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Prelims.indd i 5/15/2012 10:20:01 AM
 Textbook of

Microbiology
and
Immunology

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 “This page intentionally left blank"

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 Textbook of

Microbiology
and
Immunology
2nd Edition

Subhash Chandra Parija

MBBS, MD, PhD, DSc, FRCPath
FAMS, FICPath, FICAI, FABMS, FISCD, FIAVP, FIATP, FIMSA
Professor and Head
Department of Microbiology
Jawaharlal Institute of Postgraduate Medical Education and Research
Puducherry, India

ELSEVIER
A division of
Reed Elsevier India Private Limited

Prelims.indd iii 5/15/2012 10:20:02 AM

 Textbook of Microbiology and Immunology, 2/e
Parija

ELSEVIER
A division of
Reed Elsevier India Private Limited

Mosby, Saunders, Churchill Livingstone, Butterworth-Heinemann and

Hanley & Belfus are the Health Science imprints of Elsevier.

© 2012 Elsevier
First Edition 2009
Second Edition 2012

All rights are reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by
any means, electronic, mechanical, photocopying, recording, or otherwise without the prior permission of the publisher.

ISBN: 978-81-312-2810-4

Medical knowledge is constantly changing. As new information becomes available, changes in treatment, procedures, equipment
and the use of drugs become necessary. The author, editors, contributors and the publisher have, as far as it is possible, taken care to
ensure that the information given in this text is accurate and up-to-date. However, readers are strongly advised to confirm that the
information, especially with regard to drug dose/usage, complies with current legislation and standards of practice. Please consult
full prescribing information before issuing prescriptions for any product mentioned in this publication.

Published by Elsevier, a division of Reed Elsevier India Private Limited.

Registered Office: 305, Rohit House, 3 Tolstoy Marg, New Delhi-110 001.
Corporate Office: 14th Floor, Building No. 10B, DLF Cyber City, Phase II, Gurgaon–122 002, Haryana, India.

Sr. Commissioning Editor: Shukti Mukherjee Bhattacharya

Managing Editor (Development): Shabina Nasim
Development Editor: Shravan Kumar
Copy Editor: Shrayosee Dutta
Manager Publishing Operations: Sunil Kumar
Manager Production: NC Pant

Typeset by Mukesh Technologies Pvt. Ltd., Pudhucherry, India.

Printed and bound at EIH Unit Ltd. Press, Manesar.

Prelims.indd iv 5/15/2012 10:20:03 AM

 To my father
Late Shri Managovinda Parija
and mother
Late Smt Nishamani Parija

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 “This page intentionally left blank"

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 Preface to the
Second Edition

Like the first edition, the second edition of Textbook of Microbiology and Immunology consists of six sections. Section I and II deal with
general micro biology and immunology, respectively. Section III, IV, and V deal with bacteriology, virology, and mycology, respec-
tively. Lastly, Section VI deals with applied microbiology and includes epidemiology and control of community infections, hospital
infections, antimicrobial chemotherapy, water analysis, and immunization.

Medical microbiology is a rapidly changing and evolving field. The threat of emerging and reemerging pathogens and the changing
epidemiology of known pathogens have made it imperative that we keep abreast of the changes and developments in the field if
we are to efficiently deal with them.

The second edition of the Textbook of Microbiology and Immunology has been revised thoroughly and revamped to include the
latest information in the field of medical microbiology. Special emphasis has been placed on molecular methods of diagnosis,
which have revolutionized the diagnosis of infectious diseases and have made it possible to detect hitherto unknown and unculti-
vable pathogens from clinical specimens. The problem of antimicrobial resistance with respect to emerging mechanisms, changing
epidemiology, and newer ways of detection has been dealt with in detail.

This text aims at not only providing basic information about microbiology and immunology, but also deals with the clinical appli-
cations of microbiology in the study of infectious diseases. In each chapter, key points are highlighted. Clinical case studies have
been included so that students can evaluate their understanding of the various microbes. Photographs and pertinent line diagrams
have been included to aid the learning process. The book has been trimmed down so as to include the necessary information with-
out overburdening the students.

This textbook aims at providing comprehensive information about microbiology and it’s applications for medical students, para-
medical students and workers in the field of infectious diseases. I sincerely hope the book serves this purpose and also helps in
creating interest towards the subject among students.

Readers’ views and suggestions for further improving the book in the coming years are welcome. Suggestions may kindly be
e-mailed at [email protected].

Subhash Chandra Parija

Prelims.indd vii 5/15/2012 10:20:03 AM

Prelims.indd viii 5/15/2012 10:20:03 AM
 Preface to the
First Edition

The intent of the book is to provide an up-to-date information on microbial diseases which are emerging as an important health
problem world wide. This book has been written to provide a comprehensive coverage of basic and clinical microbiology, including
immunology, bacteriology, virology, and mycology, in a clear and succinct manner. The book also intends to provide an accurate
presentation of clinically relevant information to the learners of medical microbiology.

Textbook of Microbiology and Immunology consists of six sections. Section I and II deals with general micro biology and immunology,
respectively. Section III, IV, and V deals with bacteriology, virology, and mycology, respectively. Lastly, Section VI deals with applied
microbiology and includes epidemiology and control of community infections, hospital infections, antimicrobial chemotherapy,
water analysis, and immunization.

Emphasis, throughout the text, is made on the clinical applications of microbiology to study infectious diseases. Cultivation and
identification of each organism along with pathogenesis of diseases, clinical manifestations, diagnostic laboratory tests, treatment,
and prevention and control of resulting infections are thoroughly updated to include most recent advances in the field. Details are
summarized in the tabular format. Clinical cases are provided in most of the chapters. The book is profusely illustrated with line
diagrams and photomicrographs both black & white and color.

I believe this book will be a useful source of comprehensive information for students mainly the undergraduate students of
medicine, allied sciences, and others who are interested in medical microbiology.

I welcome reader’s views and suggestions for further improvement of the book in the future edition. Suggestions may kindly be
e-mailed at [email protected] or at [email protected].

Subhash Chandra Parija

Prelims.indd ix 5/15/2012 10:20:03 AM

 Acknowledgements

I am grateful for the valuable professional help and support provided by the staff at Elsevier, New Delhi, namely, Mr Sumeet
Rohatgi, Mr Vidhu Goel, Ms Shabina Nasim, and Mr Shravan Kumar during a period of last more than one year. It has been really
a wonderful and learning experience while working in particular with Ms Shabina Nasim and Mr Shravan Kumar; their profes-
sional contributions are immense for the development of manuscript to the present book format. I gratefully acknowledge all my
colleagues, friends, and students for their valuable advice, constructive criticism, and assistance in preparation of the manuscript.

I owe special debt of profound gratitude to my mother late Smt Nishamani Parija and father late Shri Managovinda Parija without
whose encouragement the book would not have been possible. I am indeed grateful to my wife Smt Jyothirmayee Parija for all her
support throughout the period of preparation of the manuscript of the book.

It is my pleasure to thank my niece Er Kukumina Parija, son-in-law Er Subhasis Ray, nephew Er Shri Rajkumar Parija, daughter-
in-law Ms Smriti Parija, and daughters, Ms Dr Madhuri Parija, son-in-law Dr Ajay Halder, Ms Er Mayuri Parija and son-in-law
Er Shailesh Nandan for their untiring secretarial help towards the preparation of the manuscript.

Subhash Chandra Parija

Prelims.indd x 5/15/2012 10:20:03 AM

 Contents

Preface to the Second Edition vii 6. Laboratory Identification of Bacteria

Preface to the First Edition ix and Taxonomy 41
Acknowledgements x
Color Photos CP1  Introduction 41
 Identification of Bacteria 41
 Bacterial Taxonomy 45
SECTION I GENERAL MICROBIOLOGY
7. Bacterial Genetics 47
1. History of Microbiology 3
 Introduction 47
 Introduction 3  Chromosomal Substances 47
 Historical Background 3  Mutations 48
 Microorganisms as a Cause of Disease 3  Extrachromosomal DNA Substances 49
 Study of Viruses 6  Transfer of DNA Within Bacterial Cells 50
 Phenomenon of Immunity 7  Transfer of DNA Between Bacterial Cells 51
 Chemotherapeutic Agents 8  Recombination 54

2. Morphology and Physiology of Bacteria 9 8. Genetic Engineering and

 Introduction 9 Molecular Methods 55
 Size of Bacteria 9  Introduction 55
 Microscopy 9  DNA: An Amazing Molecule 55
 Study of Bacteria 12  Genetic Engineering 55
 Structure and Functions of  Nucleic Acid Probes 57
Bacterial Cell Envelope 14  Polymerase Chain Reaction 57
 Growth and Multiplication of Bacteria 21  Recombinant DNA Technology 59
 Bacterial Nutrition 23  Genetically Modified Organisms 60
 Gene Therapy 60
3. Sterilization and Disinfection 24
 Introduction 24 9. Antimicrobial Agents: Therapy
 Definition of Frequently Used Terms 24 and Resistance 61
 Sterilization 24
 Introduction 61
 Disinfection 29
 Mechanisms of Action of
Antimicrobial Drugs 61
4. Culture Media 34  Resistance to Antimicrobial Drugs 64
 Introduction 34  Basis of Resistance 65
 Ingredients of Culture Media 34  Antibiotic Sensitivity Testing 68
 Types of Culture Media 34  Antibacterial Assays in Body Fluids 71

5. Culture Methods 38 10. Microbial Pathogenesis 72

 Introduction 38  Introduction 72
 Methods of Culture 38  Types of Microorganisms 72
 Anaerobic Culture 39  Infection 72

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 xii CONTENTS

 Stages of Pathogenesis of Infections 73 17. Immune Response 134

 Stages of an Infectious Disease 82
 Introduction 134
 Humoral Immunity 134
SECTION II IMMUNOLOGY  Cell-Mediated Immunity 138
 Transfer Factor 142
11. Immunity 85  Immunological Tolerance 142
 Introduction 85 18. Immunodeficiency 143
 Types of Immunity 85
 Introduction 143
12. Antigen 90  Primary Immunodeficiencies 143
 Secondary Immunodeficiencies 147
 Introduction 90
 Determinants of Antigenicity 90 19. Hypersensitivity 149
 Antigenic Specificity 91
 Species Specificity 92  Introduction 149
 Isospecificity 92  Type I (Anaphylactic) Hypersensitivity 149
 Autospecificity 92  Type II (Cytotoxic) Hypersensitivity 152
 Organ Specificity 92  Type III (Immune-Complex) Hypersensitivity 153
 Heterophile Specificity 92  Type IV Delayed (Cell-Mediated)
 Haptens 93 Hypersensitivity 154
 Superantigens 93  Type V (Stimulatory Type) Hypersensitivity 155

13. Antibodies 94 20. Autoimmunity 156

 Introduction 156
 Introduction 94
 Tolerance 156
 Immunoglobulins 94
 Pathogenesis of Autoimmunity 156
 Abnormal Immunoglobulins 100
 Animal Models of Autoimmunity 158
 Autoimmune Diseases 158
14. Antigen–Antibody Reactions 101
 Introduction 101 21. Immunology of Transplantation
 General Features of Antigen–Antibody and Malignancy 161
Reactions 101
 Stages of Antigen–Antibody Reactions 102  Introduction 161
 Types of Antigen–Antibody Reactions 102  Transplant Immunology 161
 Tumor Immunology 164
15. Complement System 116
22. Immunohematology 167
 Introduction 116
 Introduction 167
 The Complement System 116
 ABO Blood Group System 167
 Activation of Complement 116
 Rh Blood Group System 168
 Regulation of Complement System 119
 Blood Transfusion 168
 Biological Effects of Complement 120
 Hemolytic Disease of Newborn
 Deficiency of Complement 121
(Erythroblastosis Fetalis) 169
 Biosynthesis of Complement 121
 ABO Hemolytic Diseases 170
 Quantitation of Complement 121

16. Structure and Function of SECTION III BACTERIOLOGY

Immune System 122
 Introduction 122
23. Staphylococcus 173
 Lymphoid Tissues and Organs 122  Introduction 173
 Lymphatic Circulatory System 124  Staphylococcus 173
 Cells of the Lymphoreticular System 124  Staphylococcus aureus 173
 Major Histocompatibility Complex 131  Coagulase-Negative Staphylococci 181

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 CONTENTS xiii
 Micrococcus 182  Edwardsiella 261
 Planococcus 182  Citrobacter 261
 Stomatococcus 182  Klebsiella 261
 Enterobacter 263
24. Streptococcus and Enterococcus 183  Hafnia 264
 Serratia 264
 Introduction 183
 Proteus 265
 Streptococcus 183
 Morganella 267
 Streptococcus pyogenes 184
 Providencia 268
 Streptococcus agalactiae 191
 Erwinia 268
 Other Hemolytic Streptococci 191
 Viridans Streptococci 192
 Enterococcus 192
32. Salmonella 269
 Introduction 269
25. Pneumococcus 194  Salmonella 269
 Salmonella Gastroenteritis 280
 Introduction 194
 Salmonella Bacteremia 280
 Streptococcus pneumoniae 194

26. Neisseria 201 33. Shigella 281

 Introduction 281
 Introduction 201
 Shigella 281
 Neisseria gonorrhoeae 201
 Neisseria meningitidis 207
 Other Neisseria Species 211
34. Yersinia 286
 Introduction 286
27. Corynebacterium 213  Yersinia pestis 286
 Yersinia enterocolitica 291
 Introduction 213
 Yersinia pseudotuberculosis 292
 Corynebacterium diphtheriae 213
 Other Pathogenic Corynebacterium Species 220
35. Vibrio, Aeromonas, and Plesiomonas 294
 Other Coryneform Genera 221
 Introduction 294
28. Bacillus 222  Vibrio cholerae 294
 Noncholera Vibrios 302
 Introduction 222
 Vibrio mimicus 302
 Bacillus anthracis 222
 Halophilic Vibrios 302
 Anthracoid Bacilli 229
 Other Vibrio Species 303
 Aeromonas 304
29. Clostridium 231  Plesiomonas 304
 Introduction 231
 Clostridium 231 36. Campylobacter and Helicobacter 305
 Clostridium perfringens 232  Introduction 305
 Clostridium tetani 237  Campylobacter 305
 Clostridium botulinum 242  Helicobacter 308
 Clostridium difficile 245  Helicobacter pylori 308
 Other Helicobacter Species 311
30. Nonsporing Anaerobes 247
 Introduction 247 37. Pseudomonas, Burkholderia,
 Anaerobic Cocci 247 and Moraxella 313
 Anaerobic Bacilli 247
 Introduction 313
 Pseudomonas 313
31. Coliforms 251
 Pseudomonas aeruginosa 313
 Introduction 251  Other Pseudomonas Species 318
 Escherichia 252  Burkholderia 318
 Escherichia coli 252  Moraxella 319

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 xiv CONTENTS

38. Haemophilus, Pasteurella, 45. Mycoplasma and Ureaplasma 386

and Actinobacillus 321  Introduction 386
 Introduction 321  Mycoplasma pneumoniae 386
 Haemophilus 321  Genital Mycoplasma Species 391
 Haemophilus influenzae 321  Ureaplasma urealyticum 391
 Other Haemophilus Species 326  Atypical Pneumonia 392
 Pasteurella 327
 Actinobacillus 327 46. Actinomycetes 393
 HACEK Group of Bacteria 328
 Introduction 393
39. Bordetella and Francisella 330  Actinomyces 393
 Nocardia 395
 Introduction 330  Rhodococcus 397
 Bordetella 330  Gordonia and Tsukamurella 398
 Bordetella pertussis 330  Thermophilic Actinomyces 398
 Bordetella parapertussis 334  Tropheryma whippelii 398
 Bordetella bronchiseptica 335  Dermatophilus 398
 Francisella tularensis 335  Oerskovia 398

40. Brucella 338

47. Miscellaneous Bacteria 399
 Introduction 338
 Brucella 338  Introduction 399
 Listeria monocytogenes 399
 Erysipelothrix rhusiopathiae 400
41. Mycobacterium tuberculosis 345  Alcaligenes faecalis 400
 Introduction 345  Chromobacterium violaceum 400
 Mycobacterium tuberculosis 346  Flavobacterium meningosepticum 400
 Calymmatobacterium 400
42. Nontuberculous Mycobacteria 358  Streptobacillus and Spirillum 401
 Streptobacillus moniliformis 401
 Introduction 358  Spirillum minus 401
 Photochromogens 358  Legionella 401
 Scotochromogens 359  Legionella pneumophila 402
 Nonphotochromogens 359  Bartonella 404
 Rapid Growers 360  Capnocytophaga 406
 Gardnerella vaginalis 406
43. Mycobacterium leprae and
Mycobacterium lepraemurium 362
48. Rickettsia, Orientia, Ehrlichia,
 Introduction 362 and Coxiella 407
 Mycobacterium leprae 362
 Mycobacterium lepraemurium 370  Introduction 407
 Genus Rickettsia 407
 Typhus Fever Group 408
44. Treponema, Borrelia, and Leptospira 371
 Rickettsia prowazekii 408
 Introduction 371  Rickettsia typhi 410
 Treponema 371  Spotted Fever Group 411
 Treponema pallidum 371  Rickettsia rickettsiae 411
 Nonvenereal Treponematosis 377  Other Rickettsial Species in the Spotted
 Nonpathogenic Treponemes 378 Fever Group 413
 Borrelia 378  Rickettsia akari 413
 Borrelia recurrentis 378  Genus Orientia 413
 Borrelia vincenti 381  Orientia tsutsugamushi 413
 Borrelia burgdorferi 381  Genus Ehrlichia 414
 Leptospira 381  Genus Coxiella 415
 Leptospira interrogans Complex 382  Coxiella burnetii 416

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 CONTENTS xv

49. Chlamydia and Chlamydophila 418 56. Papovaviruses 469

 Introduction 418  Introduction 469
 Chlamydia 418  Human Papillomaviruses 469
 Chlamydia trachomatis 419  Polyomaviruses 471
 Chlamydophila 423
 Chlamydophila pneumoniae 424 57. Herpesviruses 473
 Chlamydophila psittaci 424  Introduction 473
 Herpes Simplex Virus 473
SECTION IV VIROLOGY  Herpesvirus Simiae: B Virus 479
 Varicella Zoster Virus 479
50. General Properties of Viruses 429  Epstein–Barr Virus 481
 Cytomegalovirus 485
 Introduction 429  Human Herpesvirus 6 487
 Morphology of Viruses 429  Human Herpesvirus 7 488
 Replication of Viruses 433  Human Herpesvirus 8 488
 Viral Genetics 435
 Nomenclature and Taxonomy of Viruses 437 58. Adenoviruses 489
51. Pathogenesis of Viral Infections 440  Introduction 489
 Adenovirus 489
 Introduction 440  Adeno-Associated Viruses 492
 Stages of Viral Infections 440
 Viral Pathogenesis at the Cellular Level 442 59. Parvoviruses 493
 Host Response to Viral Infections 445
 Clinical Manifestations of Viral Diseases 446  Introduction 493
 Epidemiology 447  Parvovirus B19 493

52. Antiviral Agents 449 60. Picornaviruses 496

 Introduction 449  Introduction 496
 Mechanism of Action of Antiviral Drugs 449  Enteroviruses 496
 Classification of Antiviral Drugs 449  Poliovirus 496
 Coxsackieviruses 501
53. Laboratory Diagnosis of Viral Diseases 453  Echoviruses 502
 Other Enteroviruses 503
 Introduction 453
 Rhinoviruses 503
 Methods of Laboratory Diagnosis 453
 Hepatitis A Virus 504
54. Bacteriophages 460
61. Orthomyxoviruses 505
 Introduction 460
 Introduction 505
 Morphology 460
 Influenza Viruses 505
 Life Cycle 460
 Uses of Bacteriophages 462
62. Paramyxoviruses 514
 Bacteriophage Typing 462
 Introduction 514
55. Poxviruses 463  Measles Virus 514
 Parainfluenza Virus 517
 Introduction 463
 Mumps Virus 519
 Variola (Smallpox) Virus 463
 Respiratory Syncytial Virus 521
 Vaccinia Virus 466
 Nipah Virus 522
 Monkeypox 467
 Hendra Virus 522
 Buffalopox 467
 Human Metapneumovirus 522
 Cowpox 467
 Orf 467
63. Reoviruses 524
 Molluscum Contagiosum 468
 Tanapox 468  Introduction 524
 Yabapox 468  Orbiviruses 524

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 xvi CONTENTS

 Coltiviruses 525  Viral Hemorrhagic Fever 586

 Orthoreoviruses 525  Arenavirus 586
 Rotavirus 526  Coronaviruses 588

64. Rhabdoviruses 529

SECTION V MYCOLOGY
 Introduction 529
 Rabies Virus 529
71. Introduction to Mycology 593
 Rabies-Related Viruses 534
 Introduction 593
65. Arboviruses 536  Classification of Fungi 593
 Reproduction of Fungi 593
 Introduction 536  Pathogenesis of Fungal Infection 594
 Important Properties of Arboviruses 536  Laboratory Diagnosis 595
 Roboviruses 537  Antifungal Drugs 596
 Togaviruses 537
 Flaviviruses 539
 Bunyaviridae Viruses 544 72. Superficial, Cutaneous, and
 Reoviridae 546 Subcutaneous Mycoses 597
 Rhabdoviridae 546  Introduction 597
 Ungrouped Arboviruses 546  Superficial Mycoses 597
 Cutaneous Mycoses 598
66. Hepatitis Viruses 547  Subcutaneous Mycosis 600
 Introduction 547
 Hepatitis A Virus 547 73. Systemic Mycoses 603
 Hepatitis B Virus 550
 Introduction 603
 Hepatitis C Virus 555
 Coccidioidomycosis 603
 Hepatitis D Virus 557
 Paracoccidioidomycosis 604
 Hepatitis E Virus 558
 Histoplasmosis 604
 Hepatitis G Virus 559
 Blastomycosis 605
 Cryptococcosis 606
67. Retroviruses 560
 Introduction 560 74. Opportunistic Fungal Infections 608
 Retroviruses 560
 Human T-Lymphotropic Viruses 563  Introduction 608
 Endogenous Retroviruses 565  Candidiasis 608
 Other Oncogenic Viruses 565  Candida albicans 608
 Aspergillosis 610
 Aspergillus Species 610
68. Human Immunodeficiency Virus 566  Zygomycosis 612
 Introduction 566  Pneumocystosis 612
 HIV Virus 566  Pneumocystis jiroveci 613
 Penicilliosis 614
69. Slow Viruses and Prions 578  Penicillium marneffei 614
 Pseudoallescheria boydii Infection 615
 Introduction 578  Fusarium solani Infection 615
 Slow Diseases Caused by Prions 578
 Slow Diseases Caused by Conventional
Viruses in Humans 582 SECTION VI APPLIED MICROBIOLOGY
 Slow Diseases Caused by Conventional
Viruses in Animals 582 75. Normal Microbial Flora 619
 Introduction 619
70. Miscellaneous Viruses 583
 Functions of Resident Flora 619
 Introduction 583  Factors Determining the Colonization by
 Rubella Virus 583 Microbes 619
 Norwalk Virus 585  Normal Flora at Various Sites of the Body 620

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 CONTENTS xvii

76. Bacteriology of Water, Milk, and Air 623 78. Biomedical Waste Management 634
 Introduction 623  Introduction 634
 Bacteriology of Water 623  Types of Biomedical Waste 634
 Bacteriology of Milk 626  Waste Treatment and Disposal 634
 Bacteriology of Air 628
79. Immunoprophylaxis 637
77. Nosocomial Infections 629
 Introduction 637
 Introduction 629  Active Immunization 637
 Factors Affecting Hospital-Acquired  Immunization Schedule 638
Infection 629  Passive Immunization 638
 Epidemiology of Hospital-Acquired Infection 629  Combined Active and Passive Immunization 639
 Diagnosis of Hospital-Acquired Infections 632  Individual Immunization 639
 Prevention and Control of Hospital-Acquired
Infections 632 Index 641

Prelims.indd xvii 5/15/2012 10:20:03 AM

 1
History of Microbiology
population. Some historians believe that this disaster changed
Introduction European culture and prepared the way for the Renaissance.
This is just an example from many such epidemics, which while
Medical microbiology is a branch of microbiology that deals being devastating in their scope spared not even the high and
with the study of microorganisms including bacteria, viruses, mighty of the times.
fungi, and parasites of medical importance that are capable Apart from the bubonic plague, measles (now thankfully
of causing diseases in humans. It also includes the study of extinct) and smallpox too played their roles as epidemic diseases
microbial pathogenesis, disease pathology, immunology, and causing high mortality and morbidity. The first recorded epi-
epidemiology of diseases. demic of smallpox was in the year 1350 BC in Egypt. The dis-
Medical microbiology is among the most widely studied ease was unknown in the population of the New World until
branches of Microbiology. It has given mankind a chance to the Portuguese and Spanish explorers made their appearance.
fight the organisms that, at one point of time, were pure nem- Smallpox then traveled across America, devastating the previ-
esis to us. This has also provided an in-depth knowledge and ously unexposed population. It was already known at that time
in-detail understanding of the nature of pathogens that cause that the disease spreads through the skin lesions and scabs,
disease in humans. This field of microbiology has been the and that survivors of the infection were immune to reinfection
precursor to the wide gamut of immunological innovations in on further exposure. Though adopted much later in America
the field of medical science. This field not only has helped to and Europe, the practice of inoculation or variolation, whereby
develop vaccines against many invading organisms, it has also, people were intentionally exposed to smallpox to make them
in a more holistic way, given mankind a second shot at life. immune, was already being practiced in India, China, and
Deadly and debilitating diseases like smallpox, polio, rabies, Africa for centuries.
plague, etc. have been either eradicated or have become treat-
able now because of the efforts of scientists and researchers in
the field of medical microbiology.
Microbes are the most significant life forms sharing this Microorganisms as a Cause of Disease
planet with humans because of their pervasive presence.
Depending on their food sources, microbes may have either Among various causes, the causes suggested for the occurrence
beneficial roles in maintaining life or undesirable roles in of disease were the effect of supernatural phenomena like plan-
causing human, animal, and plant diseases. These microbes etary alignments and effect of bad bodily humors; the faulty
cause frequent and often severe diseases, such as AIDS, chol- environment was also implicated.
era, tuberculosis, rabies, malaria, etc. The ubiquitous pres- Even before microorganisms were seen, some investigators
ence of microbes in large numbers have given rise to the many suspected their existence and responsibility for disease. Among
mutants, which in part are responsible for emerging diseases others, the Roman philosopher Lucretius (about 98–55 BC) and
such as AIDS, Ebola hemorrhagic fever, and multidrug-resis- the physician Girolamo Fracastoro (1478–1553 AD) suggested
tant tuberculosis (MDRTB). that disease was caused by invisible living creatures. Fracastoro
was much more than an author of the popular poem on syphi-
lis. In his book “De contagione, contagiosis morbis et curatione (On
Historical Background Contagion, Contagious Diseases, and their Treatment),” pub-
lished in 1546, he proposed the revolutionary theory that infec-
Microbial diseases have undoubtedly played a major role in tious diseases are transmitted from person to person by minute
historical events, such as the decline of the Roman Empire invisible particles. He further suggested that infections spread
and the conquest of the New World. In 1347, plague or Black from person to person by minute invisible seeds, or seminaria,
Death struck Europe with a brutal force. By 1351, about 4 years that are self-replicating and act on the humors of the body to
later, the plague had killed one-third of the population (about cause disease. His theories were ahead of their time, and it took
25 million people). Over the next 80 years, the disease has about 200 years for the microscope to be invented and his theo-
struck repeatedly, eventually wiping out 75% of the European ries to be proved.
 4 GENERAL MICROBIOLOGY

