DAVID T. SMITH, M.D. James B Duke Distngmshed, Professor of Microbiology Associate Professor of Medieme Date Latsersity School of Medicine NORMAN F, CONANT, Ph.D. James B Duke Distingushed Professor of Mycology and Profescor of Mcrebiology Duke University School of Medieme HILDA POPE WILLCTT, Pb.D. Professor of Microbnology Take University School of Medicine JOSEPH W. BEARD, M.D. Professor of Surgery and James B Duke Distinguished Professor of Virology Duke University School of Medicine D. BERNARD AMOS, M.D. James 8 Duke Distinguished Professor of Immunology and Professor of Experimental Surgery Duke Lniversity School of Medreme JONN E. LARSH, JR., Sc.D. Profescor of Parasnology School of Public Health and School of Medicine ‘Unwersity of North Carolina and Duke Unsversity Scheok of Medicine JOIN F. FLANAGAN, M.D. Formerly Professor of Medicine Duke Unnersity School of Medicine Presently Member of Watson Clinic Lakeland, Flosida EUGENE 0, DAY, Ph.D. Professor of Immunology and of Experimental Surgery Duke University School of Medicine SUDAM OSTERHOUT, M.D., Ph.D. Assoclate Professor of Microbialogy Asustant Professor of Medicme Assitant Dean for Admissions. Duke University School of Medicine REBECCA Il. BUSKICY, M.D. Justructor m Pediatrics and Research Associate in Immunology Pake University Schoo} of Medseine CHESTER M, ZMUEWSKI, Ph.D. Associate Profescor of Immunology Duke University School of Medicine EDWARD GLASSMAN, Ph.D. Professor of Biochemistry and Genetres University of Nosth Carolina School of Medicine D. GORDON SHARP, Pid, Professor of Biophysics University of North Carolina School of Medicine GROVER C, HUNTER, JR., D.D.S. Professor of Periodontology and Oral Pathology Unnersity of Noth Carohina School of Dentistry JAMES J. CRAWFORD, Ph.D. Asustant Professor of Endodontcs ‘Unwersity of North Carolina School of Dentistry €. EDWARD BUCKLEY, HI, M.D. Assistant Professor of Medicine and Associate in Microbiology and Immunology Duke University School of Medicine WILLIAM BOYLE, Ph.D. Assistant Professor of Immunology Duke Unwersity School of Medicine RICHARD 0, BURNS, Ph.D. Associate Protessor of Microbiology Duke University School of Medicme ROBERT W. WHEAT, Ph.D. Associate Professor of Macrobiclogy and Immunology Asastant Professor of Biochemistry Duke University School of Medicineee tet oe oe Pt ter Pern ZINSSER MICROBIOLOGY fourteenth edition tn nee fed oc Sa et nn -CROFTS 1 of Meredith Corporation NEW *yoRKCopynght, © 1968, by MEREDITH CORPORATION Jus reserved This book, or parts thereof, must not be ed or reproduced in ay manner without writen per mssion For information address the publisher, Appleiow Century-Crofis, Divison of Meredith Corporation, 44 Park Avenue South, New York, N ¥ 10015. 688-1 Library of Congress Catalog Card Number 68-8386. Copyright ©, 1964, by Meredith Publishing Company Copyright ©, 1960, by Appleton-Century-Crofts, Inc ‘Under the ttle ZINSSER BACTERIOLOGY, this book in part was copyrighted as follows" Copynght ©, 1957, by Appleton-Century-Crofts, Inc. Under the title ZINSSER'S TEXTBOOK OF BACTERIOLOGY, this book in part was copyrighted as follows Copynght ©, 1948, 1952, by Appleton-Century-Crofts, Ine. Under the utle A TEXTBOOK OF BACTERIOLOGY, this book in part was copynghted as follows Copyright ©, 1934, 1935, 1937, 1939, by D Appleton-Century Co, Inc. Copyright ©, 1910, 1914, 1916, 1918, 1922, 1928, by D. Appleton & Co. Copyright © renewed, 1962, 1963, by Mrs Hans Zinsser. Copyright © renewed, 1942, 1944, 1946, 1955, 1956, by Mrs Ruby H. Zinsser. Copynght © renewed, 1950, by Mrs Ruby H. Zinsser and Frederick F. Russell, M.D. Copynght © renewed, 1938, by Hans Zinsser. PRINTED IN THE UNCTED STATES OF AMERICA ‘M-81965To the memory of PHILIP HANSON HISS, JR HANS ZINSSER and to STANHOPE BAYNE-JONES whose labors over more than one third of a century have made this textbook a favonte with students of medicine and public healthPreface This ts the fourteenth edition of the orginal Textbook of Bacteriology wniten by Hiss and Zinsser tn 1910 The authors visualzed, in a remarkably accurate manner, the future developments in microbiology This 1s shown in the ongmal preface which ts reproduced on the following page ‘This 1s also the sixth edition by the present authors The raprd increase in Publications in all areas and the development of subspectalties in microbiology necessitated the addition of erght younger authors These were all chosen from among active investigators and teachers of microbiology at Duke University and the University of North Carolina These two institutions are only 12 mules apart and this has facilitated conferences and exchange of ideas in an attempt to maintain an integrated approach to the subject, which 1s so diffi- cult to achieve in a multiauthored text The public in general, and to some extent the medical profession, has been encouraged by advertisers to believe that all infections are now easily con- trolled with the numerous available antibiotics Evidence 1s presented m this new edition to show that plague, cholera, gonorchea, syphilis, salmonellosis, and the atypteal types of mycobacterial infections are increasing Meningococct of the B type, and possibly the A type also, have developed resistance to the sulfonamides Staphylococcal infections, infections with the gram-negative bacilli of the intestinal tract and the opportunistic fungi are increasing at an alarming rate This increase can be attributed partly to the development of resistance to antbotics and partly to the lowering of the patient's basic re- sistance by the mcreased use of immunosuppressive drugs In the arca of immunology the introduction has been expanded to include the evolution of the anubody producing system New chapters have been added on Lectins and Antibodies, Classifications of Antigens, Complement and Hentable Immunogenie Defects ‘The chapters on Tissue Transplantation Staphylococe:, and Atypical Myco- pbacterta have been expanded and the chapters on the Enterobactena reor- gamzed A new chapter on Microbiota Associated with Dental Canes has been added ‘A new chapter on Bacteriophage has been included im the section on the viruses In the preface to the first edition of this text, written m 1910, the authors emphasized the importance of a basic approach which would include not only wuPreface the biologic characteristics of the organisms put also the reactions of the living, tissues to the bacteria and ther products This approach made the volume of equal value to the microbiologst and to the student of public health and chmeal medicine This same pomt of vew has been retained in this edition and extends to the divisions of virology mycology, and parasitology The chapters dealing with rafections have an introductory paragraph which empha- sizes the public health aspect of the discase Highly technical sections and descriptions of diseases of minor importance have been printed in small type In general the text has been written for the beginmng student of micro- biology and the bibliographies collected for the teacher, the graduate student, and the medical student who 1s going on to see patients on the wards of the hospital The bibhographtes are larger than before and include 4,154 references from the current literature of 1960-68 ‘There are 74 new illustrations in this edition which brings the total to 606 including the three full page color plates in the section on parasitology ‘We vash to thank our many colleagues and students for the critical advice and generous help in the preparation of this revision, especially those who have supphed us with new illustrative matersal We are indebted to Elon H Clark Robert L Blake, Robert King, Wayne Willams, Thurmond Elhs, and Raymond Howard for the preparation of new illustrations We wish to acknowledge the valuable assistance of our secre- fants, Mrs Millicent Moore, Mrs Jacquelyn Blalock, Mrs Barbara Burch, ue Margaret Stinnett, Mrs Judith Auman Mrs Judith Osgood, Miss Bar- 1s, and Miss Patricia Betson who have cheerfully typed and re- typed the manuscnpt many mes We are Particularly grateful to Mrs Helen Salton who has revised many of the manuscripts and who has compiled the We also wish to express our ay ppreciation to Appleton Century-Crofts for many helpful suggestons in the preparation of the manuserspt DAVID T SMITH NORMAN F CONANT HILDA POPE WILLETTPreface to the First Edition The volume here presented 1s primarily a treatise on the fundamental Jaws and techme of bactenology, as mMlustrated by their application to the study of pathogensc bacteria So ubiquitous are the bacteria and so manifold their activities that bacter: ology, although one of the youngest of sciences, has already been divided into special fields—medical, samtary, agricultural, and mdustrral—having little in common, except problems of gencrat bacterial physiology and certain funda menta{ technical procedures From no other point of approach, however, 1s such a breadth of conception attamable, as through the study of bacteria in their relation to disease proc esses in man and anmals Through such a study one must become familar not only with the growth charactenstics and products of the bacteria apart from the ammat body, thus gaming a knowledge of methods and procedures com- mon to the study of pathagenic and nonpathogemc organisms but also with those complicated reachons taking place between the bacteria and thew prod ucts on the one hand and the cells and fluids of the anumal body on the other— reactions which often manifest themselves as symptoms and lesions of disease or by visible changes 1n the test tube Through a study and comprehension of the processes underlying these reactions, our knowledge of cell physiology has been broadened, and facts of inestimable value have been discovered, which have thrown light upon some of the most obscure problems of infechon and unmunrty and have led to hitherto unsuspected methods of treatment and diagnosts Thus, through med- ical bacteriology—that highly specialized offshoot of general biology and pathology—have been given back to the parent sctences and to medicine n generat methods and knowledge of the widest appheation It has been our endeavor, therefore, to present this phase of our subject im as broad and critical a manner as possible in the sections dealing with in- fection and immunity and with methods of biological diagnosts and treatment of disease, so that the student and practioner of medicme, by becoming familar with underlying Jaws and principles, may not only be m a position to realize the meaning and scope of some of these newer discovenes and methods, but may be im better pasition to decide for themselves their proper application and limitation ‘We have not hesitated, whenever necessary for a proper understanding of PaPreface to First Edition Continued processes of bacterial nuttion of physiology, or for breadth of view im con- stdermg problems of the relation of bicteria to our food supply and environ- ment, 10 make free use of ilustrations from the more special fields of agn- cultural and samtary bactenology, and some special methods of the bacten- ology of samtation ate given in the last division of the book, dealing with the bacteria in relation to our food and environment In conclusion st may be satd that the scope and arrangement of subjects treated of in this book are the direct outcome of many years of experience in the instruction of students i medical and in advanced university courses im bactenology, and that 1t 1s our hope that this volume may not only meet the needs of such students but may prove of value to the practitioner of medicine for whom it has also been written. itis a pleasure to acknowledge the courtesy of those who furnished us with illustearons for use in the text, and our indebtedness to Dr Gardner Hopkins and Professor Francis Carter Wood for a number of the photomicrographs taken especially for this work PH HISS, IR #H ZINSSERContents PREF, ‘ACE vi PREFACE TO THE First Epition x THE GENERAL BIOLOGY OF BACTERIA AND THE GENERAL METHODS OF BACTERIOLOGY 1 Ouruive oF TRE History aNp Score oF BacTeRioLocy t David T Smuth 2° CLASSIFICATION OF BacTERia Robert W Wheat 3° GENERAL MorPHOLoGy AND REPRODUCTION oF BACTERIA 27 David T Sruth PHYSIOLOGY OF MICROORGANISMS 4 Tue Composition AND ORGANIZATION OF A BACTERIAL CELL 45 Hilda Pope Willett 3S) Tne Gevetics oF BACTERIA 56 Edword Glassman 6 Nutrition ap Grown of BACTERIA 8h Hilda Pope Willett 7) Metapotis\t OF Bacteria 102 Hilda Pope Willett 8 THe Action OF CHEMICAL AGENTS AND CHEMOTHERAPEUTIC Drtcs oN BACTERIA Huda Pope Wille 139xu PHYSIOLOGY OF MICROORGANISMS—continued 9 Sowc Errects oF Prysical AGENTS UPON BACTERIA AND VIRUSES D Gordon Sharp ECOLOGY David T Smith 10 MicrontoLocic EcoLocy AND FLORA OF THE Normat HuMAN Bopy Microbiologie Ecology 185 Bactertal Flora of the Normal Human Body 189 IMMUNOLOGY 11 Istropuction ro InsuNoLogy D Bernard Atmos and Rebecca H Buckley 12 Lectwss ano Inauxoctonins Eugene D Day 13° Axnipopy Axticen INTERACTION Eugene D Day 14° Cuassirtcations or ANTIGENS Eugene D Day 15° Covptestent © Cdward Buckley 111 16 Tue Hosts Boop Groups anp IMstunonematoLogy Chester Mo Zmiyew she 17 Tissue TRANSPLAN: TATION IMMUNITY D Dernard Amos 18 Heriaace Disorp: ERS OF IMMUNITY Rebecca H Buckley 19 TOXINS ann ANTITOXINS Wittam Hoyle 20 Atteacy Dxrid T Smah Isrictios REsistaNce any Evie stios LOGy Davit T Sith fefetom 37) Rev stance ygr Tpemology 399 Contents 171 185 207 215 228 243 255 271 290 309 328 369Contents PATHOGENIC BACTERIA David T Snuth 22 THe Preumococcus AND PNEUMONIA 23° Tue Streptococci 24° Streptococcat Diseases 25° Tue Micrococer 26 Tne Neisseria Group oF ORGANISMS AND EPIDEMIC CEREBROSPINAL Menineitis 27 NEISSERIA GONORRHOEAE AND GoNococcal. INFECTIONS 28 “HarMOpHi.us INFLUENZAE AND OTHER ORGANISMS OF THE HAEMOPHILIC Group Haemophilus ducreyt, 496 29° BorvETELLA PERTUSSIS. (WrooPiNnc CoucH) AND RELATED ORGANISMS Bordetella pertussis, 499 30 CoryNenacTERIUM DIPHTHERIAE AND DIPHTHERIA 31. Tue MycopactertAceaE 32 MycopacTERIUM TUBERCULOSIS AND TUBERCULOSIS 33. Cassirren AND UNCLASSIFIED MYCOBACTERIA 34 THe ENTEROBACTERIACEAE 35 SaMoNELLA TyPHT AND TYPHOID FEVER 36 SALMONELLA AND SALMONELLOSIS 37 SwiceLLa AND SHIGELLosIs The Shigellae, 618 The Alkalescens Dispar Group, 627 38 Ku epsieLLA PNEUMONIAE AND RELATED ORGANISMS Rinnoscleroma, 632 Ozena 632 Granuloma Inguinale, 635 413 423 A38 447 473 482 490 499 507 520 531 564 592 60% 610 618 628xv PATHOGENIC BACTERIA—conunued 39 EscHERICHTA COLI AND RELATED ORGANISMS: Examination of Water 646 Identification of Enterobactertacene, 648 40 PseupoMoNas AERUGINOSA AND PSEUDOMONIASIS 41 Vinrio CHOLERAE AND AsiaTiC CHOLERA 42 BRUCELLA MELITENSIS, BR ABORTUS, BR SUIS, AND BRUCELLOSIS 43 