aristotle and his students had drawn up a systematic, though limited and (as we now know) often erroneous classification of hundreds of plants and animals. Accumulating knowledge of living organisms slowly became arranged into a more or less orderly system and the study of life was eventually dignified with a given name: biology (Gr. bios = life; logos = study or description). Most of biology was at first largely descriptive of outward form and macroscopic (Gr. makros = large; skopion = to see) anatomy. These descriptions became the basis of classifications and taxonomy-major preoccup-ations of most early botanists and zoologists. Until the seven-teenth and eighteenth centuries chemistry and physics were almost completely separate fields of study and little used in biology. Life and living substance were commonly thought of as mysterious and beyond physical and chemical analysis.

2.1 BEGINNINGS OF MICROSCOPY Until about 1660 A.D. all knowledge of the form, structure and life processes of plants and animals was narrowly restricted to what could be seen with the naked (or very feebly assisted) human eye. Microorganisms were merely "fabulous monsters." Visual limitations

38

MICRnBIOLOGY AND BIOCHEMISTRY

of the pitiably restricted eye of man had always stood, like an impenetrable curtain, between man and the fantastic and glittering cosmos of the microscopic world. Unaided human vision fails to see objects less than about 100 p, (0.004 or 11254 inch) in diameter or to perceive as separate objects

TABLE 2.1 SOME LINEAR MEASURES COMMONLY USED IN MICROBIOLOGY 1 inch = 2.54 cm. 10 mm. 1 mm.:::: 1000 p. 1 cm. 1 p. = 0.001 mm. = 0.00003937 or 1125,400 inch = 1000 mp. 0.001 p. = 10.0 Angstrom (A) I mp' 1 A = .0001 p. = 0.0000001 mm. ;:; 1/254,000,000 inch (i.e., resolve) particles separated by distances less than about 100 p.. Microscopic linear units are shown in Table 1. 1. The development of practical, relatively high-power microscopy about the middle of the seventeenth century was like turning on a SOD-watt lamp in a pitch-dark curiosity shop. It gave men the power to see a universe of objects, living and inanimate, so minute that their very existence had never before even been suspected.

2.2 THE FIRST MICROSCOPES By the end of the thirteenth century simple lenses (magnifying glasses) had already been used in various ways for many years. Such lenses, however, did not magnify very highly. About 1590, a Dutch spectacle maker, Zacharias J anssen, used a second lens to magnify the image produced by a primary lens. This is the basic principle of the compound microscope used by every microbiologist today. Galileo invented an improved compound microscope in 1610. Robert Hooke (1635-1703) made and used a compound microscope in the 1660's and described his fascinating explorlttions of the newly discovered universe of the microscopic in his classic "Micrographia" (1665), published at request of the Royal Society in London. Although Hooke's highest magnifications were possibly enough to reveal bacteria, he apparently made no observations of them. probably because he studied mainly opaque objects in the dry state by reflected light, conditions that, as will be explained, are not optimal for observation of microorganisms. However, his pictures of "white mould" (probably a Mucor species) are very informative and accurate.

ORlGIN OF MICROBIOLOGY

39

f'iiure 2.1 : Hooke's compound microscope: drawn by himself.

A contemporary of Hooke, and the man mainly responsible for revealing the hitherto unknown and unseen world of microorganisms, did not use a compound microscope. He was the Dutch investigator, Antonj van Leeuwenhoek (1632-1723), a linen merchant by trade and a man of public and commercial affairs in the city of Delft. He was nO! a trained scientist but was self-educated, and amused himself by means of his skill and craftsmanship in glass blowing and fine metal work. He lived in relatively easy circumstances with leisure time for his avocation of making minute, simple but powerful lenses. With these he delighted in examining a great variety of objects: saliva, pepper decoctions, cork, the leaves of plants, circulating blood in the tail of a salamander , ~minal nuid, urine, cow dung. scrapings from the teeth and &0, on. ln' many of these be saw living creatures , some of which we now know were protowa and bacteria but all of which he called "animalcules." In spite of the fact that his microscopes were not compound he obtained remarkable results with them. he showed rare ingenuity and expert craftsmanship in the grinding and mounting of his simple lenses, a skill which he zealously kept to himself; and in spite of the requests of his learned friends, he refused to disclose the secret of his success. I..eeuwennoek's instruments are not true microscopes at all in

40


Figure 2.2 : Drawing of "white mould".

the sense in which we ' think of microscopes, but rather simple magnifying glasses generally consisting of a small, single, biconvex lens. The object, and not the lens, was moved into focus by means of screws.

Figure 2.3 : Antonj VllJI Leeuwenhoek .. A fanciful delineation based on a famous portrait. The picture shows accurately the size and shape of the first microscopes. the manner in which they were used. and the simple laboratory apparatus of the "Father of Bacteriology. "

To adjust the lens to the object was so long and tedious a task that it is not surprising that Leeuwenhoek used an individual lens for each object.... The magnification varied and at best did not exceed

ORIGIN OF MICROBIOLOGY

41

two hundred to three hundred diameters .... The size of objects which Leeuwenhock examined was determined by comparison. For this purpose he used at various times a grain of sand, the seed of millet or mustard, the eye of a louse, a vinegar eel, and still later hair or blood corpuscles. In this way he secured fairly accurate measurements of a great variety of objects . . . . he was forced to admit that the sand grain was more than one million times the size of one of the animalcules. Leeuwenhoek was so interested in the things he observed that, like Hooke, he wrote minutely detailed reports about them to the Royal Society in London, beginning in 1674. He was later elected a fellow of the Royal Society. Some of his observations are at once quaint and epochmaking. For example, after examining material which he scraped from between his teeth, he said: Though my teeth are kept usually very clean, nevertheless when I view them in a Magnifying Glass, I fmd growing between them a little white matter as thick as wetted flour; in this substam.:e, though I could not perceive any motion, I judged there might probably be living Creatures.

Figure 2.4 : One of Leeuwenhoek's microscopes: front, back and side views. The tiny spherical or hemispherical lens is held in the slightly raised structure in the upper part of the metal plate. The object to be examined was mounted at the tip of the sharp-pointed mounting pin. Focusing was accomplished by means of the three· thumbscrews to which the mounting pin is attached. These are approximately actual size.

I therefore took some of this flour and mix it either with pure rain water wherein were no animals; or else with some of my spittle (having no Air bubbles to cause a motion in it) and then to my great surprise perceived that the aforesaid matter contained very small living animals, which moved themselves very extravagantly. The

MICROBIOLOGY ANO:BIOCHEMISTRY

42

biggest-sort had the .shape of A. Theirnidtron was stron.g -arid nimble; and they darted themselves through the' water or spittle~ 'as a: JaCK ot Pike· does' through the water. These' were 'generally not many; 'In number.· The .second sort had the' shape of B. These sjnm 'abOut like a top, or took 'a' course sometimes on one side; as is 'shown' at C arid D: They were more in number than' the first. In the third s~rt I could n6t well distinguish the Figure, for sometimes It seem'd to' be an oval, and other times a circle. These were so small they'seem'a no bigger than E and therewithal so swift, 'that I can compare them to nothing better than a swarm of Flies or Gnats, flying and tUrning among one another in a small space. -

cPdA

'." .',;

,

Figure 2.5 : Leeuwenh;~;~ drawings bacteria. Heii; may be seen cocci, bacilli and (probably) a spirochet~. The motion of one of the bactli is clearly indicated. Today such observations are conbnonplace. But l,eeuwenhoek itas seeing them for the first time in the history of the human race! iwas as momtfuous a discovery as that of Columbus -a new world! " . ,.!. ... '

,. : Note, that, unlike Hooke, Leeuwenhoek made many of his observations' by light transmitted through the' object' and that the ri'lic:too'rganisms were Slispended in vaiiolls f\uids,:nOl ipiInobiliied oi-'l:)therwise altered by drying. ' ,

2.'3:':, MICROORGANISMS AND':~·' ,:,'
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~~r.c: 2~; ;Pasteur, -in his; J3bo'ratoiiyl' m :'!p3$leUr' ;f~~xa1tJi'niiig'~ s~(iMR cif J~h?J CfP:? fr~Il?Cf a ,; f~~i9 ; animal.,: The L mateFial ' ra n ,the ! bottblll ';Of ·tJ\~ j~r.i i's sodium liydroxide (drying agent), :.:~:,' if!," ,Ii;): ,~,:;:i;,~ :r;:::;',:' i!;i i'i -' ' qe~~:-v~)_a~ tl1~t. t~. ~~,;~; ffi~S1?-J3ttJ~J;r pe~~ ~'~i1i~ce. aG~~cPVi}l)~IP- llhat r~;~~~Tt:, l~'i?fd~f.! to 4? i ~p .h.~ , Fpe ~> ,~~: ~Wq~i J;lf ,l?PC;f'i~'"11)fjictufe and of the ·cause of" ~ow~fi , ~~':§~fl~~~d~q!W~e~ ~d~f;;~.r:-~ wines ." ~s ,a ~e~ul~o~, th~S(!;i stHdi~QRf_~!,j~e~\dl.h l~fr:;{s~~ing

~~i 'Pa~tep';~)~}f~~to~~_ t~~ ~~.

rnm,cre.;

ciJ~~f~~~ns.:J 1i)~'> ~a~. ? at ~.~ . .~Hae$ff~ 9f;s Mfi~ltri9~..~ ~J~Uff9" f!~&94:1

;' . ,I ~~ir\~ ~9.1(;~e .Ois~- ~ t::!1

o n ::t: tI1

-;3 ~

Table 3.1 Contd.

CIl

>-X174 can have multiple uses. Ultimately, assembly of mature virus particles occurs. Release

138


of virions from the cell occurs as a result of cell lysis, which involves the participation of gene E protein.