Antony van Leeuwenhoek: The Microscopist like and not de novo ( germ theory of disease). Pasteur had not
only resolved the controversy by 1861 but also had shown how
Section I

The first person to observe and describe microorgan- to keep solutions sterile.
isms accurately was an amateur microscopist Antony van Support for the germ theory of disease began to accumulate
Leeuwenhoek (1632–1723) of Delft, Holland. Leeuwenhoek in the early nineteenth century. Agostino Bassi (1773–1856)
earned his living as a draper and haberdasher (a dealer in first showed that a microorganism could cause disease when
men’s clothing and accessories), but spent much of his spare he demonstrated in 1835 that the silkworm disease was due to
time constructing simple microscopes composed of double a fungal infection. He also suggested that many diseases were
convex glass lenses held between two silver plates. His micro- due to microbial infections. In 1845, MJ Berkeley proved that
Chapter 1

scopes could magnify around 50–300 times. It is believed the great potato blight of Ireland was caused by a fungus.
that he may have illuminated his liquid specimens by placing
them between two pieces of glass and shining light on them
◗ Pasteurization
at 45-degree angle to the specimen plane. This would have
provided a form of dark-field illumination and made bacte- Pasteur for the first time demonstrated that he could kill many
ria clearly visible. In 1673, Leeuwenhoek sent detailed letters microorganisms in wine by heating and then rapidly cooling
describing his discoveries to the Royal Society of London. It the wine, a process now called pasteurization. While developing
is clear from his descriptions that he saw both bacteria and methods for culturing microorganisms in special liquid broths,
protozoa. But he did not evaluate these organisms as agents Pasteur discovered that some microorganisms require air,
of disease. specifically oxygen, while others are active only in the absence
of oxygen. He called these organisms as aerobic and anaerobic
organisms, respectively.
Theory of Spontaneous Generation
Section III

There was a considerable controversy surrounding the origin ◗ Vaccination

of microbial pathogens. Some proposed that microorganisms
In 1877, Pasteur studied anthrax, a disease mainly of cattle and
originated from nonliving things by spontaneous generation
sheep. He developed a vaccine using a weakened strain of the
even though larger organisms did not (theory of spontaneous
anthrax bacillus, Bacillus anthracis. He attenuated the culture of
generation). They pointed out that boiled extracts of hay
anthrax bacillus by incubation at high temperature of 42–43°C
or meat would give rise to microorganisms after sometime.
and inoculated the attenuated bacilli in the animals. He dem-
Needham (1713–1781) on the basis of his experiments pro-
Chapter 43

onstrated that animals receiving inoculation of such attenu-

posed that all organic matter contained a vital force that could
ated strains developed specific protection against anthrax. The
confer the property of life to nonliving matter.
success of this concept of immunization was demonstrated
by a public experiment on a farm at Pouilly-le-Fort in the year
Louis Pasteur: Father of Microbiology 1881. In that public demonstration, he vaccinated sheeps,
Louis Pasteur, French Microbiologist, is known as the father goats, and cows with anthrax bacillus attenuated strains, but
of medical microbiology for his immense contributions to the equal numbers of these animals were nonvaccinated. All the
field of medical microbiology. He first coined the term “micro- vaccinated as well as nonvaccinated animals were subsequently
biology” for the study of organisms of microscopic size. Many challenged with a virulent anthrax bacillus culture, after which
of his important contributions are discussed below. only the vaccinated animals survived whereas nonvaccinated
group of animals died of anthrax.
In 1885, he also developed the first vaccine against rabies
◗ Germ theory of disease
in humans that saved millions of human life worldwide.
Many other scientists have contributed to the theory of spon- Pasteur coined the term “vaccine” to commemorate Edward
taneous generation with their experiments, but it was Louis Jenner who used such preparations for protection against
Pasteur (1822–1895) who settled it once for all. Pasteur first smallpox. The Pasteur Institute, Paris and subsequently sim-
filtered air through cotton and found that objects resem- ilar institutions were established in many countries of the
bling plant spores had been trapped. If a piece of cotton was world for the preparation of vaccines and for the study of
placed in a sterile medium after air had been filtered through infectious diseases.
it, microbial growth appeared. Next he placed nutrient solu-
tions in flasks, heated their necks in a flame, and drew them
◗ Control of silkworm disease
out into a variety of curves, while keeping the ends of the necks
open to the atmosphere. Pasteur then boiled the solutions for Following his successes with the study of fermentation,
a few minutes and allowed them to cool. No growth took place Pasteur was asked by the French government to investigate
even though the contents of the flasks were exposed to the air. the cause of pébrine disease of silkworms that was disrupt-
Pasteur pointed out that no growth occurred because dust and ing the silk industry. After several years of work, he showed
germs had been trapped on the walls of the curved necks. If the that the disease was due to a protozoan parasite. The disease
necks were broken, growth commenced immediately. By this was controlled by raising caterpillars from eggs produced by
Pasteur proved that all life even microbes arose only from their healthy moths.
 HISTORY OF MICROBIOLOGY 5

Joseph Lister: The Pioneer of Antiseptics his findings in 1876 briefly describing the scientific method he
followed. In this experiment, Koch injected healthy mice with a

Section I
Indirect evidence that microorganisms are the agents of human material from diseased animals, and the mice became ill. After
disease came from the work of an English surgeon Joseph transferring anthrax by inoculation through a series of 20 mice,
Lister (1827–1912) on the prevention of wound infections. he incubated a piece of spleen containing the anthrax bacil-
Lister, impressed with Pasteur’s studies on the involvement of lus in beef serum. The bacilli grew, reproduced, and produced
microorganisms in fermentation and putrefaction, developed spores. When the isolated bacilli or spores were injected into
a system of antiseptic surgery designed to prevent microorgan- mice, anthrax developed.
isms from entering wounds. Instruments were heat sterilized During Koch’s studies on bacterial diseases, it became nec-

Chapter 1
and phenol was used on surgical dressings and at times sprayed essary to isolate suspected bacterial pathogens. His criteria for
over the surgical area. The approach was remarkably successful proving the causal relationship between a microorganism and
and transformed surgery after Lister published his findings in a specific disease are known as Koch’s postulates.
1867. It also provided strong indirect evidence for the role of
microorganisms in disease because phenol, which killed bacte-
ria, also prevented wound infections. ◗ Koch’s postulates
Koch’s postulates (criteria) were useful to prove the claim that
a microorganism isolated from a disease was indeed causally
Robert Koch: The Founder of Koch’s related to it. A microorganism was accepted as the causative
Postulates agent of infectious disease, only when it satisfied all the follow-
ing criteria (Fig. 1-1):
The first direct demonstration of the role of bacteria in causing
disease came from the study of anthrax by the German physi- 1. The microorganism must be present in every case of the

Section III
cian Robert Koch (1843–1910). Koch used the criteria proposed disease but absent from healthy host.
by his former teacher, Jacob Henle (1809–1885), to establish the 2. The suspected microorganism must be isolated and grown
relationship between B. anthracis and anthrax, and he published in a pure culture from lesions of the disease.

Chapter 43
Isolation of microorganism
from diseased animal

Growth medium
Identification of
microorganism by
microscopy
Isolated microorganism injected
into healthy animal

The disease is reproduced

in healthy animal

Microorganisms are isolated

from infected animal

Identical microorganisms are identified

FIG. 1-1. Koch’s postulates.

 6 GENERAL MICROBIOLOGY

3. The isolated organism, in pure culture, when inoculated Discovery of important bacterial agents
in suitable laboratory animals should produce a similar TABLE 1-1 causing human diseases
Section I

disease. Scientist Bacteria Year

4. The same microorganism must be isolated again in Hansen Mycobacterium leprae 1874
pure culture from the lesions produced in experimental
Koch Bacillus anthracis 1876
animals.
Neisser Neisseria gonorrhoeae 1879
The specific antibodies to the bacterium should be demonstra- Ogston Staphylococcus aureus 1880
ble in the serum of patient suffering from the disease. This was Loeffler Corynebacterium diphtheriae 1884
Chapter 1

an additional criterion that was introduced subsequently.

Fraenkel Streptococcus pneumoniae 1886
Most of the human bacterial pathogens satisfy Koch’s
postulates except for those of Mycobacterium leprae and Weichselbaum Neisseria meningitidis 1887
Treponema pallidum, the causative agent of leprosy and syphilis, Bruce Brucella melitensis 1887
respectively. Both these bacteria are yet to be grown in cell-free Kitasato Clostridium tetani 1889
culture media. Yersin Yersinia pestis 1890

◗ Solid medium for culture of bacteria

Koch pioneered the use of agar as a base for culture media. He (1858–1895), a medical student in Lima, Peru. He man-
developed the pour plate method and was the first to use solid aged to prove that the same organism (later identified to be
culture media for culture of bacteria. This development made Bartonella bacilliformis) caused a chronic skin lesion called ver-
ruga peruana and another serious disease called Oroya fever.
Section III

possible the isolation of pure cultures that contained only one

type of bacterium and directly stimulated progress in all areas This he did by inoculating himself with material from the
of bacteriology. Koch also developed media suitable for grow- warts of the skin lesion. He did develop Oroya fever as he had
ing bacteria isolated from the body. Because of their similarity hypothesized, but the experiment costed him his life when he
to body fluids, meat extracts and protein digests were used as succumbed to the disease. In the subsequent 50 years, numer-
nutrient sources. The result was the development of nutrient ous microorganisms were identified as the causative agents
broth and nutrient agar media that are still in wide use today. of important human diseases and their discovery elucidated
By 1882, Koch had used these techniques to isolate the bacil- (Table 1-1).
Chapter 43

lus that caused tuberculosis in humans. Koch also discovered

that cholera was caused by Vibrio cholerae. He invented the hot
air oven and steam sterilizer, and also introduced methods to Study of Viruses
find out the efficacy of antiseptics. There followed a golden
age of about 30–40 years in which most of the major bacterial In 1892, Dmitri Ivanovsky, a Russian scientist working in
pathogens were isolated. St. Petersburg, demonstrated that the sap of leaves infected
with tobacco mosaic disease retains its infectious properties
◗ Koch’s phenomenon even after filtration through Chamberland filter candles. This
was an important observation, because it provided an opera-
Koch’s phenomenon is a hypersensitivity reaction against tional definition of viruses and also an experimental technique
tuberculosis bacilli demonstrated in guinea pigs. This was first by which an agent could be considered as a virus.
demonstrated by Koch, who showed that guinea pigs already Beijerinck, a Dutch soil microbiologist, showed that the fil-
infected with tubercle bacillus, on challenge with tubercle tered sap could be diluted and then regain its strength after
bacillus or its protein, developed an exaggerated inflammatory replication in living and growing tissue of the plant. The agent
response. could reproduce itself (which meant that it was not a toxin)
but only in living tissues, not in the cell-free sap of the plant.
Self-Experimentation Studies This explained the failure to culture the pathogen outside its
host. All these observations contributed immensely to the dis-
To study diseases in a more elaborate and controlled fashion, covery of an organism smaller than bacteria (a filterable agent)
there were a few dedicated researchers who went to the extremes that is not observable in the light microscope and is able to
of self-experimentation. The discovery that hookworm infes- reproduce itself only in living cells or tissues. Beijerinck called
tation spread by fecal–oral route was first demonstrated by this agent a contagium vivum fluidum, or a contagious living
Arthur Loos in 1898. This was known during his attempts at liquid.
studying Strongyloides stercoralis by swallowing its larvae and The concept of contagium vivum fluidum or a contagious
accidentally swallowing a fecal inoculum with hookworm eggs living liquid began a 25-year debate about the nature of
instead! viruses; whether they were liquids or particles? This conflict
These attempts did not always have happy and produc- was laid to rest when d’Herelle developed the plaque assay in
tive endings as is illustrated by the case of Daniel Carrion 1917 and subsequent development of electron microscopy by
 HISTORY OF MICROBIOLOGY 7
Ruska (1934), when the first electron micrographs of tobacco foot, by traditional tikadars who were invited into home (this
mosaic virus (TMV) were taken in 1939. The viruses were professional niche was later blacklisted by colonial-era medical

Section I
accepted as particles. practitioners).
Loeffler and Frosch (1898) described and isolated the first The method was significantly improved by the English phy-
filterable agent from animals, the foot-and-mouth disease virus sician Edward Jenner in 1798. Jenner was intrigued by the fact
of cattle. Walter Reed and his team in Cuba (1902) recognized that milkmaids who had contracted the mild disease cowpox
the first human filterable virus, yellow fever virus. Landsteiner were subsequently immune to smallpox, a disfiguring and often
and Popper (1909) demonstrated that poliomyelitis was caused fatal disease. He believed that introducing fluid from a cowpox
by a filterable virus and also successfully transmitted the infec- pustule into people (i.e., inoculating them) might protect them

Chapter 1
tion to monkeys. Goodpasture (1930) used chick embryos for from smallpox. To test this idea, he inoculated an 8-year-old
cultivation of viruses. boy with fluid from a cowpox pustule and later intentionally
The term virus (taken from the Latin for slimy liquid or poison) infected the child with smallpox. As predicted, the child did not
was at that time used interchangeably for any infectious agent develop smallpox. Pasteur followed this up with development
and so was applied to TMV and then further to all agents of of vaccines for chicken cholera, anthrax, and rabies. Although
the class. Pasteur proved that vaccination worked, but he did not under-
stand how.
d’Herelle and Twort: Founders of
the Principles of Modern Virology Mechanisms of Immunity
Twort and d’Herelle (1915) independently observed a lytic The experimental work of Emil von Behring and Shibasaburo
phenomenon in bacterial cultures, which they attributed to Kitasato in 1890 gave the first insight into the mechanism of

Section III
viruses. d’Herelle named these viruses as bacteriophages. He immunity. They demonstrated that serum contained elements
developed the use of limiting dilutions with the plaque assay to that protected against infections thus laying the foundation
titer the virus preparation. He suggested that the appearance of for the identification of humoral immunity. In recognition
plaques in the plaque assay show the viruses to be particulate, of this work, von Behring received the Nobel Prize in Medicine
or corpuscular. in 1901.
d’Herelle also demonstrated that the attachment (adsorp- In 1884, even before the discovery that a serum component
tion) of the virus to the host cell is the first step in the pathogen- could transfer immunity, Elie Metchnikoff demonstrated that

Chapter 43
esis of a virus infection. The attachment of a virus occurred only cells also contribute to the immune state of an animal. He
when bacteria sensitive to the virus were mixed with it, demon- observed that certain white blood cells, which he termed phago-
strating the host range specificity of a virus at the adsorption cytes, were able to ingest (phagocytose) microorganisms and
step. He described the process of cell lysis and subsequently the other foreign material. Noting that these phagocytic cells were
release of infectious virus particles. He developed many other more active in animals that had been immunized, Metchnikoff
techniques that are still used in virology. d’Herelle was in many hypothesized that cells, rather than serum components, were
ways one of the founders of the principles of modern virology. the major effector of immunity. The active phagocytic cells
identified by Metchnikoff were most likely blood monocytes
and neutrophils.
Phenomenon of Immunity
Specificity of the Antibody Molecule
The earliest written reference to the phenomenon of immunity
One of the greatest enigmas facing early immunologists was
can be traced back to Thucydides, the great historian of the
the specificity of the antibody molecule for foreign material
Peloponnesian War. Describing a plague in Athens, in 430 BC,
or antigen. Following theories were proposed to explain this
he wrote that only those who had recovered from the plague
mechanism of specificity:
could nurse the sick because they would not contract the
disease a second time. 1. The selective theory: The earliest conception of the
The earliest known smallpox inoculation took place in selective theory dates to Paul Ehrlich in 1900. In the
China, perhaps as early as the fifth century AD. The Chinese 1930s and 1940s, the selective theory was challenged by
method was reported to the Royal Society by an English mer- various instructional theories, in which antigen played a
chant, John Lister, during early 1900s. A Jesuit priest, Father central role in determining the specificity of the antibody
d’Entrecolles, provided details of the method, which he said molecule.
was to collect scabs from the pustules and blow a powder made 2. The instructional theory: According to the instructional
from them into an infant’s nose. The scabs or a thread coated theories, a particular antigen would serve as a template
with the pus could be stored, but the operation was usually around which the antibody would fold. This concept was
done face-to-face with a sick patient. The same method was first postulated by Friedrich Breinl and Felix Haurowitz in
used in Japan beginning in 1747. In precolonial India, a tika the 1930s and redefined in the 1940s in terms of protein
or dot would be made on a child, usually on the sole of the folding by Linus Pauling.
 8 GENERAL MICROBIOLOGY

3. The clonal selection theory: The instructional theories the late nineteenth century into the 1930s. Early in the 1920s,
were formally disproved in the 1960s, during which there was an excitement about the potential of the newly
Section I

information was beginning to appear regarding the struc- identified phage viruses to kill bacteria. The discovery of an
ture of DNA, RNA, and protein. These information offered antibacterial factor in the exudates of the fungus Penicillium by
new insights into the vexing problem of how an individ- Sir Alexander Fleming at St. Mary’s Hospital in 1928 was there-
ual could make antibodies against almost anything. In fore not totally unexpected. He accidentally discovered that
the 1950s, selective theories resurfaced as a result of new a substance produced by the fungus destroyed the pyogenic
experimental data and through the pioneering contribu- bacteria, staphylococci. This initiated the beginning of the anti-
tions of Niels Jerne, David Talmadge, and F Macfarlane biotics era. Other similar antibiotics were discovered in rapid
Chapter 1

Burnet, who refined into a theory that came to be known succession. The sulfonamide drugs discovered subsequently
as the clonal selection theory. offered cures for a wide range of bacterial infections.
Introduction of antibiotics in medicine raised a lot of expec-
tations among both doctors and patients. Certain terrifying
Chemotherapeutic Agents infectious diseases (e.g., rheumatic fever, syphilis, pneumonia,
tuberculosis) and unpleasant skin conditions (e.g., carbuncles)
Until the 1930s, there had been no chemical treatment avail- became easily treatable, and their disappearance appeared to
able to fight bacterial infections in general. Prevention was the be certain. Surgeons could risk more dangerous operations
main means of protecting patients, and an obsession with the and the use of drugs that compromised immune systems.
threat of germs and the moral responsibility to avoid infection Patients who had once turned to many kinds of alternative
was deeply instilled in Western cultures. At the same time, there medicine, or refused treatment, now entrusted themselves to
were repeated hopes for a wonder drug. Louis Pasteur’s pupil antibiotics.
Section III

Paul Vuillemin coined the term “antibiosis” in 1889 to denote a Medical uses of antibiotics on human patients were harder
process by which life could be used to destroy life. to limit—even though, from the 1940s, the fear that public
Paul Ehrlich was an exceptionally gifted histological chemist enthusiasm would promote the selection of resistant strains
and invented the precursor technique to Gram-staining of bac- did lead—to some constraints. In Britain, the Penicillin Act of
teria. He demonstrated that dyes react specifically with various 1948 was explicitly intended to, for the first time, limit through
components of blood cells and the cells of other tissues. He prescription the public’s access to a drug that was not a poison.
began to test the dyes for therapeutic properties to determine Nonetheless, during the 1950s, a penicillin-resistant strain of
Chapter 43

whether they could kill the pathogenic microbes. He developed staphylococcus aureus termed 80/81 infected hospitals and mater-
Salvarsan, an arsenical compound in 1909. The compound nity wards across the world. Newborn babies in hospital crèches
known as “magic bullet” was capable of destroying T. pallidum, were infected and postoperative infections proved common.
the causative agent of syphilis. This treatment proved effective By the late 1990s, although many variants of older drugs
against syphilis. This work was of epochal importance, stim- had been produced, new families of antibiotics were, however,
ulating research that led to the development of sulfa drugs, not being discovered. However, a sustained effort is now being
penicillin, and other antibiotics. He, therefore, is known as the made to develop more effective antibiotics to treat a wide range
father of chemotherapy. of infections.
The field of medical microbiology has been enriched by
contributions of many eminent microbiologists both in the
Antibiotics past and in the recent times. The work of many of them has
The word “antibiotic” did not follow immediately, but the drug been recognized worldwide by the award of Nobel Prizes
pyocyanase, a weakly effective antibiotic, was marketed from (Table 1-2).

TABLE 1-2 List of recent Nobel Prize winners

Year Name/Names of scientists Research done
1993 Kary Mullis Polymerase chain reaction
1996 Peter C Doherty and Rolf M Zinkernagel Cell-mediated immune defenses
1997 Stanley B Prusiner Prion discovery
2005 Barry J Marshall, J Robin Warren Discovery of Helicobacter pylori and its role in gastritis and peptic ulcer
disease
2008 Herald Zur Hausen Discovery of Human Papilloma viruses causing cervical cancer
2008 Francoise Barre Sinoussi, Luc Montagnier Discovery of Human immunodeficiency virus
2011 Ralph M. Steinman Discovery of dendritic cell and its role in adaptive immune response
2011 Bruce A. Beutler, Jules A. Hoffmann Discoveries concerning activation of innate immunity
 Morphology and 432
Physiology of Bacteria
Mycobacterium Leprae
naked eye, because the limit of resolution with the unaided eye
Introduction is about 200 microns. So, the study of bacteria requires the use
of microscopes. A microscope is an instrument that uses one or
All living beings can be classified into three kingdoms: Plant, more lenses to produce a magnified image of an object that is
Animal, and Protista. Microorganisms are a heterogeneous invisible to the unaided eye.
group of several distinct living structures of microscopic size,
classified under the kingdom Protista. The kingdom Protista
includes unicellular organisms, such as bacteria, fungi, proto- Types of Microscopy
zoa, and algae. Based on the differences in cellular organization
and biochemistry, the kingdom Protista has been divided into The following types of microscopy are used for the examina-
three groups: prokaryotes, eukaryotes, and the most recently tion of microorganisms including bacteria:
described archaebacteria.
1. Prokaryotes: Bacteria and blue green algae are prokary- ◗ Light microscopy
otes. Bacteria are unicellular free living organisms having
both DNA and RNA. They are capable of performing all Light microscopy, as the name suggests, uses natural or
essential processes of life, e.g., growth, reproduction, and artificial transmitted light as the source of light. Resolving
metabolism. They do not show any true branching except power of microscope is an important component of light
Actinomycetales, the higher bacteria. Bacteria lack chloro- microscopy. It is the ability of the lens system to distinguish
phyll unlike blue green algae, which contain chlorophyll. two closely placed objects as distinct and separate entities. It
2. Eukaryotes: Fungi, algae other than blue green, protozoa, is dependent on the wavelength of light used to illuminate
and slime moulds are eukaryotes. the object and on the numerical aperture of the microscope.
3. Archaebacteria: These are more closely related to eukary- It is about half of the wavelength of light being used. For
otes than prokaryotes. They however do not include any example, the smallest particle which can be resolved by
human pathogens. yellow light with a wavelength of 0.4 ␮m is about 0.2 ␮m.
Proper use of condenser that focuses light on the plane of the
Differences between prokaryotes and eukaryotes have been object facilitates optimization of the resolving power of the
summarized in Table 2-1. microscope. Resolving power of the microscope is enhanced
further by adjusting the medium through which light passes
between the object and objective lens. The use of immersion oil,
Size of Bacteria whose refractive index is similar to that of the glass, improves
the resolution of the microscope. The numerical aperture of
Bacteria are microscopic and very small in size. The size the microscope is defined as the light gathering power of the
of bacteria is measured in units of length called microns. microscope. Different types of light microscopy include (a)
A micron (micrometer, ␮m) is the unit of measurement used bright-field microscopy, (b) dark-ground microscopy, (c) phase-
in bacteriology. contrast microscopy, and (d) interference microscopy.
1 micron (␮m) ⫽ 1/1000 millimeter (mm). 1. Bright-field microscopy: Bright-field microscopy (always
1 nanometer (nm) ⫽ 1/1000 micron (␮m). referred to as ordinary light microscopy) is the most common
1 Angstrom unit (A⬚) ⫽ 1/10 nm (nanometer). form of light microscopy that uses a compound light micro-
Bacteria of medical importance measure 2–5 ␮m (length) scope. A compound light microscope primarily consists of a
⫻ 0.2–1.5 ␮m (width). compound lens system that contains a number of objective
lenses, such as lenses of low power (⫻10), high power (⫻40),
and oil immersion (⫻100). It also contains a fixed ocular (eye
Microscopy piece) lens, usually of ⫻10 or ⫻5. Final magnification of an
object is the multiplication of lens power of the objective with
Microscopy is an important component of diagnostic micro- that of the eye piece (Fig. 2-1). The bright-field microscopy has
biology. Bacteria being very small cannot be visualized by the many uses.
 10 GENERAL MICROBIOLOGY

Differences between prokaryotic and Eye

TABLE 2-1
eukaryotic cells
Section I

Structure Prokaryotes Eukaryotes

Ocular lens
Nucleus
Nuclear membrane Absent Present
Nucleolus Absent Present
Chromosome One, circular More than one,
linear Objective lens
Chapter 2

Location Free in the cytoplasm, Contained in

attached to mesosomes membrane bound
structure
Specimen
Replication Binary fission Mitotic division
Extrachromosomal Plasmid Inside the
DNA mitochondria
Condenser lens
Cytoplasm
Cytoplasmic organelles Absent Present
like mitochondria,
Golgi apparatus, and Source of light
endoplasmic reticulum
Cytoplasmic streaming Absent Present FIG. 2-1. Principle of compound light microscope.
Section III

Lysosomes Absent Present

Eye
Ribosomes—protein 70S (50S ⫹ 30S), free 80S (60S ⫹ 40S),
production site in cytoplasm or bound attached to rough
to cell membrane endoplasmic
reticulum Ocular lens

Chemical composition
Cell wall Present Absent, except for
Chapter 43

fungi that have

chitinous cell wall
Sterols Absent Present Objective lens
Only light reflected
Muramic acid Present Absent by specimen enters
Energy production site Electron transport Within the objective lens
chain located in the cell membrane bound Specimen
membrane mitochondria

■ It may be used to examine either wet films or “hanging drop” Condenser lens
Opaque disc
for demonstration of the motility of flagellated bacteria
(e.g., Escherichia coli, Pseudomonas aeruginosa, etc.) and protozoa
(e.g., Trichomonas vaginalis, Giardia intestinalis, etc.). The wet prep- Source of light
aration is also useful for demonstration of microorganisms in
urine or feces, and also for detection of fungi in the skin. FIG. 2-2. Principle of dark-ground microscopy.
■ It is useful for demonstration of the structural details.
■ It is also useful for measuring approximate size of the bacte-
ria, fungi, and protozoa in stained preparations. appear brightly stained against a dark background. The dark-
ground microscopy has following uses:
2. Dark-ground microscopy: The dark-ground microscopy
■ It is useful for demonstration of very thin bacteria (such as,
makes use of dark-ground microscope, a special type of
spirochetes) not visible under ordinary illumination, since
compound light microscope. The dark-field condenser with
the reflection of the light makes them appear larger. This
a central circular stop, which illuminates the object with
is a frequently used method for rapid demonstration of
a cone of light, is the most essential part of the dark-ground
Treponema pallidum in clinical specimens.
microscope. This microscope uses reflected light instead
■ It is also useful for demonstration of motility of flagellated
of transmitted light used in the ordinary light microscope
bacteria and protozoa.
(Fig. 2-2). It prevents light from falling directly on the objective
lens. Light rays falling on the object are reflected or scattered 3. Phase-contrast microscopy: Phase-contrast microscopy
onto the objective lens with the result that the microorganisms makes use of a specific optical system that converts differences
 MORPHOLOGY AND PHYSIOLOGY OF BACTERIA 11
Eye
Observer

Section I
Ocular lens Fluorescent light

Light wave
splitting mirror
Diffraction plate
Undiffracted light
unaltered by specimen Exciter filter
Objective lens

Chapter 2
Refracted or diffracted
light by specimen
Excitation light Light
source
Specimen

Excitation light
Condenser lens
Annular diaphragm

Source of light Fluorochrome stained

microorganisms present
in the specimen
FIG. 2-3. Principle of phase-contrast microscopy.