FRANCISELLA TULARENSIS AND TULAREMIA 44 PasTEURELLA PESTIS AND PLAGUE Pasteurella pestis, 689 Bacteria of the Hemorrhage Septicemia Group, 699 45 Bacit.us anTwRacts aND ANTHRAX (MILZBRAND, CHARBON) AG AnaeRovic BACILLI AND Wounp INFECTIONS Clostridium perfringens, 713 Other Clostridia Associated with Tratte matee Ioyucies, 727 47 CyostRipiu TETANI aND TETANUS 48 Crostaipium sotutinuM, CLOSTRIDIUM PARABOTULINUM, AND Foop PossONING 49 ActNopaciivus MALLET AND GLANDERS MEDIOIDOSIS AND AGTINOBACILLOSIS 50 Miscettanrous Bacteria of Mepicat. IMPORTANCE, Listersa monocytogenes, 751 Etysipetothnix vasidiosa, 754 Bac- ‘oo Streptobacillus moniliformis 758 Bartonella bacilhformis, Si Mucrostots Associaten wit DENTAL Caries Grover C. Hunter, Jr, and James J. Cran {ord MYCOPLASMATALES David T. Smith 52. Mycortas\ta AND L-Tyre For\s of Bacteria Contents 637 650 655 668 6719 689 703 713 738 744 164 781Contents SPIROCHAETALES David T Srmuth 33 34 55 36 37 SPIROCHAETACEAE TREPONEMA PALLIDUM AND SYPIIILIS ‘Tue Spirnocuetes or RELAPSING FEVER, V, '» VINCENT S ANGINA AND OTH DISEASES me Relapsing Fever 816 Fusospsrochetal Symbiohe D, Spirochetosis of Fowls 824 ” rsease S24 THe Leptospira GROUP—INFECTIOUS JAUNDICE (WEIL § DISEASE) AND Otuer Leptospiroses SPIRILLUM ANUS AND Rat-Bite FEVER RICKETTSIALES David T Smuh 38 59 60 61 62 63 RICKETTSIACEAE AND CHLAMYDIACEAE THE Typius Group oF RICKETTSIOSES Epideruc Typhus 842 Bnills Disease 849 Mure Typhys 849 Tue Spotten Fever Group oF RICKETTSIOSES Rocky Mountam Spotted Fever 856 Rickettsilpox 860 Bouton newse Subgroup Boutonneuse Fever 861 South Afrwan Tick Bite Fever 861 Kenya Tick Typhus 861 Abyssimaa Tick Typhus 861 Indian Tick Typhus 861 Siberian Tick Bue Typhus 862 Maculatum Disease 862 TsuTsucaMUsH! RICKETTSIOSES Q Fever Trench Fever 870 Other Rickettstoses 871 THE CHLAMYDIACEAE Psuttacosis Ornithosis 873 Lymphogranuloma Venereum 875 Tra choma 876 Inclusion Conjunctivitis 878 Anunal Types of Bed soma 879 VIRALES 64 Puysicat, MoRPHOLOGIC AND CHEMICAL PROPERTIES OF VIRUSES Joseph W Beard xv 799 803 816 826 841 855 864 867 872 883xvi VIRALES—conmnued 65 Biovocic PHENOMENA OF ViRUS DISEASES Joseph W. Beard 66 Vinuses oF BACTERIA Ruchard O Burns 67 Poxviruses John F Flanagan Smallpox and Vaccinia, 972 Molluscum Contagiosum, 980 Pox Diseases of Mammals, 981 68 Ravirs Jolin F Flanagan 69 ‘Tue Henretic VinusES oF Man Suydam Osterhout Varicella—Herpes Zoster 988 Herpes Simplex, 990 Herpetic In- fections of Ammals, 991 70 Measurs Suydam Osterhout Measles, 992 Rubella, 995 Exanthem Subitum, 996 71 Tur Myxoviaus Groue David T Smith Influenza, 998, Parainflvenza Viruses, 1004, 72 Mumps John F. Flanagan Mumps, 1007 Other Virus Diseases of the Sal Ty Glan 5. vary Glands, 1009 Newcastle Dr Mv: ny . r site Disease Vitus, 1010 Lymphocytic Choriomeningitis Virus, 73 avenovruscs John F. Flanagan PICORNAVIRUSES 74 Pouovruses . John F, Flanagan 75 Rwsovinusts John F, Flanagan Contents 919 956 972 983 988 992 998 1007 1011 1018 1023Contents PICORNAVIRUSES—continued 76 Coxsackie Virus, Echo Virus, REOVIRUS, AND THE ASEPTIC MENINGITIS SyNDROME 1026 David T Smith Coxsackie Viruses 1026 ECHO Viruses 1030 Reoviruses, 1032 The Aseptic Mentngits Syndrome, 1032 77 Tue Hepatrmis Viruses 1035 David T Smith Infecnous Hepatitis, 1035 Serum Hepatius, 1040 78 ArtuRopop-Borne Virus Diszases oF MAN 1044 David T Seuth Serologic Group A 1045 Equine Encephalomyelitis, 1045 Vene- zuelan Equine Encephalomyelius, 1047 Serologic Group B 1049 Yellow Fever, 1049 Dengue Fever, 1052 West Nile Fever 1052 St Lows Encephalins, 1053 Japanese B Encephalius 1034 Rus- sian Spnng and Summer Encephalitis, 1055 Louping Il 1056 Murray Valley Encephalitis, 1056 Miscellaneous Arthropod Borne Viruses, 1057 Colorado Tick Fever, 1057 Sandfly Fever, 1057 Raft Valley Fever, 1057 79 MisceLLANeous DIsEAsrs OF SUSPECTED OR KNOWN ViRaL ETIOLOGY 1061 David T Smuth Infectious Mononucleosts 1061 Epiderme Hemorrhagic Fever, 1062 Cat Scratch Disease Warts, Scrapre Kuru, Cytomegaloviruses, Re- spiratory Syncytial Virus, 1063 80 Viaus Diseases oF PLANTS, INSECTS, AND ANPUALS. 1069 Joseph W Beard MEDICAL MYCOLOGY Norman F Conant 81. IntRopuction To MepicaL MycoLocy 1085 82 AcnNomycosis AND ACTINOMYCETES 1089 Actinomycosis, 1090 Nocardiosts 1095 Achnomyconc Mycetoma, 1099 Erythrasma, 1102 ‘Trchomycosts, 1103 1106 83 Funaus Diseases INVOLVING THE INTERNAL ORGANS ocoscosss, 1106 Candidiasis, 1110 Blastomycosis_ (North ee 11S South American Blastomycous 1145 | Coceidi- odomycosis 1122 Histoplasmoscs 1127 Geotrichosis, 1131 Phy- comycosis, 1133 Cladosporwsts 1137Contents XVI MEDICAL Mi COLOGY—comnued 84 Foncus DIscASEs INVO! VING SKIN AND SUBCUTANTOUS ‘Tissurs 1149 Sporoinchons 144) Chromoblk mycaws 1152 Maduromycoss 1156 Rhinosporid oss 4198 85 Tut DermaTomycosrs 1162 The Dermatophyits 1162 Pudra 1175 Tinta Veruicotor 1176 86 Miscenrancous Mycoses 1180 Aspergillosis 1180 Pemieiihioss 1183 Mycotic Keratus 1183 MEDICAL PARASITOLOGY John E Larsh, dr 87 INTRODUCTION TO MEDICAL PARASITOLOGY 187 88 PRoTOZOA AND PROTOZOAN INFECTIONS 1188 Intestinal Oral and Gemutal Protozoa 1189 The Amebae 1189 Entamoeba histolyica 1189 The Cihated Protozoan (Batantudium colt) 1194 The Flagellated Protozoa 1195 Blood and Tissue Protozoz 1201 Flagellsted Protozoa 1201 ‘The Trypanosomal Pasasites 1201 The Letshmanial Parasites 1203 The Malanial Parasites 1204 Toxoplasma Orgamsm 1209 89 Heumarus anp HELMINTic INFECTIONS 1212 Nematodes 1212 Intestinal Nematodes infective for Man in the Egg Stage 1213 Ascaris lumbricondes 1213 Visceral Larva Migrans 1213 Trichuris tichwura 1216 Emterobiws vermeulans 1217 Intestinal Nematodes Infective for Man m a Larval Stage 1219 Hookworms 1219 Cutaneous Larva Migeans 1223 Strongyloides stercorals 1224 Trichinella spirals 1226 Tissue Nematodes 1229 Wucherena bancroftr 1229 Cestodes 1231 Intestinal Ces todes 1232 Tissue Cestodes 1235 Trematodes 1237 The Schis tosomes 1237 90 Anturorops 1243 ‘The Role of Arthropods in the Transmission of Human Diseases 1243 Ticks 1244 Mites 1246 Fleas 1247 Bioodsucking Lice 1247 Cockroaches 1247 True Bugs 1249 Fhes 1249 ms INDEX, 1255ZINSSER MICROBIOLOGYTHE GENERAL BIOLOGY OF BACTERIA AND THE GENERAL METHODS OF BACTERIOLOGY I Aee-No &72 Outline of the History and Scope of Bacteriology The history of many concepts now em- bodied in the doctrines of bacteriology 1s an account of attempts to solve the problems of the ongin of hfe, the putrefaction of dead organic materials, and the nature of com- municable changes in the bodies of living men and animals The vistble aspects of these phenomena were as apparent and as Interesting to ancient observers as they are to modern biologists In the past, notions of ulumate causes were derived from the avail- able factual knowledge colored by the theo- Jogic and philosophic tenets of the tyme The early history of what has became the scrence of microbiology 1s to be found, therefore, in the writings of the priests, philosophers, and scientists who studied and pondered these basic biologic problems Fortunately for the student of medical bac- teriology, Bulloch (8) published in 1938 a smaif, concisely writen Gook ented Te Hustory of Bactenology, contaming the re- Sults of his extensive studies Among ancient peaples epidemic and even endemic diseases were regarded as super~ natural in origm and sent by the gods as punishment for the sins of man The treat- ment and, more important, the prevention of these diseases were sought by sacrifices and lustrations to appease the anger of the gods Since man 1s willful, wanton, and sinful by nature there was never any difficulty m find- Ang a particular set of sins to justify a specific epidemic The Egyptians apparently had a vague hotion that disease could be transmuted by touch, certainly the Hebrews at the time of Moses believed that leprosy was contagious ‘They thought it could be contracted by per+ Sonal contact from the clothes of the patient and even by hiving in the home of a former leper (Leviticus 13 and 14) Although recog- nizing that leprosy could be transmitted by direct contact, the Hebrews also believed it could be created de novo as in the case of Minam who angered the Lord by speaking against Moses (Numbers 12 9) The estab- lishment of quarantine by Marseilles and Venice in the fourteenth century was perhaps imifiuenced as much by the religious feeling that a period of punfication was needed as by the pragmatic idea that such a period of ssolation would prevent the spread of disease At the ume of the stege of Troy the Greeks behteved in the divine origin of epidemic dis- eases, but as they fost faith in their gods they also lost faith in the divine origin of disease By 430 pc Thucydides had concluded that certain plagues were contagious Hippocrates rejected the theory of divine conga but dul aot coaster the possiitity of contagion He recognized two factors that were mvolved in the development of disease —an intnosic factor of “constitution” of the patent, and an extrinsic factor, or “miasm,” of air modified in some unknown way so that the aur itself became deleterious The belief in the disease-producing effect of bad air thrived im the Middle Ages and has persisted to modern times as shown by the medical words “influenza” and “malaria” Although the Middle Ages were charac- tenzed by a series of destructive epidemics, the behef that disease 1s caused by a hung, contagious agent grew slowly A knowledge of the natural history of syphuhs influenced Fracastorus, who clearly stated the theory that the agent of communicable diseases was living, a “contagium vivum ” In bis book on contagious diseases, published in 1546, Fra-2 Ch 1 Outline of the History and Scope of Bacteriology castonus (32) described the transmission of disease by direct contact by intermediary inanimate fortes, and through the ax ad distans He called the agents of disease semmana morbi ising getms and ex pressed the opimian that the sceds of these agents passing from one infected anima) produced the same disease in another which received them These essentially true state ments were unconvincing because they were not based upon a demonstration of the physt cal reality of the hypothetical mvisibic organ ysms Even after the discovery of bacteria confirmation of the theares of Fracastozius was long delayed Although Fracastorius as we now know had indeed proposed the true germ theory of disease no further progress could be made until microscopes were invented to visualize these germs and unui the question of spon- taneous generation of bactena was settled According to Smger (69) and Disney (18) the compound microscope was invented im dependently by Zaccharms Jansen in Hol land and Galteo in Italy about 1609 (33) Bacteria were discovered by Antony van Leeuwenhoek (1632 1723) m 1676 This Statement has been placed beyond refutation, through available historical records hy the great biography of van Leeuwenhoek pub Mgt “m croscope" Dobelt Antony wun Leeuwenhoek and Hes Lint fare Courtesy of Harcourt Brace & World ine Leeuwerhocchs {From yshed by Dobell (19) in 1932 In addition. to translations from the “Nether-Dutch” of all of the important parts of the letters of Leeuwenhoek im which bacterta and protozoa are described this book contains convincing evidence that Leeuwenhoek was in fact the father of protozoology and bacteriology ” The microscope” with which he saw bac- teria was a simple biconvex lens, of short focal length clamped between two metal plates The preparation under examination 1 a drop of fluid or thn glass tube was fixed wpon a metal point and moved into focus by means of screws One type of the instrument 1s shown in Figure 1, copied trom a drawing made by Dobell It1s probable that Leeuwen- hoeks best glasses gave a magnification of 300 diameters He never disclosed the secrets of all his methods, and it is not known with certainty bow he obtamed the necessary ilu- mination of his preparations Dobell suggests that Leeuwenhoek probably Int upon some simple means of dark ground illumination The first written descriptions of bacteria are in his letter to the Royal Society of London, dated October 9, 1676 (Dobells No 18) Here he gives recognizable descriptions of bactera m vanous waters and in ‘pepper water’ in which he was searching with his magnifying glass for the cause of the hotness of pepper Clearer descriptions of several forms of bacteria are given in his letter to the Royal Society dated September 17, 1683 (Wobclis No 39) In this he described the ammatcules he found in the feces of man and anmals and in the tartar from his teeth A drawing of bacteria accompamed. this letter ‘The orginal has been lost but the record has been preserved in the plate illustrating the ‘Latin translation of this letter published in 1695 (78) We reproduce a photograph of this famous drawing here, as Figure 2 it shows beyond question that Leeuwenhock saw the chief forms of bacteria-—cocci, rods, filaments, and spirochetes (14) Leeuwen- hoek believed that the air was the source of tus microscopic creatures and that they ex- asted in this medium in the form of seed or germs It 1s doubtful if he ever heard of Fra+ castorius' book which was published 137 years before, and certainly neither he not any of his immediate successors associated these ‘seed with the ‘seed which caused diseaseClassification of Bacteria St Aa , oD a ne fr “ oft Gee Fig 2 Leeuwenhoek s picture of bacteria fram the human mouth Dobeils adentificatsons are as follows A, a monle Bacillus B, Selenomonas sputigena E, mucsococes F, Lepothrix buccans G, probably Sprrachaeta buccalis (From van Leeu wenhoek ) In 1678 Hooke confirmed Leeuwenhock’s discovery, and during the early part of the eighteenth century the “little ammals” were seen and described by a number of mucros- copists The difficufties of assigning them a place and giving them a name are exempli- fied in Linnaeus’ creation in 1758 of the genus Vermes which he called “Chaos” (44) A few years later, Wrisberg named these orgamsms Jnfusoria No doubt this helped Lianacus to give them a name, for sn the eleventh edition of his Systema naturae (44), in 1767, according to Bulloch (8), he formed a class called Chaos ifusoriem That he was still confused as to the nature of bac- teria and protozoa 1s indicated by lus listing them with “the ethereal numbus floatmg tn the month of flowermg” In some respects the classification of bacteria 18 chaotic even now The first smportant classification of the Infusoria was made by O F Muller (50) m 1773 and 1786 The terms Vibrio and Monas were mtroduced by Muller Some 50 years later, Ehrenberg (25), without really under- standing the nature of bacteria, began to Publish classifications of them In 1829 he established the genus Bacterrum, using a term