5.11 SINGLE-STRANDED FILAMENTOUS DNA BACTERIOPHAGES Quite distinct from q,X174 are the filamentous DNA phages, which have helical rather than icosahedral symmetry. 'ijJ.e most studied member of this group is phage M13, which infects Escherichia coli. but related phages include f1 and fd. As with the small RNA viruses, these filamentous DNA phages only infect male cells, entering after attachment to the male-specific pilus. Interestingly, even though these phages are linear (filamentous) they possess circular DNA. The DNA is not self-complementary, however, so that the two adjacent halves of the molecule which run up and down the virus particle form loops at the ends but exhibit very little if any base pairing. Phage M13 has found extensive use as a cloning vector and DNA sequencing vehicle in genetic engineering. The virion of M 13 is only 6 run in diameter but is 860 run long. These phages have the additional unique property of being released from the cell without killing the host cell. Thus, a cell infected with phage M13 or fd can continue to grow, . all the while releasing virus particles. Virus infection causes a slowing of cell growth, but otherwise a cell is able to coexist with its virus. Plaques are thus seen only as areas of thinner cell growth in the bacterial lawn. Many aspects of DNA replication in filamentous phages are similar to that of q,X 174. The unique property, release without cell killing, can be briefly discussed. The release from the cell occurs by a budding process in which the virus particle is always released from the cell with the end containing the A protein first. Interestingly, the orientation of the virus particle across the cell membrane is the same for its entry and exit from the cell. There is no accumulation of intracellular virus particles; the assembly of mature virus particles occurs on the inner cell membrane and virus assembly is coupled with the budding process. Several features of these phages make them useful as cloning and DNA sequencing vehicles. First, they pave single-stranded DNA, which means that sequencing can be carried out by the Sanger dideoxynucleotide method. Second, as long as infected cells are kept in the growing state they can be maintained indefinitely with cloned DNA, so that a continuous source of the cloned DNA is available. Third, there is an intergenic space which does not code for protein and can' ~ teplaced by variable amounts of foreign DNA.

139

MICROBIOLOGY OF VIRUSES A protein

B protein

~~. D protein

Gene 3: A protein Gene 8: B protein Genes 7,9: C protein Gene 6: D protein

intergenic space

Figure 5.15 : The filamentous single-stranded DNA bacteriophage fd. Orientation of the proteins and genes in the virion. Note the intergenic space which contams the origin of DNA synthesis. Gene cloning is done in this intergenic space.

cell wall

cell membrane coat proteins embedded in cell membrane

phage DNA: ss circle

environment

cytoplasm

Figure 5.16 : Illustration of the manner by which the virion of a filamentous singlestranded phage (such as M13 or fd) leaves an infected cell without lysis. The A protein passes first through the membrane at a site on the membrane where coat protein molecules have first become imbedded. The intracellular circular DNA is coated with dimers of another protein, gp5, \\'hich is displaced by coat protein as the DNA passes through the intact membrane.

5.12 DOUBLE-STRANDED DNA BACTERIOPHAGES Many· bacterial viruses have genomes containing double-stranded DNA. Such viruses were the first bacterial viruses discovered, and have been the most extensively studied. With such a range of doublestranded DNA viruses, a wide variety of replication systems are present. In the present section, we discuss the best studied and most representative of the group, T4 and 17. The simpler, 17, will be discussed first.

140


Bacteriophage 1'7 Bacteriophage 17 and its close relative T3 are relatively small DNA viruses that infect Escherichia coli. (Some strains of Shigella and Pasteurella are also hosts for phage 17.) The virus particle has an icosahedral head and a very small tail. The virus particle is fairly complex, with 5 different proteins in the head and 3-6 different proteins in the tail. One tail protein, the tail fiber protein, is the means by which the virus particle attaches to the bacterial cell surface. Only female cells of Escherichia coli can be infected with 17; male cells can be infected but the multiplication process is terminated during the latent period. The nucleic acid of the 17 genome is a linear double-stranded molecule of 39,936 base pairs. The complete genome has been sequenced, and the sequence information has permitted discernment of gene structure and features of gene regulation. About 92 percent of the DNA of 17 codes for proteins. At least 25 separate genes have been characterized, but not all genes are separately coded on the DNA. Gene overlap occurs for several genes. through translation in different reading frames and through internal reinitiation with one or more genes in the same reading frame. Further genetic economy is achieved by internal frame shifts within certain genes to yield longer proteins. When the phage particle attaches to the bacterial cell, the DNA is injected in a linear fashion, with the genes at the "left end" of the genetic map entering the cell first. Several genes at the left end of the DNA are transcribed immediately by a cell RNA polymerase, using three closely spaced promoters, generating a set of overlapping polycistronic mRNA molecules. These mRNA molecules are then cleaved by a specific RNase of the cell at 5 sites, thus generating smaller mRNA molecules which code for one to four proteins each. One of these proteins is an RNA polymerase that copies doublestranded DNA. Two other early mRNA molecules code for proteins which stop the action of host RNA polymerase, thus turning off the transcription of the early genes as well as the transcription of host genes. Thus, a" host RNA polymerase is used just to copy the first few genes and to make the mRNA for the phage-specific RNA polymerase, and this phage specific RNA polymerase is then involved in the major RNA transcription processes of the phage. This 17 RNA polymerase uses a new set of promoters that are distributed along the left-center and center portions of the genome. It is thus seen that regulation of 17 has both negative and positive control: negative, by means of the formation of proteins that stop host RNA polymerase

141

MICROBIOLOGY OF VIRUSES

and thus shut off transcription of the early T7 genes that are recognized by this enzyme, and positive, by means of the formation of the new gene disignation

function

left end Early promoters - _ 0.3 _

Overcomes host restriction

0.7 •

Protein kinase

1

RNA polymerase

•

1.1

UnknOwn --Stert of DNA replication DNA ligase

Promoters - - - - 1.3 1.7 _

Nonessential

2 _ 3 _ 3.5 -

Inactivate host RNA polymerase Endonuclease Lysozyme

4

Hellcase. prlmase

11

I ____ 8_._ _ 5

promoter

Early transcription

DNA polymerase Exonuclease

7 8

•

Protein in phage particle Head protein

9

•

Head assembly protein

promoter - - - - _

Protein for DNA replication

Major head protein Tall protein Tall protein

promoter - - - - - .

promoter

1 14 _

Protein in phage particle Head protein

15

Head protein

11

_____161

Phage maturation

Head protein

17

Tail protein

18 •

DNA maturation

19 _

DNA maturation

Right end

Figure 5.17 : Genetic map of phage T 7, showing gene numbers, approximate sizes, and functions of the gene products.

RNA polymerase which recognizes the rest of the T7 promoters. We also note that T7 is an example of a virus which strongly affects host transcription and translation processes, by producing proteins

142


which turn off transcription of host genes. The virus also has genes coding for enzymes which degrade host cell DNA, and nucleotides from such degraded DNA end up in ViI'Us.'progeny. Obviously, such a virus has profound pathological effects on its host cell. As seen in the genetic map, the genes after gene 1.1, transcribed by the T7 RNA polymerase, code for proteins that are involved in 17 DNA synthesis, the formation of virus coat proteins, and assembly. Three classes of T7 proteins are formed: class I, made 4-8 minutes after infection, which use the cell RNA polymerase; class II, made 6-15 minutes after infection, which are made from T7 RNA polymerase and are involved in DNA metabolism; class III, made from 6 minutes to lysis, which are transcribed by T7 RNA polymerase and which code for phage assembly and coat protein. This sort of sequential pattern, commonly seen in many large double-stranded DNA phages, results in an efficient channeling of host resources, first toward DNA metabolism and replication, then on to formation of virus particles and release of virus by cell lysis. DNA replication in T7 begins at an origfn of replication at which DNA synthesis is initiated, and DNA synthesis proceeds bidirectionally from this origin. In both directions, an RNA primer is involved, but the enzyme involved in the synthesis of this primer is different for primer synthesis in the leftward and rightward direction. In the rightward direction, the RNA primer is synthesized by 17 RNA polymerase. whereas in the leftward direction, a virus-specific enzyme, T7 primase (gene 4 protein) is used. Both primers are then elongated by T7 polymerase. Replicating molecules of T7 DNA can be recognized under the electron microscope by their characteristic structures. Because the origin .of replication is near the left end, Y-shaped molecules are frequently seen, and earlier in replication, bubble-shaped molecules appear. . A structural feature of the T7 DNA which is important in DNA replication is that there is a direct tenninal repeat of 160 base pairs at the ends of the molecule. In order to replicate DNA near the 5' terminus, RNA primer molecules have to be removed before replication is complete. There is thus an unreplicated portion of the T7 DNA at the 5' terminus of each strand. The opposite single 3' strands on two separate DNA molecules, being complementary, can pair with these 5' strands, forming a DNA molecule twice as long as the original T7 DNA. The unreplicated portions of this end-toend bimolecular structure are then completed through the action of

143


DNA polymerase and DNA ligase, resulting in a linear bimolecule, called a concatamer. Continued replication can lead to concatamers of considerable length, but ultimately a cutting enzyme slices each concatamer at a specific site, resulting in the formation of virussized linear molecules with repetitious ends. We thus see that 17 has a much more complex replication scheme than that seen for the other bacterial viruses discussed earlier.

5.13 LARGE DOUBLE-STRANDED DNA BACTERIOPHAGES One of the most extensively -studied groups of DNA viruses is the group sometimes called the T-even phages, which include the phages T2, T4, and T6. These phages are among the most complicated in terms of both structure and manner of multiplication replication. In the present section, we will discuss primarily bacteriophage T4, the phage of this group for which the most information is available. The virus particle of phage T4 is structurally complex. It consists of an icosahedral head which is elongated by the addition of one or two extra bands of protein hexamers, the overall dimensions of the .

N~

~\~C~~Ylation--HOH,c6o I

H

Figure 1.18 : The unique base in the DNA of the T-even bacteriophages, 5-hydroxymethylcytosine. The site of glucosylation is shown.

head being 85 x 110 nm. To this head is attached a complex tail consisting of a helical tube (25 x 110 nm) to which are connected a sheath, a connecting "neck" with "collar" and "whiskers," and a complex base plate with pins, to which are attached long jointed tail fibers. All together, the virus particle has over 25 distinct types of proteins. As we noted, the DNA of T4 has a total length about 650 times longer than the dimension of the head. This means that the DNA is highly folded and packed very tightly within the head. The genome structure of T4 is quite complex. The DNA is large, with a molecular weight of about 120 x 10, and is chemically distinct from cell DNA, having a unique base, 5-hydroxymethylcytosine instead of cytosine. The hydroxyl groups of the 5-hydroxymethylcytosine are

144


modified by addition of glucosyl residues. This glucosylated DNA is resistant to virtually all restriction endonucleases of the host. Thus, this virus-specific DNA modification plays an important role in the ability of the virus to attack a host cell. Over 160 separate genes have been recognized in T4, of which the functions of 120 are known. These genes code not only for the complex array of coat proteins, but for a variety of enzymes and other proteins involved in the replication process itself.