Section III
FIG. 2-4. Principle of fluorescence microscopy.
in phase in an organism into differences in intensity of light
thereby producing light and dark contrast in the image (Fig. 2-3). ■ It is used for direct demonstration of antigen of a patho-
The optical system includes a special condenser and objec- gen in clinical specimens by direct fluorescent antibody test
tive lens which can be fitted to an ordinary light microscope (e.g., direct detection of Neisseria gonorrhoeae, Corynebacterium
to convert it into a phase-contrast microscope. The phase-con- diphtheriae, etc. directly in clinical specimens).
trast microscopy has following uses: ■ It is also used for the estimation of antibodies in the serum

Chapter 43
■ It is immensely useful for examination of living micro- by indirect fluorescent antibody test (IFA) (e.g., IFA in lepto-
organisms particularly protozoa (e.g., T. vaginalis, Entamoeba spirosis, syphilis, brucellosis, etc.).
histolytica, etc.)
■ It is useful for examining the internal structures of a living ◗ Electron microscopy
cell by improving the contrast and differentiating structures Electron microscopy utilizes a beam of electrons instead of a
within the cell that differs in their thickness and refractive beam of light used in the light microscopy. The electron beam
index. is focused by electromagnets, analogous to the lenses used in
4. Interference microscopy: This is another specialized appli- the light microscopy. The object to be examined is kept on the
cation of light microscopy used for demonstrating cell organ- path of the beam that scatters the electrons and produces an
elles. It is also useful for quantitative measurement of the image which is focused on a screen (Fig. 2-5).
chemical constituents of the cells, such as proteins, lipids, and The resolving power of the electron microscope is extremely
nucleic acids. high, theoretically 100,000 times than that of a light micro-
scope. This is because the electron microscope uses electrons
◗ Fluorescence microscopy whose wavelength is approximately 0.005 nm as compared to
5000 nm wavelength of the visible light. As mentioned earlier,
Fluorescence microscopy is based on the principle that the spec- the resolving power is half of the wavelength. In practice, the
imens stained with fluorescent dye when exposed to ultraviolet resolving power of the electron microscope, however, is about
light result in emission of longer wavelength of light (i.e., vis- 0.1 nm. There have been many developments in electron
ible light) (Fig. 2-4). The bacteria stained with fluorescent dye microscopy that include (a) shadow casting, (b) scanning elec-
appear as a brightly glowing object against a dark background. tron microscopy, (c) immunoelectron microscopy, and (d)
Fluorescence microscopy needs a fluorescence microscope freeze-etching, etc.
fitted with an ultraviolet light source. Auramine O, acridine orange,
and rhodamine are fluorescent dyes used to visualize bacteria. ■ Shadow casting is an important technique that is carried out
The resolving power of a fluorescence microscope is increased by depositing a thin layer of platinum or other metal on
due to the short wavelength of ultraviolet light. Auramine O, the microorganism to be examined. This platinum-coated
acridine orange, and rhodamine are fluorescent dyes used to organism, on bombardment with electron beams, scatters
visualize bacteria. Fluorescence microscopy is widely used in the electron and produces an image that is focused on a
diagnostic microbiology in the following ways: fluorescent screen.
 12 GENERAL MICROBIOLOGY

V Differences between electron

TABLE 2-2
A microscope and light microscope
Section I

C Electron gun
U Characteristics Light microscope Electron microscope
U
Cathode M Source Visible light Electron beam
ray tube for Medium of Air High vacuum
viewing
transmission
Nature of lens Glass Electromagnet
Condenser Focusing mechanism Lens position Current to the magnetic
Cathode lenses
Chapter 2

ray tube for is adjusted lens is adjusted

Scanning
photography coil mechanically
Scanning
circuit Changing the Switch the objective Adjust the current to
Primary magnification lens the magnetic lens
electrons
Source of contrast Differential light Scattering of electrons
Detector
Photo multiplier absorption
Secondary
electrons Specimen mount Glass slide Metal grid

VACUUM Specimen Best resolution 0.2 ␮m 0.5 nm

Specimen Highest practical 1000–1500 Over 100,000
holder magnification
FIG. 2-5. Principle of electron microscopy. Affordability Cheaper Expensive
Section III

■ Scanning electron microscopy is another development that Study of Bacteria

provides a three-dimensional image of the object as well as
high resolution. Bacteria can be studied either in unstained (wet mount) or
■ Immunoelectron microscopy is a method to enhance sensi- stained preparation.
tivity and specificity by reacting the specimen with specific
■ The wet mount preparation is useful for demonstration of
antiviral antibody that results in clumping of viral particles.
motility of bacteria by light microscopy or demonstration of
Chapter 43

In this method also, antibody may be conjugated with gold

spirochetes by dark-ground microscopy.
to visualize and determine the location of specific antigenic
■ Stained preparations are used to demonstrate structural
determinants in a specimen.
details of the bacteria by producing color contrast.
■ Freeze-etching is the method by which live organisms can be
visualized unlike in traditional methods of electron microscopy
in which living cells cannot be examined. This method is use-
Staining Methods
ful for the study of cellular ultrastructure of the microorgan- The common staining techniques used in diagnostic microbi-
isms in the living state. This method is based on rapid cooling ology are discussed below:
of specimens by deep-freezing in liquid gas and the subsequent
formation of carbon platinum replica of the specimen. ◗ Simple stains
Electron microscope is widely used for: Methylene blue and basic fuchsin are the simple stains that
provide color contrast but impart the same color to all the
■ Rapid detection of viruses directly in clinical specimens. This bacteria in a smear.
is especially useful for detection of noncultivable viruses.
■ Ultrastructural study of various microorganisms. ◗ Negative staining
Differences between electron microscope and light microscope Bacteria are mixed with dyes, such as Indian ink that produces
are summarized in Table 2-2. a uniform dark-colored background against which the
unstained organisms stand out in contrast. This is used for
demonstration of bacterial capsule that are usually not stained
◗ Newer microscopic methods by simple stains. India Ink method for demonstration of the
These include the following: fungus Cryptococcus neoformans is a common example.
■ Confocal microscopy: This is useful to obtain high
resolution images and for three dimensional reconstruction ◗ Impregnation stains
of biological models. Cells and structures that are too thin to be visualized by
■ Scanning probe microscopy: This measures surface fea- the light microscope can be rendered visible by impregna-
tures by moving a sharp probe over the object’s surface. tion of silver on their surface. Silver impregnation method
There are two types of scanning probe microscope: (a) scan- is a common method used for staining spirochetes, such as
ning tunneling microscope and (b) atomic force microscope. T. pallidum, Leptospira, Borrelia, etc.
 MORPHOLOGY AND PHYSIOLOGY OF BACTERIA 13

◗ Differential stains Acid fastness is due to the (a) high content of lipids, fatty
acids, components of mycolic acid, and (b) higher alcohols

Section I
Differential stains impart different colors to different bacteria or
found in the cell wall of the Mycobacterium. Acid fastness also
different bacterial structures. The commonly used differential
depends on integrity of the cell wall. The ZN smear is best
stains include Gram stain, acid-fast stain, and Albert’s stain.
used for staining:
Gram stain: Gram stain was devised by Christian Gram, a Danish
microbiologist, in 1884, as a convenient method for classifying bac- ■ M. tuberculosis, M. leprae, Nocardia, Actinomyces, and
teria. The Gram staining method essentially consists of four steps: ■ Oocysts of intestinal coccidian parasites (such as Cryptospo-
ridium, Cyclospora, Isospora, etc.).
(i) Primary staining with basic dyes, such as methyl violet,

Chapter 2
crystal violet, etc. Albert’s stain: Albert’s stain is used for staining the volutin
(ii) Application of mordant in the form of dilute solution of iodine. granules of C. diphtheriae. These granules have an affinity for
(iii) Decolorization with ethanol, acetone, or aniline. basic dyes and are called metachromatic granules. These gran-
(iv) Counterstaining with acidic dyes, such as carbol fuchsin, ules are stained bluish-black against green protoplasm on
safranine, or neutral red. staining by Albert’s stain.

Key Points Shape of Bacteria

Gram staining is an essential procedure that is used in the
identification of bacteria. The stain differentiates bacteria into Depending on their shape, bacteria are classified into several
two broad groups: types (Fig. 2-6):
■ Gram-positive bacteria are those that resist decoloriza- 1. Cocci: The cocci (kokkos, berry) are oval or spherical cells.

Section III
tion and retain the primary dye-iodine complex, appear- These may be arranged in pairs (e.g., pneumococci, menin-
ing violet. They have a relatively thick amorphous wall and gococci, and gonococci), tetrads (micrococci), chains (e.g.,
more acidic protoplasm which are believed to retain the
basic violet dye and iodine complex within the cell.
streptococci), and clusters (e.g., staphylococci).
■ Gram-negative bacteria are decolorized by organic sol- 2. Bacilli: The bacilli (bacillus, rod) are rod shaped. These bacilli
vents and take counterstain, appearing red. The decoloriz- may show either of the following arrangement:
ing agent, such as acetone or ethanol, used during staining
disrupts this membran-ous envelope, and the dye and (a) Coccobacilli: Length of the bacteria is approximately the same
iodine complex is washed out of Gram-negative bacteria.

Chapter 43
as its width, e.g., Brucella.
(b) Streptobacilli: These are arranged in chains, e.g., Streptobacillus.
There are certain groups of bacteria, such as Mycobacterium tuber- (c) Comma shaped: They exhibit curved appearance, e.g., Vibrio.
culosis and Mycobacterium leprae, that cannot be considered typi- (d) Spirilla: They exhibit rigid spiral forms, e.g., Spirillum.
cal Gram-negative or Gram-positive bacteria. This is because
3. Spirochetes: Spirochetes (spira, coil; chaite, hair) are slender,
these bacteria either do not take up the Gram stain or they
flexuous spiral forms, e.g., Treponema.
have a different type of envelope. These mycobacteria possess a
waxy envelope containing complex glycolipids that make them 4. Actinomycetes: Actinomycetes (actin, ray; mykes, fungus) are
impervious to the Gram stain. Gram stain also provides useful branching filamentous bacteria resembling fungi. They possess
information on the structure of bacterial cell envelope. a rigid cell wall.
Acid-fast stain: The acid-fast stain was discovered by Ehrlich,
who found that after staining with aniline dyes, tubercle bacilli
resist decolorization with acid. The method, subsequently, was
modified by Ziehl and Neelsen, hence is widely known as Ziehl–
Neelsen (ZN) stain.
Coccus Coccobacillus Bacillus

Key Points
Acid fast staining method consists of following methods:
■ Fixed smears are first stained by a strong carbol fuchsin
with the application of heat. Heating facilitates entry of Spirochete Fusiform bacillus Vibrio
phenolic carbol fuchsin stain into the bacteria.
■ It is then decolorized with 5–20% (depending on the bacte-
ria to be stained) sulfuric acid.
■ It is then counterstained with a contrasting dye, such as meth-
ylene blue. The acid-fast bacilli (AFB) retain the red color of
carbol fuchsin and appear bright red in stained smears. Pus
cells and epithelial cells present in the smear, on other hand, Spirillum
take up the blue color of the counterstain and appear blue.
FIG. 2-6. Different morphological types of the bacteria.
 14 GENERAL MICROBIOLOGY

cell wall consists primarily of teichoic and teichuronic acids.

Structure and Functions of These two components account for up to 50% of the dry weight
Section I

Bacterial Cell Envelope of the wall and 10% of the dry weight of the total cell.
1. Teichoic acids: Teichoic acids are polymers of polyribitol
The outer layer or cell envelope provides a structural and physi- phosphate or polyglycerol phosphate containing ribitol and
ological barrier between the protoplasm (inside) of the cell and glycerol. These polymers may have sugar or amino acid substi-
the external environment. The cell envelope protects bacteria tutes, either as side chain or within the chain of polymer.
against osmotic lysis and gives bacteria rigidity and shape. The Teichoic acids are of two types—wall teichoic acid (WTA) and
cell envelope primarily consists of two components: a cell wall lipoteichoic acids (LTA). They are connected to the
Chapter 2

and cytoplasmic or plasma membrane. It encloses the proto- peptidoglycan by a covalent bond with the six hydroxyl of
plasm, which consists of (i) cytoplasm, (ii) cytoplasmic inclu- N-acetylmuramic acid in the WTA and to plasma membrane
sions (mesosomes, ribosomes, inclusion granules, vacuoles), lipids in LTA.
and (iii) a single circular DNA (Fig. 2-7).

Cell Wall Key Points

Teichoic acids have many functions:
Prokaryotic cells almost always are bounded by a fairly rigid
■ They constitute major surface antigens of those Gram-
and chemically complex structure present between the cell
positive species that possess them. In Streptococcus pneu-
membrane and capsule/slime layer called the cell wall. moniae, the teichoic acids bear the antigenic determinants
Peptidoglycan is the main component of the cell wall and is called Forssman antigen. In Streptococcus pyogenes, LTA is
responsible for the shape and strength of the cell. It is a disac- associated with the M protein that protrudes from the cell
Section III

charide and contains two sugar derivatives—N-acetylglucos- membrane through the peptidoglycan layer. The long M
protein molecules together with the LTA form microfibrils
amine and N-acetylmuramic acid—joined together by short
that facilitate the attachment of S. pyogenes to animal cells;
peptide chains. N-acetylmuramic acid carries a tetrapeptide ■ They are also used as antigen for serological classification
side chain consisting of D- and L-amino acids (D-glutamic acid of bacteria;
and L-alanine) with mesodiaminopimelic acid (Gram-negative ■ They serve as substrates for many autolytic enzymes;
bacteria) or L-lysine (Gram-positive bacteria). Tetrapeptide side ■ They bind magnesium ion and may play a role in supply of
this ion to the cell;
chains are interconnected by pentaglycine bridges. Most
Chapter 43

■ They play a role in normal functioning of the cell wall and

Gram-negative cell walls lack interpeptide bridge. Cell wall provide an external permeability barrier to Gram-positive
provides shape to the cell and protects bacteria from changes bacteria; and
inosmotic pressure, which within the bacteria cell measures ■ Membrane teichoic acid serves to anchor the underlying cell
5–20 atmospheres. membrane.
Bacterial cells can be classified into Gram-positive or
Gram-negative based on the structural differences between
2. Teichuronic acid: Teichuronic acid consists of repeat units
Gram-positive and Gram-negative cell walls. The cell walls of
of sugar acids (such as N-acetylmannuronic or D-glucuronic
the Gram-positive bacteria have simpler chemical structures
acid). They are synthesized in place of teichoic acids when
compared to Gram-negative bacteria.
phosphate supply to the cell is limited.
Gram-positive cell wall also contains neutral sugars (such
◗ Gram-positive cell wall as mannose, arabinose, rhamnose, and glucosamine) and
The Gram-positive cell wall is thick (15–80 nm) and more acidic sugars (such as glucuronic acid and mannuronic acid),
homogenous than that of the thin (2 nm) Gram-negative cell which occur as subunits of polysaccharides in the cell wall.
wall. The Gram-positive cell wall contains large amount of
peptidoglycan present in several layers that constitutes about ◗ Gram-negative cell wall
40–80% of dry weight of the cell wall (Fig. 2-8). The Gram-positive
The Gram-negative cell wall is much more complex than
the Gram-positive cell wall. Peptidoglycan content in the
Single circular super- Gram-negative cell wall is significantly less than the Gram-
coiled chromosome positive cell wall. Only 1–2 layers of peptidoglycan (2–8 nm)
Ribosome Plasmid Flagellum are present just outside the cell membrane. The Gram-negative
cell wall outside the peptidoglycan layer contains three main
components—(a) lipoprotein layer, (b) outer membrane, and (c)
lipopolysaccharides (Fig. 2-9).
Lipoprotein layer: The lipoprotein layer is mainly composed of
Plasma membrane Cell wall
Braun’s lipoprotein. Braun’s lipoprotein is a small lipoprotein
that is covalently joined to the underlying peptidoglycan and
FIG. 2-7. Schematic diagram of structure of a bacteria. embedded in the outer membrane by its hydrophobic end.
 MORPHOLOGY AND PHYSIOLOGY OF BACTERIA 15

Section I
Lipoteichoic acid

Peptidoglycan
Cell wall
Teichoic acid

Chapter 2
Cytoplasmic
membrane

Structural and
enzymatic proteins

FIG. 2-8. Schematic diagram of the cell wall of the Gram-positive bacteria.

Lipopolysaccharide

Section III
Porin proteins (Pore)

Outer membrane

Lipoprotein

Chapter 43
Periplasmic space Nutrient-binding protein
Peptidoglycan

Cytoplasmic membrane Carrier protein

FIG. 2-9. Schematic diagram of the cell wall of the Gram-negative bacteria.

The lipoprotein stabilizes the outer membrane of the Gram- (b) Outer membrane proteins (OMPs): These include the
negative cell wall. following:
Outer membrane: The outer membrane is a bilayered
structure; its inner part resembles in composition with that of ■ Omp C, D, F, and PhoE&LamB are the four major
the cell membrane, while its outer part contains a distinctive proteins of the outer membrane that are responsible
component called lipopolysaccharide. The outer membrane for most of the transmembrane diffusion of maltose
and plasma membrane appear to be in direct contact at many and maltodextrins.
sites in the Gram-negative wall. The outer membrane has a ■ Tsx, the receptor for T6 bacteriophage, is responsible for
variety of proteins as follows: the transmembrane diffusion of nucleosides and some
amino acids.
(a) Porins: The outer membrane has special channels consist- ■ Omp A protein anchors the outer membrane to the
ing of protein molecules called porins. These porins have peptidoglycan layer. It is also the sex pilus receptor in
many functions: F-mediated bacterial conjugation.
■ They permit the passive diffusion of low-molecular The outer membrane also contains proteins that are involved
weight hydrophilic compounds, such as sugars, amino in the transport of specific molecules, such as vitamin B12 and
acids, and certain ions; iron-siderophore complexes; it also contains a limited num-
■ They exclude hydrophobic molecules; and ber of minor proteins, such as enzymes, phospholipases, and
■ They serve to protect the cell. proteases.
 16 GENERAL MICROBIOLOGY

Differences between Gram-positive and

TABLE 2-3 Gram-negative bacteria cell wall
Section I

Characters Gram-positive Gram-negative

cell wall cell wall
O-polysaccharide Thickness 15–80 nm 2 nm
Lipid content 2–5% 15–20%
Teichoic acid Present Absent
Variety of amino acid Few Several
Chapter 2

Core Core
Aromatic amino acid Absent Present
Action as endotoxin No Yes
Sulfur-containing amino acid Absent Present
Lipid A Lipid A
Treatment with lysozyme Protoplast Spheroplast
Smooth form LPS Rough form LPS
(or lipo-oligosaccharide)

Differences between Gram-positive and Gram-negative cell

FIG. 2-10. Structure of a lipopolysaccharide. walls are summarized in Table 2-3.

Periplasmic space
Lipopolysaccharides: Lipopolysaccharides (LPS) are complex Periplasmic space is a distinct space between cell membrane
molecules present in the outer membrane of the Gram-negative
Section III

and outer membrane (innermost layer of Gram-negative cell

bacteria. Structurally, the LPS consist of three main components— wall) in Gram-negative bacteria. This space is filled with a loose
lipid A, the core oligosaccharide, and the O polysaccharide or layer of peptidoglycan matrix. The periplasmic space of Gram-
O-antigen (Fig. 2-10). negative bacteria contains many proteins that participate in
■ Lipid A: This consists of phosphorylated glucosamine disac- nutrient acquisition, and many hydrolytic enzymes, beta-
charide units, to which a number of long-chain fatty acids lactamases binding proteins, and enzymes that participate in
are attached. This also consists of hydroxymyristic acid, a the peptidoglycan synthesis. Polymers of D-glucose, called
unique fatty acid, which is associated with endotoxic activity membrane-derived oligosaccharides, appear to play a role in
Chapter 43

of the LPS. There is a little variation in the structure of the osmoregulation. The periplasmic space is less distinct in Gram-
lipid A among different species of the Gram-negative bacte- positive cell walls.
ria. However, it remains the same within the bacteria of the
same species. ◗ Cell wall of acid-fast bacilli
■ Core oligosaccharide: The core oligosaccharide includes two The cell wall of acid-fast bacilli, such as M. tuberculosis, contains
characteristic sugars—ketodeoxyoctanoic acid (KDO) and large amounts of waxes known as mycolic acids. The cell wall is
a heptose—both joined together by lipid A. This is genus composed of peptidoglycan and an outer asymmetric lipid
specific and similar in all Gram-negative bacteria. Lipoo- bilayer. The inner lipid bilayer contains mycolic acids linked to
ligosaccharides (LOS) are smaller glycolipids. They have an arabinoglycan protein and the outer layer contains other
relatively short, multiantennary (i.e., branched) glycans extractable lipids. This hydrophobic structure renders these
present in bacteria (e.g., Neisseria meningitidis, N. gonorrhoeae, bacteria resistant to many harsh chemicals including deter-
Haemophilus influenzae, and Haemophilus ducreyi) that colo- gents and strong acids. During staining, if dye is introduced
nize mucosal surfaces. They exhibit extensive antigenic and into these cells by brief heating or treatment with detergents,
structural diversity even within a single strain. LOS is an they resist decolorization by sulfuric acid or acid alcohol, and
important virulence factor. Epitopes on LOS have a termi- are therefore called acid-fast organisms.
nal N-acetyllactosamine (Gal(␤)l-4-GlcNAc) residue, which
is immunochemically similar to the precursor of the human
◗ Atypical forms of bacteria
erythrocyte i antigen. Sialylation of the N-acetyllactosamine
residue in vivo provides the organism with the environmen- Atypical forms of bacteria include (i) cell wall deficient forms,
tal advantages of molecular mimicry of a host antigen and (ii) pleomorphic bacteria, and (iii) involution forms. Many
the biologic masking thought to be provided by sialic acids. agents, such as antibiotics, lysozyme, and bacteriophages inter-
■ O polysaccharide or O-antigen: It is the portion extending fere or inhibit the synthesis of bacterial cell wall components,
outwards from the core. It has several peculiar sugars and resulting in the formation of defective bacteria.
varies in composition between bacterial strains, conferring 1. Cell wall deficient forms: The cell wall could be removed by
species-specific antigen specificity. It is exposed to host– hydrolysis with lysozyme or by blocking peptidoglycan
immune system. Gram-negative bacteria may thwart host synthesis with an antibiotic such as penicillin. These defective
defenses by rapidly changing the nature of their O side bacteria are believed to play a role in the persistence of pyelone-
chains to avoid detection. phritis and other chronic infections. Cell wall deficient forms
 MORPHOLOGY AND PHYSIOLOGY OF BACTERIA 17
without cell walls or with deficient cell walls may be of various Cytoplasm
types, such as—protoplasts, spheroplasts, mycoplasma, and

Section I
L-forms. Bacterial cytoplasm is a colloidal suspension of a variety
of organic and inorganic solutes in a viscous watery solu-
■ Protoplasts: These are defective unstable forms of bacteria tion. The matrix is largely formed by nearly 70% water.
with an intact cytoplasmic membrane but without any cell Cytoplasm contains all the biosynthetic components
wall. In hypertonic media, these are produced from Gram- required by a bacterium for growth and cell division, together
positive cells on treatment with lysozyme. with genetic material. Prokaryotic cytoplasm, unlike that of
■ Spheroplasts: These are defective forms derived from Gram- eukaryotes, lacks endoplasmic reticulum and mitochon-

Chapter 2
negative bacteria in the presence of EDTA (ethylenediami- dria. It also does not show any protoplasmic streaming.
netetraacetic acid). The EDTA disrupts the outer membrane Bacteria lack a true cytoskeleton. The cytoplasm consists
allowing access of lysozyme and resulting in formation of of ribosomes, mesosomes, and intracytoplasmic inclusions
spheroplasts. Spheroplasts are osmotically fragile and still bodies.
retain outer membrane and entrapped peptidoglycan.
■ Mycoplasma: These are naturally occurring bacteria without Ribosomes: The cytoplasmic matrix often is packed with ribo-
cell wall. They do not possess any definite shape. They are somes. Ribosomes look like small, featureless particles at low
very minute in size measuring 50–300 nm in diameter. magnification in electron micrographs. They are smaller than
■ L-forms: This is named after Lister Institute, London, where their eukaryotic counterpart with sedimentation of 70S, com-
the abnormal form of Streptobacillus moniliformis was first pared with 80S in eukaryotes. They consist of two subunits of
demonstrated. The L-forms do not exhibit any regular size 30S and 50S, giving a net 70S. Ribosomes are important
and shape. They may be spherical or disc shaped, about 0.1– because:
20 ␮m in diameter. They are difficult to cultivate and usu-

Section III
■ They serve as the sites of protein synthesis; matrix ribo-
ally require a medium that is solidified with agar as well as somes synthesize proteins destined to remain within the
having the right osmotic strength. They are produced more cell, whereas plasma membrane ribosomes make proteins
readily with exposure to penicillin than with lysozyme. Some for transport to the outside.
bacterial species produce L-forms spontaneously. L-forms in ■ They are also the sites of actions of several antibiotics, such
the host may produce chronic infections and are relatively as amino glycosides, macrolides, and tetracyclines.
resistant to antibiotic treatment.
Mesosomes: These are vesicular convoluted or multilaminated