formed from the Greek word Baxiqoov the diminutive of Baxtoov, stenfying a staff ‘The whole subject of bacteriology has taken its common name from the prommence of the rod forms 3 The transference of the bacteria to the plant kingdom was first proposed by Ferdi- nand Cohn in 1854 Cohn’s (15) publica~ tions after 1872 marked a great advance in the systematic study of bacteria His personal influence as a teacher and chrector of investi- gators equaled that of his written works Under Cohn’s mudance, the botanic port of view became dominant m bacteriology in 1857 Nagel: had noted the telationsmp of bacteria to fungi and had mtroduced the term Schizomycetes, a word which has re mamed for the scientific designation of these organisms Primitive classifications of bactena were established and accepted before they were associated with disease One early concept of the ongin of disease, however, involved 2 belief sn a communicable agent, such as that responsible for the leavening of bread, the making of wine, and the brewing of beer Robert Boyle suggested in 1663 that some diseases, such as “Feavers and others,” aight be a kind of fermentation But fer- mentations and putrefactions often occurred without any known cause and were, there- fore, attributed to spontaneous generation The problem of spontaneous generation 15 one facet of the larger problem of the ongin of life That phase of the problem which deals with small ammals, worms, and insects could be studied without the aid of a mscro- scope, but, backed by Arstotle’s authority, this conception of spontaneous generation, or abiogenesis, dominated thought through- out the Middle Ages Samson's middle re- lating to the production of bees and honey fram the carcass of a hon {Judges 14 8, 14) was matched in 1648 by van Helmont’s direc- tions for generating mice from preces of dirty cloth and fermentmg gram What appeared to be particularly astonishing to van Helmont was that the mice generating from tags and grain were sexually mature and capable of copulating with muce descended from natural parents (77) Similar fanciful notions of the generation of snsects were put to cxperimen- tal test by Francesco Redr shortly after the middie of the seventeenth century Redr proved conclusively that the maggots which appeared in decomposing meat were devel- oped from the eggs of flies and showed by experunents that when meat was screened by guuze so that fies cauld not deposit ther4 Ch 1 Qutlne of the History and Scope of Bacteriology eges in at no visible worms OF insects Were generated, although the meat putrefied Redis cozclusion that bfe comes from hfe, supported by experiments which anyone could repeat should have decided the issue The wort of Reds and his followers (61) finally dispoted of the theory of spontancous generation of hving things of macroscopic size yet ts advocates did not surrender but continued to believe im spontantous gencra tron at the microscopic level The old con- troversy about spontaneous generation now transferred to the microscopic level had to be settled before acceptable conclusions could be reached regirding other problems Needham the Welshman (52) found fpacteria in hus infusion of animal and vege lable matenals even after boing and exclud ing atr from the vessels Needham and Buffon were assailed by the great TItahan naturalist Abbé Spallanzant (70) who pointed out the technical errors im their work (1765-1776) He also discovered bacteria which grew in the absence of air, and forms which resisted boiling which we now know as spores His work would have settled the question of spontaneous generation had Necdham not shifted the grounds for the controversy by chiming that the excessive heatung produced: chemuicat changes in the solutions which made spentincous generation impossible Schulze (67), Schwann (68), and Schroder and von ‘Dusch (66) supported Spailanzant with nu- merous new experments including the intro- twchian of cotton stoppers to filter bacteria, rom the air ‘The question was not definitely settled ntl the years Immediately following 1860 when Pasteur conducted a series of exper- merits which were important not only in in controvertiblsy refuting the doctrine of spon- fancous geacration of bacteria but also in etablishing the principles of screntific inses~ uration whnh hive influenced bactenolagie tescarch since brs time: Pasteur attacked the problem from two posts of tsew In the first instance he dem- eevratad that when air was filtered throuch eter Bool ienumerable microorganiims were depo ted upon the filler A single shred of toch contaminated flee dropped into a fork of preswuily sterdired neteive Guat sctind to ibate a raped sad tuxuriant rowh «l encrterasom In the second experiment he succeeded mm showing that similar stenhized ‘ putrescible” liquids, when Jeft in contact with air, would remam un- contaminated provided the entrance of dust particles was prevented This he accom- plished by devising flasks, the necks of which had been drawn out into fine tubes bent in the form of a U The ends of these U-tubes, being left open, permitted the sedimentation ‘of dust from the aur as fat as the lowest angle of the tube, but, in the absence of an air current, no dust was carried up the second arm into the liquid In such flasks he showed that no contammation accurred but that it could be ammediately induced by slanting the entice apparatus untd the liquid was allowed to run into the bent arm of the U-tube Fimally, by exposing a series of flasks con- taming sterile yeast infusion at different at- mospheric levels, in places im which the air was subject to varying degrees of dust con- tamination, he showed an inverse relation- ship between the purity of the air and the contamination of hus flasks with mrcroorgan- asms (56, 57, 58) (Fig 3) ‘The doctrine of spontaneous generation of bacteria had thus received sts final refutation, except in one particular It was still puzzling why complete sterty was sometimes not ob- tained by the application of defimte degrees of heat In England, as im France, the chief ‘opponent of the doctrine of spontaneous gen- eration was a man of ideas, great experi- mental skall, keen insight, and exact method ‘Ths was John Tyndall (75) who, starting {rom experiments to remove motes from alr, made many important discoveries in bacte- tology He demonstrated independently the great heat resistance of spores previously discovered by Cohn in 1871 (15) and de- ‘ised the method of fractional sterilization. By means of lectures and demonstrations, and by a notable book published in 1882, Tyndall, as Bulloch states, “gave the final blow to the doctrine of spontaneous zenera- ton" and opened the road for the advance- ment of the germ theory of disease (8) Meanutile Pasteur, while mvestigatmg Spontancaus generation, had been carrying Of experiments on the subject of fermenta- tion alang the lines sugresied by Cagmard- Latour (10} As a result of these experi ments, he not only confirmed the opinions bath of this author and of Schwann concernDiscovery of Disease-Producing Organisms 5 —— Fig 3 One of Pasteur S$ onginal flasks (From Bulloch The History of ‘jacteriology. Oxford Unt versity Press ) ing the fermentation of beer and wine by yeast, but also showed that a number other fermentations, such as those ‘of lactic and butyric acid, as well as the putrefactive decomposition. of organic matter, were di- rectly duc to the ‘The importance of his contnbution to bacte- nology can hardly be ‘overemphasized, I~ deed, as Rene Dubos (22) has so aptly sand an his Louss Pasteur, Free Lance of Scuence (1950) the domestication of microbial hfe began with Pasteur ‘Proof that putrefaction 1s caused by hving agents stimulated Listet (34, 45), who was a bacteriologist as well as 2 Surgeon, to apply carbolic acid dressings to operative wounds Thus were instituted the antiseptic princi- ples Jater largely replaced by aseptic methods which have made modern surgery possible The history of bacteriology in surgery from. the time of Lister to the present era has been reviewed by Meleney (48) Almost 150 years passed after Leeuwen- hoek’s discovery of bacteria and protozoa before any definite applications of ths new knowledge were made im the study of the cause of disease In a remarkable series of investigations during the first quarter of the nineteenth century Agostino Bassi proved al- most conclusively that a fungus, later named in his honor Botryits bassiana, was the cause of a disease of silkworms called “mal segno” in Italy and “muscardine” im France Basst’s biographer, Calandruccio (11), called Bassi the founder of the theory of parasitism In 1839 Schoenlein (65) found the causa tive fungus in the Jestons of favus, and in 1846 Eichstedt (28) discovered a fungus 10 the skin scrapings from patients with pity- riasis versicolor and noted the contagrousness of the disease Rayer and Davaine had seen rod-shaped organisms 10 the blood of ani mals dead of anthrax in 1850 Rayer (60) recalled expersments made by Barthelemy in 1825 showing that anthrax was moculable im series m sheep By 1863 Davame (16) had proved expermmentally that the disease could be transmitted by blood and the sede ment of laked blood contaming these rods ‘put could not be transmit which the rods were absent and 1872 Obermeir (55) at the Charité in under Virchow’s eye, Was carefully hus discovery of the relationship He demon- presence of 2 strated for the first time the the blood of pathogeme microorgamsm 10. man It must be remembered that during these carly years of etiologic research investigators: had pare cultures to work with only by acci- dent and did not know when contaminants were present 31 the materzals they used much speculation ‘and loose think- ing and a considerable amount of equivocal ork that hindered the development of bac- teriology Henle (36), the future teacher of Robert Koch, affirmed his belief un the anzmateIn 1876 Robert Koch who had been a pupil of Jacob Henle came to Breslau at the invitation of Cohn to demonstrate the results of has studies on anthrax In Cohn’s labora- tory hoch exhibited tus culture methods, showed the life evele of the anthrax bacillus from spore to spore and proved beyond doubt the ability of cultures of this orgamsm to produce the disease Koch s classic paper ‘on the etiology of anthrax, published in 1876, was the first of a great series of enlighten ing contributions by him and his pupils It inavgurated a new era of research in bac- teriolory (40) Koch reahzed the importance of method and techmaquc and was not long in introduc- ang many of the procedures which are now everyday, indispensable practices in labora- tones of bacteriology Trom Weigert, Bhr- Ach and Satomonsen he learned methods of stamng bacteria with aniline dyes (27) In his work on anthrax his chief culture medium had been sterile aqueous humor from the eyes of animals He had seen the advantages of the otder opaque solid media made from Totato, beets, starch, bread, egg white, and flesh but rethzed that they were incapable of pring all the desired snformation about Sacteral colomes In order 10 separate one. pects of bacterium from another, in 1881 hoch devised a transparent solid medium by miuing gelatin with Lofller's peptone solution During a penod of 13 years, from 4870 ta 1883. snd especsliy dang the veven years aficr 1876, fundamental dis- covers went bard in hand with advances in teska que and improvements in appara ts (41, 42) *P Ch 1 Outline of the History and Scope of Bacteriology The period of great technical advance, 1871 to 1884, saw the gradual mtroduction of methods of filtration of fiuids contarming pacteria It was desirable to test the possible toxicity of filirates, freed from cells of the organssms When it became apparent that filter papers, porous clay cups, and packed asbestos fibers would not retain bacteria, the logical step of making tighter filters was taken Chomberland (13) devised a success- ful fiter from a cup of unglazed porcelain in 1884 The Berkefeld type of filter, made of Kieselguhr was inteaduced by Nordtmeyer (53) in 1891 The Chamberland-Pasteur filters made possible the immediately realized discavery of bactetial toxins There seems to have been no suspicion at that time that these filters would later appear to be sieves capa ble of separating vistble from invisible forms of Ife and to be a sort of dividing partition ‘between biologic concepts it was found, unexpectedly, that the agents of some of the communicable diseases passed through these filters im invisible forms In 1892 Iwanowski (37) showed that the ultra- muczascopic agent of mosaic disease of tobacco plants was present m the filtered juice of diseased leaves In 1899 Beyerinck independently confirmed this discovery He conceived of the smvisible self-perpetuating agent of mosaic disease as a “contagram fluidum vivum™” A few years later Loffler and Frosch showed that foot and mouth dis- ease also was due to a filterable virus Since then many diseases of animals and plants have been added to this list by the demon- Stration of the filterabsluy of their etiologic agents (71) By means of filters a remarkable material causing transmissible lysis of bacteria was Ascovered by Twort (74) sn 1915 and independently by @Herelle (17) m 1917 ‘This matenal, called “bacteriophage” by S'Herelle, 1s now recognized as a virus of bactena While Koch and his students were con- eentrating on more precise technical meth- ‘ods and deseribin; ig the etiologic agents of in fection, Pasteur and his assocates were lay- ing the foundation f Tanuroten for modern immunology hike baer - cient story, tenology, has an an- @ prekmmary period of ap- prowmate realization and, after a lapse of many years, @ rapid advancement throughDiscovery of Allergy scienific expermentation upon anumals Many ancient peoples immunized them selves against the venom of serpents by in troducing small quantsties of the poison 1nto cuts or scratches in the skin Variolization the moculation against smallpox with dried material from dermal lesions, was discovered in China over 20 centuries 280; and from there it spread along the trade routes throughout Asia It 1s the achievement of Edward Jenner that a practice of the people was converted by controlled observations into a scientific principle of prophylaxis About 1778 Jenner began his study of the immunity to smallpox which seemed to exist 11 milkmaids who had been infected with cowpox He published bis observations and experiments i 1798, €s tabhshing a method of protection against smallpox and a generally applicable prin ciple of active ymmunization by the use of attenuated virus (4, 38, 62) Almost 100 years passed between the period of Jenners investigations and the fffective foundation of the scicnee of immu nology by Pasteur In 1877 Pastevt became interested in the recovery of animals from infection He found that fowls moculated with old cultures of the bacterum cholera recovered after 2 mid were subsequently refractory to infection with virulent organisms He penetrated at once to the core of this phenomenon Re- ferring to these old cultures as “attenuated virus,” he recognized ‘the relationship of his discovery to the immunity against smallpoy consequent upon jennerian vacemation, 30" gave the general term “yaccme” to the vat ous attenuated organisms he later used 10 induce immunity to several e bacterial dts- ases ‘After his studies 00 chicken cholera Pas- teur began his investigation of anthrax, USNS as a prophylactic @ vaccine made from cul- tures of the anthrax bacillus attenuated by growth at 42° to 43° C In 1881, at the dra- matic demonstration of the power of his vaccine to protect sheep again! Pounlly-le-Fort, Pasteur gained an nary triumph (22, 23. 