Figure 5.19 : Simplified genetic map of T4. Late genes with morphogenetic functions (coat proteins and assembly), and genes with functions in DNA replication are identified. Note that although the genetic map is represented as a circle, the DNA itself is actually linear.

The genetic map of T4 is generally represented as a circle, even though the DNA itself is linear. This "genetic circularity" arises because the DNA of the phage exhibits a phenomenon called circular permutation. This arises because in different T4 phage particles, the sequence of bases at each end differs (although for a given molecule the same base sequence occurs at both ends). This structure, a consequence of the way the T4 DNA replicates (see later), results in


145

an appearance of genetic circularity even though the DNA itself is lineat. mRNA synthesis and regulation in bacteriophage T4 In bacteriophage T4, the details of regulation of replication are more complex than those of T7, but involve primarily positive control. T4 is a much larger phage .than T7 and has many more genes and phage functions. In addition, the DNA of T4 contains the unusual base, 5hydroxymethylcytosine and some of the OH groups of this base are glucosylated. Thus enzymes for the synthesis of this unusual base and for its glucosylation must be formed after phage infection, as well as formation of an enzyme that breaks down the normal DNA precursor deoxycytidine triphosphate. In addition, T4 codes for a number of enzymes that have functions similar to those host enzymes in DNA replication, but are formed in larger amounts, thus permitting faster synthesis of T4-specific DNA. In all, T4 codes for over 20 new proteins that are synthesized early after in fection. It also codes for the synthesis of several new tRNAs, whose function is presumably to read more efficiently T4 mRNA. Overall, the T4 genes can be divided into three groups, for early, middle, and late proteins. The early proteins are the enzymes involved in DNA replication. The middle proteins are also involved in DNA replication. For instance, a DNA unwinding protein (DNA gyrase) is formed which destabilizes the DNA double helix, forming short single-stranded regions at which DNA synthesis can be initiated. The late proteins are the head and tail proteins and the enzymes involved in liberating the mature phage particles from the cell. In T4, there is no evidence for a new phage-specific RNA polymerase, as in T7. The control of T4 mRNA synthesis involves the production of proteins that modify the specificity of the host RNA polymerase so that ·it recognizes different phage promoters. The early promoter, present at the beginning of the T4 genome, is read directly by the host RNA polymerase, and involves the function of host sigma factor. Host RNA polymerase m~ves down the chain until it reaches a stop signal. One of the early proteins blocks host sigma factor action. The early protein combines with the RNA polymerase core enzyme, and when this protein builds up, initiation of early phage genes is stopped. The RNA polymerase cores are now available to combine with new phagespecific activators, which control the transcription of the middle and late genes. The middle genes are generally transcribed along the same DNA strand as the early genes, but the late genes are transcribed along the opposite strand.


146

head host

-==

! ®

I

tail endplate IiiiiJ

l l !

®

Iiiiil

head precursor

~

IiiiiI

endplate joined to core

DMA packaged into head

j

~ t t

l

-1 ~l·':· 1~ -1 j~ sheath protein added

1

~.=~ head and tail joined

stabilized tail

tail fibers added complete infective particle

fr,'.

tsJrj" host

\~

~ ::\~\~\..J L

-I-i-

Figure 5.20 : Steps in the assembly of a T4 bacterial virus.


147

DNA replication The process of DNA replication in T4 is similar to that in TI, but in T4, the cutting enzyme which forms virus-sized fragments does not recognize specific locations on the long molecule, but rather cuts off head-full packages of DNA irrespective of the sequence. Thus each virus DNA molecule not only contains repetitious ends, but the nucleotide sequences at the ends of different molecules are different, although each molecule contains the complete sequence of viral genes. As shown, the cutting process results in the formation of DNA molecules with permuted sequences at the ends. Assembly and lysis In the case of T4, assembly of heads and tails occur on independent pathways. DNA is packaged into the assembled head and the tail and tail fibers are added subsequently. Somehow, the DNA is packed tightly and inserted into the empty phage head. Exit of the virus from the cell occurs as a result of cell lysis. The phage codes for a lytic enzyme, the T4 lysozyme, which causes an attack on the peptidoglycan of the host cell. The burst size of the virus (the average number of phage partides per cell) depends upon how rapidly lysis occurs. If lysis occurs early. then a smaller burst size occurs, whereas slower lysis leads to a higher burst size. The wild type phage exhibits the phenomenon of lysis inhibition, and therefore has a large burst si,?:e, but rapid lysis mutants, in which lysis occurs early, show smaller burst sizes.

5.14 TEMPERATE BACTERIAL VIRUSES: LYSOGENY Most of the bacterial viruses described above are called virulent viruses, since they usually kill the cells they infect. However, many other viruses, although also often able to kill cells, frequently have more subtle effects. Such viruses are called temperate. Their genetic material can become integrated into the host genome and is thus duplicated along with the host material at the time of cell division, being passed from one generation of bacteria to the next. Under certain conditions these bacteria can spontaneously produce virions of the temperate virus, which can be detected by their ability to infect a closely related strain of bacteria. Such bacteria, which appear uninfected but have the hereditary ability to produce phage, are called lysogenic. With most temperate phages, if the host simply makes a copy of the viral DNA, lysis does not occur; but if complete virion pruticles are produced, then the host cell lyses. In a lysogenic bacterial culture at anyone time, a small fraction of the cells, 0.1 to 0.0001 percent,

148


produce virus and lyse, while the majority of the cells do not produce virus and do not lyse. Although only rarely do cells of a lysogenic strain actually produce virus, every cell has the potentiality. Lysogeny can thus be considered a genetic trait of a bacterial strain. The temperate virus does not exist in its mature, infectious state inside the cell, but rather in a latent form, called the provirus or prophage state. In considering virulent viruses we learned tha~ the DNA of the virulent virus contains information for the synthesis of a number of enzymes and other proteins essential to virus reproduction. The prophage of the temperate virus carries similar information, but in the lysogenic cell this information remains dormant because the expression of the virus genes is blocked through the action of a specific repressor coded for by the virus. As a result of a genetic switch, the repressor is inactivated, virus reproduction occurs, the cell lyses, and virus particles are released. A lysogenic culture can be treated so that most or all of the cells produce virus and lyse. Such treatment, called induction, usually involves the use of agents such as ultraviolet radiation, nitrogen mustards, or X rays, known to damage DNA and activate the SOS system. However, not all prophages are inducible; in some temperate viruses, prophage expression occurs only by natural events. Although a lysogenic bacterium may be susceptible to infection by other viruses, it cannot be infected by virus particles of the type for which it is lysogenic. This immunity, which is characteristic of lysogenized cells, is conferred by the intracellular repression mechanism under the control of virus genes. It is sometimes possible to eliminate the lysogenic virus (to "cure" the strain) by heavy irradiation or treatment with nitrogen mustards. Among the few survivors may be some cells that have been cured. Presumably the treatment causes the prophage to detach from the host chromosome and be lost during subsequent cell growth. Such a cured strain is no longer immune to the virus and can serve as a suitable host for study of virus replication. How is it possible to determine whether a strain is lysogenic? A sensitive host is necessary-that is, a strain closely related to the presumed lysogenic strain but not infected with the prophage. In practice, a large number of related strains are obtained, either from nature or from culture collections. The presumed lysogenic strain is cultured in a suitable medium where it grows, infrequently releasing phage. The titer of phage particles in an induced lysogenic culture

149


~

~altachment \ cell (host)

Injection

lytic infection

~I!I ~. ~

!

#\

\ : ~ '" Q ~.J coat proteIns synthesized; virus particles assembled

lysogenic cycle

viral DNA rephcates

lysogenIC induction

normal cell growth

Figure 5.21 : Consequences of infection by a temperate bacteriophage. The alternatives upon infection are integration of the virus DNA into the host DNA (lysogenization) or replication and release of mature VIruS (lysis). The lysogenic cell can also be induced to produce mature virus and lyse.

is typically lO'-lO$/ml. Irradiation can be used to attempt to induce the prophage to replicate. After further incubation, the culture is filtered to remove live bacteria, and the filtrate is tested against the various test strains using the agar overlay technique described for use in assaying virus particles. If plaques are seen, it can be assumed that virus particles are present and that the strain is lysogenic. Sometimes a large number of strains must be tested to fmd a sensitive host. Most bacteria isolated from nature are lysogenic for one or more viruses. Consequences of temperate virus infection What happens when a temperate virus infects a nonlysogenic organism? The virus may

150


inject its DNA and initiate a reproductive cycle similar to that described for virulent viruses, with the infected cell lysing and releasing more virus particles. Alternatively, when the virus injects its DNA, lysogenization may occur instead: the viral DNA becomes incorporated into the bacterial genetic material and the host bacterium is converted into a lysogenic bacterium. In lysogenization the infected cell thus becomes genetically changed. Sensitive cells can undergo either lysis or lysogenization; which of these occurs is often determined by the action of a complex repression system, as will be described below. We thus see that the temperate virus can have a dual existence. Under one set of conditions, it is an independent entity able to control its own replication, but when its DNA is integrated into the host genetic material, replication is then under the control of the host.

\(4'\ I": ~

"': ~ .:,

~.:~;,;-?r~ ~W1~I~ ~ ~\:;:.~ :~ :N~:'?:f;~;'·" : .•

Figure 5.22 : Electron micrograph by negative staining of a lambda bacteriophage . particle.