Chapter 43
2. Pleomorphic bacteria: Pleomorphic bacteria (e.g., Yersinia structures formed as invagination of the plasma membrane
pestis) may show considerable variation in size and shape called into the cytoplasm. Mesosomes are of two types—septal and
pleomorphism. lateral. The septal mesosome attached to the bacterial DNA is
3. Involution forms: The involution forms are those that on believed to coordinate nuclear and cytoplasmic divisions dur-
ageing of culture show swollen and aberrant forms, especially ing binary fission. The function of lateral mesosomes still
in high salt concentration. remains to be known. Mesosomes are analogous to the mito-
chondria of eukaryotes and are the principal sites of respiratory
◗ Demonstration of cell wall enzymes in bacteria.
The cell walls can be demonstrated by (a) differential staining Intracytoplasmic inclusion bodies: Intracytoplasmic inclu-
procedure, (b) electron microscopy, (c) plasmolysis, (d) micro- sion bodies are present in the protoplasm of bacteria. Their
dissection, (e) mechanical rupture of the cell, and (f) serological main function is believed to be of storage. This occurs when
test by exposure to specific antibodies. their main constituent element is present in excess in the cul-
ture medium. Since inclusion bodies are used for storage, their
Cell Membrane quantity can vary depending on the nutritional status of the
cell. They are the sources of carbon, inorganic substances, and
Cell membrane or plasma membrane is a thin (5–10 nm)
energy. Some inclusion bodies also function to reduce osmotic
semipermeable membrane that acts as an osmotic barrier. It lies
pressure.
beneath the cell wall separating it from the cell cytoplasm. Cell
They may be of two types: (i) organic inclusion bodies, which
membrane primarily contains phospholipids and proteins. It also
usually contain either glycogen or polyhydroxybutyrate, and
contains enzymes associated with DNA biosynthesis, cell wall
(ii) inorganic inclusion bodies, which may be of polyphosphate
polymers, and membrane lipids. Bacterial plasma membranes
granules or sulfur granules. Examples of intracytoplasmic
usually have a higher proportion of protein than eukaryotic
inclusion bodies include metachromatic granules or volutin
membranes. They usually differ from eukaryotic membranes in
granules, starch inclusions, and lipid inclusions. Volutin
lacking sterols, such as cholesterol, except in Mycoplasma. The cell
granules, typically present in C. diphtheriae, can be demonstrated
membrane has following functions:
by Albert’s stain. Similarly, starch granules present in the
■ It acts as a semipermeable membrane regulating the inflow bacteria can be demonstrated on staining with iodine. Lipid
and outflow of metabolites to and from the protoplasm. inclusion found in Mycobacteria is demonstrated by Sudan
■ It helps in electron transport and oxidative phosphorylation. black dye.
 18 GENERAL MICROBIOLOGY

Nucleus However, Bacillus anthracis has a capsule comprising of polyamino

acids, such as D-glutamic acid. The D-glutamic acid is probably
Section I

The bacterial nucleus is neither enclosed in a nuclear membrane analogous to the negatively charged polysaccharide capsule.
nor associated with any nucleolus. It is haploid and replicates
by simple fission. The nucleus of the bacteria consists of a
single circle of double-stranded deoxyribonucleic acid (DNA), Key Points
arranged in a supercoiled circular structure. It measures about The capsule has various functions:
1000 ␮m when straightened.
The chromosome is located in an irregularly shaped region ■ It contributes to invasiveness of bacteria by protecting the
bacteria from phagocytosis.
Chapter 2

called nucleoid, but often referred to as bacterial chromosome ■ It also prevents bacteria from generating immune response
because of the analogy with the eukaryotic structure. The in infected hosts.
nucleoid is visible through the light microscope after staining ■ It facilitates adherence of bacteria to surfaces. Streptococcus
with the Feulgen stain, which specifically reacts with DNA. mutans, for example, owes its capacity to the glycocalyx to
In actively growing bacteria, the bacterial DNA can account ■ adhere tightly to tooth enamel to its glycocalyx.
■ It plays a role in the formation of biofilms.
for up to 20% of the volume of the bacterium and has projec- ■ The glycocalyx layer of the capsule may also play a role in
tions that extend into the cytoplasmic matrix. Careful electron resistance to desiccation.
microscopic studies often have shown the nucleoid to be in
contact with either the mesosome or the plasma membrane.
Many bacteria also possess smaller circles of extrachromo- Demonstration of capsule
somal DNA called plasmids. The plasmids are double-stranded
DNA molecules, usually circular, that can exist and replicate The capsule is fully hydrated and can be demonstrated by light
Section III

independently. Plasmids are not required for host growth and microscopy in either living or stained bacteria as follows:
reproduction, although they may carry genes that confer the Special capsular staining methods: These include Welch method
bacterium with properties such as antibiotics resistance or the and M’Faydean capsule stain. Welch method uses copper as
capacity to produce toxins or enzymes. mordant. This involves treatment of fixed smear with hot
crystal violet solution followed by rinsing with copper sulfate
Capsule and Slime Layer solution. The latter is used to remove excess stain because
conventional washing with water would dissolve the capsule.
Many bacteria, both Gram-positive and Gram-negative, possess
Chapter 43

The copper salt also gives color to the background, with the
a gel-like layer outside the envelope when growing in their result that the cell and background appear dark blue and the
natural environments. When a gel-like layer forms a well- capsule of the bacteria a much paler blue. M’Fadyean capsule
defined condensed layer around the bacterial envelope, it is stain, using polychrome methylene blue stain, is a frequently
called a capsule and is demonstrable by a light microscope. used method for demonstration of capsule of B. anthracis.
When this gel-like layer is narrower, detectable only by indirect
serological methods or by electron microscope but not by light Negative staining with India ink: Also known as wet India ink
microscope, it is called a microcapsule. An amorphous viscid method. It is the simplest way to demonstrate capsule. It is
colloidal material secreted by some bacteria extracellularly is carried out by mixing a suspension of bacteria with an equal
termed as loose or free slime or glycocalyx. volume of Indian ink on a slide, covering with a cover slip, and
then examining it under microscope. The capsule appears as a
◗ Capsule clear zone around the cell. This method is useful for improving
visualization of encapsulated bacteria in clinical samples, such
The capsule is mostly made up of polysaccharides, often referred
as blood or cerebrospinal fluid.
to collectively as exopolysaccharides. Exopolysaccharides are some-
times neutral homopolysaccharides (e.g., the glucans and fruc- Serologic methods: Since capsules are antigenic, they can be
tans of many oral streptococci) or negatively charged (Table 2-4). demonstrated by serologic methods. Quellung’s reaction is

TABLE 2-4 Chemical composition of capsules of various bacteria

Organism Polymer Chemical subunits
Bacillus anthracis Polypeptide D-glutamic acid
Enterobacter aerogenes Complex polysaccharide Glucose, fucose, glucuronic acid
Neisseria meningitidis Homopolymers and heteropolymers Partially O-acetylated N-acetylmannosaminephosphate
Streptococcus pneumoniae Complex polysaccharide (many types) Rhamnose, glucose, glucuronic acid
Streptococcus pyogenes (group A) Hyaluronic acid N-acetylglucosamine, glucuronic acid
Streptococcus salivarius Levan Fructose
 MORPHOLOGY AND PHYSIOLOGY OF BACTERIA 19
such a serological method for demonstration of capsule. Filament
When a suspension of bacterium is mixed with its specific

Section I
anticapsular serum and methylene blue and examined under Flagellar hook
microscope, the capsule becomes very prominent and appears Outer membrane
swollen due to increase in refractoriness. This method is useful
for rapid identification of capsular serotypes of S. pneumoniae, N. Peptidoglycan
meningitidis, H. influenzae, Yersinia, Bacillus, etc. Rod
Periplasmic space
◗ Slime layer

Chapter 2
Plasma
Slime layer (S-layer) is a structured paracrystalline protein layer membrane
Basal plates
shown by electron microscopy. These are generally composed
of a single kind of protein molecule, sometimes with carbohy- FIG. 2-12. Structure of a bacterial flagellum.
drates attached. They are resistant to proteolytic enzymes and
protein-denaturing agents. The slime layer protein protects the
body by a complex structure consisting of a hook and a basal
cell from wall-degrading enzymes and bacteriophages. It plays
body. The basal body bears a set of rings, one pair in Gram-
an important role in the maintenance of cell shape, and it may
positive bacteria and two pairs in Gram-negative bacteria,
be involved in cell adhesion to host epidermal surfaces.
through which the bacteria rotates either in a clockwise or an
anticlockwise direction. Above the base of filament is the hook, a
Surface Appendages short curved structure between the external filament and basal
The surface appendages of the bacteria include flagella and body. This part produces a propeller-like repulsion from the

Section III
fimbriae or pili. revolving f lagellum (Fig. 2-12).
Spirochetes are motile bacteria but without any external
◗ Flagella flagella. They are motile due to the presence of an axial fila-
ment. Axial filament consists of a bundle of flagellum-like
Bacterial flagella are thread-like appendages intricately
structures that lie between the cell surface and an outer sheath,
embedded in the cell envelope. These structures are responsible
and connects one end of the cell to the other. They are some-
for conferring motility to the bacteria. The arrangement of
times called the endoflagellates.
flagella varies between different bacterial species. Depending

Chapter 43
on the arrangement, flagella can be of the following types: Function: Flagella have the following functions:
■ They are primarily responsible for motility of bacteria by
■ Monotrichous (single polar flagellum), e.g., Vibrio cholerae.
chemotaxis.
■ Lophotrichous (multiple polar flagella), e.g., Spirilla.
■ They may play a role in bacterial survival and pathogenesis.
■ Peritrichous (flagella distributed over the entire cell), e.g.,
They are highly antigenic, they possess H antigens, and some
Salmonella Typhi, E. coli, etc.
of the immune responses to infection are directed against
■ Amphitrichous (single flagellum at both the ends), e.g.,
these proteins. The flagella of different bacteria differ
Spirillum minus (Fig. 2-11).
antigenically. Flagellar antibodies are not protective but
Structure: The flagella are 3–20 ␮m in length and 0.01–0.03 ␮m help in serodiagnosis.
in diameter. The main part of the filament is made up of protein Demonstration of flagella: The flagella can be demonstrated
subunits called flagellin arranged in several helices around a cen- by direct and indirect methods. The direct methods include
tral hollow core. The flagellum is attached to the bacterial cell direct demonstration of capsule by electron microscope. These
also include demonstration of capsule after staining by special
staining methods, such as Ryu’s method and Hugh–Leifson’s
method. Since flagella are very thin structures, these staining
methods are used to demonstrate flagella by increasing their
thickness by mordanting with tannic acid.
Indirect methods of demonstration of flagella include
demonstration of motility of the bacteria by (a) dark-ground
microscopy, (b) hanging drop method, or (c) observing spread-
ing type growth on semisolid media, such as mannitol motility
Peritrichous medium.

◗ Pili (fimbriae)
Pili or fimbriae are synonymous for most purposes. They
Monotrichous Lophotrichous
are hair-like filaments that extend from cell surface and are
FIG. 2-11. Arrangement of the bacterial flagella. found almost exclusively on Gram-negative bacteria. They are
 20 GENERAL MICROBIOLOGY

composed of structural protein subunits termed pilins. Minor DNA Septum

proteins termed adhesins are located at the tips of pili and are
Section I

responsible for the attachment properties.

Structure: The pili are shorter and straighter than flagella,
although the basic structure is same. Like flagella, it consists
of helics of protein called pilins, arranged around a hollow core Endospore
but without a motor. They are 0.5 ␮m long and 10 nm thick. coat
They are antigenic in nature. Pili hemagglutinate RBCs of
guinea pigs and are specifically inhibited by mannose, on the
Chapter 2

basis of which they are classified into four types as follows:

■ Type 1: These occur in E. coli, Klebsiella, Shigella, and Salmonella.
They are mannose sensitive. Peptidoglycan Cytoplasmic
membrane
■ Type 2: These are present in Salmonella Gallinarum and
Salmonella Pullorum, devoid of any hemagglutinating or
adhesive properties.
■ Type 3: These are present in some strains of Klebsiella, Serratia,
etc. They agglutinate RBC only after heating and are man-
nose resistant.
■ Type 4: These are mannose resistant and occur in Proteus.
Endospore
Sex pili: A specialized kind of pili called sex pili is responsible
Section III

released free
for the attachment of donor and recipient cells in bacterial
conjugation. These pili are longer (10–20 ␮m) and vary 1–4 in FIG. 2-13. Schematic diagram showing process of bacterial sporulation.
number. The sex pili are of two types:
(i) F pili: They specifically adsorb male specific RNA and DNA double membrane structure whose facing surfaces correspond
bacteriophages. They are encoded by sex factor F and fertil- to the cell wall-synthesizing surface of the cell envelope. The
ity inhibition–positive resistance factors (fi ⫹ R factors). growing points move progressively toward the pole of the cell
so as to engulf the developing spore. Two spore membranes
Chapter 43

(ii) I pili: They adsorb male specific filamentous DNA phages,

encoded by col factor and fi ⫺ R factor. then engage in the active synthesis of special layers that form
the cell envelope: the spore wall and the cortex, lying outside
Function: Pili play a major role in the adherence of symbiotic the facing membranes. In the newly isolated cytoplasm, or core,
and pathogenic bacteria to host cells, which is a necessary step many vegetative cell enzymes are degraded and are replaced by
in initiation of infection. Transfer of bacterial DNA takes place a set of unique spore constituents. During the process of
through sex pili during the process of conjugation. sporulation, each cell forms a single internal spore; the spore
Demonstration of pili: The pili can be detected: germinates to produce a single vegetative cell (Fig. 2-13).
■ Directly by electron microscope and
■ By agglutination of RBCs of guinea pigs, fowl, horses, and ◗ Spores
pigs. They agglutinate human and sheep RBCs weakly. The Morphology: The endospores are a highly resistant resting
hemagglutination can be specifically inhibited by D-mannose. phase of bacteria. The spore shows following structures.
Some of the Gram-positive bacteria do not possess typical pili 1. Core: The core contains a complete nucleus (chromosome),
but instead possess a fine fibrillar arrangement of proteins all of the components of the protein-synthesizing apparatus,
on their surfaces known as fibrils. These fibrils bind to the and an energy-generating system based on glycolysis. The
host surfaces. M-protein of S. pyogenes is an example of Gram- heat resistance of spores is due in part to their dehydrated
positive bacteria possessing fibrils. state and in part to the presence of large amounts (5–15% of
the spore dry weight) of calcium dipicolinate in the core.
2. Spore wall: This is the innermost layer surrounding the
Sporulation inner spore membrane. It contains normal peptidoglycan
Sporulation is a primitive process of differentiation with formation and becomes the cell wall of the germinating vegetative cell.
of endospores, a highly resistant resting phase of some of the bac- 3. Cortex: It is the thickest layer of the spore envelope containing
teria (e.g., spores of aerobic Bacillus spp. and anaerobic Clostridium unusual peptidoglycan. It is extremely sensitive to lysozyme,
spp.). The organism survives in spores, a dormant state, for longer and its autolysis plays a role in spore germination.
period of starvation and other adverse conditions. 4. Protein coat: It is composed of a keratin-like protein con-
Sporulation process begins in nutrition deprived conditions. It taining many intramolecular disulfide bonds; this layer
begins with the formation of an axial filament. The process confers relative resistance to antibacterial chemical agents
continues with infolding of the membrane so as to produce a due to its impermeability (Fig. 2-14).
 MORPHOLOGY AND PHYSIOLOGY OF BACTERIA 21
Exosporium

Section I
1
Protein coat

Outer membrane

Core
2
Cortex

Inner membrane

Chapter 2
Spore wall
4

FIG. 2-14. Diagrammatic representation of bacterial spore.

8
The spores may vary among different species depending on the
position, shape, and relative size of the spores. For example,
spores may be central, subterminal or terminal; may be oval or FIG. 2-15. Schematic diagram showing binary fission of the bacteria.
spherical in shape; and may be bulging or nonbulging.
Demonstration of spores: Spores are most simply observed (Fig. 2-15). The growth rate of a bacterium is therefore measured

Section III
as intracellular refractile bodies in unstained cell suspensions by measuring the change in bacterial number per unit time.
or as colorless areas in cells stained by conventional methods,
such as Gram staining. Spores are commonly stained with Generation Time
malachite green or carbol fuchsin. On staining by modified
ZN stain (using 0.25–0.5% sulfuric acid instead of 20% sulfuric Generation time is the time required for a bacterium to give rise
acid), the spores appear as red acid-fast bodies. to two daughter cells under optimum conditions. The
generation time for most of the pathogenic bacteria, such as
Properties of spores: Bacterial spores are resistant to ordinary
E. coli, is about 20 minutes. The generation time is longer (i.e.,

Chapter 43
boiling, disinfectants, and heating. Spores of all medically
20 hours) for M. tuberculosis and longest (i.e., 20 days) for M.
important bacteria are destroyed by autoclaving at 121⬚C for
leprae. A bacterium replicates and multiplies rapidly producing
15 minutes. The process of conversion of a spore into vegetative
millions of cells within 24 hours. For example, E. coli in about
cell under suitable conditions is known as germination. The
7 hours can undergo 20 generations and produce 1 million
germination process occurs in three stages: activation,
cells, in about 10 hours undergo 30 generations and produce
initiation, and outgrowth.
1 billion cells, and in 24 hours produces 1021 cells (Fig. 2-16).
However, in actual practice, the multiplication of bacteria is
Key Points arrested after a few cell divisions due to exhaustion of nutrients
and accumulation of toxic products.
The spores have certain uses in clinical microbiology:
■ The spores of Bacillus stearothermophilus are used as indica- Bacterial Count
tor of proper sterilization by autoclaving. These spores are
destroyed at a temperature of 121⬚C for 10–20 minutes, the Microbial concentrations can be measured in terms of (i) cell
time required for autoclaving. concentration (the number of viable cells per unit volume of
■ The spores of certain bacteria, such as B. anthracis are mis- culture) or (ii) biomass concentration (dry weight of cells per
used as agents of bioterrorism. unit volume of culture). The number of bacteria at a given time
can be estimated by performing a total count or viable count.
Total count: This denotes the total number of bacteria in the
sample, irrespective of whether they are living or dead. This is done
Growth and Multiplication of Bacteria by counting the bacteria under the microscope using counting
chamber or by comparing the growth with standard opacity tubes.
Bacterial growth can be defined as an orderly increase of all the
Viable count: This usually indicates the number of living
chemical components of the cell. Cell multiplication is a con-
or viable bacteria. This count can be obtained by dilution or
sequence of growth that leads to an increase in the number of
plating method.
bacteria making up a population or culture. Most bacteria divide
by binary fission in which the bacteria undergo cell division to ■ In dilution method, several tubes with liquid culture media
produce two daughter cells identical to the parent cell. Bacterial are incubated with varying dilutions of sample and the
growth can be equated to cell number: one bacterium divides viable count is calculated from the number of tubes
into two, these two produce four, and then eight, and so on showing bacterial growth. This method is widely used in
 22 GENERAL MICROBIOLOGY

Time (hour) Lag Log Stationary Phase of

Single bacterial cell phase
Section I

phase phase decline

Number of microorganism
0 Total count

Agar plate Viable count

Chapter 2

Time

FIG. 2-17. Bacterial growth curve.

8

Visible bacterial Growth rate during different phases of

colony TABLE 2-5 bacterial growth curve
Section III

Phase Growth rate

12 Lag Zero
Log or exponential Constant
Stationary Zero

FIG. 2-16. Generation time of the bacteria. Decline Negative (death)

Chapter 43

microbiological testing of water for presumptive coliform population doubles during every generation. They multiply at
count in drinking water. their maximum rate. The bacterial cells are small and uniformly
■ In the plate method, a sample is diluted and small volume of stained. The microbes are sensitive to adverse conditions, such
it is spread on the surface of an agar plate. The number of as antibiotics and other antimicrobial agents.
colonies that grow after a suitable incubation time indicates 3. Stationary phase: After log phase, the bacterial growth almost
viable count of the bacteria. stops completely due to lack of essential nutrients, lack of water
oxygen, change in pH of the medium, etc. and accumulation of
their own toxic metabolic wastes. Death rate of bacteria exceeds
Bacterial Growth Curve the rate of replication of bacteria. Endospores start forming dur-
When a broth culture is inoculated with a small bacterial inocu- ing this stage. Bacteria become Gram variable and show irregular
lum, the population size of the bacteria increases showing a clas- staining. Many bacteria start producing exotoxins.
sical pattern. The bacterial growth curve shows the following four 4. Decline phase: During this phase, the bacterial population
distinct phases (Fig. 2-17): declines due to death of cells. The decline phase starts due to
(a) accumulation of toxic products and autolytic enzymes and
1. Lag phase: After a liquid culture broth is inoculated, the
(b) exhaustion of nutrients. Involution forms are common in
multiplication of bacteria does not start immediately. It takes
this stage. Growth rate during different phases of bacterial
some time to multiply. The time between inoculation and
growth curve is summarized in Table 2-5.
beginning of multiplication is known as lag phase. In this
The continuous culture is a method of culture useful for
phase, the inoculated bacteria become acclimatized to the
industrial and research purpose. This is achieved by using a
environment, switch on various enzymes, and adjust to the
special device for replenishing nutrients and removing bacte-
environmental temperature and atmospheric conditions.
rial population continuously so that bacteria growth is not
During this phase, there is an increase in size of bacteria but no
inhibited due to lack of nutrients or due to accumulation of
appreciable increase in number of bacterial cells. The cells are
toxic bacterial metabolites.
active metabolically. The duration of the lag phase varies with
the bacterial species, nature of culture medium, incubation
temperature, etc. It may vary from 1 hour to several days. Factors Affecting Growth of Bacteria
2. Log phase: This phase is characterized by rapid exponential A variety of factors affect growth of bacteria. These are discussed
cell growth (i.e., 1 to 2 to 4 to 8 and so on). The bacterial below:
 MORPHOLOGY AND PHYSIOLOGY OF BACTERIA 23

◗ Oxygen ◗ Light

Section I
Bacteria on the basis of their oxygen requirements can be classi- Depending on the source of energy bacteria make use of, they
fied broadly into aerobic and anaerobic bacteria. may be classified as phototrophs (bacteria deriving energy from
Aerobic bacteria: They require oxygen for their growth. They sunlight) or chemotrophs (bacteria deriving energy from chem-
may be: ical sources).

■ Obligate aerobes—which can grow only in the presence of ◗ Osmotic pressure

oxygen (e.g., P. aeruginosa).
Microbes obtain almost all their nutrients in solution from
■ Facultative aerobes—which are ordinary aerobes but can also

Chapter 2
surrounding water. Hence factors such as osmotic pressure and
grow without oxygen (e.g., E. coli). Most of the pathogenic
salt concentration of the solution affect the growth of bacteria.
bacteria are facultative aerobes.
Bacteria by virtue of mechanical strength of their cell wall are
■ Microaerophilic bacteria—those bacteria that can grow in
able to withstand a wide range of external osmotic variations.
the presence of low oxygen and in the presence of low (4%)
Organisms requiring high osmotic pressures are called osmo-
concentration of carbon dioxide (e.g., Campylobacter jejuni).
philic bacteria. Sudden exposure of bacteria to hypertonic solu-
Some fermentative organisms (e.g., Lactobacillus plantarum) tion may cause osmotic withdrawal of water, leading to osmotic
are aerotolerant but do not contain the enzyme catalase or shrinkage of the protoplasm ( plasmolysis). On the other hand,
superoxide dismutase. Oxygen is not reduced, and therefore sudden transfer of bacteria from concentrated solution to dis-
hydrogen peroxide (H2O2) and nascent oxygen (O2⫺) are not tilled water may cause excessive imbibition of water leading to
produced. swelling and bursting of cell ( plasmoptysis).
Anaerobic bacteria: Obligate anaerobes are the bacteria that

Section III
can grow only in the absence of oxygen (e.g., Clostridium botulinum
Clostridium tetani, etc.). These bacteria lack superoxide dismutase
Bacterial Nutrition
and catalase; hence oxygen is lethal to these organisms.
The minimum requirements for growth of bacteria include
water, a source of carbon, a source of nitrogen, and certain
◗ Carbon dioxide inorganic salts. These are required for synthesis of proteins,
The organisms that require higher amounts of carbon dioxide enzymes, etc. For example,

Chapter 43
(CO2) for their growth are called capnophilic bacteria. They ■ Nitrogen is required for synthesis of proteins, DNA, RNA,
grow well in the presence of 5–10% CO2 and 15% O2. In candle and ATP.
jar, 3% CO2 can be achieved. Examples of such bacteria include ■ Sulfur is required for certain amino acids and vitamins,
H. influenzae, Brucella abortus, etc. and phosphorous is required for nucleic acids, ATP, and
phospholipids.
◗ Temperature ■ In addition, inorganic ions, such as potassium, sodium, iron,
The optimum temperature for most of the pathogenic bacteria magnesium, calcium, and chloride are required to facilitate
is 37⬚C. The optimal temperature, however, is variable; depend- enzymatic catalysis and to maintain chemical gradients
ing on their temperature range, growth of bacteria is grouped across the cell membrane.
as follows: Some bacteria grow in a variety of simple media. E. coli and
■ Psychrophiles: These bacteria are cold loving microbes that other members of the family Enterobacteriaceae are examples
grow within a temperature range of 0–20⬚C. Most of soil and of bacteria that can grow in a variety of simple media contain-
water saprophytes belong to this group. ing the inorganic salts and with a source of energy, the simplest
■ Mesophiles: These are moderate temperature loving microbes being glucose. The inorganic salts in the media provide major
that grow between 25⬚C and 40⬚C. Most of pathogenic bac- essential elements of carbon, hydrogen, oxygen, nitrogen, phos-
teria belong to this group. phate, and sulfur. These chemicals are usually present in the
■ Thermophiles: These are heat loving microbes. They can grow media and are not added specifically.
at a high temperature range of 55–80⬚C. B. stearothermophilus Some bacteria, such as H. influenzae and other related
is an example. bacteria, on other hand, are very fastidious and have certain
growth requirements. They require certain amino acids,
vitamins, and other growth factors that are supplied by
◗ pH adding yeast extract and meat digests to the media. They also
Most pathogenic bacteria grow between pH 7.2 and 7.6. Very require addition of blood or serum for their growth.
few bacteria, such as lactobacilli, can grow at acidic pH below Certain lower forms of bacteria even fail to grow in cell-free
4.0. Many food items, such as pickles and cheese, are prevented culture media and require living cells for their growth.
from spoilage by acids produced during fermentation. T. pallidum and M. leprae are two pathogenic bacteria that
V. cholerae is an example of the bacteria that can grow at an cannot grow in any artificial culture media, but can only be
alkaline (8.2–8.9) pH. cultured when inoculated into living animals.
 Sterilization and 3
Disinfection
Chemical agents called antiseptics are applied directly to the
Introduction exposed body surfaces (e.g., skin and mucous membranes),
wounds, and surgical incisions to destroy or inhibit vegetative
Microbes are ubiquitous and many microorganisms are pathogens. Examples of antisepsis include (a) preparing the
associated with undesirable consequences, such as food spoil- skin before surgical incisions with iodine compounds, (b)
age and disease. Therefore, it is essential to kill a wide variety of swabbing an open root canal with hydrogen peroxide, and (c)
microorganisms or inhibit their growth to minimize their ordinary hand washing with a germicidal soap.
destructive effects. The goal is twofold: (a) to destroy pathogens Sanitization is any cleansing technique that mechanically
and prevent their transmission and (b) to reduce or eliminate removes microorganisms (along with food debris) to reduce the
microorganisms responsible for the contamination of water, level of contaminants. A sanitizer is a compound (e.g., soap or
food, and other substances. detergent) that is used to perform this task. Cooking utensils,
dishes, bottles, cans, and used clothing that have been washed
and dried may not be completely free of microbes, but they are
Definition of Frequently Used Terms considered safe for normal use. Air sanitization with ultraviolet
lamps reduces airborne microbes in hospital rooms, veterinary
Sterilization is defined as a process by which an article, sur- clinics, and laboratory installations.
face, or medium is freed of all living microorganisms either It is often necessary to reduce the numbers of microbes on
in the vegetative or in the spore state. Any material that has the human skin through degerming procedures. This process
been subjected to this process is said to be sterile. These terms usually involves scrubbing the skin or immersing it in chemi-
should be used only in the absolute sense. An object cannot be cals, or both. It also emulsifies oils that lie on the outer cutane-
slightly sterile or almost sterile; it is either sterile or not sterile. ous layer and mechanically removes potential pathogens from
Although most sterilization is performed with a physical agent, the outer layers of the skin. Examples of degerming procedures
such as heat, a few chemicals called sterilants can be classified are (a) surgical hand scrub, (b) application of alcohol wipes to
as sterilizing agents because of their ability to destroy spores. the skin, and (c) cleansing of a wound with germicidal soap and
A germicide, also called a microbicide, is any chemical agent water. The concepts of antisepsis and degerming procedures
that kills pathogenic microorganisms. A germicide can be used clearly overlap, since a degerming procedure can be simultane-
on inanimate (nonliving) materials or on living tissue, but it ously treated as an antiseptic and vice versa.
ordinarily cannot kill resistant microbial cells. Any physical or
chemical agent that kills “germs” is said to have germicidal Sterilization
properties.
Disinfection refers to the use of a chemical agent that Methods of sterilization can be broadly classified as:
destroys or removes all pathogenic organisms or organisms
capable of giving rise to infection. This process destroys vegeta- 1. Physical methods of sterilization, and
tive pathogens but not bacterial endospores. It is important to 2. Chemical methods of sterilization.
note that disinfectants are normally used only on inanimate
objects because they can be toxic to human and other animal Physical Methods of Sterilization
tissue, when used in higher concentrations. Disinfection pro- Physical methods of sterilization include the following:
cesses also remove the harmful products of microorganisms
(toxins) from materials. Examples of disinfection include (a) 1. Sunlight
applying a solution of 5% bleach to examining table, (b) boil- 2. Heat
ing food utensils used by a sick person, and (c) immersing ther- 3. Filtration
mometers in an isopropyl alcohol solution between use. 4. Radiation
In modern usage, sepsis is defined as the growth of micro- 5. Sound (sonic) waves
organisms in the body or the presence of microbial toxins in
blood and other tissues. The term asepsis refers to any practice ◗ Sunlight
that prevents the entry of infectious agents into sterile tissues Direct sunlight is a natural method of sterilization of water in
and thus prevents infection. tanks, rivers, and lakes. Direct sunlight has an active germicidal
 STERILIZATION AND DISINFECTION 25
effect due to its content of ultraviolet and heat rays. Bacteria fungi, it does not kill endospores or thermoduric species
present in natural water sources are rapidly destroyed by (mostly nonpathogenic lactobacilli, micrococci, and

Section I
exposure to sunlight. yeasts). Milk is not sterile after regular pasteurization. In
fact, it can contain 20,000 microbes per milliliter or more,
◗ Heat which explains why even an unopened carton of milk will
Heat is the most dependable method of sterilization and is eventually spoil on prolonged storage. Newer techniques
usually the method of choice unless contraindicated. As a rule, have now been used to produce sterile milk that has a stor-
higher temperatures (exceeding the maximum) are microbicidal, age life of 3 months. In this method, milk is processed with
ultrahigh temperature (UHT) of 134°C for 1–2 seconds.