76) : ‘Living, ‘attenuated VITUS» however, W not without its dangers Anew advance ‘upon a safer, but possibly Jess effective, ine we made in ths country between 1884 an 7 1886 bv D & Salmon and Theobald Smuth (63) whe showed that immunity could be producea by imyections of heat killed cul- tures of the hog cholera bacillus From successes in immunization agamst bacterial dieases Pasteur transferred his m- terest to the study of rabies He recognized that rabies wes due to an ultramicroscopic virus whcse imcubation period could be shortened b, passage through rabbits and whose virulence could be further attenuated by desiccation of the spinal cords of exper mentally infected ammals Results were acineved rapiduy and by 1886 several thou- sand victims of the bites of dogs and wolves had received the Pasteur treatment In com- memorauon of this trumph, the Pasteur In- suiute was erected in Pans in 1888 The success of Pasteur im immunizing against rabies. prowded a pattern of approach which with many modifications, has resulted in effective vacemnation agarmst typhus fever, yellow fever, and pohomyelits ‘During these years Pasteur had noted the differences in resistance exhibited by differ ent species of anmmals had seen that lowering the body temperature decreased the resist- an amma} to infection, and had concerved of immunity as bemg a sort of exhaustion process which, by depletion of certain essential materzals, rendered the body unfavorable 5 2 ‘medium for the growth of microorganisms Little, however, had been vd in the search for a specific €X- planation of the mechansm of ammunity (4) Durmg the last decade of the nineteenth century phenomenal progress was made in the study of specrfic serologic reactions and their interpretation Two schools of mmu- nologie doctrine grew up In one, protection was altributed wholly to the action of the Pood and tissue fluids 1n the other, certain cells of the pody were regarded as the agents of defense against infection Bordet (7) at- tempted to explain the serologic reaction 1n ‘of physical chemustry while Ehrlich sepported 2 chemical explanation for both ‘unizing process and the mechanism of reactions ‘of antigens with the products of mmunization (26, 56) Ehrhch’s famous or receptor theory (27) domi- amunologic thought for the next 25 Both Bordet and Ehriich supported * ance of years8 Ch 1 Outhne of the History and Scope of Bacteriology the humoral theory of smmunty (7), and they were soon 19 conflict with the cellular and phagocyte theory of immunity which Matchmihofi had been developing since 1881 (49) Itis now apparent that there was much truth m both pomnts of view, but since blood and tissue fiuids are the products of cells, the distinctions draws were somewhat artificral The first decade of the twentieth century witnessed the development of a new phase of immunology, one that deals with states ‘of specific hypersensitivity, referred to some- ‘ames as ‘ anaphylaxis and sometimes as * al- lergy” The study of hypersensitivity in man actually began with Jenner s observation of the accelerated vaccmal reaction in certain previously vaccinated persons In 1837 the physiologist, Magendie (46) described the sudden death of dogs which had been re- peatedly imyected with egg albumin, and in 1894 Flexner reported that ammals which were apparently uninjured by an mitial dose of dog serum would succumb to a second dose administered after a lapse of some days or weeks The first systematic study of ana- phylaxis was undertaken by Richet and Porter (59) m 1902 The publication of Arthus (1) 11 1903 and the * phenomenon of ‘Theobald Smith” communicated to Bhrhch in 1904 served to focus attention upon the curious state of hypersensitivity which de- velops after a certain interval of time follow- img the parenteral injection of protems Koch tubetculin stimulated interest in the tuberculin hike type of allergy But the eflec- Uve Gevclopment of this field of investigation, in telation to man took place after 1905 when von Parquet and Schick (80) published thetr observations on serum sickness Smce that tine there has been an enormous ac- cumulation of knowledge on the numerous causes of hypersensitiveness, the protean manifestauons of hypersensitiveness, and the telationship of allergy and anaphylaxis to smmunity and other features of infection The discatery in 1949 by Hench and Ken- alt (35) that cortisone and the adrenocorti- Cotsophic hormone (ACTH) rapidly and completely imbubtt allergic reactions of varie ‘ous kinds opened new fields for mvestigation and suppled new therapeutic agents for the control of a vanety of diseases winch were formerly partially or completely resistant to all forms of therapy The history of smmunology has been sum- matwed briefly here because this science sprang from bacteriology and 15 still inti mately connected with it, It 18 becoming more and more apparent, however, that im- munology and serology have an independent status While they are mseparably associated with bacteriology, they are important sub- divisions of the fields of biochemistry and physiology One direction of this independ- ent destiny has been clearly indicated in Landstemner's and Heidelberger's work on the chemical basis of the specificity of um- mune reachons (5) Domagks (20) report in 1935 on the dramane effect of prontosil on streptacaccie imfections was soon confirmed by Levaditr and Vaismian (43) in France and Colebrook and Kenny in England In 1936 Tréfouel, Trefovel, Nutt, and Dovet (73) from France, and Buttle, Gray, and Stephenson (9) an England discovered independently that prontost (20) was converted in the body to sulfantlamide, the active chemical agent ‘The success of the sulfonamdes catalyzed new interest in chemotherapeutic agents and has resulted im the discovery of the sulfones, which are partially effective in the treatment of leprosy, and of para-ammosalieyhe acid and isonicotmic acid, which are remarkably effective agamst the tubercle bacillus ‘The long, tortuous story of the antibiotres reviewed by Waksman (81, 82) sn 1941 and 1944 can be divided into the fruitless geriod, beginning with Pasteur and ending with Blum m 1925, and the frustful period, begining with Fleming's discovery of test tube pent- cillin in 1929 (30) (Fig 4} Finally, as_a result of the stimulus of World War I, Chain and Florey and thew associates (12, 31) revived Fleming's pem- clin and demonstrated 1s practical clintcal value As the result of milltons of tests with thousands of organisms, we now have Waks- man’s streptomycin (81, 82), Meleney’s bacitracm (47), Burkholder’s chloromycetin (25a), Duggar’s auteomycin (24), and Fine lay’s terramycin (29) ‘There 18 every reason for believing that newer and more specific and potent chemo- therapeutic and antibiotic drugs will be dis- covered in the coming years However, the new therapy has created new problems,Bacterial Genetics Fig 4 Photograph of Flemings original culture which was accidentally contaminated by Penuithum notatem (From Fleming Brit J Exp Path such as resistance of the organisms, sensi- tization of the patient to the drugs, modifica- ons of the clinical syndromes, and the crea- tion of new syndromes by disturbance in the normal ecologic flora of the body The biochemical approach to the problem of infectious disease which began as a trickle 30 years ago has now reached flood tide Studies on both saprophytic and pathogenic bacteria have revealed with monotonous reg- ularity that bacteria, with few exceptions, employ the same enzyme systems and meta- bole pathways as the cells of anunals Ta Dubos’ stimulatmg monograph on Brochem- teal Determinants of Microbiat Diseases (21), the path for the future 1s defined as @ search for “a combination of minor and subtle peculiarities of the infective agents which permit them to survive, multiply, and cause damage 1m specific in vivo environ- ments” The pecular and specific reactions of the host, both chemical and smmunologic, Thust receive simultaneous and equal atten- tion The importance of the unsolved bio- logic problems in mfectious disease was Teviewed by the philosopher bacteriologist E GD Murray in 1955 (51) 10 226 1929) The 1954 Nobel prize winners Enders, Weller, and Robbms, perfected the tissue culture methods for vires cultivation which has revolutionized the study of viruses This was followed by the 1958 Nobel prize win- ners Lederberg, Beadle, and Tatum, whose new techniques have catalyzed the study of microbial genetics, and the 1960 Nobel pnze winners Macfarlane Burnet and Peter Brian Medawar, whose studies have lad a foundation for a parnal understanding of the smmunologic control of transplanted ussue (Table 1) Bacterial genetics has become a mayor division of microbiology The phenomenon of antigenic relanonships between species and general bacterra, as well as the more practical problem of varying degrees of viru lence, depends upon the bacterial gene An extension of these studies to the viruscs has brought hope that chemical compounds might be found and synthesized which would block some essential enzymatic reactions in the virus (72) without injarmg the cells of the host The genetic material of a virus ob- viously has to possess the information for the symthesis of the protein coat of the virus,10 Ch 1 Outhme of the History and Scope of Bacteriology Table 1 The Nobel Poze Winners in the General Field of Microbiology 101 Emii son Behnng 1907 Ronald Ross 1905 Robert hoch 1997 Charles LA. Laveran $508 Lhe Metchnko Paul Ehrlich 1913 Charles Richet 1919 Jules Border 1928 Charles Nr ollie 1930 Aart Landstemer 1939 Gerhard Domagk 1935 Alexander Fleming Ernst Bons Chain Howard Walter Florey 1951 Max Theiler 1982 Selman Abraham Waksman 1983 John F Enders Thomas H_ Welter Frederick C Robbins 1938 Joshua Lederberg George Wells Beadle Edward Lawre Tatum 1960 Macfarlane Burnet Peter Brian Medawar 1965 André Lwoff Jacques Monod Francois Tacob 1966 Peyton Rous and there 1s now evidence that the enzymes which are py esponsible for the synthesis of nucleic atids of animal viruses may, at least tn some instances, be Rew vitus-controlled Proteins (3) Tamm and Eggers (72) have Teported their success in demonstrating in. lophertion in the cell of certain picomaviruses © two substances used were fuanidine hydrochtoride and 2a hydroxybenzyt) benzimidazole (HBB) ¢ first practical pplication of this type of esUration was made by Kang; Mare tola, and Dohiman in 1962 (39), te they succeeded In curing. SPontaneoy US Viral. induced hermetic Kerautss in may i T’xation of S-todo-2 ~e-oxyuridine Ta the more feneral Feld of Benetics, Wi Diphtheria antitoxm Malana Tuberculosis and other diseases Protozoa causing diseases Antibodies and phagocytosis Anphytans Complement fixation and immunity Typhus fever Blood groups Antibacterial effects of Prontosil Peniewlin Yellow fever vaccine Streptomycin Tissue culture method for growng Poho and other viruses Microbial genetics Tmmunologic control of transplanted tissues Lysogeny in bactena Regulator gene concept Tumors induced by viruses ‘ons (83) has demonstrated that bacteria are the tools which the biochemusts are uti- ling to learn the structure and the method of Controlling the synthesis of the chromo- some itself under control of the Principles of micro- biology and immunology It is not an exage Eeration to state that modern ways of hfe Oe their sec ‘urity to the guardianship of Microbiologists Y the applicationReferences REFERENCES 1 Arthus, MoM ‘55 817, 1903 2 Avstrain, R, 3 Baltimore, D, et at & Dis, 49 843, 1963 4 Baron, J The Life of Edward Jenner, MD London, H Colburn, 1827 5 Bayne Jones, S_ Science, 73 599, 1931 2 Benrmg. 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J Debesral Disease Cambridge, Mass. yard University Press, 1954 27 a Louis Pasteur, Free Lanes of Science Boron, Litle, Brown and Co, 1959 23 Duclaux, E Pasteur, the History ‘of a Mind, ‘py Erwin F Smith, ‘Philadelphia Compt rend Soc de biol, Bact Rev, 24 261, 1960 ‘Proc Nat Acad Sci Acad d sc de C 3 Davaine, Ongin and Development of the translated 1920 24 Dusgar, BOM Ann NY Acad Sete 31 177, 1948 25 Ehrenberg CG Die Infusronsthierchen als vollkommene Organusmen Leapee 183 25a Ehrich, J. et al Scvence, 106 417, 1947 36 Ehriich, P "lim Jahrb » 6 299, 1897 37 _—— Collected Studies oF Tramumity, 1805 ated by © Bolduan, New York, 1906 tate teat Neue Notz. a ¢ Beh d Natur A Heslkunde, 39 270 1846 Fialay, A Cet al Science, It 85, inne A. Bat 1 Exp Path, 10 226, 28 1950. 