Regulation of lambda reproduction One of the best studied temperate phages is lambda, which infects E. coli, and our knowledge of the molecular mechanisms involved in lysogenization and lytic processes in this phage is very adQnced. Morphologically, lambda particles iook like those of many other bacteriophages. The virus particle has an icosahedral head 64 nm in diameter, and a tail 150 nm which has helical symmetry. Attached to the tail is a single 23 nm long fiber. In addition to the major proteins of the coat, there are a number of minor coat proteins. The nucleic acid of lambda consists of a linear double-stranded


151

molecule of 31 x 1()6 molecular weight. At the 5' terminus of each of the single-strands is a single-stranded tail of 12 nucleotides in length which are complementary (the ends of the DNA are'said to be cohesive). Thus, when the two ends of the DNA are free in the host cell, they associate and form a double-stranded circle. In the circular form the DNA contains 48,502 base pairs, and its complete sequence is known. Lysis or lysogenization? If lysogeny occurs, then the phage genes are maintained stably in the lysogenic state until a switch occurs and they are converted with high efficiency into a second state in which lytic growth occurs. This process is called lysogenic induction. How does the genetic switch from lysogeny to lysis occur? The lambda genome has two sets of genes, one controlling lytic growth, the other lysogenic growth. Upon infection, genes promoting both lytic growth and lysogenic integration are expressed. Which pathway succeeds is determined by the competing action of these early gene products and by the influence of host factors. To understand how this works, we need to present the genetic map of lambda. The genetic map, although actually linear, can thus be oriented as a circle (because of the cyclization via the cohesive ends mentioned above). The lambda map consists of several operons, each of which controls a set of related functions. Some of the phage genes are transcribed by RNA polymerase from one strand of the double helix, and others are transcribed from the other strand. Upon injection, transcription of the phage genes which code for the lambda repressor occurs, and if repressor builds up before lytic functions are expressed, lytic reproduction is blocked. The repressor protein blocks the transcription of all later lambda genes, thus preventing expression of the genes involved in the lytic cycle. In a lysogenic cell, a single phage gene is expressed continuously, the gene which codes for the lambda repressor protein. This repressor protein, which is coded for by a gene called cI, binds to two operators on the lambda DNA and thereby turns off the transcription of all the other genes of the phage genome. This is the negative control function of the lambda repressor. In addition, the lambda repressor turns on its own synthesis. This is the positive control function of the lambda repressor. Thus, by promoting its own synthesis, the lambda repressor ensures that no other genes except the gene coding for itself is made. In a lysogenic cell, there will usually be only one copy of the lambda genome, but about 100 active molecules of

152

MICROBIOLOGY AND BIOCHEMISTRY "

repressor protein. Therefore, there is almost always excess repressor to bind to lambda DNA and prevent the transcription of the genes necessary for lambda growth and reproduction.

L1

~

§ E

.5

5'G VIrion

i

3'

DNA ends

Figure 5.23 : Genetic and molecular map of lambda.

Lytic growth of lambda How, then, does lambda virus multiplication occur? In a lysogenic cell, multiplication of lambda occurs only after the repressor is inactivated. As we have noted, agents which induce lysogenic cells to produce phage are agents which damage DNA, such as ultraviolet irradiation, X rays, or DNAdamaging chemicals such as the nitrogen mustards. Upon DNA damage, a host defense mechanism called the SOS response is brought into play. An array of 10-20 bacterial genes is turned on, some of which help the bacterium survive radiation. However, one result of DNA damage is that a bacterial protein called RecA (normally involved in genetic recombination) is turned into a special kind of protease which participates in the destruction of the lambda repressor. With lambda repressor destroyed, the inhibition of expression of


153

lambda lytic genes is abolished. We should note that the protease activity of RecA, brought about by DNA damage, nonnally plays an important role in the cell's response to DNA damaging agents, by participating in the breakdown of a host protein, LexA, which represses a set of host genes involved in DNA repair. Induction of bacteriophage lambda is thus an indirect consequence of the SOS response. Once the lambda repressor has been inactivated, the positive and negative control exerted by this repressor are abolished, and new transcriptional events can be initiated. The most important transcriptional event is that involved in the synthesis of another lambda protein called Cro. coded by a gene called ero. The gene ero is located almost adjacent to the gene cl which codes for lambda repressor. The key to the genetic switch lies in the close proximity of the regulator genes for repressor and cro protein. These two genes are transcribed in opposite directions, beginning at different start points. DNA damage

~ 1I I I IiI .-~

____

_

host genome

activation of SOS response

!

activation of host RecA protein; conversion of RecA protein to a protease

protease cleavage of lambda repressor protein (cl)

lytic response Figure 5.24 : Activation of the host SOS response leads to lysis of a lysogenic cell.

In the region separating these two genes are two kinds of sites, promoters and operators, to which each of the proteins of the switch can bind. When lambda repressor is bound to its operator, it covers


154

leftward transcription -

rightward transcription

C~RM r-,--O-R----.I PRcro~

Figure 5.25 : Two back-to-back promoters in the region of cI and cro control the genetic switch. When cI is present, it activates its own synthesis and blocks transcription of cro. When cI is inactivated, transcription of cro can occur, resulting in the lytic cycle. The cI (repressor) protein combines with the operator, OR'

the ero promoter, whereas when Cro is bound, it covers one of the cl promoters. As we have noted, the direction in which transcription occurs on a DNA double-strand (and hence which of the two strands is read) depends upon the promoter. A promoter essentially points the RNA polymerase in the proper direction. In the case of lambda, the cl promoter points RNA polymerase "leftward," whereas ero promoter points the polymerase "rightward." The lambda system provides one of the best studied examples of a genetic switch, in which one or the other of two competing genetic functions occurs. Which of the two genetic functions gets the upper hand will depend initially on chance events, but once one of the two functions has become established, it prevents the action of the other function. Only under unusual circumstances, such as when induction occurs, would the dominant genetic function be superseded. Integration Integration of lambda DNA into the host chromosome occurs at a unique site on the E. coli genome. Integration occurs by insertion of the virus DNA into the host genome (thus effectively lengthening the host genome by the length of the virus DNA). The cohesive ends of the linear lambda molecule find each other and form a circle, and it is this circular DNA which becomes integrated into the host genome. To establish lysogeny, genes cl and int must be expressed. As we have noted, the cI gene product is a protein which represses early transcription and thus shuts off transcription of all later genes. The integration process requires the product of the int gene, which is a site-specific topoisomerase catalyzing recombination of the phage and bacterial attachment sites. During cell growth, the lambda repression system prevents the expression of the integrated lambda genes except for the gene c/, which codes for the lambda repressor. During host DNA replication, the integrated lambda DNA is replicated along with the rest of the host genome, and transmitted to progeny cells. When release from repression occurs, the lambda productive cycle occurs. '


155 m'

att

m

lambda DNA

l

m'

cyclizes at cohesive ends

m.m'

l

m'

--- ---

site-specific nuclease creates staggered ends of . / phage and host gal phr . / bio ch/A ---·----==~::::::JI _~====-

":=::==.=-,==

I t

gal

phr

,==

-_:::::::.:.-_-=----------

integration of lambda DNA and closing of gaps m' by DNA ligase

m

i

bio

ch/A

i

Figure 5.26 : Integration of lambda DNA into the host. Integration always occurs at a specific site on the host DNA, involving a specific attachment site (att) on the phage. Some of the host genes near the attachment site are given. A sitespecific enzyme (integrase) is involved, and specific pairing of the complementary ends results in integration of phage DNA.

Replication Replication of lambda DNA occurs in two distinct fashions during different parts of the phage production cycle. Initially, liberation of lambda DNA from the host results in replication of a circular DNA, but subsequently linear concatamers are formed, which replicate in a different way. Replication is initiated at a site close to gene 0 and from there proceeds in opposite directions (bidirectional

156


symmetrical replication), tenninating when the two replication forks meet. In the second stage, generation of long linear concatamers occurs, and replication occurs in an asymmetric way by rolling circle replication. In this mechanism, replication proceeds in one direction only, and can result in very long chains of replicated DNA. This mechanism is efficient in permitting extensive, rapid, relatively uncontrolled DNA replication; thus it is of value in the later stages of the phage replication cycle when large amounts of DNA are needed to form mature virions. The long concatamers formed are then cut into virus-sized lengths by a DNA cutting enzyme. In the case of lambda, the cutting enzyme makes staggered breaks at specific sites on the two strands, twelve nucleotides apart, which provide the cohesive ends involved in the cyclization process. Lambda is one of the agents of choice for use as a cloning vector for artificial construction of DNA hybrids with restriction enzymes. It has several features that make it an excellent system for genetic engineering. One feature of lambda that makes it of special use for cloning is that there is a long region of DNA, between genes Jand au, which does not seem to have any essential functions for replication, and can be replaced with foreign DNA. Temperate viruses as plasmids Another class of temperate viruses have quite a different mechanism for maintenance of the prophage state. In this group, viruses resemble plasmids. They do not actually become integrated into the host chromosome, but instead replicate in the cytoplasm as circular DNA molecules. Among such viruses is bacteriophage PI of Escherichia coli. Although in broad features, such viruses resemble the temperate viruses such as lambda just discussed, at the level of virus replication they are, of course, quite different. Interestingly, although the plasmid prophage is not physically connected to the host DNA, phage DNA replication is closely coordinated with cell division, since only one copy of the prophage is present per host chromosome.' The phage repressor is somehow involved in this regulation process.

5.15 A TRANSPOSABLE PHAGE: BACTERIOPHAGE MU One of the more interesting bacteriophages is that called Mu, which has the unusual property of replicating as a transposable element. This phage is called Mu because it is a mutator phage, inducing mutations in a host into which it becomes integrated. This mutagenic property of Mu arises because the genome of the virus


157

can become inserted into the middle of host genes, causing these genes to become inactive (and hence the host which has become infected with Mu behaves as a mutant). Mu is a useful phage because it can be used to generate a wide variety of mutants very easily. Mu can be used in genetic engineering. A transposable element is a piece of DNA which has the ability to move from one site to another as a discrete element. Transposons are found in both procaryotes and eucaryotes, and play important roles in genetic variation. There are three types of transposable elements: insenion elements, transposons, and viruses like Mu. (Also the retroviruses discussed later, are a group of animal viruses which have features of transposable elements). An insenion element is the simplest type of transposable element: it carries no detectable genes and simply moves itself around. A transposon is a genetic element with a unique piece of DNA, usually coding for one or more proteins, to which is attached, at each end, an insertion element. The insertion element at each end of the transposon is identical, although the DNA sequence may be either a direct repeat or an inverted repeat. Mu is a very large transposable element, carrying insertion elements and a number of Mu genes involved in Mu multiplication. Structurally, bacteriophage Mu is a large doublestranded DNA virus, with an icosahedral head, a helical tail, and 6 tail fibers. The DNA of Mu is arranged inside the virus head as a linear doublestranded molecule. It has a molecular weight of 25 X 106 (3839 kilobases). The genetic map of Mu is shown in Figure 6.37a. It can be seen that the bulk of the genetic information is involved in the synthesis of the head and tail proteins, but that important genes at each end are involved in replication and immunity. At the left end of the Mu DNA are 50-150 base pairs of host DNA and at the right end are 500-3000 base pairs of host DNA. These host DNA sequences are not unique and represent DNA adjacent to the location where Mu had become inserted into the host genome. When a Mu phage particle is formed, a length of DNA containing the Mu genome just large enough to fill the phage head is cut out of the host, beginning at the left end. The DNA is rolled in until the head is full but the place at the right end where the DNA is cut varies from one phage particle to another. For that reason, as shown on the genetic map, there is a variable sequence of host DNA at the righthand end of the phage (right of the attR site) which represents the host DNA that has become packaged into the phage head. Each phage