Chapter 3
whereas lower temperatures (below the minimum) tend to have
inhibitory or microbistatic effects. Two types of physical heat
2. Sterilization at a temperature of 100°C: Sterilsation at
are used in sterilization—moist and dry heat.
a temperature of 100ºC includes (a) boiling and (b) steam
Sterilization by moist heat sterilizer at 100°C.
Moist heat occurs in the form of hot water, boiling water, or Boiling: Simple boiling of water for 10–30 minutes kills
steam (vaporized water). In practice, the temperature of moist most of the vegetative forms of bacteria but not bacterial
heat usually ranges from 60 to 135°C. Adjustment of pressure spores. Exposing materials to boiling water for 30 minutes
in a closed container can regulate the temperature of steam. kills most nonspore-forming pathogens including resis-
Moist heat kills microorganisms by denaturation and coagula- tant species, such as the tubercle bacillus and staphylo-
tion of proteins. Sterilization by moist heat can be classified as cocci. Sterilization by boiling is facilitated by addition of
follows: 2% sodium bicarbonate to water. Since boiling only once at
100°C does not kill all spores, this method cannot be used

Section III
1. Sterilization at a temperature ⬍100°C for sterilization but only for disinfection. Hence, it is not
2. Sterilization at a temperature of 100°C recommended for sterilizing instruments used for surgical
3. Sterilization at a temperature ⬎100°C procedure. The greatest disadvantage of this method is
4. Intermittent sterilization that the items sterilized by boiling can be easily recontami-
1. Sterilization at a temperature ⬍100°C: Pasteurization is nated when removed from water after boiling.
an example of sterilisation at a temperature ⬍100°C. Steam sterilizer at 100°C: Usually, Koch’s or Arnold’s
steam sterilizer is used for heat-labile substances that tend

Chapter 43
Pasteurization: Fresh beverages (such as milk, fruit juices,
beer, and wine) are easily contaminated during collection to degrade at higher temperatures and pressure, such as
and processing. Because microbes have potential for spoil- during the process of autoclaving. These substances are
ing these foods or causing illness, heat is frequently used to exposed to steam at atmospheric pressure for 90 minutes
reduce the microbial load and to destroy pathogens. during which most vegetative forms of the bacteria except
Pasteurization is a technique in which heat is applied to liq- for the thermophiles are killed by the moist heat.
uids to kill potential agents of infection and spoilage, while 3. Sterilization at a temperature > 100°C: This method is
at the same time retaining the liquid’s flavor and food value. otherwise known as sterilization by steam under pressure.
This technique is named after Louis Pasteur who devised A temperature of 100°C is the highest that steam can
this method. This method is extensively used for steriliza- reach under normal atmospheric pressure at sea level.
tion of milk and other fresh beverages, such as fruit juices, This pressure is measured at 15 pounds per square inch
beer, and wine which are easily contaminated during collec- ( psi), or 1 atmosphere. In order to raise the temperature of
tion and processing. steam above this point, it must be pressurized in a closed
chamber. This phenomenon is explained by the physical
principle that governs the behavior of gases under pres-
Key Points sure. When a gas is compressed, its temperature rises in
Two methods of pasteurization are followed: flash method direct relation to the amount of pressure. So, when the
and holder method. pressure is increased to 5 psi above normal atmospheric
■ In the flash method, milk is exposed to heat at 72°C for 15–20 pressure, the temperature of steam rises to 109°C. When
seconds followed by a sudden cooling to 13°C or lower. the pressure is increased to 10 psi above normal, its tem-
■ In the holder method, milk is exposed to a temperature of perature will be 115°C and at 15 psi (a total of 2 atmo-
63°C for 30 minutes followed by cooling to 13°C or lower, spheres), it will be 121°C. It is not the pressure by itself
but not less than 6°C.
that is killing microbes, but the increased temperature it
produces. This forms the principle of sterilization by
The flash method is preferable for sterilization of milk steam under pressure. Such pressure–temperature combi-
because it is less likely to change the flavor and nutrient nations can be achieved only with a special device that can
content, and it is more effective against certain resistant subject pure steam to pressures greater than 1 atmo-
pathogens, such as Coxiella and Mycobacterium. sphere. Health and commercial industries use an autoclave
Although pasteurization inactivates most viruses and for this purpose and a comparable home appliance is the
destroys the vegetative stages of 97–99% of bacteria and pressure cooker.
 26 GENERAL MICROBIOLOGY

Autoclave: It is a cylindrical metal chamber with an airtight steam from circulating freely around the contents and
door at one end and racks to hold materials. The lid is impedes the full contact that is necessary. The holding
Section I

fastened by screw clamp and rendered airtight by an time varies from 10 minutes for light loads to 40 minutes
asbestos washer. It has a discharge tap for air and steam at for heavy or bulky ones; the average time being 20 minutes.
the upper side, a pressure gauge and a safety valve that can
be set to blow off at any desired pressure. Heating is usually
carried out by electricity. Steam circulates within the jacket
Key Points
and is supplied under pressure to the inner chamber where Uses of autoclave: The autoclave has many uses, which are
materials are loaded for sterilization (Fig. 3-1). The water in given below:
Chapter 3

the autoclave boils when its vapor pressure equals that of ■ It is a good method to sterilize heat-resistant materials,
surrounding atmosphere. Following the increase of such as glassware, cloth (surgical dressings), rubber (gloves),
pressure inside the closed vessel, the temperature at which metallic instruments, liquids, paper, some media, and some
heat-resistant plastics.
the water boils inside the autoclave also increases. The ■ It is also useful for sterilization of heat-sensitive items,
saturated steam that has a higher penetrative power, on such as plastic Petri plates that need to be discarded.
coming in contact with a cooler surface condenses to ■ It is useful for sterilization of materials that cannot withstand
water and releases its latent heat to that surface. For the higher temperature of the hot-air oven.
example, nearly 1600 mL steam at 100°C and at atmospheric
pressure condenses into 1 mL of water at 100°C and However, the autoclave is ineffective for sterilizing
releases 518 calories of heat. The gross reduction in volume substances that repel moisture (oils, waxes, or powders).
of steam sucks in more steam to the area and this process Types and uses of various moist heat sterilization methods
continues till the temperature of that surface is elevated to are summarized in Table 3-1.
Section III

that of the steam. Sterilization is achieved when the steam

Sterilization controls: Various sterilization controls are used
condenses against the objects in the chamber and gradually
to determine the efficacy of sterilization by moist heat.
raises their temperature. The condensed water facilitates
These include (a) thermocouples, (b) chemical indicators,
moist conditions that ensures killing of microbes.
and (c) bacteriological spores as mentioned below:
Sterilization condition: Experience has shown that the most
efficient pressure–temperature combination for achiev- (a) Thermocouples are used to record temperatures
ing sterilization by autoclave is 15 psi, which yields 121°C. directly in autoclaves by a potentiometer.
Chapter 43

It is possible to use higher pressure to reach higher tem- (b) Brown’s tube is the most commonly used chemical
peratures (for instance, increasing the pressure to 30 psi indicator of moist heat sterilization in the autoclave. It
raises the temperature by 11°C), but doing so will not contains red solution that turns green when exposed to
significantly reduce the exposure time and can harm the temperature of 121°C for 15 minutes in an autoclave.
items being sterilized. It is important to avoid over packing (c) Bacillus stearothermophilus spores are used as the
or haphazardly loading the chamber, because it prevents indicators of moist heat sterilization in the autoclave.
This is a thermophilic bacterium with an optimum
temperature of 55–60°C, and its spores require an
exposure of 12 minutes at 121°C to be destroyed. The
efficacy of the autoclave is carried out by placing paper
strips impregnated with 106 spores in envelopes and
keeping those envelopes in different parts of the load
inside the autoclave. These strips after sterilization
are inoculated into a suitable recovery medium and

TABLE 3-1 Types and uses of moist heat sterilization

Method Uses Comments
Water bath For sterilization of serum, Only disinfection possible.
below 100oC body fluids, and vaccines Spores would be spared
Water bath For sterilization of glass, Some spores will still be
at 100oC metal, and rubber items spared at this temperature
Arnold steamer: For sterilization of culture Preserves properties of
steaming at media containing sugar media
100oC and gelatin
Autoclave: For sterilization of culture Kills all the vegetative as
steam under media and operation well as spore forms of
pressure theater as well as bacteria
FIG. 3-1. Autoclave. laboratory materials
 STERILIZATION AND DISINFECTION 27
incubated at 55°C for 5 days. Spores are destroyed if
the sterilizing condition of the autoclave is proper.

Section I
4. Intermittent sterilization: Certain heat-labile substances
(e.g., serum, sugar, egg, etc.) that cannot withstand the high
temperature of the autoclave can be sterilized by a process
of intermittent sterilization, known as tyndallization.
Tyndallization is carried out over a period of 3 days and
requires a chamber to hold the materials and a reservoir for
boiling water. Items to be sterilized are kept in the cham-

Chapter 3
ber and are exposed to free-flowing steam at 100°C for
20 minutes, for each of the three consecutive days. On the
first day, the temperature is adequate to kill all the vegetative
forms of the bacteria, yeasts, and molds but not sufficient
to kill spores. The surviving spores are allowed to germi-
nate to vegetative forms on the second day and are killed on
re-exposure to steam. The third day re-ensures killing of all
the spores by their germination to vegetative forms.
Intermittent sterilization is used most often to sterilize
heat-sensitive culture media, such as those containing sera
(e.g., Loeffler’s serum slope), egg (e.g., Lowenstein–Jensen’s FIG. 3-2. Hot-air oven.
medium), or carbohydrates (e.g., serum sugars) and some

Section III
canned foods. 3. Hot-air oven: The hot-air oven provides another means of
dry heat sterilization and is the most widely used method.
The hot-air oven is electrically heated and is fitted with a
Sterilization by dry heat fan to ensure adequate and even distribution of hot air
Sterilization by dry heat makes use of air with a low mois- in the chamber. It is also fitted with a thermostat that
ture content that has been heated by a flame or electric heat- ensures circulation of hot air of desired temperature in the
ing coil. In practice, the temperature of dry heat ranges from chamber. Heated, circulated air transfers its heat to the

Chapter 43
160°C to several thousand degrees Celsius. The dry heat kills materials inside the chamber. While sterilizing by hot-air
microorganisms by protein denaturation, oxidative damage, oven, it should be ensured that the oven is not overloaded.
and the toxic effect of increased level of electrolytes. Dry heat The materials should be dry and arranged in a manner
is not as versatile or as widely used as moist heat, but it has which allows free circulation of air inside the chamber. It is
several important sterilization applications. The temperature essential to fit the test tubes, flasks, etc., with cotton plugs
and time employed in dry heat vary according to the particular and to wrap Petri dishes and pipettes in a paper. Steriliza-
method, but in general they are greater than with moist heat. tion by hot-air oven requires exposure to 160–180°C for
Sterilization by dry heat includes sterilization by (a) flaming, 2 hours and 30 minutes, which ensures thorough heating
(b) incineration , and (c) hot air oven: of the objects and destruction of spores (Fig. 3-2).

1. Flaming: Sterilization of inoculating loop or wire, the tip

of forceps, searing spatulas, etc., is carried out by holding
Key Points
them in the flame of the Bunsen burner till they become Hot-air oven is used in laboratories and clinics for heat-
resistant items that are not sterilized well by moist heat. They
red hot. Glass slides, scalpels, and mouths of culture tubes
are used for sterilization of:
are sterilized by passing them through the Bunsen flame
without allowing them to become red hot. ■ Glasswares (syringes, Petri dishes, flasks, pipettes, test tubes, etc.).
■ Surgical instruments (scalpels, scissors, forceps, etc.).
2. Incineration: Incineration is an excellent method for
■ Chemicals (liquid paraffin, sulfonamide powders, etc.); and
safely destroying infective materials by burning them to ■ Oils that are not penetrated well by steam used in moist
ashes. It has many uses: heat sterilization.

■ Incinerators are used to carry out this process and are

regularly employed in hospitals and research labs to Thermocouples, chemical indicators, and bacteriological
destroy hospital and laboratory wastes. spores of Bacillus subtilis are used as sterilization controls to
■ The method is used for complete destruction and dis- determine the efficacy of sterilization by hot-air oven.
posal of infectious material, such as syringes, needles,
culture material, dressings, bandages, bedding, animal ◗ Filtration
carcasses, and pathology samples. Filtration is an excellent way to reduce the microbial popula-
■ This method is fast and effective for most hospital wastes, tion in solutions of heat-labile material by use of a variety of
but not for metals and heat-resistant glass materials. filters. Filters are used to sterilize these heat-labile solutions.
 28 GENERAL MICROBIOLOGY

Filters simply remove contaminating microorganisms from

solutions rather than directly destroying them. The filters are TABLE 3-2 Types and uses of radiation for sterilization
Section I

of two types: (a) depth filters and (b) membrane filters. Types Uses Comments
1. Depth filters: Depth filters consist of fibrous or granular Ionizing radiation For sterilization of Though expensive
materials that have been bonded into a thick layer filled with administered using pharmaceuticals and fraught with
twisting channels of small diameter. The solution contain- Cobalt-60-based like antibiotics, safety risks, it is very
ing microorganisms is sucked in through this layer under instruments hormones, sutures; effective due to better
and prepacked penetration power
vacuum and microbial cells are removed by physical screen-
disposable items, such
Chapter 3

ing or entrapment and also by adsorption to the surface of as syringes, infusion

the filter material. Depth filters are of the following types: sets, catheters, etc.
■ Candle filters: These are made up of (a) diatomaceous Nonionizing radiation Only for disinfection Hazardous and not
earth (e.g., Berkefeld filters) or (b) unglazed porcelain administered through of clear surfaces in as effective as ionizing
(e.g., Chamberlain filters). They are available in differ- UV lamps OTs, laminar flow radiation
ent grades of porosity and are used widely for purifica- hoods, etc.
tion of water for drinking and industrial uses.
■ Asbestos filters: These are made up of asbestos such as mag-
nesium silicate. Seitz and Sterimat filters are the examples Air also can be sterilized by filtration. Two common exam-
of such filters. These are disposable and single-use discs ples are surgical masks and cotton plugs on culture vessels
available in different grades. They have high adsorbing that let air in but keep microorganisms out. Laminar flow
capacity and tend to alkalinize the filtered fluid. Their use biological safety cabinets are most widely used air filtration
systems in hospitals and industries. In this method, air is
Section III

is limited by the carcinogenic potential of asbestos.

■ Sintered glass filters: These are made up of finely pow-
passed through high-efficiency particulate air (HEPA) filters
dered glass particles, which are fused together. They have that remove nearly 99.97% of 0.3 ␮m particles from the
low absorbing property and are available in different filtered air.
pore sizes. These filters, although can be cleaned easily,
are brittle and expensive. ◗ Radiations
2. Membrane filters: Membrane filters are made up of (a) The ionizing and nonionizing radiations are the two types of
Chapter 43

cellulose acetate, (b) cellulose nitrate, (c) polycarbonate, (d) radiation used for sterilization (Table 3-2).
polyvinylidene fluoride, or (e) other synthetic materials.
1. Ionizing radiations: Ionizing radiation is an excellent
These filters are now widely used and have replaced depth
sterilizing agent with very high penetrating power.
filters for last many years. These filters are circular porous
These radiations penetrate deep into objects and destroy
membranes and are usually 0.1 mm thick. Although a wide
bacterial endospores and vegetative cells, both prokaryotic
variety of pore sizes (0.015–12 ␮m) are available, mem-
and eukaryotic. These are, however, not that effective
branes with pores about 0.2 ␮m are used, because the pore
against viruses. Ionizing radiations include (a) X-rays,
size is smaller than the size of bacteria. These filters are
(b) gamma rays, and (c) cosmic rays. Gamma radiation from
used to remove most vegetative cells, but not viruses, from
a cobalt-60 source is used for sterilization of antibiotics,
solutions to be filtered. In the process of filtration, the
hormones, sutures, catheters, animal feeds, metal foils,
membranes are held in special holders and often preceded
and plastic disposables, such as syringes. This has also
by depth filters made of glass fibers to remove larger parti-
been used to sterilize and “pasteurize” meat and other
cles that might clog the membrane filter. The solution is
food items.
then pushed or forced through the filter with a vacuum or
Irradiation usually kills Escherichia coli O157:H7,
with pressure from a syringe, peristaltic pump, or nitrogen
Staphylococcus aureus, Campylobacter jejuni, and other patho-
gas bottle, and collected in previously sterilized containers.
gens. Since there is no detectable increase of temperature
in this method, this method is commonly referred to as
Key Points “cold sterilization.” Both the Food and Drug Administration
Membrane filters remove microorganisms by screening them and the World Health Organization have approved food
out in the way a sieve separates large sand particles from small irradiation and declared it safe.
ones. These filters have many uses:
■ They are used to sterilize pharmaceutical substances, oph-
thalmic solutions, liquid culture media, oils, antibiotics,
Key Points
and other heat-sensitive solutions. Gamma radiation from a cobalt-60 source is used for steriliza-
■ They are used to obtain bacterial free filtrates of clinical tion of antibiotics, hormones, sutures, catheters, animal feeds,
specimens for virus isolation. metal foils, and plastic disposables such as syringes. This has
■ They are used to separate toxins and bacteriophages from also been used to sterilize and ‘pasteurize’ meat and other food
bacteria. items.
 STERILIZATION AND DISINFECTION 29
2. Nonionizing radiations: Nonionizing radiations include cold merely retards the activities of most microbes. Although it
infrared and ultraviolet radiations. is true that cold temperatures kill some microbes, gradual cool-

Section I
ing, long-term refrigeration, or deep-freezing does not adversely
■ Infrared radiations are used for rapid and mass steriliza- affect most of the microorganisms. In fact, freezing tempera-
tion of disposable syringes and catheters. tures, ranging from ⫺70 to ⫺135°C, provide an environment
■ Ultraviolet (UV) radiation with wavelength of 240–280 nm that can preserve cultures of bacteria, viruses, and fungi for lon-
is quite lethal and has a marked bactericidal activity. It ger periods. Some psychrophiles grow very slowly even at freez-
acts by denaturation of bacterial protein and also inter- ing temperatures and can continue to secrete toxic products.
feres with replication of bacterial DNA. S. aureus, Clostridium species (spore formers), Streptococcus species,

Chapter 3
UV radiation is used primarily for disinfection of closed areas and several types of yeasts, molds, and viruses are the pathogens
in microbiology laboratory, inoculation hoods, laminar flow, that can survive for several months in the refrigerated food items.
and operating theaters. It kills most vegetative bacteria but not
spores, which are highly resistant to these radiations. However, Key Points
it does not penetrate glass, dirt films, water, and other sub-
Lyophilization is a process of freezing and drying. It is the
stances very effectively. most common method of preserving microorganisms and
Since UV radiations on prolonged exposure tend to burn other cells in a viable state for many years. Pure cultures are
the skin and cause damage to the eyes, UV lamps should be frozen instantaneously and exposed to a vacuum that rap-
switched off while people are working in such areas. idly removes the water (it goes right from the frozen state
into the vapor state). This method avoids the formation
of ice crystals that would damage the cells. Although not
◗ Sound (sonic) waves all cells survive this process, lot many of them survive after

Section III
High-frequency sound (sonic) waves beyond the sensitivity of the reconstitution of lyophilized culture.
human ear are known to disrupt cells. Sonication transmits vibra-
tions through a water-filled chamber (sonicator) to induce pres- As a general rule, chilling, freezing, and desiccation are not
sure changes and create intense points of turbulence that can stress considered as methods of disinfection or sterilization because
and burst cells in the vicinity. Sonication also forcefully dislodges their antimicrobial effects are erratic and uncertain, and one
foreign matter from objects. Heat generated by the sonic waves (up cannot be sure that pathogens subjected to these procedures
to 80°C) also appears to contribute to the antimicrobial action. have been killed.
Gram-negative rods are most sensitive to ultrasonic vibra-

Chapter 43
tions, while Gram-positive cocci, fungal spores, and bacterial
spores are resistant to them. Ultrasonic devices are used in Disinfection
dental and some medical offices to clear debris and saliva from
instruments before sterilization and to clean dental restorations. Disinfection is the process of inactivating microorganisms by
However, most sonic machines are not reliable for regular use direct exposure to chemical or physical agents. Differences
in disinfection or sterilization. between sterilization and disinfection have been summarized
Biological controls used for testing the efficacy of steriliza- in Table 3-4.
tion techniques are summarized in Table 3-3. ■ Disinfectants are products or biocides that destroy or
inhibit the growth of microorganisms on inanimate objects
Chemical Methods of Sterilization or surfaces. Disinfectants can be sporistatic but are not nec-
Several chemical agents are used as antiseptics as well as dis- essarily sporicidal.
infectants. All these chemical agents (e.g., alcohols, aldehydes, ■ Antiseptics are biocides or products that destroy or inhibit
etc.) are described later in detail under disinfection. the growth of microorganisms in or on living tissue.
The effects of cold and desiccation: The main benefit of Antiseptics and disinfectants are used extensively in hospitals
cold treatment is to slow the growth of cultures and microbes in for a variety of topical and hard surface applications. They are
food during processing and storage. It is essential to know that an essential part of infection control practices and aid in the
prevention of nosocomial infections.

Biological controls used for testing Properties of Ideal Disinfectant

TABLE 3-3
efficacy of sterilization techniques
Technique Control organism An ideal disinfectant or antiseptic has the following
Autoclave Geobacillus stearothermophilus
characteristics:
Hot-air oven Bacillus subtilis 1. Ideally, the disinfectant should have a wide spectrum of
Ionizing radiations Bacillus pumilus antimicrobial activity. It must be effective against a wide
variety of infectious agents (Gram-positive and Gram-
Ethylene oxide Bacillus globigii
negative bacteria, acid-fast bacteria, bacterial endospores,
Bacillus subtilis
fungi, and viruses) at high dilutions.
 30 GENERAL MICROBIOLOGY

Differences between sterilization and ■ Type of microorganism: Vegetative cells are more suscep-
TABLE 3-4 disinfection tible than spores. Spores may be resistant to the action of
Section I

Sterilization Disinfection disinfectants.