29 30 1929 31 32 33 34 35 3 a 37 38 3 8 40 41 a2 4 44 45 46 4 a 48 4 & i i) 52 5: 3 34 55 56 57 59 61 il Florey, HW, et al Antbi , ott Qrford University Pres, 1949 roues London, racastorius, H De contagiomibus et - ssauosis morbis et eorum curatione, libri tres Venice, 1546, Consulted sa the German trans: y Viktor Fossel, in Median seis, 1910 the Klasnker der age, SH The M Gage, 8H croscope, 15th ed Ithaca, Godlee, J" Lord Lister London, 1917 fench, P_S, et al Proc Staff Meet, Ma Clin, 24 181, 1949 vmeyve lente Henit 1, Faihelogeche Unterschungen Ber- Iwanowski, D Bull d Acad Is imp @ St Petershourg, 13 237, 1852 Guoted from Centre £ Bait $ 250, 1899 moe jenner, E An Inquiry into th Jenne the Causes and Eifets of the Vanolte Vaccine Lonéon, Kaufman, HE, et 235, 1962 a Koch, R Bert Kosh, B Bete x Biol d Pfanz, 2, 2 277 Gesammelte Werke and E Pfuhl, Leipzig, 1912 ed by G Gafiky Zar Zuchtung von path Srgamamen, Mitt ad. Kawerhenen Gesunde heitsamte 1 1-48, 1881 jesund- Levaditt, C, and. Vaisma Com, See de biol, 119 946, ee trend innaeus, C_— System Holiiae, 1758, 820 sat uae 1h ed Lister J ie Collected Pi Maron Les vols Oxford, 19090, PE fagendte, F Lectures on th Jaton, Philadelphia 1839 ¢ Blood Trans- Meleney, F E, and Jobo: 133 675, 1947 son, BOTAM A, ‘Treause on Surgical Infe Fok Ontaed University Press, ae xeithakoff, B Liimmunté dans tes mala Mefchamcucuses, Paris 1901 Translated by FG Binnse, Cambridge, 1905 ¥ Muller, OF Ammaleula infusona, Hau. mae, 1786 . ~ Murray, E. G D Trans Ray Soc 49 Series IN 1955 Canada, Arch Ophthal, 68 Needham T Philosoph Trans, 490 61s, 1749 Quoted by Bulloch . Nordimeyer, H Ztschr F Hye. 10 145, 1891 Nuttall, G H F_ Parastology, 16 214 1924 Obermerer, O HF Centralbl fd med ‘Wissensch , 11 14S, 1873 Pasteur, L. Aon de chim et phys, 64 5-110, 1862 18r oeuvres de Pasteur Pans 1922-1928 Porter, J L. Bact. Rev, 25 389, 196f Portier, P, and Richet C Compt rend Soc de biol, 54 170 1902 Rayer Compt. rend 2141 144, 1850 Redi, F degl'insetty, Sth ed, Soc de bil, alla generazione Espenienze intorno Le 1688 A trans: Florence.12 Tatton of this book was published by M Bigslow, Chicago 1909 62 Roddis, LH Edward Jenner ard the Dis covery of Smallpox Vacunation Menasha, Woscorsin 1930 63 Salmon DE and Smith T Proc Biol Soc Washington 3.29 1886 64 Schatz A etal Proc Soe Exp Diol Med, 55.66 1944 65 Schoeniem Arch f Anat Physiol u Wis sensch Mrd 82 1839 66 Schroder Hand von Dusch T Ann d Chery u Pharmacie 89232 1884 67 Schule F Ann d Phys u Chem, 39 487, 1836 68 Schwann T Ann d Phys u Chem 41 184 1837 69 Singer, C The dawn of microscopical dis govery, J Roy Micr Soc, 317340 1915 70 Spallanzant L Saggio di osservaziont mi Sroscopiche Modena 1765 This reference 1s quoted from Bulloch 71 Stanley W OM The ssolation and Properties of tobacco mosate and other virus’ proteine Harvey Leet, 1938, Ser XXXHE, p 170 2 7. 74 25 7 8 7 2 80 a1 82 83 2 Classification of Bacteria MICROBIAL TAXxONOwY Bactena (the Schizomycetes or fission 2s ike the blue-green algae (the Afyx- ophyceae Cyanophyceac) and fung (Eumy- are usually class ina thint Veruses, since they included This Eited by ihe darinst and Germ: st, Hacekel, 6 AS A sol Problem of whether to melude qe? 12 the Cetrent evolution or af they resi ulted fror BAe of dierent ancestors rn by conver. Ch, 2° Classification of Bacteria Tamu, I, and Eggers, H Sctence, 142 24, 1963 Trefouel, J, et al 120 756, 1936 Iwort F W_ Lancet, 2 1241, 1915 Tyndall, J Essays on the Floating Matter of the Air im Relation to Putrefaction and In- fection, New York, 1882 Vallery Radot, R- The Life of Pasteur, trans- lated by RL Devonshire, New York, 1926 van Helmont J B Ortus medicmae id est initia physteae inaudita, Tract 21, Imago fer- menti impraegnat massam semmme, Amster- dam, Elavir, 1648, Part 9, P13 van Leeuwenhoek, Antony Arcana naturae, detecta Delft, 1695, p 42 Villemin JA Compt rend Acad d sc, 61 1012 1015, 1865 von Pirquet, C F, and Schick, B Die Ser- vmkrankhert Leipag 1905 Waksman,S A Bact Rev, 5 231, 1941 ——— Proc Soc Exp Biol Med , 57 244, 1944 Wilkins, MoH F Science, 140 941, 1963 Compt rend Soc de biol, | of - ated cate eseeates of primiuvely difteren. Th differentiated my systems of plan cy do not form wtticellular tissue ts and animals the highly and organBacterial Classification The Protists can be subdivided into eucaryotic (true nucleus) and procaryotc cells The eucaryotrc cells resemble plant and ansmal cells in haying membrane bound nuclei and cytoplasmic organelles, a mitotic apparatus, and multiple chromosomes The procaryotic cells, which include the bacteria and the blue-green algae, have been shown by electron microscopy to contam no mem- brane-bound organelles, a smgle, circular naked chromosome, and a semingid cell wall of umque composition A presumed phylogenetic relationship among the Pro- usts is shown in Fig 1 PLANTS PROTISTS Blue green Algae Bacteria Fig t 13 BACTERIAL CLASSIFICATION When studying large numbers of micro- organisms, it 18 obvious that some taxonomy of interrelated classification and nomen- elature system must be used for the pur- pose of identifying, nammg, and describing different organisms and their properties and relationships Such classifications can be artificial and utiditarran in affording rapid identification, they can be descnptively phe netic mm comparing simudar or dissimiar features without regard for genetic relation- ships, or they can altempt to show patterns ANIMALS Protozoa Euearyote Cells (Bucaryotie ancestors) Procaryotic Cells | | > | I \/ (Procaryotic ancestors) ! Presumed phylogenetic relationstups between protists4 jorms Using the model of the ancestral tree the problem of establichira ames of evolu tuonary relationships between bacterial Strains at this instent mm time is somewhat like trying to devcrmine the height branch Points, and exact form and structure of a Wee from the trunk on up by observing only a transverse thin section sliced through the increasingly twisted —ramifying diverging and converging branches Because there has Been no way to discern which features bac- teria developed early and. late, or which fea- tures are the result of divergent rather than convergent evolution, it 1s impossible ta de- velop a true phylogenctic taxonomy Recent developments in comparative biochemistry indicate that investigation of stable meta- bolic patterns and cell Polymer structures May yield information of evolutionary sig- nificance One example is the observation that the biosynthesis of the amino acid Iysine is via a common intermediate (pre- sumably a, ¢ daminopimelic acid) in bac, tena, actinomycetes, blue-green algae, water molds, green algae, observation that almo: Mycetes, and blue Sreen algae produce a semungd cell wall structure of almost unt- form composition called mu 4) Iewoutd be difficult to 4 More knowl = tomes availble, it is ae ie Rot surprising that opines change with fegard to taxonomi¢ Ch 2 Classification of Bactena positions This ts reflected in the fact that Beizeys Manual of Determnanve Bac- retiology, prepated by many contributors us} generally widely accepted in the United “tates has shown substantial changes in the scven editions which have attempted, since 1923, to construct a phylogenetic system based on morphologte and physiologic fea- sures This manual also offers a determina- uve key for identification of microorganisms as well as a wealth of descriptive material The International Society for Mucrobi- ology established m 1948 and amended m 1953 a code of nomenclature based on spec es genus, tribe, family (-aceae), order ales), and class (-aceae), ete » for the pur- Pose of constructing classifications A. spe- eres 1s given a Latin binomial name for Senus and species in the Linnean tradition of naming plants and animals However, there ts no entirely satisfactory classifica- tion for bacteria, and none has been ac- cepted for international use New systems are being proposed from time to time (5, 8, 9, 16, 19, 27, 28, 32-37) Throughout this book an attempt has been made to fol- low the terminology proposed in the seventh edition of Bergey's Manual Recognizing that this classification 1s not perfect and that changes will be made in succeeding editions, it seems wiser to use a recorded terminology rather than to introduce new names for old organisms The well-known trivial or common names such as the tU- berele bacillus (Mycobacterium tuberculo- sis) and the typhoid bacillus (Salmonella typht) will continue to be used as before in the body of the text It should be noted that when a Latin generic (genus) name 1s Vernaculanized m English, it 1s not capital- wed or italicized A diagrammatic Presentation of some of the groupings occurring in the seventh ¢di- tion of Bergey's Manual 1s outhned in Table 1 THE BACTERIAL “species” According to Present definitions each dis- Kind of bacterrum may be considered a Species A clearer defimtion 1s difficult For example, many bacteral groups may be comprised of continuous varietics of strains which form interrelated constellations (again an analogy with the ancestral tree) Within12903, ‘anowuoN. aigoeuy vynwed 4 auollaA, ‘waDeUuassIaN, arqorsy sno0030000) TRON, sna2020S uu, OMSHINASOLOHUNON VIMHLOVd TALLYDSN WED 2jnowuou 20 t,380y snoysupaad —pasnposd suodsopwg——— Ye Oe bua yim 20, ‘31q0u3y sonyiuo g = opin TanpO}DEAI aigoise kyjexusg —sauaorKsov0U 7 Thay auaguuaiseqeu.Lo, spos 3y310§ aouzyrydip 3 wantaajzequKsoD, seaeutiod spiae onaze atuoidosg aanpasd ‘re fe 1s0WIY 3131003509194 unniayseqivordold — ae22uuIzequonon 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SAUNLVa DUSNSLOVEYHD, SaaS INYLYOUINT vena, Sarna co penumuog—T O°. 718 a Constellation Clusters of high density areas (not Necessarily at dead ce, ter) contain big. types from which a strain may be arbitrarily el many marginal Anterrelated Stra, found Examples of groups Which form Constel- lations of simular strains include the Entero. bacteriaceae, Be a feetic acid bac. teria, and 22) 0S May be at the accepted Species levels The “gen Pecies” has bee; SUBEested for Such Organisms (22, 27, 28) However, Species designations mM such Soups ha Made for ther good Teasons a, 18 doubttuy thay established Species wy] abolished And even at the SPecies level, interfertiity Paticrns are not clear Ung, ‘unately, est of snterfert yy A which Tequires mult Sompatibilities of cell SUrface stry Te, DNA hom BY, ete for conjugal DN, tomosome ransfer, hag Not been Completely helpfuy cfining the hms of ap, al species f, Se only a muted ny; f bacterial eS have en show; le of sex, Tecombina. ton One reaso, 18 that unoby, bacteria} virus (phage) ‘nfection (see } geny, iP 35) ma Cause Modrfication Of host cell Surface op DN ts and the Production of Hucleases wh, degrade Ron. mad fed DY < viral hose cel] Modifi. fstiang, Chap Creating an “'solation Prchaanm™ tween (daughter ell) steams Aty het extrem, rita spe. crs 4 defined OUP of ba, era capa. He of Sharing 3 Eene pool, then for ftample, the roblem of BSCxual pone Warafer by Phage (1p. ton by bacteray Satuics) OF MUN be ted (seg 5,65) thw erelore not ‘Uprising tg find thay 19 pteseny Clas3: atom including Bergey's Veet, PCI are guy Sbutanly defined Ch. 2 Classification of Bactex which dos bya descriptive array of eee or ge Aot include the tests of inte: used ford transfer However, the ee acters # Scnbing different ‘kinds’ im of phenoine Species encompass a spectrur art a $20 Xpressions which desenbe n P ater ling of the potential al weeratos ase du 4 particular gene pool ‘ance of bat clude the Morphologic pee pattern f terial cel] shape, size, flagell Joon not Thotile, capsule occurrence, co ng prope Phology, and Pigmentation, Siang ey tes, including the gram, capsul Sates, ‘nett ‘ast, and flagellar visuahzing s| quire alte Patterns, including growth m sagas and Conditions, ability to fermen viele Utilize Unique chemicals, energy ie prod Patterns, ang characteristic metal end since ‘Ucts, Macromolecular composition antigen ture, for example, the specific ules, Pattern of Surface macromo Sour of the chemical Composition and oructure soutet cell wall, and DNA, the ecology a ® habitat, and the ability to parasiti ¢ Pathogenic the lack of a defintive unitizing ae for species designation in bacteria hn derstandably Fesulted in variation a adit ae ttch groups are divided, dep cuter Upon whether the imvestigator 1s a “spl att ” ¢ “splitter” category 10 have divided the Salmonella 3p. ing 7200 species on the basis of seroty The Validity of the separations 1s pl bane !8Y, a thar different host suscep bilities app, fo correlate with the d ne ences in Pacteriat cell surface Polysacchané? Compos; 2nd structure of the ae s Organisms Such iierences One monosacchaniee gc Broup, re one md UMpers,” on the other hand, subdivided ¢f, lebsiella, Sucre ‘ect into “number ht “types” for eac! € Microbial tax- with the prac- Eee Mentifying fea th stable and character bility. together with rapid Y Tesult ing bacterial popu- Fapidly changes Characteristics m Jelding tg cavn ‘Avironmentay selection pres-Use of Ecologic Features sures on [laboratory media For this reason stock or type cultures are usually stored in a viable, minimal or nongrowth state (eg, freeze-dried) for future comparative use Representatives of type cultures of species are maintained in collection centers, such as the American Type Culture Collection, Washmgton, DC, and are generally avail- able for comparison NUMERICAL TAXONOMY Because of the difficulties in constructing Phylogenetic classifications which are based on only a few arbitrary weighted characters, descriptive taxonomy has been revived in the form of computerized numerical com- parison of large nymbers of diagnostic fea~ tures for many strains First devised by Michel Adanson in the eighteenth century, this sysiem gives equal weight to all features chosen for comparison Designations of groups are then made on the basis of degree of similarity, quantitatively defined by simi- lanty coefficients (S values) If characters chosen for numerical taxonomy conform to features presently used for describing bac- tera] species then some agreement between the two systems 1s certam to occur And it would appear that the neo-Adansonian ap- pfoach has yielded quantitatively described groupings, which are generally similar to present classifications arrived at by usual means Significant regroupings may also re- sult from further development and applica- thon of this approach (9, 19, 33) PHYLOGENY BASED ON DNA HOMOLOGY Since genetic information is encoded :n DNA base sequence, it 1s logical to assume that as organisms drift apart by mutation, selection in different environments, and re- combination (re, evolution) their genes would code for different structures and func tions corresponding to changes in DNA base sequence Thus either or both the chemical composition and base sequence would be expected to change Analyses by both chemical and physico- chemical methods show that DNA base composition 2s constant and characteristic for each organism (4, 5, 13, 15, 20 22) This 1s stated in terms of the mole fraction of guanosine plus cytosine (ic, GtC/ 19 G+C+A+T) content im DNA from vanous organisms Such analyses show that DNA of an individual strain yields a constant GC content Hawever, such values vary for dif- ferent organisms from 30 to 75 percent GC content (Table 2) At the level of descrip tively defined genera it 1s to be noted that several species do exhibit smilar DNA base compositions (e g, genera of the enterobac- teria, including Escherichia, Shigeila Sal- monella, and others, such as the lactobacilt streplococc:, and pnevmococear) In other species, large differences occur, notably in several Bacillus species and in Sporosarcina indicating a need for realignment of their classification (22) or a lmut to the applica- tron of GC composition to classification Thus, similar base composition as 2 mimmal requirement but 1s obviously not conclu- sive evidence for DNA homology, since all vertebrates, including man, contain 40-44 mole percent GC content as do