158


particle arising from a single infected cell will have a different amount of host DNA, and the host DNA base sequence of each particle from the same cell will be different. In some cases, completely empty Mu heads become filled with purely host DNA. Such particles can transfer host genes from one cell to another, a process called transduction. posrtive acbvat90r of late mANA synthesIS Immunity

invertible G segment (host range)

lysis

I

head and tail genes

Integration replication host DNA

,

lie

I

variable and

rL,

(host DNA)

.---------'-------..IrL,

rJ.., Iys

AB

C

DEHFGITJKLMVNP

Rsuu's'l I

I allL

allR

transposase

(a)

l

I - - - - , I G C C G A A G C A G C G T T G~_ _ _--1 .CGGCTTCGTCGCAAC. L---I

1-----41

GC CGA

L -_ _ _~CGGCTTCGTC

I----,IG C C G A A G C A G CGGCTTCGTC

'-------'

•

1

L---I

A G C A G C G T T GI..._ _ _-I GCAAC

'-----I

A G C A G C G T T G~_ _ _--1 TCGTCGCAAC '-------' repair of DNA leads to formation of S-base·pair duplication

Figure 5.27 : Bacteriophage Mu. (a) Genetic map of Mu. (Confusingly, there are two G's, the G gene and the invertible G segment. These are different O's.) (b) Integration of Mu into the host DNA. showing the generation of a five-base-pair duplication of host DNA.


159

A~ shown in the genetic map, a specific segment of the Mu genome called G (distinct from the G gene) is invertible, being present in either the orientation designated SU, or in the inverted orientation U'S'. The orientation of this segment determines the kind of tail fibers that are made for the phage. Since adsorption to the host cell is controlled by the specificity of the tail fibers, the host range of Mu is determined by which orientation of this invertible segment is present in the phage. If the G segment is in the orientation designated G+· then the phage particle will infect Escherichia coli strain K12. If the G segment is in the G- orientation, then the phage particle will infect Escherichia coli strain C or several other species of enteric bacteria. The two tail fiber proteins are coded on opposite strands within this small G segment. Left of the G segment is a promoter that directs transcription into the G segment. In the orientation G +, the promoter directing transcription of S and U is active, whereas in the orientation G-, a different promoter directs transcription of genes S' and U' on the opposite strand. Upon infection of a host cell by Mu, the DNA is injected. In contrast with lambda, integration into the host genome of Mu is essential for both lytic and lysogenic growth. Integration requires the activity of the A gene product, which is a transposase enzyme. At the site where the Mu DNA becomes integrated, a 5 base pair duplication of the host DNA arises at the target site. This host DNA duplication arises because staggered cuts are made in the host DNA at the point Mu becomes inserted, and the resulting single-stranded segments are converted into the double-stranded form as part of the integration process. Lytic growth of Mu can occur either upon initial infection, if the c gene repressor is not formed, or by induction of a lysogen. In either case, replication of Mu DNA involves repeated transposition of Mu to multiple sites on the host genome. Initially, transcription of only the early genes of Mu occurs, but after gene C protein, a positive activator of late RNA synthesis, is expressed, the synthesis of the Mu head and tail proteins occurs. Eventually, expression of the lytic function occurs and mature phage particles are released. Because Mu integrates at a wide variety of host sites, it can be used to induce mutants at many locations. Also, Mu can be used to carry into the ceil genes that have been derived from other host cells, a form of in vivo genetic engineering. In addition, modified Mu phage have been made artificially in which some of the harmful functions

160


of Mu have been deleted. These phages, called Mini-Mu, are deleted for significant portions of Mu but have the ends of the phage in - nonnal orientation. Mini-Mu phages are usually defective, unable to form plaques, and their presence must be ascertained by the presence of other genes which they carry. One set of Mini-Mu phages containing the [3-galactosidase gene of the host (called Mudlac, d for defective) can be detected in 'the integrated state if the lac gene is oriented properly in relation to a host promoter. Under these conditions, the hOst cell will form the enzyme [3-galactosidase, which can be detected in colonies by a special color indicator. f3galactosidase-positive colonies from a agalactosidase-negative host are thus an indication that Mud-lac infection has occurred. We thus see that phage Mu provides a useful tool for geneticists, as well as being an interesting bacteriophage in its own right.

5.16 GENERAL OVERVIEW OF ANIMAL VIRUSES We have discussed in a general way the nature of animal viruses in the first part of this chapter. Now we discuss in some detail the structure and molecular biology of a number of important animal viruses. Viruses will be discussed which illustrate different ways of replicating, and both RNA and DNA viruses will be covered. One group of animal viruses, those called the retroviruses, have both an RNA and a DNA phase of replication. Retroviruses are especially interesting not only because of their unusual mode of replication, but because retroviruses cause such important diseases as certain cancers and acquired immunodeficiency syndrome (AIDS). Before begilming our discussion of the manner of replication of animal viruses, we should mention first the important differences which exist between animal and bacterial cells. Since virus replication makes use of the biosynthetic machinery of the host, these differences i1:t cellular organization and function imply differences in the way the viruses themselves replicate. Bacteria, being procaryotic, do not show compartmentation of the biosynthetic processes. The genome of a bacterium relates directly to the cytoplasm of the cell. Transcription into mRNA can lead directly to translation, and the processes of transcription and translation are not carried out in separate organelles. Animal cells, being eucaryotic, show compartmentation of the transcription and translation processes. Transcription of the genome into mRNA occurs in the nucleus, whereas translation occurs in the cytoplasm. The messenger RNA in the eucaryote is usually modified by adding to it


promoters

161

B

c

~A t:.::::~p:.;::L_ _ _..lI_ _ _ _

....L_ _ _ _..........1 bacterial

genome

cistrons (genes)l

operator

A

c

B

polycistronic mRNA

formation of polyribosome and direct translation into protein (a) procaryote intron

"

L-_ _ _...J®t..::...;:"a._ _ _ _... ~;;:.~.;:",;: ......_ _ _~1

eucaryotic genome

(~----+::"::'+-----+::"::'~~----J) f§SJ ,

primary RNA transcript

,, ,,

~ ,

I , I , I ,I

,

l l

I I I I I I , I

I

I

I

I

k "~-....1...------L..------4(

,

poly AI tail

J ---------

5'cap

• •

RNA processing removes introns

capping and polyadenylation nuclear membrane transfer from nucleus to cytoplasm

monocistronic mRNA translated

(b) eucaryote

Figure 5.28 : Comparison of protein synthesis processes in procaryote and eucaryote. (a) Procaryote. (b) Eucaryote.

162

MICROBIOLOGY AND 6IOCHEMISTRY

a poly (A) tail of 100 to 200 adenylic residues at the 3' end and a methylated guanosine triphosphate, called the cap .. at the 5' end. The cap is required for binding of the mRNA to the ribosome and the poly A tail may be involved in subsequent RNA processing and transfer of the mature mR:NA 'from the nucleus to the cytoplasm. All of the protein-synthesizfqg,machinery of the eucaryotic cell, the ribosomes, tR"NA molecules, and accessory components, is in the cytoplasm, "~nd the mature mRNA associates with the proteinsynthesizing apparatus once it leaves the nucleus. The genes of eucaryotes are often split, with noncoding regions called introns separating coding regions (exons). TraPscription of both the coding and noncoding regions of a gene occurs and an RNA, called the primary RNA transcript, is formed and is subsequently converted into the mature mRNA by a mechanism called RNA processing, in which the noncoding regions are excised (the cap and tail remain after RNA processing is complete). After processing, the mature mRNA is translated into protein. One important distinction between eucaryotic and procaryotic mRNA is that procaryotic mRNA is generally polycistronic, with more than one coding region present in a single mRNA molecule, whereas eucaryotic mRNA· is monocistronic. In procaryotes, during the translation process the ribosomal machinery moves down the mRNA past a stop site and initiates translation of another gene without ever leaving the mRNA. A-lthough eucaryotic mRNA is usually monocistronic, this does not mean that only a single type of protein molecule results from the translation of a eucaryotic mRNA. Frequently, the eucaryotic mRNA codes for a single, large multifunctional protein, called a poly-protein, which may subsequently be cleaved by a specific protease into several distinct enzymes. In other cases, the polyprotein may remain as a single multifunctional polypeptide. We might also note another important difference between animal and bacterial cells. Bacterial cells have rigid cell walls containing peptidoglycan and associated substances. Animal cells, on the other hand, lack cell walls. This difference is important for the way by which the v:rus genome enters and exits the cell. In bacteria, the protein coat of the virus remains on the outside of the cell and only the nucleic acid enters. In animal viruses, on the other hand, uptake of the virus often occurs by endocytosis (pinocytosis or phagocytosis), processes which are characteristic of animal cells, so that the whole virus particle enters the cell. The separation of animal virus genomes from their protein coats then occurs inside the cell.


163

Classification of animal viruses Most of the animal viruses which have been studied in any detail have been those which have been amenable to cultivation in cell cultures. As seen, animal viruses are known with either single-stranded or doublesttanded DNA or RNA. Some animal viruses are enveloped, others are naked. Size varies greatly, from those large enough to be just visible in the light microscope, to those so tiny that they are hard to see well even in the electron microscope. In the following sections, we will discuss characteristics and manner of multiplication of some of the mqst important and best-studied animal viruses.

@) ~

~

(al

whole virus particle (en\I8Ioped)

uptake into animal cell

by e~OSls

and loss of

.

f:i;\

~

viral envelope nucleocapsid

-

uncoating of capsid

/

processes of

............ virus multiplication

virus

nucleic acid

(bl

Figure 5.29 : Uptake of an enveloped virus particle by an animal cell. (a) The process by which the viral nucleocapsid is separated from its envelope. (b) Electron micrograph of adenovirus particles entering a cell. Each particle is about 70 om in diameter.