■ Physiological state of the cell: Young and metabolically
Definition Freeing an article, surface, or Process that reduces active cells are more sensitive than old dormant cells.
medium from all living organisms the number of
Nongrowing cells may not be affected.
including viruses, bacteria and contaminating
their spores, and fungi and their microorganisms, liable ■ Environment: The physical or chemical properties of the
spores. to cause infection to a medium or substance influence rate as well as efficiency of
level which is deemed disinfectants, e.g., pH of the medium and presence of
Chapter 3

no longer harmful to extraneous materials.

health. Spores are not
killed.
Types of Disinfectants
Uses Objects or instruments coming in Objects or
direct contact with a break in skin instruments coming Disinfectants include the following: (a) phenolic compounds,
or mucous membrane or entering in direct contact with (b) halogens, (c) alcohols, (d) aldehydes, (e) gases, (f) surface
a sterile body area. mucous membrane active agents, (g) oxidizing agents, (h) dyes, (i) heavy metals, and
but tissue is intact or
(j) acids and alkalis.
via intact skin.
Examples Surgical instruments, needles, Endotracheal
◗ Phenolic compounds
syringes, parenteral fluid, tubes, aspirators,
arthroscopes, media, reagents gastroscopes, bed In 1867, Joseph Lister employed phenolic compounds to reduce
and equipments in laboratory use. pans, urinals, etc. the risk of infection during operations. Phenolic compounds are
Section III

the most widely used antiseptics and disinfectants in laboratories

and hospitals worldwide. They are bactericidal or bacteriostatic
2. It should act in the presence of organic matter.
and some are fungicidal also. They act by denaturing proteins
3. It should not be toxic to human or corrosive. In prac-
and disrupting cell membranes. They are effective in the pres-
tice, this balance between effectiveness and low toxicity
ence of organic material and remain active on surfaces long after
for animals is hard to achieve. Some chemicals are used
application. Different phenolic compounds are as follows:
despite their low effectiveness, because they are relatively
nontoxic. ■ Phenol: It is effective against vegetative forms of bacteria,
Chapter 43

4. It should be stable upon storage and should not undergo Mycobacterium tuberculosis, and certain fungi. It is an excellent
any chemical change. disinfectant for feces, blood, pus, sputum, etc. It has a low
5. It should be odorless or with a pleasant odor. degree of activity as compared to other derivatives. It is not
6. It should be soluble in water and lipids for penetration suitable for application to skin or mucous membrane.
into microorganisms. ■ Cresol: Cresols are more germicidal and less poisonous
7. It should be effective in acidic as well as in alkaline media. than phenol but corrosive to living tissues. They are used
8. It should have speedy action. for cleaning floors (1% solution), for disinfection of surgical
9. If possible, it should be relatively inexpensive. instruments, and for disinfection of contaminated objects.
Lysol is a solution of cresols in soap.
Action of Disinfectants ■ Halogenated diphenyl compounds: These compounds
include hexachlorophene and chlorhexidine. They are highly
Disinfectants act in many ways as discussed below. effective against both Gram-positive and Gram-negative
1. They produce damage to the cell wall and alter permeabil- bacteria. They are used as skin antiseptics and for the clean-
ity of the cell membrane, resulting in exposure, damage, or ing of wound surfaces.
loss of the cellular contents. Hexachlorophene has been one of the most popular antiseptics
2. They alter proteins and form protein salts or cause coagu- because once applied it persists on the skin and reduces growth
lation of proteins. of skin bacteria for longer periods. However, it can cause brain
3. They inhibit enzyme action and inhibit nucleic acid syn- damage and is now used in hospital nurseries only after a
thesis or alter nucleic acid molecules. staphylococcal outbreak.
4. They cause oxidation or hydrolysis.
◗ Halogens
Factors Influencing Activity of Disinfectants Halogens are fluorine, bromine, chlorine, and iodine—a group
of nonmetallic elements that commonly occur in minerals, sea
Various conditions influencing the efficiency of disinfectant
water, and salts. Although they can occur either in the ionic
are as follows:
(halide) or nonionic state, most halogens exert their antimicro-
■ Temperature: Increase in temperature increases the bial activity primarily in their nonionic state, but not in the
efficiency of disinfectants. halide state (e.g., chloride, iodide).
 STERILIZATION AND DISINFECTION 31
These agents are highly effective disinfectants and anti- antiseptic before surgery and also occasionally as a treatment for
septics, because they are microbicidal and not just microbi- burnt and infected skin. A stronger iodine solution (5% iodine and

Section I
static. They are also sporicidal with longer exposure. For these 10% potassium iodide) is used primarily as a disinfectant for plas-
reasons, halogens are the active ingredients in nearly one-third tic items, rubber instruments, cutting blades, and thermometers.
of all antimicrobial chemicals currently marketed. Chlorine Iodine tincture is a 2% solution of iodine and sodium iodide
and iodine are the only two routinely used halogens because in 70% alcohol that can be used in skin antisepsis. Because
fluorine and bromine are dangerous to handle. iodine can be extremely irritating to the skin and toxic when
Chlorine and its compounds: Chlorine has been used for dis- absorbed, strong aqueous solutions and tinctures (5–7%) are no
infection and antisepsis for approximately 200 years. The major longer considered safe for routine antisepsis.

Chapter 3
forms used in microbial control are (a) liquid and gaseous chlo- Iodine tablets are available for disinfecting water during emer-
rine and (b) hypochlorites. In solution, these compounds com- gencies or for destroying pathogens in impure water supplies.
bine with water and release hypochlorous acid (HOCl), which Iodophors: Iodophors are complexes of iodine and a neutral
oxidizes the sulfhydryl (S–H) group on the amino acid cysteine polymer, such as polyvinyl alcohol. This formulation permits
and interferes with disulfide (S–S) bridges on numerous enzymes. the slow release of free iodine and increases its degree of pen-
The resulting denaturation of the enzymes is permanent. etration. These compounds have largely replaced free iodine
Gaseous and liquid chlorine are used almost exclusively for solutions in medical antisepsis because they are less prone to
large-scale disinfection of drinking water, sewage, and wastewa- staining or irritating tissues.
ter from sources, such as agriculture and industry. Chlorine ■ Betadine, povidone, and isodine are the common iodophor
kills not only bacterial cells and endospores but also fungi and compounds that contain 2–10% of available iodine. They are
viruses. Treatment of water with chlorine destroys many patho- used to prepare skin and mucous membranes for surgery
genic vegetative microorganisms without unduly affecting its

Section III
and in surgical hand scrubs.
taste. Chlorination at a concentration of 0.6–1.0 part of chlorine ■ They are also used to treat burns and to disinfect equipments.
per million parts of water makes water potable and safe to use. ■ A recent study has shown that betadine solution is an
effective means of preventing eye infections in newborn
infants, and it may replace antibiotics and silver nitrate as
Key Points the method of choice.
Hypochlorites are perhaps the most extensively used of all
chlorine compounds. They are used for:

Chapter 43
◗ Alcohols
■ Sanitization and disinfection of food equipment in dairies,
restaurants, and canneries; Alcohols are among the most widely used disinfectants and
■ Treatment of swimming pools, spas, drinking water, and antiseptics. They are bactericidal and fungicidal but not spori-
even fresh foods; cidal. They have no action against spores and viruses. Ethyl
■ Treatment of wounds; and alcohol and isopropyl alcohol are the two most popular alcohol
■ Disinfection of equipments, beddings, and instruments.
germicides. They are effective at a concentration of 60–70% in
water. They act by denaturing bacterial proteins and possibly
Common household bleach is a weak solution (5%) of sodium by dissolving membrane lipids. They are used as skin antisep-
hypochlorite that is used as an all-around disinfectant, deodor- tics. Isopropyl alcohol is used for disinfection of clinical ther-
izer, and stain remover. It is frequently used as an alternative mometers. A 10–15 minute soaking is sufficient to disinfect
to pure chlorine in treating water supplies. However, the major thermometers. Methyl alcohol is effective against fungal spores.
limitations of chlorine compounds are that they are:
◗ Aldehydes
(a) Ineffective if used at an alkaline pH,
(b) Less effective in the presence of excess organic matter, and Formaldehyde and glutaraldehyde are the two most commonly
(c) Relatively unstable, especially if exposed to light. used aldehydes that are used as disinfectants. They are highly
reactive molecules that combine with nucleic and alkylating
Iodine and its compounds: Iodine is a pungent black chemical molecules. They are sporicidal and can also be used as chemical
that forms brown-colored solutions when dissolved in water or sterilants.
alcohol. Iodine rapidly penetrates the cells of microorganisms,
Formaldehyde: Formaldehyde is usually dissolved in water or
where it apparently disturbs a variety of metabolic functions.
alcohol before use. In aqueous solution, it is bactericidal, spo-
It acts by interfering with the hydrogen and disulfide bonds of
ricidal, and also effective against viruses. Formalin solution is
proteins (similar to chlorine). It kills all types of microorgan-
40% aldehyde in aqueous solution. It is used to:
isms if optimum concentrations and exposure times are used.
Iodine activity, unlike chlorine, is not as adversely affected by ■ Preserve fresh tissue specimens,
organic matter and pH. The two primary iodine preparations ■ Destroy anthrax spores in hair and wool,
are free iodine in solution and iodophors. ■ Prepare toxoids from toxins,
Free iodine in solution: Aqueous iodine contains 2% free ■ Sterilize bacterial vaccines, and
iodine in solution and 2.4% sodium iodide. It is used as a topical ■ Kill bacterial cultures and suspensions.
 32 GENERAL MICROBIOLOGY

Glutaraldehyde: A 2% buffered solution of glutaraldehyde is ends. Due to their amphipathic nature, detergents solubilize
an effective disinfectant. It is less irritating than formaldehyde and are very effective cleansing agents. They are different
Section I

and is used to disinfect hospital and laboratory equipments. from soaps, which are derived from fats. Surface active agents
Glutaraldehyde usually disinfects objects within time frame of are of four types:
10 minutes but may require as long as 12 hours to destroy all
1. Cationic surface active agents: The cationic detergents
spores. Glutaraldehyde is especially effective against tubercle
are effective disinfectants. Cationic detergents like benzal-
bacilli, fungi, and viruses. It can be used for cleaning cysto-
konium chloride and cetylpyridinium chloride kill most
scopes and bronchoscopes, corrugated rubber anesthetic tubes
bacteria but not M. tuberculosis, endospores, or viruses. They
and face masks, plastic endotracheal tubes, metal instruments,
Chapter 3

do have the advantages of being stable and nontoxic, but

and polythene tubing.
they are inactivated by hard water and soap. These are
often used as skin antiseptics and also as disinfectants for
◗ Gases disinfection of food utensils and small instruments. Qua-
Various gaseous agents are used for sterilization of large volume ternary ammonium compounds, such as cetrimide are the
of heat-sensitive disposable items and also instruments. most popular cationic detergents. They act by disrupting
Ethylene oxide, formaldehyde gas, and betapropiolactone are microbial membranes and possibly by denaturing proteins.
frequently used gaseous agents. 2. Anionic surface active agents: These include soaps prepared
Ethylene oxide: Ethylene oxide is a colorless liquid used for either from saturated or unsaturated fatty acids, which act
gaseous sterilization. It is active against all kinds of bacteria, better at acidic pH. The soaps prepared from saturated fatty
spores, and viruses. It kills all types of microorganisms by acids are more effective against Gram-negative organisms,
inhibiting proteins and nucleic acids. It is both microbicidal whereas those prepared from unsaturated fatty acids are
Section III

and sporicidal. It is a highly effective sterilizing agent because more active against Gram-positive bacilli and Neisseria.
it rapidly penetrates packing materials, including plastic wraps. 3. Nonionic surface active agents: These are nontoxic and
It is used to sterilize disposable plastic Petri dishes, sutures, some of them may even promote the growth of bacteria.
syringes, heart-lung machine, respirators, and dental equip- 4. Amphoteric or ampholytic compounds: These are active
ments. Ethylene oxide is highly inflammable and carcinogenic. against a wide range of Gram-positive and Gram-negative
Extensive aeration of the sterilized materials is necessary to bacteria and also against a few viruses. These are known as
remove residual ethylene oxide gas, which is toxic. “Tego” compounds.
Chapter 43

Formaldehyde gas: The formaldehyde gas is used for

(a) the fumigation of operation theaters, wards, sick rooms, ◗ Oxidizing agents
and laboratories; and (b) the sterilization of instruments and This group includes halogens, hydrogen peroxide, potassium
heat-sensitive catheters, clothing and bedding, furniture, permanganate, and sodium perborate. They are good disinfec-
books, etc. The formaldehyde gas is produced by adding 150 tants and antiseptics but are less effective in the presence of
gm of potassium permanganate to 280 mL formalin in 1000 organic matter. Hydrogen peroxide, used as 3% solution, is a
cu ft of room volume. The room to be sterilized is completely weak disinfectant. It is useful for cleaning of the wounds and
closed and sealed at least for 48 hours after fumigation with for mouth wash or gargle. Potassium permanganate is bacteri-
formalin gas. Sterilization is achieved by condensation of gas cidal in nature and active against viruses also.
on exposed surface. The gas is toxic when inhaled and is irri-
tant to eye, hence its effect should be nullified by exposure to ◗ Dyes
ammonia. It is highly inflammable and carcinogenic.
The dyes that have been used extensively as skin and wound
Beta-propiolactone: Beta-propiolactone (BPL) is a condensa- antiseptics include (a) acridine dyes and (b) aniline dyes. The
tion product of ketone and formaldehyde. It is active against acridine dyes include acriflavine, euflavine, proflavine, and
all microorganisms and viruses. It is more efficient than aminacrine. They show more activity against Gram-positive bac-
formaldehyde for fumigation purpose. In the liquid form, teria than against Gram-negative organisms. They act by interfer-
it has been used to sterilize vaccines and sera. BPL destroys ing with the synthesis of nucleic acids and proteins in bacterial
microorganisms more readily than ethylene oxide but does not cells. The yellow acridine dyes, acriflavine and proflavine, are
penetrate materials well and may be carcinogenic. For these sometimes used for antisepsis and wound treatment in medi-
reasons, BPL has not been used as extensively as ethylene oxide. cal and veterinary clinics. Aniline dyes (such as gentian violent,
Recently, vapor-phase hydrogen peroxide has been used to crystal violet, and malachite green) are also more active against
decontaminate biological wastes. Gram-positive bacteria than against Gram-negative organisms.
They are also effective against various fungi, hence are incorpo-
◗ Surface active agents rated into solutions and ointments to treat fungal skin infec-
Surface active agents, such as detergents are the substances tions, such as ringworm.
that alter energy relationship at interfaces producing a The dyes, nevertheless, have limited applications because
reduction in surface tension. Detergents are organic mol- they stain and have a narrow spectrum of antimicrobial activ-
ecules that serve as wetting agents and emulsifiers because ity. They also have no activity against tubercle bacilli. Their
they have both polar hydrophilic and nonpolar hydrophobic actions are also inhibited by the presence of organic matter.
 STERILIZATION AND DISINFECTION 33

◗ Heavy metals ◗ Phenol coefficient (Rideal–Walker) test

Section I
Soluble salts of mercury, silver, copper, arsenic, and other heavy Rideal and Walker designed the phenol coefficient test to
metals have antibacterial activity, both bactericidal and bacte- compare the performance of a disinfectant with that of phe-
riostatic. They combine with proteins, often with their sulfhy- nol for the ability to kill Salmonella typhi. Phenol coefficient is
dryl groups and inactivate them. They may also precipitate cell determined by dilution of the disinfectant in question which
proteins. Silver compounds are widely used as antiseptics. sterilizes the suspension of S. typhi in a given time divided by
Silver sulfadiazine is used for burns. Silver nitrate is used as a the dilution of phenol which sterilizes the suspension in the
prophylactic agent in ophthalmia neonatorum in newborn same time.

Chapter 3
infants. Copper sulfate is an effective algicide in lakes and In this test, a series of dilutions of phenol and the experi-
swimming pools. Mercuric chloride is used as disinfectant. mental disinfectant are inoculated with the test bacteria
These compounds, however, are increasingly replaced by other S. typhi and S. aureus, then placed in a 20°C or 37°C water bath.
less toxic and more effective germicides. These inoculated disinfectant tubes are then subcultured on a
regular fresh medium at 5 minute intervals, and the subcul-
tures are incubated for two or more days. The highest dilutions
◗ Acids and alkalis that kill the bacteria after a 10 minute exposure, but not after
Acids (such as sulfuric acid, nitric acid, hydrochloric 5 minutes, are used to calculate the phenol coefficient. The
acid, and benzoic acid) and alkalis (like potassium and reciprocal of the appropriate test disinfectant dilution is
sodium hydroxide and ammonium hydroxide) are germi- divided by that for phenol to obtain the coefficient. Suppose
cidal in nature. They kill microorganisms by hydrolysis that the phenol dilution was 1/90 and maximum effective dilu-
and altering the pH of the medium. They are rarely used as tion for disinfectant X tested was 1/450, then the phenol coef-

Section III
disinfectants. ficient of X would be 5.
Organic acids are widely used in food preservation
because they prevent spore germination and bacterial and
fungal growth, and because they are generally regarded as Key Points
safe to eat. Acetic acid, in the form of vinegar, is a pickling The higher the phenol coefficient value, the more effective the
agent that inhibits bacterial growth. Propionic acid is com- disinfectant under these test conditions. A value greater than
monly added into breads and cakes to retard molds; lactic 1 means that the disinfectant is more effective than the phenol.
The test, however, does not show the action of disinfect-
acid is added to sauerkraut and olives to prevent growth of