many micro- organisms Obviously then, different organ- isms with even symilar DNA base compo- sitions must have different or heterologous base sequences The measurement of DNA homology has recently been quanufied by several procedures which determine the de- gree of formation of molecular hybrids from two DNA strands of different origin A few of the measurements presently available are summarized in a simplified manner in Table 3 USE OF ECOLOGIC FEATURES It should be noted that the microbiologist makes good use of ecologic traits Ecologic criteria are seldom used in describing higher Ife forms, whereas bacterial species may often be diagnosed or isolated by techniques based on ecologic features Microbiologists have long used selective culture techmques to ssolate specific bacteria These techniques depend upon the abuhty of certam organisms to survive or thrive m certam environments The corollary 1s often used by the medical microbiologist Specific pathogenic organ- isms are frequently assoctated with, recog- med by, and even named for the diseases they cause (eg, Diplococcus pneumoniae Corynebactertum diphtheriae, Clostridium tetam Bacillus anthracis, Neisseria mentm- gititidts, Shigella dysenteriae, etc ) 3Size of Microorgamsms 21 Teble J DNA Homologier among Some Bactena DNA Source % RELATEDVESS = REFERENCE A Eschencloa coh “e ia Shigella dysenteriae 7 4 Alchsella (nerobacter) acrogenes 4s 4 it Salmonetia typhimurcum 38 av Proteus sulgaris 18 1 Serrana marcescens 7 Ht Psexdomonas aeruginosa t 5 Bacillus subishis i 3 Brutella neotamar 8 4 B (Wo No memnginndus) Neuserca meningitidis 100 15 Neisseria gonorrhoeae 80 15 Neisserca perflaaa 35 8 Nessseria subflaver 48 15 Nersseria catarrhalis 1S 15 Newsena caviar 20 B Mima sp 5 15 Herellea 5 15 Escherichia cots 3 i or is Monkey \idney cellular plant-lhhe organisms without chloro- phyll which usually muluply by binary devi sion The individual cells in colonies arc phystologicaily independent, although they ate influenced by environmental changes produced by neighbormg cells The sndi- vidual cells which are spheres are called cocer, the straight rods bacilli, and the curved rods spitilia (Fig 2) ‘The cocct are, when fully developed and free, perfectly sphencal, but when two or more afe in apposition, they may be slightly flattened along the tangentsal surface, givin an oval appearance The bacith ave straight rods whose length vanes from 2 to 10 times their width and with ends which may be gently rounded hike those of Salmonella typhosa or squared like Bacillus anthracis ‘The spinila vary from small, comma shaped. orgamsms with a single curve to longer smuyous forms with from 4 to 20 curves which suggest the appearance of an ant- mated corkscrew The turns 1 typical spt rilg are always m three planes and ate best described as helicodal Among the known mucroorgamsms the bacilly outnumber by far the other forms Many variations from these fundamentat types Occur even under normal conditions and will be discussed sm subsequent chapters SIZE OF MICROORGANISMS There ts considerable vartation im the size of different microorganisms Cote; may Fig 2 Morphology 1 simele cover 2 cocci 19 pass 3 cocer tm chains 4 cocer an clusters 5 Focen in tetrads 6 coccobacillt 7, club-shaped ba fail @ bacill: with rounded ends 9 bacille with Square ends 10 fusiform bacills 11 whrios 12, Spinlla V3 Borrela 16 Treponema \S Lepto- spirocinta (035 2) to the minute vires’ of polto- myelitis (0027 x) Even the larger viruses cannot be properly studied by the ordinary ght of darkfield mucroscopes although they can be photographed by ultraviolet light The invention of the electron microscope by Kroll and Ruska, m 1931 (10) opened up @ new dimension for the study of the mor- Phology of the bacterial cell and provided, for the first ume, a glimpse of the size, shape, and structure of viruses (Fig 3) The early studies by Marton (23) and Krause (18) Were followed by the more detailed explora- tions of Mudd and Anderson (25), Knays: (17), Wyckott (38), and Johnson (14) Fig 3° Electron micrograph show, wath rabbit papitioma virus Ch 2 Classification of Bacteria (Fig 3) Magnifications up to 100,000 have been obtained and particles as small as 10 my have been photographed Improved electron microscope technology in the areas of fixing, embedding in non- shrinking plastics, and cutting and sta:ning of thin sections has resulted in a rebirth of imterest in fine structure of microbial cells Negative staining techmque with electron- dense materials (¢ g, phosphotungstie acid) has allowed the development of knowledge of cell surface structure (3) The smallest free-hving microorgamsms must be somewhat larger than the small \iruses because they must have a comple- ment of enzymes and genes for growth and Teproduction, while the viruses use a part of the host cells’ enzymes for growth The smallest independent organisms probably occur in the Mycoplasmatales order, where the individual organism may be as small as medium-sized viruses Morowitz and Tour- tellotte (24) have calculated that the small- est self-duplicating unit would have to be 500 angstrom units and contain 150 large molecules (Fig 4) The smallest free-living organism actually observed 1s M ladlaw, which has twice the diameter (1,000 ang- stroms) and eight times the volume with 1,200 large molecules M. gallisepticum, with a large diameter of 2,500 angstroms, ing a small bacil} % 22,100 (Electron meroerph bret Ean rp) nad fied G ShayStaining Reactions Fig $ Relanve size of pleurogneumonee tke organism =PPLO elementary body and smallest hypothetical kwing cell (From Morowitz and Tourtellotte Serentific Amencan 206 117 4962) has 20 000 large molecules and encompasses an automatic metabolic and reproductive system STAINING REACTIONS Certain differential stains such as the gram stain and the acid fast stam are of aid m Classification After starmmg by Grams method some organisms are found to be violet and some red Afi of the stamable spiral orgamsms about one third of the coce: and one half of the bacill appear red Grams more or less fortuitous and em pincal discovery that some organisms could be stained violet and others red furnishes one of the simplest and most useful tech Riques in aif merobiolagy 2} A thin film of orgamisms on a glass shde 1s fixed with heat and then treated wrth gentian or crystal violet for one mute rmsed quickly with water and covered with Grams sodine 50 lution for 1 minute Following another quick 23 rinse in water the slides decolorized with alco hol or a muxture of acetone and ether washed thoroughly and counterstamed with safranin for 10 seconds washed with water and dried In the first step all organisms are stained Vio Jet and alt assume a dirty bluish brown color after Creatment with the iodine solution The rodides serve as a mordant to fix the violet dye 1n certain types of organisms so itis not washed out by the alcohol or ether acetone decolorizing agents The saframin stains req those organisms which have been decolonzed A little practice and judgment are requured in applying the de colonizing solution to prevent snadeqnate or ex cessive decolonization ‘The organisms which retain the violet dye are referred to as gram pasuwve and those which are decolonzed and counterstained with the red dye are called gram negative The phystochem teal reactions which produce the differenual ‘Staining are not completely explained An smtact cell waif is essential for retaning the crystaf violet stam Gerhardt and his asso ciates (11) have shown that crushed ceffs and Protoplasts of cells which were originally gram Positive stam gram negative after damage or destruction of the cell wall Spontaneous auto! ysis of a portion of the cel! wall probably explains the frequent occurrences of gram negative organisms in pure cultures of gram positive organisms (Fig 5) These increase fapidly as the culture ages The vatious theones. purporting to explain the mechanism by which grag positive organisms retain the crystal violet have been reviewed and eniticized (26 29 31) Although bpids, polysaccharides RNA and certain proteus can all retain the erystal wolet 1odine complex these are not sufficiently dif ferent in number and amount to explain the binding of the blue stain by the gram posinve organsm the predommance of mucopephides in the gram posttives and lipids im the gram negatives might play an accestory role how ever (29) Later work by Salton (30) shows chat the Bram postiveness of organisms core sportds to @ decreased leakage of Pg: com pouads ‘The studies of Bartholomew and Funkelstem <2) shown ia Figure 5 demonstrate that crys tal violet stains the cell wall as well as the protoplasm of both gram positive and gram negative baci: (Fig 5 AE) The E col cells wich are gram negative can actually bind more crystal violet per gram of cell weight than B subtils a typical gram positive organism (2) ‘The organisms remain the same size after treat meat with the ;odine (Fig 5B) wnless there 1s excessive Washing with water between the crys tal violet and the 1odine reagent: With moderateCh 2 Classification of Bacteria MM oo caer? 7a a) ‘eayaiat VIOLETStainmg Reactions washing the blue dye is washed from the cell wall but not from the protoplasm, with exces sive washing of the cell the dye 1s washed out and the organism appears gram negative With brief decolonization with atcohol the blue dye 33 removed from the gram negate organism but not from the gram positive one (Fig 5 C) On counterstammng with safranin the gram Regative organism stains red Brief washing with water does not wash the red stain from the orgamsm but prolonged washing will do so. with the bacilli appearing thinner and thinner (2) Even the brief washing necessary to clear the shde of excessive safranin has apparently removed the safranin from the cell wall so the mae ree peers eee er 7 Rugese Lorre as ore) Eos 25 gram negative cell in Figure $ Dis not as wide as that in Figures 5 A ond 5 B Finkelstein and Bartholomew (7) have found that the binding of crystal violet by both gram positive and gram negative microorganisms can be inter preted as obeying the Freundlich and Langmuir Tass of absorption Treatment with iodine produces a crystal violet iodine complex in both gram positive and gram negative organisms Salton (29} visual ized the differential decolonization as depend img upon changes in the cell wall pores during the process of dehydration with alcohol The afcohol wash after treatment with 1odine dis solves away much of the lipid from the cell Raised rn Fig 6 Types of colony formation showsng structs edges re of colomes elevation and nature atCOLONY FORMATIONS, Microscopic unicellular bacteria multiply rapidly and form Macroscopic colomes in 24 to 48 hours when supphed with adequate food temperature moisture and a solid or semisolid supporting base With some spe cies such as Mycobactermum tuberculosis a longer period of incubation 35 required and some form colonies which are so small that a hand fens or even the low power of the microscope 1s required for visualization Theoretically a colony 1s composed of the descendants of a single cell but it may de velop from a clump of cells or from two or single colonies tained ments have been classified by Graham Smith (12) as loop-forming folding snapping and Ch 2 Classification of Bacteria shpping Colones of organisms of the loop- forming yroup usually have wavy edges (B ai thracis) Those of the folding and snap- Pint group (Past pestis C diphiheriae) have serrat.d or crenated edges and those of the slipping group (E colt Proteus vulgaris) have smooth or lobate edges with a spread mr g owth (Fig 6) Motile organisms in flud medm give a smooth homogencous growth but nonmotile forms may at times produce definite colonies im undisturbed fluid medi Other solid media such as po- tatocs bread coagulated serum silica gel and ussuc from animals, may form the base for the development of definite colonies It 8 important to remember that the same microorganism will produce colomes which vary greatly in morphology depending upon their location in a dish of poured medium on the surface in the depth of the medium or between the medium and the bottom of the dish REFERENCES Bartholomew J W and Mutwer T Bact Rev 161 1952 2 and Finkelsten H J Bact 75 77 1958 3 Bayer ME and Anderson T F Proc Nat Acad Sci 54 1592 1965 4 Brenner DJ et al J Bact 94 486 1967 5 Deley J and Friedman § J Bact 88 937 1964 6 Edwards P Ro and Ew ng WH Hentfica tion of Enterobacterracea Minneapolis Minn Burgess Publ Co 1962 Finkelstein Hand Bartholomew J W J Bact 80 14 1960 Floodgate G D Focht D D 90 1314 1965 {0 Freund ch MM Science 142 183 1963 EL Gerhardt Pp etal I Bact 72721 1956 12 Graham Smith G8 1910 _ woo Bact Rev 26 277 1962 and Lockhart WR J Bact Parasitology 3 17 13 Hoyer BH et al 14 Jobnson FH et af 1S Kingsbury DT yy 16 Kluyver AJ and Vai f Bakt 1 17 Knays: G 18 Krause F Sctence 144 959 1964 J Bact 46 167 1943 fact 94 870 1967 in New CB Centralbl Abt 94 369 1936 etal J Bact 53 525 1947 Naturw ssenschaften 25 817 J Bact 85 1061 1963 J Bact Rev 31 21S 1967 21 ——— and Bolton B Proc Nat Acad 50 156 1963 22 Marmur Tet al 329 1963 19 Liston J et af 20 McCarthy B Ann Rev Mcrobiol 17Structure of Bacteria 23 Marton, L Bull Acad Belg Classe des Scr, 23 672 1937 Morowiz, H I, and Tourtellotte, ME Sct Amer , 206 117, 1962 Mudd’ S, and Anderson, T F J Immun, 42 251, 1941 Normore, WM, and Umbret, WOW J Bact , 90 1500, 1965 Ravin, A W Bact Rev, 24 201, 1960 ‘Amer Naturahst 97 307 1963 Salton, M RM Bact Rev, 25 77, 1961 I Gen Microbiol , 30 223, 1963 ‘The Bacterial Cell Wall Elsevier New York, 1964 24 25 26 27 28 29 30 31 32 33 uM 35 36 37 38 3 27 Skerman VB D Bact Rev, 13 175, 1949 Sneath P H A Ann Rev Microbiol, 18 335, 1964 Stamer, RY The ‘Academic Press, 1964 Doudoroff, M, and Adelbers E. A The Microbial World 2nd ed Englewood Cliffs NJ, Prentice Hall Inc, 1963 Van Nel C B Cold Spring Harbor Sym posta on Quantitanwve Biology, 11 285 1946 Vogel, HJ Symp Evolutionary Biochem . Intern Congr Biochem 15th (London Per amon 1961) ‘Wyckoff, RW G Electron Microscopy New York, Interscience Publishers 1949 Bacteria New York, General Morphology and Reproduction of Bacteria When unstamed, most bacteria are trans- parent, colorless, homogeneous, OF granular bodies with a low refractive index, approxi- mately that of water The bacterial cell con- sists of four morphologically distinct: struc tures—protoplasm, cytoplasmic membrane, cell wall, and capsule——each varying in chemical, physiologic, and antigenic constit- The protoplasm of the or- or potentially fluid, collord ‘an extremely thin, ductile, cytoplasmic membrane The intact cytoplasmic membrane with ils contents 15 usually referred to as 2 protoplast The cyto- plasmic