Consequences of virus infection in animal cells Viruses can have varied effects on cells. Lytic infection results in the destruction of the host cell. However, there are several other possible effects following viral infectioA of animal cells. In the case of enveloped viruses, release of the viral particles, which occurs by a kind of h¢dingprocess, may be s10w and the host cell may not be lysed. The cell may remain alive and continue (0 produce virus over a long /period of time. Such infections are referred to as persistent infections.

164


Viruses may also cause latent infection of a host. In a latent infection, there is a delay between infection by the virus and the appearance of symptoms. Fever blisters (cold sores), caused by the herpes simplex virus, result from a latent viral infection; the symptoms reappear sporadically as the virus emerges from latency. The latent stage in viral infection of an animal cell is generally not due to the integration of the viral genome into the genome of the animal cell, as is the case with latent infections by temperate bacteriophages. Viruses and cancer A number of animal viruses have the potential to change a cell from a normal cell to a cancer cell. This process, called transformation, can be induced by infections of animal cells with certain kinds of viruses. One of the key differences between normal cells and cancer cells is that the latter have different requirements for growth factors . Rapidly growing cells pile up into accumulations that are visible in culture as foci of infection. Because cancerous cells in the animal body have fewer growth requirements, they grow profusely, leading to the formation of large masses of cells, called tumors. The term neoplasm is often used in the medical literature to describe malignant tumors. Not all tumors are seriously harmful. The body is able to wall off some tumors so that they do not spread; such noninvasive tumors are said to be benign. Other tumors, called malignant, invade the body and destroy normal body tissues and organs. In advanced stages of cancer, malignant tumors may develop the ability to spread to other parts of the body and initiate new tumors, a process called metastasis. How does a normal cell become cancerous? The process can be broken down into several stages. In the first step, initiation, genetic changes in the cell occur. This step may be induced by certain chemicals, called carcinogens, or by physical stimuli, . such as ultraviolet radiation or X rays. Certain viruses also bring about the genetic change that results in initiation of tumor formation. Once initiation has occurred, the potentially cancerous cell may remain dormant, but under certain conditions, generally involving some environmenta1 alteration, the cell may become converted into a tumor cell, a process called promotion. Once a cell has been promoted to the cancerous condition, continued cell division can result in the formation of a tumor. Although the ability of viruses to cause tumors in animals has been proved for many years, the relationship of viruses to cancer in

MICROBIOLOGY OF VIRUSES nonenveoped

165 enveloped

o

ssDNA

paNOllirus

~

dsDNA

papovavirus

dsDNA

poxvirus

adenovirus

dsDNA herpesvirus

iridovirus ~

(a) DNA viruses

-

-o

100nm

ssANA

......-...

.... ANA

--- ®

(I .....,...,...

@ANA -...

@dsANA ........ ~

100rwn

(b)

ANA_

-~ ...

® .• -.-•

....-us

~

g= f§

~

C l)

Photochemolithotrophic

Photosynthetic bacteria

o-l

~

GENERAL METABOLISM

173

and animals which consume organic materials and oxygen generated by phototrophs. Energy losses associated with the vital activity of all organisms on the Earth are being compensated for by the energy of solar radiation. It should be noted that the cells of man and animals utilize highly reduced, i.e. hydrogen-containing, compounds (carbohydrates, lipids, proteins, etc.) as energetic materials. Hydrogen is an energetically valuable material. Its energy in a transformed form is stored in the ATP chemical bonds in the cells of heterotrophic organisms.

6.3 ENERGETICS OF BIOCHEMICAL REACTIONS To get a deeper insight into the metabolic and energetic processes, knowledge of general principles of chemical energetics appears to be of help. In the living cell, all chemical reactions contributing to the metabolism obey the laws of energetics. The first law of energy conservation states that the energy of a chemical reaction can be neither annihilated nor generated from nothing, it can merely be converted from one form into another. In terms of this law, it becomes possible to defme the energy balance of a chemical process. Spontaneous chemical processes can proceed only in one direction towards a state of equilibrium; the state of equilibrium having been reached, the process is brought to termination. The second law of thermodynamics (energetics) enables one to predict the direction of biochemical processes. According to this law, any spontaneous process takes the route corresponding to a maximum of entropy under the given conditions until an equilibrium state for the reaction is reached. Entropy conveys a measure of the disorder in a system. An increase in entropy in the course of a reaction prevents the return of the reaction to the initial state, since for that to occur a diminution in entropy would be required. A spontaneously disordered system is never capable of turning into an ordered one. Therefore, all reactions that proceed with a concomitant increase in entropy are irreversible. For their reversal, an additional energy is needed to be spent to compensate for the losses in entropy change, i.e. to bring the system from the disordered into the ordered state. However, from the pract~cal standpoint it appears expedient to use the so-called free energy which, in contrast to entropy, is amenable to measurement in the course of a reaction. The free energy defmes a portion of the total energy of a system that can be converted to work at pressure and temperature kept constant. The free energy is

174


denoted AG. The portion of total energy of a chemical process that cannot be converted to work at pressure and temperature kept constant is called the bound energy and is expressed as the product TAS, where T is the absolute temperature and AS is the entropy change of a system under the given chemical reaction conditions. The sum of the changes in free and bound energies is called enthalpy (internal energy of a system) or heat energy content of a system and is denoted OH. Enthalpy can be measured experimentally and is equal to the amount of heat released in the given process. The enthaply change is defined by the equation MI = /lG + T /l S Otherwise stated, /lG=MI - T /l S The energetic state of any system, including that of a cell and an organism, can be defmed in terms of this very important equation. The free energy is expressed in kilojoules per mole of substance, kJ/ mol. The free energy is a very convenient parameter for defining the spontaneity or nonspontaneity of a chemical process. For spontaneous processes, the free energy is seen to decrease, i.e. /lG has a negative value. Such reactions are called exergonic, i.e. proceeding with a release of energy. These reactions supply energy to the cells. The processes for which the free energy is increased (i.e. /lG has a positive value) cannot proceed spontaneously. They require energy supply from the outside. Such processes are referred to as endergonic, i.e. proceeding with an expenditure of energy. In a state of equilibrium, /lG = O. The free energy of chemical reactions may be estimated both under the standard conditions and under real, or physiological, conditions. The standard free energy, /lGo , of a biochemical reaction is defined as a free energy change under the standard conditions, i.e. at the concentration of reactants I mol/litre, temperature 25°C < 298 X), and pH 7. If water is involved either as a starting compound or as a reaction product, its concentration is taken equal to 1.0 mol/litre, although the true concentration of water in dilute aqueous solutions is close to 55 mol/litre. The standard free energy is found as a difference between the sum values of free energy for the end products and the initial reactants. The value of /l Gp for a biochemical reaction proceeding under the

175

GENERAL METABOLISM

physiological conditions is estimated with allowance for the actual concentrations of components involved. With reference to the free energy as a characteristic of metabolism one may say that catabolic reactions proceed with a release of energy and anabolic ones, with a consumption of energy' The anabolic reactions can proceed only as closely coupled to the catabolic reactions. High-energy, or macroergic,compounds act as energetic mediators between these two types of reactions.

6.4 HIGHT-ENEGRY AND LOW-ENERGY PHOSPHATES: GENERAL CONSIDERATIONS Commonly, all compounds are classified into high-energy and lowenergy ones. As a conventional borderline between these two classes, the free energy of about 20 kJ/mol for the phosphate bond hydrolysis has been taken. This value used for the purpose of characterization of biochemical processes should not be confused with the bond energy which is conceived as an energy required for disruption of the bond between two neighbouring atoms in any molecule. Cleavage of the phosphate bond results, alongside energy release, in the formation of inorganic phosphates. Energetic characteristics for some of the compounds of interest are listed in the following Table. TABLE 8.2 Standard Free Energies, AGo, for Hydrolysis of Some High-energy and Low-energy Compounds, and Free Energies for Hydrolysis of Compounds Under Physiological Conditions, AGp. Compound

- AGpI

kJ/mol

High-energy compounds Phosphoenol pyruvate l,3-Diphosphoglycerate Creatine phosphate ATP Acetyl-CoA

61.7 49.2 42.5 30.4 30.4

66.7 41.7 50.0 (Table 8.2 Contd.)

Compound

ADP Pyrophosphate (H4P20,)

_ AGO,

- AGpI

kJ/mol

kJ/mol

28.3 28.3

50.0 50.0

176

UDP-glucose Glucose I-phosphate Fructose 6-phosphate AMP

Glucose 6-phosphate a-Glycerol phosphate


24.2 20.8 Low-energy compounds 15.8 14.1 13.8

23.8

9.2

It shouij be emphasized that ATP, the key mediator in energy metabolism, is not the most energy-rich species. ATP is found in the middle of. energy scale. The most frequently occurring route is cleavage of the end phosphate from ATP: ATP ~ ADP + Hl04 (1) The end phosphate adds water and is transferred onto another compound, causing thereby the phosphorylation of the latter. An alternative route for the phosphate bond energy release is exemplified by pyrophosphate cleavage of ATP: ATP ~ AMP + H4 Pp? (2) This type of reaction is less frequent in biological processes. Its distinctive feature is the formation of pyrophosphate, which ranks among energy-rich materials. Hydrolysis of pyrophosphate (3) releases roughly as much energy as hydrolysis of the ATP end phosphate bonds. In biological processes, energy-rich pyrophosphate bonds are seldom used for synthesis of other compounds because of the heat energy released by pyrophosphate hydrolysis. ADP may be used as a high-energy reactant in biochemical processes. The cleavage of the ADP end phosphate bond (4) releases the same amount of energy as that generated by splitting the end phosphate bond in ATP. At first glance, it may appear that ATP can be adequately replaced by ADP in chemical reactions, in particular in the phosphoryla9 ion of other compounds. However, as evidenced by the available experimental data, such a possibility has never been realized in biological processes. At least such reactions are as yet unknown. It is known, however, that ADP can be hydrolyzed to lowenergy AMP and phosphate with heat release. For a long time, there , have been difficulties in determining the free energy for the ATP phosphate bond. The standard free energy for hydrolysis of the ATP phosphate bond, AGo, is equal to about -30.4 kJ/mol. This value has

GENERAL METABOLISM

177

been derived under standard conditions, i.e. at concentrations I M for initial reactants and end products, pH 7.0, temperature 37°C, and excess of Mg2+ ions. It stands to reason that in the cell under physiological conditions, the concentrations of initial reactants, end products, and Mg" ions are substantially inferior to the standard valueS; variations in pH are also possible. Therefore, the real free energy for hydrolysis of the end phosphate bond in ATP and ADP, and of the phosphate bonds in pyrophosphate is close to -50.0 kJ/mol. To be noted, the values of ~Go for other compounds differ from the standard value, but not necessarily towards higher energies.