Chapter 43
ant in natural conditions, i.e., in the presence of organic
anaerobic bacteria, especially the clostridia; and benzoic and contaminants.
sorbic acids are added to beverages, syrups, and margarine to
inhibit yeasts.
Activities of commonly used disinfectants against various
◗ Chick Martin test
microorganisms are summarized in Table 3-5.
It is a modification of Rideal–Walker test, in which the
disinfectant acts in the presence of organic contaminants (e.g.,
Testing of Disinfectants dried yeast, feces, etc.) to simulate the natural conditions.
The efficiency of disinfectants can be determined with the help
of several tests. These are: ◗ Capacity (Kelsey–Sykes) test
The capacity (Kelsey–Sykes) test determines the appropriate use of
■ Phenol coefficient (Rideal–Walker) test dilutions of the disinfectants. It measures the capacity of a disin-
■ Chick Martin test fectant to retain its activity when repeatedly used microbiologi-
■ Capacity (Kelsey–Sykes) test cally. The disinfectant is assessed by its ability to kill bacteria by
■ In-use (Kelsey–Maurer) test demonstrating growth or no growth on recovery culture media
but not by comparison with phenol. The test is performed under
both clean and dirty conditions, hence shows the effectiveness of a
TABLE 3-5 Activities of commonly used disinfectants
disinfectant in the presence of organic material.
Disinfectant Fungi Bacteria Spores Enveloped Nonenveloped
viruses viruses ◗ In-use (Kelsey–Maurer) test
Phenol Good Good Nil Good Variable The “in-use” (Kelsey–Maurer) test is a test that determines
Hypochlorite Good Good Fair Good Good whether the chosen disinfectant is effective, in actual use, in
hospital practice and also for the period of its use. The
Alcohols Good Good Nil Good Variable
effectiveness of the disinfectant is determined by its ability to
Aldehydes Good Good Good Good Good inactivate a known number of standard strain of a pathogenic
Glutaraldehyde Good Good Good Good Good staphylococci on a given surface within a certain given time.
In-use test allows a more accurate determination of effective-
Iodophor Good Good Nil Good Good
ness of a disinfectant compared to phenol coefficient test.
 4
Culture Media
Introduction Key Points
Agar has a unique property of melting at 98°C and solidifying
Laboratory diagnosis of an infection is usually confirmed by at 42°C. The jellifying property of agar varies depending on the
isolating and culturing microorganisms in artificial media. type of agar used (e.g., New Zealand agar is more jellifying than
Bacteria and fungi are cultured in either liquid (broth) or on Japanese agar). Agar does not have any nutritive value and is
solid (agar) artificial media. Koch pioneered the use of agar as also not affected by growth of bacteria.
a base for culture media. He developed the pour plate method
and was the first to use solid culture media for culture of
bacteria. At first, Koch cultured bacteria on the sterile surfaces Peptone
of cut, boiled potatoes. This was unsatisfactory, because Peptone is another important ingredient of culture media. It is
bacteria would not always grow well on potatoes. He then tried a complex mixture of partially digested proteins. It is obtained
to solidify regular liquid media by adding gelatin. Separate by digestion of lean meat or other protein materials (such as
bacterial colonies developed after the surface had been streaked heart muscle, casein, fibrin, or soya flour) with proteolytic
with a bacterial sample. The sample could also be mixed with enzymes (such as pepsin, trypsin, or papain).
liquefied gelatin medium. When the gelatin medium hardened,
individual bacteria produced separate colonies. Despite
its advantages, gelatin was not an ideal solidifying agent Key Points
because it was digested by many bacteria and melted when the ■ Peptone is an important source of nutrition for bacteria to
temperature rose above 28°C. A better alternative was provided grow.
by Fannie Eilshemius Hesse, the wife of Walther Hesse, one of ■ It contains peptones, proteoses, amino acids, inorganic
Koch’s assistants. She suggested the use of agar as a solidifying salts (phosphates, potassium, and magnesium), and cer-
tain accessory factors, such as nicotinic acid and riboflavin.
agent—she had been using it successfully to make jellies for
■ Neopeptone and proteose peptone are special types of
sometime. Agar was not attacked by most bacteria and did not peptone with high nutritive value.
melt until it reaches a temperature of 100°C. One of Koch’s
assistants, Richard Petri, developed the Petri dish (plate), a
container for solid culture media. Other Ingredients
Other common ingredients of media include water, sodium
chloride and other electrolytes, meat extract, yeast extract, malt
Ingredients of Culture Media extract, blood, and serum. Meat extract is available commercially
as Lab-Lemco and contains inorganic salts, carbohydrates, certain
The basic constituents of culture media include the following: growth factors, and protein degradation products. Blood or
serum is usually used for enriching culture of bacteria. Usually,
Agar Agar or Agar 5–10% defibrinated sheep or human blood is used.
Agar is the main component that is used universally for prepa-
ration of solid media. It is obtained from a variety of sea weeds Types of Culture Media
and after necessary processing is usually available as powder or
as long shreds. Agar is chiefly composed of:
Culture media can be classified in several ways:
■ A long-chain polysaccharide, consisting of D-galactopyranose
units; Liquid Media, Semisolid Media, and
■ A variety of inorganic salts, minute quantities of protein-like Solid Media
materials, traces of long-chain fatty acids; and
■ Minerals, such as calcium and magnesium. ◗ Liquid media
Agar is usually used in a concentration of 2–3%. Agar is hydro- Liquid media provide greater sensitivity for the isolation of
lyzed at high temperatures and at high acid or alkaline pH. small numbers of microorganisms. Examples of liquid media
Other documents randomly have
different content
ihrem großen, blauen Blick, es war, als kennten sie sich lange, als
seien sie bis jetzt nur durch einen Zufall getrennt gewesen.
Sie stammte aus dem äußersten Nordwesten Deutschlands, aus
einer hochangesehenen Patrizierfamilie, die ganz nach alten
Traditionen lebte. Sie hatte sich freigemacht, gegen den Willen ihres
Vaters war sie Malerin geworden. Erst fast mit ihr deshalb verfeindet,
söhnte er sich allmählich mit dem Schritte aus, als er die Erfolge sah.
An seinem Vaterglück fehlte nun nichts weiter, als daß seine Tochter
nach Hause gekommen wäre und geheiratet hätte. Sie dachte nicht
daran, obgleich ihr der Gedanke selbst nicht abliegend und fremd
war, denn sie liebte ihre Vaterstadt und das ganze Land da droben
mehr als alles andre was sie sah, und als letztes Lebensziel schwebte
ihr ein breites Familienglück in großem Stil vor, wie man es auf
prunkvollen holländischen Bildern gemalt sieht. Aber dieses Ziel lag
noch weit ab. Mehrere heftige Erlebnisse endeten mit
Enttäuschungen, ohne ihre feste Natur im mindesten zu beirren. Sie
lernte Pitt Sintrup kennen, und seine verträumte Unklarheit, das
Passive, Widerstandslose seines Wesens zog ihre eigene, dem Leben
zugewandte, auf das Leben wie zum Sprung gerichtete Seele an.
Pitt, dem noch kein Mädchen so entgegengekommen war, fühlte
seine eigene Empfindung mit fortgerissen. Er erzählte ihr sein
ganzes Leben, und während sie kein Wort verlor, wurde in ihr der
Wunsch, diesen Menschen zum Leben, zum Erwachen zu bringen,
was vor ihr noch keiner gelungen war, immer heftiger. Sie sahen sich
täglich, und während er wähnte, die Erfüllung könne noch in weiter
Ferne liegen, war für Herta selbst alles längst entschieden. Dann
kam ihre heftige Einigung. —
Ihm begann ein neues Leben an ihrer Seite. Die Vergangenheit
war ausgelöscht, eine unbekannte Frische kam über ihn, seinem
Dasein schien ein wirklicher Inhalt gegeben, es folgte eine Zeit des
Glückes, der Ausgeglichenheit, der heitersten Ruhe, wie er sie nie für
sein Leben erhofft hatte. — Die ersten Tage ging er herum wie im
Traum. Unfaßlich war ihm alles: Wie war es möglich, daß er wirklich
ein Mädchen sein eigen nannte, das er liebte und von dem er sich
geliebt wußte! Bisher hatte er mit dem Gefühl der Liebe nur etwas
Trauriges und Entsagendes verbunden, und nun empfand er, wie
alles andere neben ihr gering erschien! Mit welchem Stolze zeigte er
sich an ihrer Seite! —
Wie lange ihr enges Verhältnis zu Pitt dauern würde, wußte sie
selbst nicht; es reizte sie das Schicksal dieses Menschen, dem sie
sich in allem was das Leben anging, überlegen fühlte, in die Hand zu
nehmen, und jetzt, wo sie ihn näher kennen lernte, ward ihr dies
Gefühl befestigt. Ihr selbst war ein Leben ohne Arbeit, ohne
Schaffen unmöglich. Nur ein paar Tage waren sie zusammen im
Süden, dann verlangte sie zurück in ihre Tätigkeit. Pitt gewöhnte
sich daran ihr in allem sich zu fügen. Sie verlangte, daß er selber
arbeitete, und er tat es. Zu Anfang hatte er fast Scheu, ihr seinen
Beruf zu gestehen, da er glaubte sie werde ihn verachten. Aber das
tat sie gar nicht, im Gegenteil, und sie sagte: Mein Vater würde dich
mehr achten als die meisten andern, die er durch mich kennt, denn
nach seiner Schätzung taugen im Grunde nur die Menschen etwas,
deren Beruf sich direkt wieder auf die Menschen richtet!
Sie setzte es durch, daß Pitt sein Examen bei der nächsten
Gelegenheit machte. Er sagte, er fühle sich nicht sicher. Sie rechnete
nach, wieviel Semester er nun allmählich hinter sich hatte, und
meinte dann: wenn er sich jetzt noch nicht sicher fühle, würde er
sich niemals sicher fühlen. Er solle den Versuch machen, mehr als
durchfallen könne er nicht, und das sei nicht schlimm. Sie sprach
auch mit einem seiner Professoren, den sie kannte und auf einer
Gesellschaft traf. Sie erfuhr, Pitt Sintrup gelte in den Seminaren als
der beste Kopf, der mit Leichtigkeit die feinsten Fragen zu spalten
vermochte, aber leider die ganze Jurisprudenz wie eine Bagatelle
ansehe. Nun setzte sie ihren ganzen Einfluß hinter ihn, und Pitt
empfand es so wohltuend, daß ihn jemand trieb; zum Scheine
weigerte er sich immer noch und trieb sie dadurch zu immer
heftigerer Forderung. Die hörte er so gern, und mit Vergnügen sagte
er dann endlich lächelnd: Nun also — in Gottes Namen! — Und nach
ein paar Monaten machte er das Examen und etwas später bekam er
auch den Doktortitel, da Herta sagte, es käme nun auch nicht mehr
darauf an, ob er noch etwas mehr arbeite. Dann aber gab es Kämpfe
was nun werden sollte. Referendar werden wollte er nicht, er wollte
überhaupt nun nichts mehr mit der ganzen Juristerei zu tun haben;
wußte aber auch nichts anderes. Ihr war diese Eröffnung ganz neu,
sie lachte und nannte ihn verrückt. Dann begannen tagelange
Bearbeitungen, die damit endeten, daß Pitt sich zu allem bereit
erklärte. Die Aussicht in eine fernere Zukunft schien ihm nach diesen
Gesprächen auch nicht mehr so entsetzlich wie früher: Hertas
Familie dort oben in dem kleinen Hansastaat hatte die größten,
einflußreichsten Verbindungen, durch sie würde es ihm leicht
werden, eine gute einträgliche Stelle zu bekommen, die er durch
eigene Energie vielleicht nie erreicht hätte, und großen Eindruck
machte es ihm, daß er dann kein königlicher Angestellter war,
sondern daß er unter einer Republik diente, deren Oberhaupt, wie er
erst jetzt erfuhr, Hertas Vater selber war. — Herta lächelte für sich,
als er ihr eines Tages erklärte: Jetzt sitze ich wirklich drin, in diesem
Maulwurfsnest; — er meinte damit das Amtsgericht. Und was wird
dann eigentlich später aus uns beiden? fragte er manchmal; ich
denke, du nimmst dir dann ein Haus für dich und wir besuchen uns
wann wir mögen! — Laß uns nicht daran denken, sagte sie, bis
dahin hat es sich schon entschieden, ob wir dauernd zusammen
bleiben wollen oder nicht; und wollen wir, so ist es nicht anders
möglich, als daß wir den Schritt tun, den die meisten tun, die
zusammen bleiben wollen. — Pitt verzog unwillkürlich das Gesicht,
wie wenn er etwas Bitteres schmecke. — Wäre dir der Gedanke so
schrecklich? fragte sie. — O nein, antwortete er schnell, durchaus
nicht, ich machte dies Gesicht eben nur aus alter Gewohnheit. Herta
sprach öfter von der Möglichkeit solcher Aussichten, ja manchmal,
wie zum Scherz, malte sie sich ihre und Pitts nähere Zukunft aus. Sie
sprach von einem schönen Haus, das sie sich bauen würden. Sie
wußte schon den herrlichsten Platz dafür. Oben im zweiten Stock
sollte ein riesiges Atelier sein mit einem Ausblick auf das ferne flache
Land. Oft redete sie von diesen Dingen, auch von den Menschen mit
denen sie dort verkehren würden, von ihren Eltern, von ihren
Verwandten. Pitt sah dann im Geist die lange Reihe der Ahnenbilder,
von denen sie ihm erzählte, die zu Haus im großen Saale hingen,
und er wunderte sich über ihren festen Zusammenhang mit ihrer
Heimat und allem was mit ihr verbunden war. Ihm selber fehlte
solcher Zusammenhang gänzlich, und bei ihren Worten überschlich
ihn zuweilen ein dumpfes Unbehagen. Gegen einzelne Menschen,
von denen sie immer wieder sprach, empfand er nach und nach
geradezu eine Abneigung.
Wie ist es nur möglich, sagte er einmal, daß ein Mensch so stark
verwandtschaftlich empfinden kann! — Es kommt eben nur auf die
Verwandten an! entgegnete sie. Das ließ er gelten, obwohl er wußte,
daß da noch ganz andere, tiefere Unterschiede mitspielten. Aber er
sprach nichts davon aus, da er fühlte, daß, wenn sie dann darüber
stritten und er Herta schließlich recht gäbe, eine Einigung doch nur
ganz oberflächlich sein würde. Und zugleich rührte diese Frage
überhaupt an Tiefen, die er nicht sehen, die er vergessen wollte.
Du möchtest wohl, sagte sie ein andermal, als sie wieder von
ihrem Luftschloß redete, daß wir unser Haus verschlössen und gar
niemand hereinließen? — Er lächelte, und zugleich durchzog ihn eine
halb peinliche Empfindung bei der Vorstellung an dies Haus, in dem
sie allein sein würden. Er kannte es ja auch noch gar nicht, für ihn
war es vorläufig ein fremdes Haus wie jedes andere. Herta freilich
kannte es auch noch nicht, und doch sprach sie von ihm als ob sie
schon darin wäre. Sie zeigte ihm nun auch Bilder ihrer Eltern und
ihrer Geschwister: Lauter blonde, großgewachsene Menschen der
reinsten Rasse. Alle hatten unter sich Ähnlichkeit, wie die
verschiedenen Blätter desselben Baumes. — Sie wollte nun auch
Bilder seiner Familie sehen, und Pitt holte aus einem Kasten einige
halb vergessene Photographien, die ihm seine Mutter mitgab, wie er
ins erste Semester ging. — Deinem Vater bist du nicht ähnlich, sagte
Herta, und deiner Mutter auch nicht; wie bist du nur zu diesem Kopf
gekommen! — Sie malte ihn jetzt noch einmal, und das Bild wurde
besser als das erste. Ihm erschien es, bei aller Ähnlichkeit, zu laut in
der Empfindung. Aber er sprach dies nicht aus, sondern betrachtete
es nur, in Nachsinnen verloren. — Es wäre besser, sagte er endlich,
wenn die Menschen ihren ganzen Körper aufgäben und nur aus Kopf
beständen! Wieviel Aufregung und Gräßliches würde ihnen erspart
bleiben. — Und das sagst du zu m i r?! rief sie und sah ihn
verwundert an. Sie begegnete seinem stillen Blick, der sie nicht zu
sehen schien. Möchtest du, fuhr sie fort, daß wir beide körperlos
wären? Ist dir dein Körper so wenig lieb? Er antwortete nicht. — Was
für einen Unsinn redest du! Mein Körper ist mir das liebste auf der
Welt, viel lieber als mein Kopf und alles was drin steckt an Kunst und
Gedanken! Und lieber möchte ich auf alles, alles verzichten als den
kleinsten körperlichen Makel haben; du redest wie ein alter Mann
und sollst dich schämen! — Er lachte und zuckte die Achsel, und wie
er sie so blühend vor sich sah, den lebensvollen Blick jetzt halb
vorwurfsvoll auf ihn geheftet, dachte er: Mein Gott, wie recht hat
sie, und wie dankbar bin ich, daß sie nicht körperlos ist! und schloß
sie heftig in seine Arme. — Siehst du nun wie dumm du bist! Sie
hielt ihn fest umschlungen, so lange und so bewegungslos, daß
endlich seine Gedanken abirrten, bis er die körperliche Ermüdung
des Stehens empfand und sich langsam aus ihren Armen befreite.
Höre, sagte sie eines Tages, morgen kommen meine Eltern her;
sie reisen nach Italien, und bleiben nur einen einzigen Tag hier. Ich
möchte, daß du sie sähest. — Pitt durchfuhr dies sehr unbehaglich.
— Was werden deine Eltern denken? fragte er ausweichend. — Sie
werden sich die Wahrheit denken, sagte Herta; sie wissen, daß ich
kein Kind mehr bin und daß ich nach meinen eigenen Vorschriften
lebe. Und sie haben sich damit abgefunden. Meine Mutter weiß
übrigens über dies Bescheid; sie weiß über alles Bescheid, was mich
angeht, und vieles wäre noch viel schwerer gewesen in meinem
Leben, wenn ich mich nicht stets an sie gehalten hätte. — Pitt war
über diese letzte Eröffnung sehr erstaunt. Ich glaubte, sagte er,
deine Mutter sei so konventionell! — Herta lächelte und sagte: Du
wirst sie kennen lernen und Achtung vor ihr gewinnen! Ich glaube,
du achtest die Frauen überhaupt viel zu wenig. — Vor meinem Vater
allerdings, fuhr sie fort, vor meinem Vater suche zu bestehen! und
sie sprach die letzten Worte mit Nachdruck; — übrigens machst du
dir von ihm vielleicht ebenfalls ein falsches Bild. Mein Vater hat den
allergrößten Blick für Menschen und Dinge, es ist etwas Großzügiges
in seinem ganzen Wesen. Nur gegen seine nächsten Angehörigen
empfindet er wie ein gewöhnlicher Bürgersmann. In bezug auf mich
habe ich es ihm aber abgewöhnt. Nimm dich zusammen, wenn du
mit ihm sprichst, es ist auch gut für dich, für deine spätere Zukunft,
wenn er jetzt ein günstiges Bild von dir gewinnt. Ich kann mich doch
auf dich verlassen, daß du keinen Unsinn redest? Da er nicht
antwortete, fragte sie ihn halb unruhig: Du fühlst dich doch jetzt
ganz deiner sicher, nicht wahr?
Pitt liebte solche Fragen nicht. Herta tat sie zuweilen. Dann
erschien sie ihm jedesmal etwas fremd. — Er legte den Arm um sie,
küßte ihr glänzendes duftendes Haar und sagte: Sei ohne Sorge, ich
werde schon vor ihm bestehen. —
Pitt zog sich zu Hause um, zögernd, langsam, trat vor den Spiegel,
sah lange hinein — und mit einem plötzlichen Entschluß entkleidete
er sich wieder, zog seinen gewöhnlichen Anzug an und machte einen
stundenweiten Spaziergang, anstatt sich mit Hertas Eltern und ihr
selbst zu treffen.
Damit verletzte er sie sehr. — Hast du denn absolut nicht den
Wunsch gehabt sie kennen zu lernen? — Er schüttelte den Kopf. —
Manchmal verstehe ich dich nicht! — Ich dich auch nicht! antwortete
er, und sah sie nicht an dabei. — Er ist schwerer zu lenken als ich
glaubte; dachte sie, man muß Geduld mit ihm haben und nicht zuviel
auf einmal wollen. Ich rede immer viel zu viel von allem, anstatt ihn
ohne Worte zu lenken.
Es war ihr ein Bedürfnis von Zeit zu Zeit von ihm bestätigt zu
hören, wie sehr er sich innerlich umgewandelt fühlte. Es erfüllte sie
mit Stolz, daß sie es vermocht hatte diesen schwankenden
Menschen aufzurichten und ihm das Gefühl seiner Kraft
zurückzugeben. Dies Bewußtsein bildete einen Teil ihrer Liebe, die
dadurch wieder etwas Kameradschaftliches bekam; überall war sie
die selbstverständlich Leitende, und er war so gewohnt ihr zu folgen,
daß es doppelt auffällig war, wenn er einmal den eigenen Willen
durchsetzte.
Jeden Morgen — ob es nun gutes oder schlechtes Wetter war —
holte sie ihn in der Frühe zum Spazierengehen ab; er war immer
schon längst fertig und bereit, ehe sie ankam; das frühe Aufstehen
tat ihm gut, er wunderte sich, einen wie großen Teil seines Lebens er
früher verschlafen hatte. Nach einer Stunde trennten sie sich dann;
sie ging an ihre Arbeit, er an die seine. Sie machte ihm nicht gerade
Freude, war ihm aber auch nicht unangenehm. Sein Gedächtnis, das
in den letzten Zeiten etwas nachzulassen drohte, war frisch wie in
seinen frühesten Jahren.
 Manchmal überraschte er sich selbst, indem er einen tiefen
Seufzer ausstieß und in eine Ecke starrte. Er lächelte; — wie doch
alte Gewohnheiten in einem festsitzen! dachte er; wenn ich jetzt
nicht zufrieden und glücklich bin, so habe ich kein Recht auf Glück!
—
Herta war immer frisch, immer schaffensfreudig, immer voller
Pläne für ihre eigene Kunst. Er sah jetzt auch viele ihrer früher
gemalten Bilder. Eines nach dem andern stellte sie vor ihn hin, und
erzählte wo sie dieses, wo sie jenes gemalt hatte, merkte aber, daß
ihn dies nicht sonderlich interessierte, obgleich er sich Mühe gab
ihren Erinnerungen zu folgen und selbst etwas von ihren Gefühlen zu
empfinden. — Du hast recht, sagte sie; all dies hat ja im Grunde mit
den Bildern nichts zu tun und es wäre schlimm, wenn man ihnen von
diesen Gefühlen etwas anmerkte; aber in der Erinnerung habe ich
sie doch; dafür bin ich auch kein Mann.
Höre, sagte sie eines Morgens zu ihm, mir scheint, du wirst jetzt
faul; gestern hast du mich eine Viertelstunde vor deinem Hause
warten lassen, und heute ebenfalls; weißt du, das geht nicht; wenn
du das öfter tust, mußt du mich künftig abholen! Du mußt dich an
eine ganz regelmäßige Tageseinteilung gewöhnen, ohne die kommt
man nicht aus im Leben. — Am nächsten Morgen war sie überrascht,
ihn vor ihrem Hause zu sehen. — Sie lachte, wie sie sein halb
zerstreutes, halb verschlafenes Gesicht sah: so war es nicht gemeint
von mir, außerdem ist es für dich doch ein Umweg! — Er nahm sich
vor, nicht mehr in solche Unregelmäßigkeit zurückzufallen, und
abends, wenn sie sich trennten, freute er sich auf den Morgen. Doch
kam es immer häufiger vor, daß er dann am Morgen mit dem Gefühl
aufwachte: Jetzt sollen wir schon wieder zusammen sein! Wir waren
doch erst eben zusammen! Manchmal schwebte es ihm auf den
Lippen zu sagen, ob sie sich nicht lieber jeden zweiten Tag zum
Spazierengehen treffen wollten, aber er wußte, daß sie ihm dann
Energielosigkeit vorwerfen würde. Fast mißmutig kam er zuweilen
die Treppe herab; aber wenn er dann dieses Mädchen vor sich sah,
das, unbekümmert um Wind und Wetter, wie ein junger, herrlicher
Baum vor ihm stand, und wenn ihn dann das Gefühl überkam: Sie
gehört mir und niemand anders — und wenn er ihre kräftige Hand
fühlte, die frisch und warm in der seinen lag, dann vergaß er das
Gefühl, das ihn zuvor beherrscht hatte. Wieder und wieder sagte er
sich, wie dankbar er dem Schicksal sein müsse, daß dieses
vollendete Geschöpf sein eigen sei. Und doch — wenn sie dann
zusammen durch die Felder gingen, wünschte er sie manchmal fort.
Mitunter fühlte er sich geradezu beklommen durch ihre nahe
Gegenwart. Er suchte sich dies Gefühl auszureden, aber es kam
wieder. Dann wurde er einsilbig und zerstreut, und sie, die sich das
nicht deuten konnte, fragte ihn, was ihm sei. Er antwortete nicht und
sah aus wie ein Mensch, von dem eine Krankheit langsam Besitz zu
nehmen droht, die ihre Vorboten vorausgeschickt, leise eine
unverstandene, allgemeine, dumpfe Angst verbreitend. — Laß mich
allein! sagte er einmal, mitten auf dem Wege stehen bleibend, ich
weiß nicht was es ist — aber ich muß allein sein, jetzt! — Wie sie
dann wirklich gehen wollte, hielt er sie wieder zurück und sagte:
nein, bleibe hier, wenn du fortgehst, fühle ich noch viel mehr Angst.
—
Ähnliche Stimmungen wiederholten sich abends, unverhüllter,
freier. — Wir sehen uns doch morgen wieder! du mußt doch jetzt
schlafen! — Sie sah ihn ganz verständnislos, mit großen Augen an:
Gerade jetzt, jetzt wollte er gehen, wo alles in ihr drängte, noch
länger mit ihm zusammen zu sein, um langsam, mit ihm zusammen,
sich wieder in die Wirklichkeit zurückzufinden? — Er zögerte und
blieb, oder ging, je wie es ihn trieb.
Ich weiß es nicht, sagte sie manchmal und betrachtete ihn
sinnend, zuweilen bist du mir ganz nah, und auf einmal habe ich das
Gefühl, du seist mir in Wirklichkeit ganz fern. — Er widersprach
alsdann mit großer Heftigkeit, denn ihre Worte machten ihm innere
Angst, sie sprachen nur das aus, was er selber fühlte und nicht
fühlen wollte.
Ich glaube, sagte er, als er einmal in etwas freierer Stimmung
neben ihr herging, wir sehen uns zu oft, wir dürfen unser Gefühl
nicht abstumpfen. — Das verstand sie nicht: Mein Gefühl wird nicht
abgestumpft; und wie denkst du es dir denn wenn wir später einmal
vielleicht wirklich dauernd zusammen leben? — Da war es, wie wenn
etwas in ihm, das leise und allmählich angewachsen war, mit heftiger
Bewegung an das Licht drängte. — Wie und wann dieses
Schreckliche begonnen hatte, wußte er nicht mehr; er hatte es vor
sich selber abgeleugnet, aber es meldete sich stärker und immer
stärker und ließ sich nicht mehr bezwingen; was er seit geraumer
Zeit schon ahnte, und nicht ahnen wollte, sah er mit immer
quälenderer Deutlichkeit: daß sein Glück den Höhepunkt
überschritten hatte, daß es mit langsamem Schritte abwärts ging.
Die erste große Zeit, wo alles unfaßlich, neu, als reiches Geschenk
über ihn gekommen war, diese erste Zeit war längst vorüber, das
Größte war ihm zur Gewohnheit geworden. Manchmal schien es ihm,
als sei alles von Anfang an eine Täuschung gewesen, als wäre er im
Grunde stets allein geblieben. Aber nun wehrte er sich mit
Verzweiflung gegen sich selbst, denn was sollte werden, wenn alles
wirklich so war wie er es dachte, und wenn er dann wieder allein
sein würde?
Wenn er Herta wiedersah, konnte er ihr zu Anfang kaum in die
Augen blicken, es war, wie wenn er seinen Makel offen auf der Stirne
trüge.
Was ist es denn?! Was hast du denn?! fragte sie, wenn er ihr so
stumm gegenübersaß und in eine Ecke starrte. — Er sah sie an wie
ein hilfloses Tier.
Langsam begann sie die Wahrheit zu erkennen. — Liebst du mich
nicht mehr? fragte sie einmal. — Er antwortete nicht. — Weißt du es
selber nicht? — Ich liebe dich, wie ich noch nie einen Menschen
geliebt habe. So sprach er und suchte in diesem Wort selbst einen
Halt. — Was ist es dann, was kann es dann sein, das dich so
furchtbar niederdrückt? — Ich glaube, sagte er, es ist die Angst vor
der Zukunft, wie du sie manchmal hinstellst, dort oben, in deinem
Lande — das Haus — deine Familie — wir selbst, auf immer
verbunden — — Es überlief sie kühl. Vor all dem hast du Angst? Es
braucht ja nie zu sein! sagte sie halblaut und ihre Augen nahmen
einen halb traurigen, halb stumpfen Ausdruck an. — Ja, sagte er und
sprang auf, aber das alles kann ja wieder vergehen, du weißt doch
wie ich bin, es ist alles so schnell über mich gekommen, ich kann
mein Wesen nicht in einem Nu von Grund aus ändern, du hast soviel
Geduld mit mir gehabt und mußt sie weiter haben, du weißt doch
selbst am besten, wie sehr du mich schon geändert hast, das hätte
außer dir niemand, niemand vermocht! — Sie trat dicht zu ihm
heran, und wie er ihre Arme fühlte, war es, als sei alles Schlimme
vergessen.
Aber am nächsten Tage war es wieder da. — Wenn ich jetzt nicht
glücklich bin, so habe ich kein Recht auf Glück! Dieses Wort, das er
sich schon früher vorgesagt hatte, verlor seine suggestive Kraft, es
kam ihm phrasenhaft und hohl vor. Er ging wie in einem bösen
Traum umher. — Bin ich denn verrückt geworden, sprach er zu sich
selbst, wie und wann ist denn dies alles gekommen? Liebe ich sie
denn wirklich nicht mehr? Aber sogar diese Selbstgespräche verloren
an unbewußter Ehrlichkeit, er hörte sich wie einen andern, er wußte
kaum selber mehr, was echt, was unecht an ihm war. — Mehr und
mehr ahnte Herta die Wirklichkeit. Es begann eine Zeit der Kämpfe
für sie, der ewigen Selbstverleugnung, der Überwindung und der
angespanntesten Geduld. Noch immer glaubte sie, alles könne
vorübergehend sein. Manchmal empfand sie es selber, daß es besser
sei, sie sähen sich nicht so oft, und sie hielt ihn ein paar Tage fern.
Wenn sie dann wieder zusammen kamen, war sie doppelt liebevoll,
während für ihn die Ferne eine andere Wirkung hatte: Sie näherte
sein Gefühl nicht, sie entfernte es nur mehr. Ihr Stolz begann
allmählich zu leiden. Sie begann zu fühlen, daß auch dieses Erlebnis
zu einem Ende führen würde, nicht durch sie, sondern durch Pitt
selber, und dies gab ihr ihre Kraft zurück. Mehr und mehr lehnte sich
ihr eigenes Wesen gegen das seine auf, das ihr im Grunde so sehr
fremd war. — Ich weiß es, sagte sie, daß du mich nicht mehr liebst;
du bestreitest es, du sagst, dein Gefühl für mich sei so wie sonst,
und du habest nur Angst vor der Zeit, wo wir vielleicht einmal
verbunden sein würden. Ich will nicht sagen, daß ich mit einem
Menschen, den ich liebe, nur dann zusammen leben kann, wenn ich