membrane, which varies from 5 to 10 mp in thickness, 15 composed manly of Iipd and protein (1,102) Tuas physiologically active, permitting In a selective manner the entrance and exit of smail motecules By the usual staining, meth- ods this membrane stains more deeply than the cytoplasm and resists decolorization longer When the cell 15 plasmolyzed, the ‘membrane contracts with the cytoplasm, draws away from the cell wall, and can be photographed readily with the electron microscope It has been identified m thin sections of bacteria (iil) and asolated from ruptured protoplasts by differential centrif- ugation (138) Morphotogn structure of » facil wt. A. Tr ructeun C. cytepian granule. D yxcuole E, oytop!samt. membrane Fy ret wall G slime Layer or caprole H Eosellurs23 ‘The cell wal is no stamed by ordmary bactenal stains unless previously treated with the proper mordant (Fig 7) (81 122) and is not vistble when living ceis are exammed in the dark field but it 1s delineated clearly 19 electron micrograghs (25) In completely plasmolyzed celis the tough cell wall re mans asa ghost After mechanical (109) OF enzymatic disruption the cell walls of cocet resemble grape hulls (Fig 3) (6 10 101) as do the cell walls of organisms from the psittacosis and nickettstat groups of or ganisms (73) Electron mucrographs often reveal a fine Structure which vaties with the specific or Banism Spherical subunits have been re vealed in the walls of some while others. show a fibrous structure (Fig 4) (69, 97, r mee ro | Vig 2 Docitlur cereus ceystat stain showing cell plates bur ror cell wat “From Clark Webb and Chance J ‘Bact 73 at 1959 ia 3 Gerera! Morphology and Reproduction of Bactena 109) The spherical units have been de- scrived 1° hollow spheres (124), globes (113) or hexagonal lattice structures (56) Important differences exist between the chemical composition of the walls of gram positive and gram negative orgamsms The cell walls of the gram positive bactena are usually less complex than those of gram negative species although gram positive bac teria also contam components other than the basal structure (41) The murein or mucopeptide layer is present in both gram- Positive and gram negative organisms This component 1s responsible for the rigidity of the cell wall and the maintenance of the characteristic shape of the orgamsm Its Uniqueness provides several points of attack for antibacterial agents since its synthesis Tequires enzymatic pathways not present in mammalian systems In addition to the mucopeptide layer, gram negative species also contain large amounts of kpid polysaccharide and pro- tem complexes The antigemce specificity and endotoxic properties of gtam negative or- ganisms can be traced to certain compo nents in the cell wall The antigens of the cell wall are Unique, and 1t us possible that in some instances they may replace the whole bodies of the organisms as mmmu- nizing agents GROWTH OF CELL WALL Tt was assumed for Benerations that the cell wall grew in a uniform manner about its entire circumference untit Bisset (13, 15) demonstrated that cell wall growth in septate acl 1s immediately adjacent to the sep tum These observations have been con firmed by Bisset and Pease (17) for spinlla and by Cole and Hahn (37) for Strepto this mode of growth by Quorescent staming (Fig 5), but witen the same ‘ehmaue Was applied to the growth Pht growth a ? cn was found to be no! @ general ult a tercalation to" a ramIcroscopic 1 SLIME LAYER OR CAPSULE Under normal conditions the cell wall 18 SStrounded by a loosely attached slime layerSlime Layer or Capsule Fig 3 Electron micrograph of cell wall of Streptococcus Joecalis (After Bibb and Straughn J Bact, 84 3094 1962 consisung of mucoid substances of high molecular weight which ace diffusing can- stantly into the medium When viscous ma- terial 1s produced in sufficient amounts and Fig 4 Fragment of envelope of L. hyalina after tryptic digestion showing fibrous structure X 160000 (Courtesy of J A. Chapman and M RI Salton) ae eee ee a ate a Fig 5 Growth of cell wall of Streptococcus pyogenes demonstrated by direct and indirect um munofluotescence Growth 1 not uniform but lim ited to two actively growing areas for each coccus (From Cols and Hahn Setence 135 722 1962 } Temains concentrated at the surface of the cell wail, it 1s called a capsule and 1s the sub- stance which gives a sttingy texture to flud cultures and a moist, glistenmg surface to colonies grown on solid media Bacteria are prolific in the production of polysaccharides Under favorable conditions they may syn- thesize as much as 60 percent of their dry weight as intracelulat polysaccharides and many times therr cellular dry weight as extra- cellular polysaccharides (141) Both cap- sule and shme Jayer are visualized readily with the wet film India ink methed (52) Using specific antiserums Tomesik (7, 61) and his associates have studied capsules of certain species of bacilli in which the major component, a polypeptide, 1s enclosed in a polysacchande framework which has septa that yom the cell wall (Figs 6, 7, 8) ‘The capsular material apparently 1s pro- duced by the hving cell wall Sumlar chem ical and antigenic substances often can be extracted from bacterra which have no demonstrable capsule or from bactenal cells30 Ch 3 General Morphology and Yeproduction of Bacteria SR aren etmppat em P t o ra met ne 8 * ae Fig 6 Electron m cro, ter um No capsule s vs ble aft Ronspec fic B cereus ammune serum, Fi Prann-Grace and Tomes k Schwe ¢ Zeschy « Bakt 21 906 1953 Fs 7 Electron fertum Polysacchand, tum war viv Ble afte rot Grace and Bakr me 8) le capsule T the add Schner ™ crograph of zraph of Rac tlus mega fet the add t on of rom Baw rf Path f Bac tus mega 1 transverse sep ‘on of homologous (From, 2ischr Baumann Pal Fig & Electton micrograph of Bac ite mots der um Polypept de capsule with rad al stra on Tah M8 ble after the addit on of acchr polyper le IMmunoserum Note absence of materia! 1 EIB ON OF the transverse septa (From Baume ake, and Tomes k Schwes Zicl> f Path # Bakt 21 906 1958) Whose capsules have been removed prev! ously by mechanical means (50) é 85 Forme dchydrorenase 47 <03 Soe Matec dehydrogenase 166 <03 So3 Mala enzyene pe <05 8 bouik dehydrogenase Ses 266 0 452Cytoplasm Diosynthesis of cell wall polymers and exo- es. Within the cytoplasm, mesosomes Cintra- cytoplasmic membranes, chondrioids) have been observed by electron microscopy af thin sections of a large number of bacteria. Such membranous organelles are generally villous or lamellated in appearance and maintain a connection between the cyto- plasmic membrane and the comparable Jayers of their boundary, thus providing art increased membrane surface area for en- zymatic activity. Their association with the region of the membrane that invaginates in septum formation suggests a role of these organelles in cell wall formation. A connec- tion between the mesosome and the nucleo- Plasm has also been repeatedly observed, and although the meaning of this observa- tion is as yet unknown there is strong sup- port for the proposal that mesosomes par- ticipate in the separation of replicating DNA (29, 32) (Fig. 6). CYTOPLASM Many types of inclusions may be observed within the bacterial cytoplasm of both liv- ing and stained preparations. In most cases the presence of vacuoles and inclusions is conditioned by the environment and physi- clogic state of the cell, Many appear to be absent during the most active phases of growth and to increase in prominence dur- ing the Jatter phases of the growth cycle. The metachromatic granules which stain red with such dyes as toluidine blue and methylene blue are found in many different types of microorganisms. The chief chem~ ical component of these granules is poly- phosphate of very high molecular weight. Lipid globules, glycogen, and other poly- saccharide inclusions may also be detected by specific cytochemical techniques (6). In addition to the granules and inclusions which may be secn in stained preparations, the use of the electron microscope with a resolving power of 20A. and the develop- ment of techniques for sectioning have en- abled us to gain further knowledge of the internal structure of the cell. Within the cell wall and membrane layers of the cell two types of somewhat amorphous material may be seen in thin sections (Fig. 7). The trans- Fig, 6. Dividing celt of Lactobacillus plan« tarum showing prominent mesosomes associated with a newly forming cross wall. The immature type of mesosome seen at the bottom is not associ- ated with nucleoplasm in contrast to the upper mature mesosomes which are surrounded by a tripletayered boundary and are conunuous with the nucleoplasms. The black bodies are polyphos- phate granules, X 94,300. (From Kakefuda, Holden, and Utech. J, Bact., 93:474, 1967.) parent material seen in and around the core of the cell corresponds in distribution to the chromatin material described by cytologists, while the outer, electron-dense material con- sists of closely packed granules, 140 to 170A in diameter, the ribosomes. Rela- tively pure preparations of granules of simi- lar dimensions may be obtained by high- speed centrifugation of lysed protoplasts or disrupted cells. Wide variations have been54 Ch 4 The Composition and Organization of a Bacterial Cell 4 a Fig 7 we ee eed ‘A divid ng cell m the process of laying down a septum between the two daughter cells Farrly deep projections of cytoplasm ito the nuclear region and of nuclear region into cytoptasmn can be observed The bacterial cytoplasm appears to consist largely of 200 A gran ales (From Chapman J Biophys Biochem Cyrol 6 221 1959 } observed m the sedimentation pattern of ribosomes obtained from different sources Not only does the magnesium concentration markedly affect the sedimentation coeffi- cient, but both the metabolic state of the cell at the time of breaking the cells and the method of breaking the cells alter the pattern Because of these wide variations which have been observed under different conditions it is difficult te compare the tibosomes from different sources Those which have been most extensively studied however, show in common particles with a sedimentation constant of 70 S which dis sotiate im low concentrations of magnesram into two relatively stable subunits of un- tqual sizc, corresponding to about 50 and 30 Sin bactena The 50 and 30 S subunits cach contain approximately 65 percent RNA. and 35 percent prote:m in such an organ wm as E colt that divides every 30 minutes the sibonucleoprotem accounts for about 30 percent of the cells dry weight Of this RNA. comprises as much as 25 percent Although appear to form a substructure which seems to be contmuous over a whole raw of these particles as a complicated fabric (29) NUCLEUS The central area of lesser electron density visualized im thin sections of bactena with the electron microscope has been shown to occupy a position corresponding to the chromatime bodies Structures observed 10 bacterial cells and suspected of being nu clear elements or their equivalent can only be identified by a positive Feulgen reaction Tt 1s difficult to demonstrate chromatin bodies by direct staining because of the high concentration of ribonucleic acid Pretreat ment, however, with hot 1 N HCI or with mibonuclease will remove all or nearly all of the RNA in a few minutes, allowing the chromatin bodies to be seen in any stage of the growth cycle of the organism Although the Peulgen reaction sdentifies the chromatin material as deoxyribonucleic acid, stainingReferences tam up to 500 molecular helices of DNA, usually oriented in the Jong axis of the bac- term Much of the controversy that has existed among cytologists concermng the nature and structure of the bacterial nucleus may be due to the absence of a limitng nuclear membrane and a resulting intimate associa- ton between cytoplasm and nucleus A}- though bacterial DNA represents only 2 to 3 percent of the cell’s weight and 1s large enough to contain a complete genome, it occupies about 10 percent of tts volume Thus loose arrangement allows for the ready diffusion of soluble materials to all parts of the nuclear structure Further discussion of bacterral DNA may be found in Chapters 5 and 7 and tm the reviews of Breger (6) and Fuhs (19) REFERENCES 1 Abram, D, et al J Bact, 90 1337, 1965 2 Aj, S J Proc 4th Int Congr Biochem, 13 215, 1959 3 Baddiley, J Fed Proc, 21 1084, 1962 4 Endeavour, 23 33, 1964 5 Bneas, E, et al Biochem J, 6 2598, 1967 6 Brieger, E, M Structure and Ultrastructure of Mrcroorgamsms New York, Academe Press, 1963 7 Bunton, C C, Ir 776, 1964 Trans NY Acad Sci, Ser I, 27 2003, 1855 In The Specificty of Cell Surfaces Soc Gen Physiol Symp Englewood Clits, NJ, Preatice Hai! Inc., 1967 J Biochem Biophys Cytol, Proc Nat Acad Scr, $2 9 539, 1961 11 Cedergren, B, and Holme T J Ultrastruct Res. 3.70, 1959 12 Cohen Bazire, G, and London J J Bact, 94 458, 1967 13 Cota Robles, EH J Bact., 85 499, 1963 14 Cummins, C S Microbial Classification, 12th Symp Soc Gen Microbiol Cambridge Unt versity Press, 1962 15 Davies D A. L. Advances Carbohyd Chem, 15 271, 1960 16 De Petns,$ J Ultrastruct Res, 19 45, 1967 17 Ellwood, D C, et al, Biochem J, 85 213, 1963 18 Engelman D M, et al Biochim Biophys ‘Acta, 135 381, 1967 19 Fubs G W Bact Rev, 29 277, 1965 20 Ghuysen J M, et al Biochem J, 6 2607, 1967 21 Haukenes, G, et al 53 425, 1961 Biochim Biophys Acta, 2 23 24 25 26 27 28 29 3 32 33 34 36 37 38 39 40 an a2 4a 45 4 & 4 4a 43 49 50 51 52 53 54 55 55 Hay, JB, et al Biochim Biophys ts 7h 188, 1963 pms Acta Hymans, W J J Gen Microbiol, 28 177, 1962 Hoemger, J F M, et al 603, 1966 Holwill, ME 3 Physiol Rev, 46 696, 1966 J Cell Biol, 31 Housewnght, R D Jn The Bacteria FC Gunsalus and RY Stanter, eds, Vol UT New York, Academic Press, 1962 Hughes D E J Gen Microbiol, 29 39 1962 no, T, and Lederberg J© Monogr Bio! 13 111, 1964 Iterson, W van Bact Rev, 29 299, 1965 ——— et al, J Cell Biol, 31 585, 1966 Toys, T M, and Stocker, BA D J Gen Microuot, 44 121, 1966 Kakefuda T, et al J Bact, 93 472, 1967 Kerndgc, DI Microbial Reaction to En viconment 11th Symp Soc Gen Mcrobiol ‘ogy Cambridge University Press 1961 Krause, RM Bact Rev, 27 369, 1963 Tee KY, etal Ano Init Pasteur 91 212, Lowy, J, and Hanson, J. Nature, 202 538, 1964 Mandelstam, J Biochem J 84 294 1962 Marmur, J et al Ana Rey Microbiol, 17 329, 1963 Marr, HG Ann Rev Microbiol, 14 241, 1960 Martner, R J, et at J Molec Biol, 28 45, 1967 Mulls, G T, and Smith BE B Fed Proc, 21 1089, 1962 Mitchell, P = Membranes and Surtaces of Cells Biochem Soc Symp 22 142 1963 Morse,S I J Exp Med 116 229 1962 Mune, H, e623) Bigchemusiry, S374 1986 Murray, R G E. 12th Symp Soc Gea Microbiol, 12 119, 1962 Neidhardt, FC ‘Ann Rey