6.5 ENERGY TRANSFER IN BIOCHEMICAL PROCESSES The biological activity of the cell is closely associated with the continuous redistribution of the energy delivered by the compounds that enter the cell. The storage of energy in the specific phosphate bonds of ATP constitutes the basis for the energy transfer mechanism in the living cell. The living cell is a nonequilibrated chemical systemthe circumstance that permits storing the energy, produced by catabolic reactions of nutrients, in the ATP phosphate bonds. The ATP energy in the cell can be converted, via tree major routes, to energy of chemical bonds, to thermal energy, and to energy for performing work. We now consider in general terms the transfer of chemical bond energy. The chemical reaction is associated with the generation of ATP energy. If the reaction is in a state of equilibrium, the energy transfer is accomplished to a 100% efficiency. Nonetheless, no ATP energy will accumulate, since at equilibrium, the free energy release is zero. Therefore, in order to provide for energy storage in the ATP phosphate bonds, the reaction must be a nonequilibrium one. This is accomplished as a portion of the chemical reaction energy is lost as heat. The remaining energy is transferred onto the ATP phosphate bonds to be stored therein. It follows, therefore, that biological generators of energy cannot attain the 100% efficiency. The third route of energy conversion leads to the performance of work. The chemical energy of ATP phosphate bonds can be spent on osmotic, electric, mechanical, and other types of work. In doing so, not all of the ATP energy is used for performing work; a portion of it is dissipated as heat. Especially much heat is produced by muscle contraction, which represents a mechanochemical mechanism for heat generation in the living organisms. Heat is of little use for performing

178


work in the living system. It is only in wann-blooded animals and man that the heat is consumed for warming and maintaining a constant temperature of the body, equal to about 37°C. All chemical processes in the living organism can proceed only with the involvement of enzymes. For this reason, prior to consider the mechanism of energy extraction and various metabolic pathways, it appears worthwhile to discuss enzymes and their functions.

7

Metalbolism of Saccharides Carbohydrate metabolism in the organism tissues encompasses enzymic processes leading either to the breakdown of carbohydrates (catabolic -pathways), or to the synthesis thereof (anabolic pathways). Carbohydrate breakdown leads to energy release or intermediary products that are necessary for other biochemical processes. The carbohydrate synthesis serves for replenishment of polysaccharide reserve or for renewal of structural carbohydrates. The effectiveness of various routes of carbohydrate metabolism in tissues and organs is defined by the availability of appropriate enzymes in them.

7.1

CARBOHYDRAGE CATABOLISM IN TISSUES

A number of routes for carbohydrate catabolism in tissues are known. They include glycolysis and its variant, glycogenolysis, which are auxiliary pathways to energy production, respectively, by breakdown of glucose (or other monosaccharides) and glycogen to lactate (under anaerobic conditions) or to CO2 and Hp (under aerobic conditions). The involvement of glycolysis and glycogenolysis in the energetic function has been discussed in detail in the foregoing section 'Bioenergetics" . There is known one more catabolic route for carbohydrates commonly referred to as the pentose phosphate cycle (also called hexose mono phosphate shunt, or phosphogluconate pathway). As a tribute to the biochemists who have played a decisive role in its investigation, the pentose phosphate cycle is also referred to as the Warburg-Dickens-Horecker pathway. The pentose phosphate cycle represents a mUltienzyme system in 179


180

which the important intermediates are, as the name implies, pentose phosphates. This cycle may be regarded as a branching, or shunt, at the glucose 6-phosphate step in the overall glycolysis. 7.1.1 Pentose Phosphate Cycle To provide for all steps of the pentose phosphate cycle, at least three glucose 6-phosphate molecules are required. Let us consider separate reactions of this cycle. 1. Dehydrogenation o( g1ueose 6-phosphate is the reaction that directs- glucose 6-phosphate via the pentose phosphate pathway; it is catalyzed by glucose-6phosphate dehydrogenase (in the schemes below, for a fuller description of the cyclic process, three glucose 6-phosphate molecules are used)

,/

H

OH

~-oH 3 HO-C-H

I

6

GI~hate

~-oHI

dehydrogenaa

·7"'3NADP +

3NADP·H+H+

H-C

I

H,C-oPO)H, glucose 6-phosphate

Glucose-6-phosphate dehydrogenase is a dimer with a molecular mass of about 135 000. Up to eight electrophoretic ally separable isoenzymes for this enzyme are known. A specific feature of the above reaction is the formation of NADP • Hr The reaction equilibrium is strongly shifted to the right, since the lactone formed is liable to hydrolysis, which is spontaneous or lactonase-assisted. 2. Hydrolysis of 6-phosphoglueonate lactone to 6-phosphoglueonate:

o C"

H-~-OH 1

3 HO-C-H

I I

H-b-OH

Lactonaae I 0 ~ 3 HO-C-H

H - t - OH H-C------!

1-

COOH

H-~-OH

H-C-OH

1

HaC-OPOaH,

H.C-OPOaH.

6-phoapbocl uconate lactone

8-pbOllphoglueonate

181

METABOLISM OF SACCHARIDES

3. Dehydrogenation of 6-phosphogluconate to ribulose 5phosphate. This reaction is catalyzed by 6-phosphogluconate dehydrogenase according to the scheme: COOH

I

3

"-r-O "o-y-"

H

"-y-o".'

. /"

""

.

6-phosphoglUCOlllte dehydrogenaR

3NADP'

+lC01

H-C-OH

.1

H2C-oP0 3H1 tJ.phosphog!uconate

D-ribulose S-phosphate

The reaction equilibrium is shifted to the right. 6-Phosphogluconate dehydrogenase is a dimer with a molecular mass of about 100 000. Several isoenzymes are known for this dehydrogenase. A specific feature of this reaction is that dehydrogenation leads to an unstable intermediate which is immediately decarboxylated on the surface of the enzyme. This is the second oxidation reaction in the pentose phosphate cycle that leads to NADP • H2 ; therefore, the conversion of glucose 6-phosphate to ribulose 5-phosphate is commonly referred to as the oxidative phase of the pentose phosphate cycle. The sequence of reactions starting from ribulose 5-phosphate to the formation of initial glucose 6-phosphate is called the nonoxidative, or anaerobic, phase of this cycle. 4. Interconversion or isomerization of pentose phosphates. Ribulose 5-phosphate is capable of a reversible isomerization to other pentose phosphates-xylulose 5-phosphate and ribose 5-phosphate. These reactions are catalyzed by two respective enzymes, viz., pentosephosphate epimerase and pentose-phosphate isomerase, according to the scheme below: H.C-OH

H.C-OH

I

c=o I

2 HO-C-H

I

H-C-OH

I

HsC-OPO,H. ]).:1,1111_

~pllOl)lllate

Pmt_pllOlphate eplmeralll

I I

C= 0

~===-~ 3 H-C-OH

I

H-C-OH

I

IItC-OPO.H. ])'rlllulOlO

5-p.~OI)IIIate

H-C=O

-

I

Pentooe-pllOlp/late H-C-OH

-

Isomerue

I

H-C-OH

I

H-C-OH

I

H.C-OPO.H. ]).r\IIoIe

5-pII01)111ate

Two other pentose phosphates (ribose 5-phosphate and xylulose 5-phosphate), which are derived from ribulose 5~phosphate, are important for the subsequent reaction of the cycle. Two molecules of

182


xylulose 5-phosphate and one molecule of ribose 5-phosphate are required for this. S. Transfer of glycolic aldehyde from xylulose S-phosphate onto ribose S-phosphate or the first transketolase reaction. The next reaction, which is catalyzed by transketolase, involves the pentose phosphates produced by the foregoing reaction (the transferable moiety is shown in the box): I

c=o I

2 HO-C-H

I

H-C=O

H.C-OH

HoC-OH Pent_pboopbate eplmerue

I

I

Pent __pboopbate H-C-OH

C= 0

I

.........I _~===-:: H-C-OH I

~====-:: 3 H-C-OH I

H-C-OH

H-C-OH

H-C-OH

I

I

I

HaC-OPOIHa

HoC-OPOIH.

BsC-OPOIH.

J>rtbal_

J>syl.l_

$-p:toopbate

$-pboopbate

A ribose 5-phosphate molecule and one of the two xylulose 5phosphate molecules are used during the first transketolase reaction. The other xylulose 5-phosphate molecule is consumed later, in the second transketolase reaction. Transketolase is a dimer with a molecular mass of 140000. Its coenzyme is thiamine bisphosphate. Mgt+ ions are required for the reaction. Both transketolase reaction products are used as substrates at the next step of the cycle. 6. Transfer of dihydroxyacetone moiety from sedoheptulose 7-phosphate onto glyceraldehyde 3-phosphate. This reaction is reversible and is catalyzed by transaldolase according to the scheme:

[H2C:'o'Hi

r-----'

I

I

I

C.O ... -,--_...1

I H,C-OH I I I I IL __c-o 1___ .-JI

I

.

Transketolase

HO-C-H

I

I

HO-C-H

I H-C-OH

I H-C-OH

H-C-OH

I

I

H-C-O

+

I H-C-OH

I H 2C-OPO)H2

H 2C- OPO)H2

H-C- OH

D·xylulote &-phosphate

H 2 C- OPO)H2 D·sedoheptulose D.gIycerilldehyde 711hotphate 3·pllosphllte

I

D·ribose S.phosphllte

Transaldolase is a dimer with a molecular mass of about 70 000. The fructose 6phosphate molecule produced by this reaction enters the glycolysis, while erythrose 4-phosphate is used as a substrate at the subsequent steps of the cycle.