Microbiol, 17 61, 1963 Nikaido H Bacterial Cell Wall—Deep Lay- ers In The Specificity of Cell Surfaces Davis BD, and Warren L, eds Englewood Cliffs, NJ, Prentice Hall Inc, 1967 Park J T J Biol Chem, 194 877, 1952 and Stromunger,J L Scrence, 125 99, 1957 Perkins, H R_ Bact Kev, 27 18, 1963 Repashe, R. Biochim Biophys Acta, 30 225, 1958 Robiaow, C F Outhne of the Visible Or ganization of Bacteria In The Cell, 4 45 New York, Academic Press, [960 Rogers, H J Biochem Soc Symp, 22 $4 Cambridge University Press, 1963 Sakon, M RJ dn The Bactena Vo! 3,1 C. Gunsalus and R Y Stanier, eds New York, Academe Press, 1960 J Gen Microbiol, 29 15, 1962 ~—— The Bacterial Cell Wall New York, Elsewer Pub Co. 196456 $7 Sanderson A R, et al Biochem Biophys Res Commun, $ 472 1961 58 Shockman, GD et al 1963 $9 Tipper, D J and Strommger, J L Proc Nat Acad Sci, 54 1133 1965 60 Weibull, C In The Bactena Vol I I C Gonsalus and RY Stanier eds New York, Academic Press 1960 Weidel W and Pelzer H Advances Enzym, 26 193, 1964 J Bact, 85 168 6 Ch. 5, The Genetics of Bacteria 62 et al J Gen Microbiol, 22 158 1960 63 Wicken A J, and Baddiley, J Biochem J, 87 54 1963 64 Wilkinson, J F J Gen Microbiol, 32 171, 1963 65 Wise, EM, Jr, and Park, J T Proc Nat. Acad Sci, 54 75, 1965 66 Work, E. J Gen Microbiol, 25 167, 1961 67 Zinder, N D, and Arndt, W F Proc Nat Acad Ser, 42 $86, 1956 5 The Genetics of Bacteria Although man has speculated on the mechanism of heredity for centuries, the science of genetics dates from the redis- covery of Mendels paper in 1900 It was not unul 1946 (108, 109, 110), however, that mating and recombination were shown exist in the oacteria Prior to that time, inability to make crosses between bac- terial strains made the mterpretation of var ations in bacterial populations very difficult This was especially true before the advent of techniques which permitted the 1solation of pure clones, when it was thought that all bacteria belonged to a few Species that had a temendous capacity for variation (pleo- morphism) General acceptance of the oc currence of variation in fixed species of bac- teria (monomorphism) did not come about until much later (1920-30) There is no doubt today that mutation and selection Account for most bactertal variation (20) The continuity of bacterial characteristics, from generation to generation 1s made pose sible only by the fact that cells have a defi- mite organization which 1s duplicated with each division In higher organisms the visi~ ble nucleus contams thread like structures, the chromosomes, which maintain their in- tegrity from generation to Beneration and are in latge part responsible for the trans- mussion of characteristics Cytologic studies wm bacteria have established beyond doubt the presence of nuclear bodies im bacteria (see Chap 3), and it seems certain that 0 bacteria, as well as in higher organisms, there ts a similar material basis for the inheritance of genes (98) Operationally a gene 1s defined as a factor which can change or mutate to alternative forms, called alleles, which result im a pet manent detectable change This new cbar- acteristic 1s transnutted to progeny cells and 1s distnbuted to other strains according t0 Precise mechanisms when gene transfer 0¢- curs Collectively all the genes of an ot ganism are known as the genome and the Particular assortment of normal and mutant genes in any strain 1s called the genotype ‘The biochemical or morphologic appeat- ance 1s called the phenotype The phenotype 1s the result of the interaction between the genotype and the environment both inside and outside the cell ‘The normal or standard stram 1s usually called the wild type (designated +) In bac- tetia the orginal strain, whether it contatns mutant genes or not, 3s called the proto- troph, while the mutant strains derived fromt wt which require additional nutrients in the medium are called auxotrophs In ths chap- ter auxotrophic genes wall be indicated with @ minus sign (—) Recently, however, ® uniform nomenclature has been proposed for bacterial mutants in which gene loctBacterial Mutations are designated by an stalicized, three letter, lower case symbol which 1s followed by a capital fetter to distinguish the gene from other genes that can mutate to produce the same phenotype (45) Different mutants of the same gene are distinguished by a num- ber following the gene symbol Wherever possible this recommended nomenclature wail be followed im this chapter However, we shall retain the + and — signs to indt- cate 2 prototroph and an auxotroph, respec- tively, far the sake of convenicnee and for the clarity of some discussions In bactena, eg, Escherichia col: and Sal- monella ty plumurium, the genes are \inked together on a circular chromosome com- posed of DNA (deoxyribonucleic acid) (see Fig 9) and exert their influences by con- 57 trolling the type and amount of protein made by the cell (see Chap 7) A gene can also be defined as that umt of DNA which controls the synthesis of a simgle protein or a single polypeptide chain However, the Tecogmition of a specific molecule of DNA as a specific gene has not been achieved Bacteral Mutations Few, if any, genes are completely stable Indeed, the production of new genes by mu- tation provides the raw material for evolu- uon through natural selection A mutation 4s a change mm a gene that results in an al- tered morphologic or biochemical pheno- type which is inherited Mutations occur spontaneously in all organisms (Table 1) Table 1 Spontancous Mutation Rates sn Different Organisms (From Sager and Ryan Cell Hereduty John Wiley & Sons) ‘Oncantsa CuaRacTeR Rate Uns Bactenophage fysis inhibition r~> 7 1x 108 Per gene * ~ T, host range h -> bk 3x 19° per ° teplication Bacteria lactose fermentation lac — Joe 2x 107 Escherichia col. phage T, sensitwity Ty s+ Ty r 2x 108 fastidine requirement hus -> his? 4x 108 Als’ -» has 2x 108 per streptomycin sensitivity tell strso> sind 1x 10° per sted > airs 1x 108 division Algae streptomycin sensitivity Chlamydomonas sies~> sire 1x 108 veihards Fungs Inositol requirement wos — mos” 8x 108 mutant Neurospora adenine requitement ade ~» ade* 4x 108 frequency crassa among asexual spores Com shrunken seeds S-> sl 1x ws Zea mays purple P~> p Ix ios mutant Frat fly yellow body ¥ > y an males 1x 108 frequency Drosophila Y> y m females 1x 108 per ‘melanogaster white eye Ww 4K 10: aemete brown eye Bw> bw 3x 108 rer ‘Mouse piebald coat color S—> s 3x 108 sexual Mus musculus dilute coat color D> d 3x 108 generation Man normal > hemophitic 3x ot Homo sapiens normal <> albino ; x ie : normal» 8 azoguanine resistant x Hparrow eels an normal > 8 azoguanosine resistant 1x 108 per eel tussue culture tren ¥ Correction of the otter mutation rales in ius table to a per gene basis would not change their order of magmiude58 and can have very mild or very severe effects, dependmg upon the cellular process which ss affected If plated on an agar me- dium, a mutant bacterimm will give rise to a clone of cells all of which are mutant These mutant cells will breed true with respect to the new phenotype but with low frequency may mutate further to new mutant types or backmutate to the orpinal parental type (Table 1) The mutation rate is usually ex- pressed as the probability of any one bac- terium or gene changing dunng a defined unit of time, usually one generation or one division time Although the spontaneous mutation rate ss relatively constant for a specific gene in a given strain, mutation rates vary for different genes and for the same gene from stram to strain (10, 66, 99, 125, 184) Indeed, it can be seen in Table 1 that the rate in the forward direction (1e, from wild type to mutant) does not com- cide with the rate in the backward or reverse direction (1¢, from mutant to wild type), some mutants are quite stable and do not show backmutation to wild type Theoretically, if each gene tends to mu- tate at a rate of about 10° bactena per division, and if there are only 200 genes per bactermm (there are probably many more), then in a culture of 10-* bacteria per ml, there will be 100 new mutant genes in the 2x 10° cells per ml found after the next division Most of these mutations, however, will be deleterious, and the cells contamnmg, them will die This potential source of ge- netic variability even in pure cultures should be kept in mind Mutations of different genes occur inde- pendently of one another This is one reason why two or more antibiotics are supphed together during drug thetapy The probabil- uy of a single bacterium having a mutation in two separate genes so that it is resistant 1o two or more drugs at the same time 1s the product of the probabilities that a muta- von for resistance in each gene will occur ‘Thus, sf the rate of mutation to streptomycin resistance 1s 10-* bacteria per generation and that to pemeillin resistance is 10-5 bac- tera per generation, then the probability that a single bacterial cell will mutate to re- sistance for both is 10-* x 10-4 or 10-11 bactena per generation, an extremely small probability (see Chap 8) Ch. 5: The Genetics of Bacteria The spontaneous rate of mutation of a gene can be raised 10 to 100 times by the use of mutagens These are very diverse and include x-rays, garama rays, ultraviolet light, neutrons, alpha particles, various chemicals (methyl mtrosoguamidine, mitrous acid, acri- dine dyes, nitrogen mustards, purine, and pyrimidine analogs), and many other agents (8) Their mechanism of action 1s not known with certainty, and we shall discuss various hypotheses after we have considered the chemical nature of the gene MORPHOLOGIC MUTANTS One method for detecting morphologic mutants consists of spreading normal bac- teria that have been treated with a mutagen on an agar plate in such a way as to cause the growth of many nonoverlapping colo- nies These colonies are examined at inter- vals for morphologic variations differmg from the normal Morphologic mutations are named for the trait affected These include colony sue, shape, texture, and color, where color 1s a part of the colony morphology (102) Many of these, such as the R, S, M, G, and L forms, have been discussed in Chapter 3 It should be noted that not all morphologre changes have been analyzed genetically to determine whether they are im fact due to true gene mutations of to some other agency (20) BIOCHEMICAL MUTANTS While morphologic mutations have use as gene markers, the mutations which affect the nutntion of the bactermm are more useful to bacterial geneticists Many mucro- orgamsms, such as the enteric coliform, Escherichna col, can grow very well on 2 medium containing a simple carbohydrate (€ g, glucose) as a carbon and energy Source, ammonium salts or nitrates as a nitrogen source, and additional salts and minerals From this simple mimimal medium these organisms can synthesize all of the orgamc compounds which they must have to grow and reproduce Since genes control chem- ical pathways by specifying the amount and the structure of the protems of the cell (see Chap 7), st 1s possible to produce a amu: tant strain in which there 1s an absence ofBiochemical Mutants an enzyme catalyzing a reaction in the path- way leading to the synthesis of a vital com- pound (eg, tryptophan) which the cell can usually manufacture This compound (tryptophan) 1s now required as a supple- ment to the munma!l mediwm or the cell will de The early methods used to detect bio-~ chemical mutauons m bactera were very similar to those originally developed by Beadle and Tatum (15) for the detection of biochemical mutations in the bread mold Neurospora crassa Although there are dif ferences m techmique due to the differences between the biolagy of molds and bacterta, this basic method can be summanzed as fol- lows (Fig 1) one stage in the cell cycle of normal cells ss exposed to 2 mutagenic agent, such as ultraviolet ight, x rays, or chemical mutagens The treated cells are then plated on a “complete” medium—that is, a Munimal medium which is supplemented with extracts derived from yeast cells, mam- malian liver, and plants, as well as with mix tures of compounds, such as many of the known biochemucals, vitamins, ammo acids nucterc acid derivatives, cte Each of the col omses of cells which grow on the “complete’ medium are tested on the mma} medium, and any colony which can grow on the mun- amal medium 1s discarded These are not deficient in a synthetic pathway Those bac- teria which grow on the complete medium Cee aay Fig. 1 General method for detecting nutriuonat mutants. 59 but which do not grow on the mmunal medium are saved for further study Many of these are mutants in which the synthesis of a specific compound present in the com- Plete medium does not take place Tests are then carried out to discover the nature of this compound, and the mutant 1s named for the required nutnent For example, a mutant stra which requites tryptophan 4s designated érp~ (tryptophan requitmg) while a strain which requires nicotine acid 3s nic~ (nicotinic acid requiring) (45) Other types of biochemical mutations are those which, unlike the wild type, cannot use Various sugars, such as lactose, maltose, or galactose, as carbon sources These mutants usually lack the ability to carry out a step an the pathway leading to the conversion of these sugars into intermediates im glycolysis These mutations are named according to the sugar which cannot be fermented For ex- ample, @ strain which cannot utilize maltose as designated mal while a stram which can not utshze lactose 1s designated lac~ Note that here the mutant is named for the sub- strate which cannot be metabolzed, while those discussed above were named for the particular product which could not be syn- thesized In most, sf not all, of these mutants, the phenotype 1s due to an effect on an enzyme so it no longer functions normally The resulting block im the metabolic path- way results im the observed nutritional phenotype In the absence of special techmques (Table 2} morphologic mutants are more easily recognized than biochemical mutants However, selection techmques can be de- vised sa that biochemical mutations can be detected with much greater ease than ts ever achieved with mutations that primarily af- fect morphology Even so, many inherited changes occur which remain overlooked be- cause methods for detecting them directly are not available This 1s especially true of genes which modify the chemistry of the organism so greatly that these mutants can- not be kept alive by some external manipu- Jaton, such as supplying a necessary chem- xcal or changing the pH or temperature Such mutant genes do irreparable damage to the cell, and the cell dies The mutants whieh we will discuss in this chapter are mamly those 11 which a change in the gene