183

7. Transfer of glycolic aldehyde from xylulose S-phosphate onto erythrose 4-phosphate or the second transketloase reaction. This reaction is related to the first transketolase reaction and is catalyzed by the same enzyme. The only distinction is that erythrose 4-phosphate acts as an acceptor for glycolic aldehyde:

r-:-----' I H,C-OH I I IL

I

I

__C-O 1___ -JI

HO-C-H

I

H-C-OH

I

HzC- OPOJH Z

O·xylulOll 5-phOlPhlte

fH2C:' O"H; I

I

H-C-O

I

I

H-C-OH

I + H-C-OH I

H-C-OH

I

HO-C-H Transketolase ' I , H- c- OH M.H I H-C-OH

H ZC-OPO JH2

O·ribose 5-phosphate

I

C.O

I

... -I---.J

I

H-C .. O

+

II

H-1- 0H H~-OPOJH2

H-C- OH

I

HzC- OPOJH Z O-sedoheptulOll O-glyceraldehyde 7 -phosphite 3·pnosphate

Fructose 6-phosphate and glyceraldehyde 3-phosphate also enter the glycolysis. Thus, in the course of reactions catalyzed by the intrinsic enzymes of the pentose phosphate cycle, two fructose 6-phosphate molecules, one glyceraldehyde 3-phosphate molecule, and three carbon dioxide molecules are produced from three glucose 6-phosphate molecules. In addition, six NADP -H2 molecules are formed. The overall scheme for the pentose phosphate cycle is: 3 Glucose 6-phosphate-6NADP+ ~ 2 Fructose 6-phosphate + Glyceraldehyde 3-phosphate + 6NADPeH2+ 3C02 Interrelation of the Pentose Phosphate Cycle and Glycolysis These two pathways for carbohydrate conversion are closely related. The products of the pentose phosphate route-fructose 6phosphate and glyceral-dehyde 3-phosphate-are likewise glycolysis metabolites; for this reason, they are involved in glycolysis and undergo conversion by glycolytic enzymes. Two molecules of fructose 6-phosphate can regenerate to two glucose 6-phosphate molecules through the agency of the glycolytic enzyme glucose-phosphate isomerase. Here, the pentose phosphate pathway functions as a cycle. The other product, glyceraldehype 3-phosphate, enters the glycolysis to be either converted to lactate (under anaerobic conditions) or oxidized to CO2 and Hp (under aerobic conditions). As can easily be estimated, the conversion of glyceraldehyde 3-phosphate to lactate 7.1.2


184 Glucose

I

Glucose 6-pLsphate

~

G

L Fructose 6-phosphate y ' F ructose 6-phosphate

e o

Fructose 1.6-bisphosphate

,

Synthesis of nucleotides and nu" Ieosides. nucleoS Dihyd-~Glyceraldehyde 3-phosphate L~r--"-.tide coenzymes. I roxyaceI ~-.;....--' polynucleotid8s. Stone I and histidine I phosphate + • Pyruvate

Ly

r

i

U

Lactate Figure 7_1 Scheme for integration of pentose phosphate shunt and glycolysis.

leads to the formation of two ATP molecules, while the combustion to CO2 and Hp produces 20 ATP molecules. It follows therefore that under physiological conditions, when the pentose phosphate pathway for carbohydrate conversion is included in the glycolysis, the overall process of glucose 6-phosphate conversion may be expressed via the pentose phosphate cycle. Under anaerobic conditions: 3 Glucose 6-phosphate+6NADP+ + 2P l ~ 2 Glucose 6-phosphate + Lactate + 2ATP + 6NADP . H2 + 3C02 Under aerobic conditions: 3 Glucose 6-phosphate + 6NADP+ + 20ADP + 20Pl ~ 2 Glucose 6-phosphate + 6NADP.H2+ 6C0 2 + 6Hp + 20ATP At, first glance, the energetic value of this conversion of glucose 6-phosphate via the pentose phosphate cycle appears to be inferior to that of the aerobic glycolysis pathway, the latter providing a maximum of 38 ATP molecules. However, it should be borne in mind that a major portion of energy is stored in NADP - H2, and 6 NADP - H2 molecules are energetically equivalent to 18 A TP molecules. Consequently, the energetic effect remains the same.

7.1.3 The Biological Function of the Pentose Phosphate Cycle The biological function of the pentose phosphate cycle involves the production of two compounds: NADP -H 2, which is a "reductive force" in the synthesis of various materials, and the metabolite ribose


185

5-phosphate, which is used as a building material in the synthesis of various specieiS. The major functions of the pentose phosph~te cycle ~e:

(1) amphibolic function: the cycle is a route to degradation of

carbohydrates and, simultaneously, to the supply of materials used in synthetic reactions (NADP • Hz and ribose 5phosphate); (2) energetic function, since the involvement of pentose phosphate cycle products (glyceraldehyde 3-phosphate) in the glycolysis produces energy; (3) synthetic function, as a major function associated with the use of NADP • Hz and ribose 5-phosphate. NADP • ~ is used: (1) in the detoxification of drugs and poisons in the monooxygenase oxidation chain of the endoplasmic reticulum of the liver; (2) in the synthesis of fatty acids and other structural and reserve lipids; (3) in the synthesis of cholesterol and its derivatives-bile acids, steroid hormones (corticosteroids, female and male sex hormones), and vitamins D; and (4) in the neutralization of ammonia under reductive amination. Ribose 5-phosphate is used in the synthesis of histidine, nucleosides and nucleotides (nucleotide mono-, di-, and triphosphates), nucleotide coenzymes (NAD, NADP, FAD, and CoA), and polymeric nucleotide derivatives (DNA, RNA, and short-chain oligonucleotides). The pentose phosphate pathway for carbohydrate conversion is primarily operative in the organs and tissues in which an intensive utilization of NADP • H2 is needed for reactions of reductive synthesis and for reactions involving ribose 5phosphate in the synthesis of nucleotides and nucleic acids. For this reason, a high activity of this pathway is observed in fat tissue, liver, mammary gland tissue (especially during lactation, since the milk fat synthesis is essential in this case), adrenal glands, gonadal glands, marrow, and lymphoid tissue. Relatively high is the activity of pentose phosphate shunt dehydrogenases in the erythrocytes. A low activity of the pentose phosphate pathway is observed in muscular tissue (heart and skeletal muscle).


186

7.2 BIOSYNTHESIS OF CARBOHYDRATES IN TISSUES In the human and animal tissues and organs, synthesis of carbohydrates occurs. Since glucose is the starting structural unit for producing other monosaccharides and for assembling polysaccharides, it is expedient to consider potential routes for the glucose synthesis in tissues and organs. Formation of glucose from nonccu:bohydrate materials is attested by the fact that, under prolonged starv'ation (in an extreme contingency or as applied in therapy), the polysaccharide carbohydrate reserves are rapidly consumed, while the glucose level in the circulating blood is maintained to supply tissues, especially brain, with energy. 7.2.1 Gluconeogenesis The synthesis of glucose from noncarbohydrate sources is referred to as the gluconeogenesis. It is feasible only in certain organism tissues. The major site for gluconeogenesis is the liver. To a lesser extent, the kidneys and intestinal mucosa are involved in this process. Mechanism for Gluconeogenesis. Since the glycolysis involves three energetically irreversible steps at the pyruvate kinase, phosphofructokinase, and hexokinase levels, the production of glucose from simple noncarbohydrate materials, for example, pyruvate or lactate, by a reversal of glycolysis ("from bottom upwards") is impossible. Therefore, indirect reaction routes are to be sought for. The Jirst indirect route in glucose synthesis involves the formation of phosphoenolpyruvate from pyruvate without the intervention of pyruvate kinase. This route is catalyzed by two enzymes. At first, pyruvate is converted into oxaloacetate. This reaction occurs in the mitochondria as the pyruvate molecules enter them, and is catalyzed IJy pyruvate carboxylase according to the scheme Pyruvate .arOOsyl. .

CH.-C-COOH+HCO.+ATP

II

°

.... HOOC-CH.-C-COOH+ADP+H.PO.

II

o

onlo_tate

This enzyme, similar to all CO2 assimilating enzymes, contains

biotin for a cofactor. Oxaloacetate is released from the mitochondria into the cytoplasm to enter gluconeogenesis. In the cytoplasm, oxaloacetate converts to phosphoenolpyruvate via a reaction catalyzed by phosphoenolpyruvate carboxylase:

187

METABOLISM OF SACCHARIDES ..._ _ _lpfnl...te e..~ ...

HOOC-CH.-C"":COOH+GTP(ATP)

-

~

oulo_late

... CHs=C-COOH+GDP(ADPHCO.

6-POaHs

pboopboonolpfnlY.te

The reaction equilibrium is shifted to the right. The major supplier of phosphate groups is GTP, but for this purpose, ATP may also be available. All of the glycolysis reactions ranging from phosphoenolpyruvate to fructose 1,6-bisphosphate are reversible, and the phosphoenolpyruvate IOOlecules fonned are consumed for producing fructose 1,6-bisphosphate by making use of the same glycolysis enzymes. The second indirect route involves the formation of fructose 6phosphate from fructose 1,6-bisphosphate without the intervention of phosphofructokinase reaction. This route is catalyzed by fructose bisphosphatase: Fructose 1,6-bisphosphate

+ H20

Fructosebispoosphatase)

Fructose 6-phosphatc

+ H2 PO4

The reaction is irreversibly shifted to the right. Fructose 6phosphate is isomerized to glucose 6-phosphate by glucose-phosphate isomerase. The third indirect route involves the formation of free glucose from glucose 6phosphate by circumventing the hexokinase reaction. This route is catalyzed by Glucose 6-phosphate

+ H2 0

Glucose 6-poosphatase

)

Glucose + H 2 PO 4

The free glucose produced by this reaction is supplied to the blood from the tissues. As exemplified by gluconeogenesis, one may easily envision the economical organization of these metabolic routes, since, apart from four special gluconeogenesis enzymes-pyruvate carboxylase, phosphopyruvate carboxylase, fructose bisphosphatase, and glucose 6phosphatase-individual glycolytic enzymes are also used in the gluconeogenesis. Noncarbohydrate Sources for Gluconeogenesis. In addition to pyruvate and lactate, which are delivered to the liver and kidneys, other noncarbohydrate compounds serve as substrates for glucose synthesis. In accordance with the gluconeogenesis scheme, it may be anticipated that all materials of noncarbohydrate nature that are


188

t.

Cl

rr-

e (") ... ru~,......

-

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