später dauernd mit ihm verbunden werde; du weißt aus meinem
früheren Leben, daß ich nicht so denke: Aber mit jemand zusammen
leben, der in einem späteren Zusammenleben nur etwas
Schreckliches, Entsetzliches erblickt, dem alles andere das nicht
aufwiegen könnte, was es an äußeren Unannehmlichkeiten im Leben
gibt — denn um die handelt es sich nur — das kann ich nicht! Von
einem solchen Menschen weiß ich: Seine Liebe ist nicht so wie sie
für mich nötig ist! — Es ist nur dieses Eine! rief er; diese Furcht vor
der Zukunft! Du nennst das äußere Unannehmlichkeiten — für mich
sind sie untrennbar vom Leben überhaupt!
Sie glaubte ihm noch halb, da sie die Sehnsucht hatte ihm zu
glauben. Seine entsetzliche, plötzlich wie wahnsinnig ausbrechende
Angst vor einem späteren, gebundenen, bürgerlichen Leben,
nachdem er eine große Zeit lang alles überwunden zu haben schien
durch ihre Liebe — war dies nicht vielleicht wirklich, wie er selber
sagte, nur wie das letzte Aufzucken eines Lichtes, das erstickt
schien, das heimlich weiter schwelte und qualmte, das nun am
Verenden war und für einen Augenblick noch aufflammte? Konnte
nicht doch alles noch gut werden?
Sie lebten noch eine Zeitlang miteinander fort, scheinbar in der
alten Selbstverständlichkeit, aber er verlor mehr und mehr von
seiner Natürlichkeit, er wurde gekünstelt, sein Bild wurde ihr zur
Karikatur. Und mehr und mehr drängte ihre gesunde Natur, sich zu
befreien von diesem Gewicht, das immer schwerer auf ihr lastete.
Eines Tages faßte sie den Entschluß, den sie seit langem erwogen
hatte, der der einzige Ausweg aus diesem Irrsal war: Alles mit einem
Hiebe durchzuschlagen.
Er beschwor sie, flehte, sie blieb fest. Er warf ihr vor, sie liebe ihn
nicht mehr. — Im Gegenteil! rief sie: da ich dich so sehr liebe, muß
ich allem ein Ende machen; ich will nicht, daß etwas, das mir hoch
steht, herabgezogen wird, bis es schließlich triviale Gewohnheit wird,
die man bestehen läßt, weil sie einmal bestanden hat; auf diese
Weise führen viele Verhältnisse unter den Menschen endlich zu einer
Ehe; von der reden wir schon lange nicht mehr, aber so wie alles ist,
bin ich mir auch zu gut, überhaupt ein solches Leben weiter zu
führen, wie wir es tun. — In ihm begannen die festen Gedanken sich
aufzulösen; das ganze Zimmer, jedes einzelne Möbel schien sich
plötzlich zu einer unerhörten Bedeutung vorzudrängen; er sah mit
einem Male, daß hier ein Bild etwas schief hing, daß dort die Kante
am Sekretär ein ganz wenig abgestoßen war, daß jener Stuhl nicht
ganz so schön mehr wäre, wenn seine Lehne sich nicht eben in
diesem ganz besonderen Winkel an den Sitz anfügte — und doch
dachte er nur an seine Angst, an sich und Herta. — Ich liebe dich,
wie ich nur überhaupt einen Menschen lieben kann, rief er, und
blitzschnell schoß der Gedanke dazwischen: die vielen i’s im Anfang
meines Satzes! Ich werde verrückt, was um Gottes willen ist dies! An
Gott glaube ich nicht einmal. — Wie d u nur überhaupt einen
Menschen lieben kannst, das ist wohl leider wahr! rief Herta, und
seine Augen richteten sich nun auf sie, indem er ihre Gestalt für
einen Augenblick fast wie einen Maßstab der ganzen Höhe des
Raumes ansah, obgleich er verzweifelt auf sie blickte. — Alles Glück,
fuhr sie fort, ist dir nur eine Selbsttäuschung, du bist überhaupt
unfähig einen Menschen zu lieben. Ich bereue nicht mit dir
zusammengelebt zu haben, aber das weiß ich: Die Erinnerungen, so
warm sie sind, werden niemals Macht über mich bekommen, ich bin
zu kräftig, als daß ich nicht alles überwinden könnte. Ich habe
Mitleid mit dir, soviel ein Mensch nur haben kann für einen andern,
aber ich muß weiter; ich kann nicht bei dir bleiben, es ist unmöglich.
Ob ich jemals einen andern heirate weiß ich nicht, aber das ist
gewiß: Du bist nicht der letzte Mensch, der in mein Leben
eingetreten ist, ich habe einen zu festen Willen zum Leben.
Alles an ihr atmete Kraft und Schönheit wie sie so sprach. Er war
vollkommen in die Gegenwart des Augenblicks zurückgekehrt; ein
Schmerzgefühl durchriß ihn, und fast mit Wollust empfand er seinen
Schmerz: ich bin nicht empfindungslos, dachte er, o Gott, wie könnte
ich sonst so empfinden! Und er stürzte an ihre Brust, in aller Angst
vor der Leere, die von neuem vor ihm lag. Er fühlte ihre Arme, aber
sie umschlossen ihn nicht mit der alten Kraft. — Ich kann nicht von
dir fort, rief er heftig, du mußt bei mir bleiben, du wirst sehen, daß
du dich in mir getäuscht hast, ich bin anders als du denkst, ich
schwöre dir, daß ich anders bin, nur laß mir Zeit, dies ist ja ein
Wahnsinn! —
Er preßte seine Wange an ihren Kopf und starrte über ihre
Schulter hinweg ins Leere, begegnete aber seinen eigenen Augen,
die ihn aus einem gegenüberhängenden Spiegel ansahen. — Herta
schwieg und er ward beruhigter. — Fasse wenigstens nicht jetzt
einen Entschluß, fuhr er fort, ohne sein Spiegelbild aus den Augen
zu lassen, nicht jetzt, wo du dir selbst nicht klar bist über alles, —
und mitten zwischen die Worte, die er sprach, schob sich der
Gedanke: Sie ahnt nicht, daß ich ihre Gestalt von hinten sehe. —
Warte wenigstens einen Tag, zwei, drei Tage. Laß mich diese drei
Tage dich nicht sehen, und dann laß mich wiederkommen, und was
du dann beschlossen hast, dem werde ich mich fügen, ohne
Widerspruch. — Die Lippen des Spiegelbildes schlossen sich:
während der Zeit war es Pitt so gewesen, als wenn der da drüben
redete und nicht er selbst, obgleich er wußte, daß er sprach. Jetzt
kehrte er den Blick hinweg, sah Herta in die Augen und — als
erwache er aus einem dumpfen Traum, packte ihn mit einemmal die
helle Angst, er umklammerte sie und brach in Tränen aus. Er fühlte
ihren Körper und wußte plötzlich mit schrecklicher Deutlichkeit: Dies
ist ein wirkliches Stück Leben, das sich von mir losreißen will, das
einzige lebendige, das ich besitze. — Sie strich über sein Haar hin.
Du hast recht! sagte sie, vielleicht sehe ich dann alles anders. —
Aber sie dachte: Es ist doch alles vorbei. Fühlte er wirklich stark zu
mir, er würde mich nicht bitten, er würde mich zwingen. In drei
Tagen werde ich ihm dasselbe sagen müssen wie heute. Er löste sich
aus ihren Armen, sie reichte ihm die Hand und geleitete ihn so zur
Tür. Sie sahen sich lange in die Augen, als wollten sie sich in die
Seelen sehen, und beide empfanden für einen Augenblick den
Schauer offener und doch verschlossener Welten. — Wie mit einem
Entschluß schlang sie heftig die Arme um ihn, und er fühlte ihre
Lippen auf den seinen.
Sowie er draußen auf der Straße war, löste sich die Spannung in
ihm. Es war ihm wie nach einer langen Krankheit, die die Krise
überstanden hat. Die augenblickliche Gefahr schien vorüber, und,
wie es bei solchen Krisen zuweilen geht, daß der Patient, noch eben
Abschied nehmend von einem Leben, das ihm, nun er es verlieren
soll, als das kostbarste Kleinod erscheint, dieses selbe Leben, wenn
es ihm zurückgeschenkt ist, als etwas Selbstverständliches hinnimmt
— — so richteten sich Pitts Gedanken schnell wieder auf die ihm
gewohnte Wirklichkeit. Daß Herta nach drei Tagen noch bei ihrem
Entschluß verharren würde, glaubte er nicht. Und er selber: Er
würde sich Mühe geben, daß sie mit ihm zufrieden war. Sie würden
miteinander sprechen, er würde ihr sagen, daß es wirklich für sie
beide besser wäre, wenn sie sich nicht so sehr oft sähen — denn
schon wieder regte sich in ihm ein kleines Unbehagen bei dem
Gedanken, daß sie vielleicht wieder tagtäglich beieinander wären —
und damit war dann alles Schlimme beseitigt. — Aber trotz aller
beruhigender Gefühle empfand er eine Leere, und dann dachte er:
Was ist eigentlich geändert gegen früher?
Der nächste Tag verging, der übernächste auch, der dritte begann.
Hatte Herta seinen Vorschlag ganz wörtlich aufgefaßt? Mehr und
mehr hatte sich diese letzten Tage eine tiefe Niedergeschlagenheit
seiner bemächtigt. — Es ist nur das Wetter schuld, — dieser ewige
Nebel! dachte er, obgleich er Nebelwetter mehr liebte als jedes
andere. —
Am Abend ging er hin zu ihr, im rötlichen Dämmer der Laternen,
deren Lichter wie flimmernde trübe Kugeln im Grau zu schweben
schienen.
Wenn sie nun auf ihrem Entschluß bestände?
Eine alte Frau öffnete auf sein Läuten. Mit schlürfendem Schritt
ging sie ins Zimmer und kam mit einem Brief zurück, den das
gnädige Fräulein für ihn hinterlassen habe. Wo ist sie denn? Kann ich
sie denn nicht selber sprechen? fragte Pitt schnell und hastig. —
Verreist! antwortete die alte Frau, und da sie nichts hinzuzufügen
hatte, zog sie sich wieder zurück und schloß die Tür.
Es war ihm, als habe er mit flacher, harter Hand einen Stoß vor die
Stirne bekommen; noch auf der Treppe, unter einer Lampe, las er
den Brief; er las ihn zweimal, dreimal. Er empfand keinen Schmerz,
keine Trauer, aber ein dumpfes, stumpfes, gespenstisches Gefühl
war in ihm. Wie ein Traumwandelnder trat er endlich auf die Straße.
Er sah nicht, welchen Weg er ging, er setzte Schritt vor Schritt, ohne
Aufhören, als sei das Gehen das einzige in der Welt was er noch tun
konnte. Die matten Laternen blieben allmählich hinter ihm, dann
befand er sich in einem endlosen stummen Grau. Endlich stieß er mit
dem Fuß gegen etwas Festes, gegen eine Bank, auf die setzte er
sich, und blieb dort sitzen, den Blick ins Nichts geheftet. —
 Achtes Kapitel.
Fox Sintrup hatte, nachdem er die Stadt Lottes und Herrn Könneckes
mit Protest verließ, bald hier, bald dort studiert. Schließlich blieb er in
einer größeren Universitätstadt, die ihm behagte. Er arbeitete wenig,
wurde aber im Laufe der Zeit ein großer Feinschmecker und
Weinkenner. Dazu unterhielt er freundschaftliche Beziehungen zu
den gewaltigsten Persönlichkeiten.
So ein Lerchenzungenragout — also es geht doch nichts über so
ein Lerchenzungenragout! — So sprach er in der Weinstube, sah alle
Freunde der Reihe nach an und fügte hinzu: Höchstens ein
Nachtigallenzungenragout, das soll noch besser sein, das kenne ich
nicht; dann erzählte er, wie er neulich beim Kriegsminister
eingeladen worden sei, verstummte aber plötzlich — scheinbar in
ratloser Überraschung. — Teufel noch mal! rief er endlich und schlug
mit der Hand auf den Tisch, indem er die Gegenübersitzenden wie in
starrer Überlegung ansah, — das habe ich ja total verbummelt!
Kellner! Briefbogen, Kuvert und einen Dienstmann! — Während der
Kellner verschwand, erklärte Fox, er sei auch für heute abend beim
Kriegsminister eingeladen, habe diese Einladung aber vollkommen
vergessen. — Schon manchmal hatten seine Freunde Zweifel an
seinen Angaben erhoben, aber was er nun tat, schlug jedes
Mißtrauen nieder. Mit Bleistift schrieb er flüchtig einen kurzen Brief,
während ihm seine Nachbarn ins Papier schauten. Er entschuldigte
sein Ausbleiben, das er um so mehr bedauere, als er nun leider
darauf verzichten müsse, mit seiner Exzellenz dem Handelsminister
über seine nationalökonomische Broschüre zu sprechen, die
demnächst erscheinen werde. — Der Brief ward gefaltet, kuvertiert,
auf den Umschlag der volle Name und die Adresse des
Kriegsministers geschrieben, und dem Dienstmann eingeschärft, das
Ganze sofort, aber sofort beim Hausdiener im Palais abzuliefern.
Sintrup verkehrt beim Kriegsminister! hieß es nun, und sein Ansehen
war wieder um eine kleine Stufe gestiegen. Niemand hatte bemerkt,
daß Fox jenen Brief nicht mit seinem Namen, sondern mit zwei
unleserlichen Anfangsbuchstaben unterzeichnete. Mochte der
Kriegsminister sich denken, was er wollte! —
Fox war und konnte jetzt alles; er hatte sehr viel gelesen, sehr viel
herumgehorcht und viel erfahren, manches Schwierige aus
gedruckten Abhandlungen auswendig gelernt. — Ja du lieber Gott,
die Menschen führen immer das Wort „Kunst“ im Munde, was ist
denn nun eigentlich Kunst? Er nagelte den andern mit seinen Augen
fest, und dann wurde mit scheinbarer intensiver Anspannung des
Geistes irgendeine komplizierte Definition geboren, die er tags zuvor
gelesen und memoriert hatte. Er schriftstellerte, es erschienen hier
und da in den Blättern in der Tat Artikel von ihm, mit seinem vollen
Namen gezeichnet. Früher schon hatte er politische Broschüren
erscheinen lassen, diese aber alle anonym, denn: — Na, Sie können
sich ja denken! Man zweifelte an seiner Autorschaft, aber
gelegentlich, wenn Freunde ihn besuchten, ließ er sie einen Blick in
die dunkle kleine Kammer tun: Da lagen die Broschüren stoßweise,
verstaubt, und er sagte bedauernd: Ja, vom Schriftstellern wird man
nicht reich, wenn man Talent hat. — Die Broschüren hatte er alle aus
irgend einem Lagerraum, wo sie vergessen lagen, aufgekauft. Er
schrieb auch Essays über veraltete, altmodische Schriftsteller, die er
ausgrub auf Bibliotheken; sie erschienen zwar nicht gedruckt, aber
die Manuskripte lagen bei ihm zu Hause, und er las sie vor; sie
klangen gut, merkwürdig echt war darin der Stil der Zeit getroffen.
Allerdings hatte er alles eigentlich abgeschrieben aus irgend einem
verschollenen Buch, das niemand kannte. Seine Bemühungen, sich
im Ansehen der Menschen aufrecht zu erhalten und es immer noch
zu steigern, waren allmählich ins Groteske gewachsen und bedurften
viel mehr Nachdenken und Aufwand als seine früheren
gelegentlichen Mystifikationen. Und da Fox im Grunde eigentlich
schwerfällig und faul war, so kostete ihn dies Treiben viel Sorge und
Selbstdisziplin. Er wurde ein Opfer seiner selbst.
 Fox galt auch als guter Rezitator; es war bekannt, daß er einst
Schauspielstunde gehabt hatte und daß sein Lehrer ihm beim
Abschied sagte: Wenn S i e nicht zur Bühne gehen, mach ich meine
Unterrichtsbude zu! — Na, zur Bühne war er nicht gegangen, und im
Vortrag der Gedichte vermied er streng alles was an die Bühne
erinnerte. Nicht scharf genug konnte er die Unsitte der meisten
Schauspieler verurteilen, die die Gedichtform mit der dramatischen
verwechseln, beim Vortrag mit den Händen agieren und wie auf der
Bühne Mimik treiben: Wie unendlich fein hatte schon Goethe diesen
Unterschied präzisiert! Auf d e n Bahnen galt es fortzuschreiten, da
galt es wieder anzuknüpfen. Unwillkürlich geriet Fox, wenn er so
redete, in die Spuren seines Bruders, dessen Gedanken er, getaucht
in die Farbe seiner eigenen Sprache, wiederholte. Er konnte dies
ohne jede Gefahr, von Pitt hatte hier niemand eine Ahnung, niemand
war dabei gewesen, wenn er ihm seine Gedichte vortrug. Fox übte
sich auch im Aufstellen barocker Behauptungen, wie sie zuweilen
von den Lippen seines Bruders kamen. Bei Pitt waren sie ein
künstlerisches Spiel, er glaubte selbst nicht an sie, hielt sie aber,
wenn man dann opponierte, mit allen Mitteln der Dialektik aufrecht;
diese fehlten Fox nun gänzlich. Die Einwände, die man ihm
entgegenwarf, nahm er nicht wie Bälle, denen man geschickt
ausweichen, denen man durch ein einziges Wort eine neue Richtung
geben, die man parieren und auf den Angreifer zurückschlagen
kann, sondern sie lagen da wie dicke Holzklötze, über die seine Füße
stolperten. Aber das schadete nichts: Mit einer Handbewegung, wie
Pitt sie liebte, wenn er seine Worte an mindere Leute
verschwendete, bis er selber ungeduldig wurde, schnitt er die
Gegenrede ab und verschanzte sich hinter eine vielsagende Miene,
die bei ihm ganz von selber dazu kam. Wie er noch mit Pitt
zusammen war, ging sein ganzes Bestreben dahin, selbständig
neben seinem Bruder zu erscheinen, seinen Einfluß zu verleugnen.
Nun zehrte er sehr von der Vergangenheit, suchte er seinen Bruder
auch in seinen Äußerlichkeiten zu kopieren, verband er dessen
Sonderlichkeiten mit dem Pompe seiner eigenen Persönlichkeit, und
niemand sah das unscheinbare kleine Schiff, das diesen stolzen
Dreimaster in seinem Fahrwasser hinterherzog. Zuweilen war es, als
wenn die Maske plötzlich rutschte: So sagte er, ehe er seine
Militärzeit antrat, einmal mit kapriziös leidender Stimme: Dienen ist
doch schlimmer als tot sein! — Nanu, Sintrup, rief einer, ich denke
du willst es mindestens bis zum Hauptmann bringen? — Da sah ihn
Fox erst unsicher an, dann sammelte er sich und antwortete im
schneidigst zurechtweisenden, kurzen Tone: Na ja?!
Fox war gern gesehen in seinen Kreisen. Er zählte nun schon zu
den alten Semestern, zu den sehr alten sogar, denn sein Examen
hätte er eigentlich seit langem machen müssen, und Herr Sintrup
wies in seinen Briefen darauf hin, Pitt säße doch nun längst in „Amt
und Würden“. Fox tröstete dann immer mit dem Hinweis auf seine
glänzende Karriere und auf die wahnsinnig vorzüglichen Anlagen
seines Kopfes. Und zunächst ließ sich auch Herr Sintrup noch
trösten, da er alles glaubte und ja auch gedruckte und ungedruckte
Bestätigungen dieser Talente erhielt. — Aber Fox brauchte enorm
viel Geld, so daß Herr Sintrup sich oft fragte, wo das noch
hinauswolle. In den vornehmen Wirtshäusern war er ein gern
gesehener und bestbedienter Gast, dem es nicht darauf ankam eine
ganze große Gesellschaft freigiebig zu bewirten, wenn er in Laune
war. Mit Stolz sah er die Spitze seiner Nase sich braun färben und
erklärte denen, die den Grund nicht wußten, mit bedauernder
Stimme, das käme vom vielen Burgundertrinken, was auch der Fall
war. Manchmal machte er sich selber Sorgen um seine vielen großen
Geldausgaben, und in der Erwägung, daß man stückweise teurer
einkauft als wenn man en gros bestellt, ließ er sich zuweilen ganze
Lieferungen kommen und legte sich auch aus demselben Grunde
einen Weinkeller an. Bezahlt wurde wenig oder nichts von diesen
Dingen, denn man kannte ihn als einen guten, sichern Kunden.
Überall erweckte er den Anschein größter Vertrauenswürdigkeit, und
er selber hielt sich für eine Art Ehrenmitglied der menschlichen
Gesellschaft. —
An festgesetzten Tagen der Woche besuchte ihn jetzt regelmäßig
ein Fräulein, welches in seiner übrigen Zeit einem durchaus
einwandfreien, anständigen Gewerbe nachging. Sie war jung und
ziemlich hübsch, und bezog ein monatliches Gehalt von ihm für ihre
Toiletten, die stets niedlich und sauber waren. Sie liebte Fox nicht
gerade, aber sie hatte ihn doch recht gern. Er fragte sie nie nach
ihrer Vergangenheit, hatte ihr aber angedroht, wenn er den Schein
eines Verdachtes merke, so werde Entsetzliches geschehen. Er habe
von seiten seiner mütterlichen Familie korsisches Blut in den Adern,
sie solle es nicht in Wallung bringen! —
Sie verehrte ihn sehr, und da sie nicht viel Temperament besaß,
ward es ihr nicht schwer sein Gebot zu halten. — Mädchen mit
Temperament, pflegte Fox zu sagen, sind nicht mein Fall; viel besser
so eine, die abwartet, wie man selbst gestimmt ist! Die haben keine
Launen und man kann immer auf sie rechnen; wenn man sich mal
trennt, geschieht es ohne Aufregung und Geschrei. — Fox war dieser
Dame zwar nicht absolut treu, aber sie bestand auch nicht darauf,
nachdem sie ihn erst darum gebeten hatte und mit den kurzen
Worten abgefertigt wurde: Männer sind einmal polygam! was sie
nicht verstand und sich erklären ließ.
Es gehörte zu Fox’ Ehrgeiz, die Mädchen, die er liebte, zu sich
heraufzuziehen, ja er sah es sogar als seine soziale Pflicht an. —
Jede Woche bekam das Fräulein ein neues Buch von seinem Regal,
bis zum nächstenmal mußte sie es durchgelesen haben und angeben
können was darin stand. Auch führte er sie in die Musikliteratur ein,
indem er ihr Lieder vorsang und wohl auch dieses oder jenes
Musikstück vorspielte, das er noch von seiner Gymnasiastenzeit her
auswendig konnte. — Er hatte jetzt Singstunden genommen, seinem
Programm der allseitigen Ausbildung folgend. Er sang mit vielem
Gefühl, und war es ein Volkslied, so wollte das Fräulein unbefangen
einstimmen, was er ihr aber, sich langsam auf dem Klavierstuhl
drehend, mit einem ausdrucksvollen Blick verbot. Dann setzte er ihr
den Unterschied auseinander zwischen Kunstgesang und
Naturgesang: Jedes für sich allein sei schön, aber beide zusammen
bildeten eine unerträgliche Einheit. Und sie nickte mit dem Kopf und
sagte, sie begreife alles. Wenn Fox sich dann auf seinem Stuhle
zurückdrehte und den Erlkönig von Schubert sang, so stand das
Fräulein leise auf, ging zu dem kleinen Schränkchen im andern
Winkel des Zimmers und entzündete eine Kerze, die sie dort brennen
ließ. War Fox mit seinem Liede fertig, drehte er sich wieder langsam
mit seinem Stuhle, diesmal nach der andern Seite, starrte das Licht,
noch halb im Reiche der Musik, aber doch wie etwas Bekanntes,
Selbstverständliches an, erhob sich, nahm es und verschwand, und
kam nach einigen Minuten wieder, während deren das Fräulein still
seine Rückkehr erwartete und solange einfach die Augen schloß. —
Selbst diesem Fräulein gegenüber war ihm sein eigentlicher und
eigener Wert nicht genügend, doch wandte er ihr gegenüber niemals
komplizierte Mittel an, um sich zu heben, sondern arbeitete nur mit
groben, die ihren Zweck vollständig erfüllten, denn sie glaubte alles,
ohne sich jedoch wesentlich dafür zu interessieren. Wenn sie so am
Tisch saßen, und er ihr von seinen grundlegenden Arbeiten auf
diesem und jenem Gebiete erzählte, nickte sie eifrig und dann immer
unmerklicher mit dem Kopfe, und erst wenn die Worte kamen: Ich
kann dir sagen, mein neues Werk wird wie eine Bombe einschlagen!
wurde sie für einen Augenblick lebendiger, da er bei dem Worte
„Bombe“ auf den Tisch schlug, was sie jedesmal etwas
zusammenfahren ließ, obgleich sie es ja eigentlich schon wußte.
Ist sie wohl etwas indolent? dachte er manchmal. Er gab sich dies
im Grunde zu, auch sah er, daß es ihm wohl nie gelingen würde sie
zu sich heraufzuziehen, aber das schadete auch nichts: Goethes Frau
hatte auch weit unter dem Olympier gestanden, mit dem er sich
übrigens in keiner Weise vergleichen wollte — und dieses Fräulein
würde er ja überdies niemals heiraten, was sie auch ganz genau
wußte und nicht erstaunlich fand.
So hatte er Monate und Jahre ein breites und durch nichts
verbittertes Dasein geführt, als ihm sein Vater eines Tages mitteilte,
er habe starke geschäftliche Einbuße erlitten, es sei die höchste Zeit,
daß Fox an sein Examen denke. Er habe ihn nun lange genug
erhalten und sei mit seiner Geduld zu Ende. — So sah er sich denn
genötigt, sich von einem jener eigens für diesen Zweck vorhandenen
Individuen für das Examen einpauken zu lassen. Ihm brummte der
Kopf bei diesem Pauken, das Fräulein mußte ihn überhören, und
wenn etwas nicht stimmte, so hatte sie die Schuld. Nach solchen
Lernereien fühlte er dann das Bedürfnis sich auszuspannen. Diese
Ausspannungen wurden sehr häufig. Die leichteren Weine wirkten
nicht mehr, er trank ganz schwere; und auch die spülte er fast wie
Wasser hinunter; am nächsten Morgen war er dann untauglich zu
jeder Arbeit, und doch mußte er immer wieder trinken; der Wein war
das einzige was einigermaßen half gegen die Last der Arbeit und die
düsteren Ideen, die allmählich in ihm aufzusteigen begannen. Er
fühlte, daß das gute Leben ein für allemal ein Ende haben werde,
zumal auch seine Gläubiger in immer größerer Zahl anfingen sich zu
regen und schließlich dreist und dreister wurden. — Fast
ununterbrochen rauchte er die schwersten Zigarren; seine Hände
begannen zu zittern, sein Blick bekam etwas Glasiges. Der Geist des
Weines, eine schwirrende Fülle von Paragraphen, der blaue Rauch
des Tabaks, das alles wirbelte in ihm durcheinander. Das Examen
kam heran, ging über ihn, ließ ihn zurück, und Fox war
durchgefallen!
Andern Tags saß Herr Sintrup im Sofa und studierte die
Kursberichte. Da wurde ihm ein sonderbarer Brief überbracht; das
Kuvert war unfrankiert, zerrissen, und mit dem Bemerk versehen:
Von der Post verschlossen. Die Buchstaben der mangelhaften
Adresse waren verklext und tanzten auf und nieder, und ebenso sah
es auf dem Briefbogen aus, auf dem irgendeine Flüssigkeit halb
klebrig eingetrocknet schien. — Aus einem Entrüstungsruf fiel Herr
Sintrup in den andern: Diese Schande, diese Gemeinheit, diese
Schamlosigkeit! Frau Sintrup trat verschlafen ein, und nun hörte sie
es: Fox war durchgefallen, und damit nicht genug: In der
Betrunkenheit hatte er diesen Brief geschrieben, in vollkommenster
Betrunkenheit! Als einen Witz teilte er seine Schande mit! Seine
Schande und seine Schulden!
Am selben Morgen starrte Fox mit ausdruckslosen Augen vom
Bette aus gegen die Decke und dachte immer: was habe ich gestern
nur an den Alten geschrieben, was war es nur — irgend etwas
Fürchterliches.
Am folgenden Tage läutete es, und dann stand Herr Sintrup vor
ihm. Er war zunächst so erregt, daß er kaum sprechen konnte; dann
ging das Donnerwetter los, Fox ließ es über sich ergehen, kleinlaut,
wortlos, ganz ohne sich zu verteidigen. Dann forderte Herr Sintrup
Aufschluß über seine Schulden; er verlangte die Rechnungen zu
sehen. Mit unsicheren Händen kramte Fox in seinen Laden und holte
Papier auf Papier hervor, drehte seine Gestalt zur Seite und hörte nur
ab und zu Töne, die sein Vater durch die Nase stieß, kurz und
wütend, so wie ein Hund, der niest. — Dann stand Herr Sintrup auf,
trat zu ihm hin und durchbohrte ihn mit seinem Blick: Kannst du mir
in die Augen sehen? Hat dir dein Vater ein solches Lebensbeispiel
gegeben? Wie ich so alt war wie du, habe ich schon lange selbst
verdient, und vorher, als ich noch Geld von zu Haus bekam —
überlegt habe ich mir jeden Pfennig, den ich ausgab, dreimal
umgedreht habe ich ihn, kaum eine Flasche Bier habe ich mir
geleistet, und wenn ich mir einen Hering spendierte, verteilte ich ihn
auf zwei Abende! Und du, und du? Sieh deinen Bruder Pitt an! Er ist
kein leuchtendes Vorbild, er hat auch ziemlich lange Zeit bis zum
Examen gebraucht, aber in punkto Geld i s t er ein Vorbild! Nie hat er
auch nur einen Pfennig mehr gebraucht als er hatte! — Das weißt du
ja gar nicht, sagte Fox etwas bissig, denn er ärgerte sich, seinen
Bruder als Beispiel vorgehalten zu bekommen, vielleicht hat er viel
mehr Schulden als ich! Im selben Augenblick aber erinnerte er sich
daran, daß er ihn kürzlich schriftlich um eine größere Summe
angegangen habe, und daß dies Geld sogleich auch eintraf; seine
Worte erschienen ihm schlecht gegen Pitt. Und deshalb fügte er
hinzu: Ich glaube ja gar nicht, daß es so ist, absolut nicht, aber
wenn es nun so wäre, wenn er nun zehnmal soviel Schulden hätte
als ich? Was wolltest du denn dann erst sagen?! — Die Logik ist ja
reizend! spottete Herr Sintrup. — Ja, bitte, antworte mir doch erst
mal, ich setze also voraus, Pitt hätte soviel Schulden, daß meine
ganz klein dagegen erscheinen! — Diese Worte kamen langsam,
pointiert heraus, in abstrahierend objektivem Tone, und doch mit
einer innerlichen stotternden Zerfahrenheit, während er seinen Vater
mit festem Blicke anzusehen strebte und sein Kopf ganz leise,
unsicher hin und her ging. — Blödsinn! rief Herr Sintrup, zeig mir
diese zehnfachen Rechnungen und dann laß uns weiter darüber
reden! Bis jetzt halte ich mich an deine eigenen. Er schlug wütend
mit der Hand auf all die Papiere. Da waren Zigarrenkistchen für
fünfzig Mark, für siebzig Mark, das flog nur so! Und die Summe für
Delikatessen, die Fox im Laufe der Zeit — meist ohne das Fräulein —
vertilgt hatte, war so hoch, daß Herr Sintrup entrüstet rief, soviel
brauche er für seinen Haushalt das ganze Jahr nicht, und seine Frau
aß doch auch gern Delikatessen! — Was denkst du nun, daß wird??
Glaubst du, ich zahle dir das alles? — Nee — sagte Fox, so in die
Enge getrieben, obgleich er eigentlich gar keinen Zweifel daran
erhoben hatte. Herr Sintrup wurde durch dies halb trocken
herausgesprochene Wort einen Moment aus dem pathetischen
Dunst, in dem er sich befand, herausgerissen, dann gab er seiner
Stimme wieder Nachdruck und fuhr fort: Das fällt mir auch gar nicht
ein, und übel wärest du dran, du Patron, wenn du nicht eine so
schwache Mutter hättest. Für diesmal bist du gerettet: Sie zahlt dir
deine Schulden und zieht das Geld von deinem Erbteil ab! Fox sah
überrascht auf, denn dies hatte er nicht erwartet. Im selben
Augenblick aber fühlte er sich wieder auf seiner alten Höhe und kam
sich nun seinem Vater gegenüber gleichsam wie ein Geschäftsmann
vor, denn das Geld wurde ja von seinem eigenen genommen, von
dem, das ihm rechtlich später sowieso zugekommen wäre. Beide
standen sich nun wieder gleichwertig gegenüber. Na — sagte er, zog
die Augenbrauen in die Höhe und ließ seinen Blick, ohne den Kopf zu
wenden, nachdrücklich zu seinem Vater hingehen: damit wäre die
Angelegenheit ja dann für alle Teile befriedigend geregelt; nun rede
aber auch bitte nichts mehr davon. Und er hoffte, dieser Blick würde
genügen, seinem Vater Eindruck zu machen. Überhaupt — setzte er
aber noch hinzu, brächtest du mir gar nicht ein so großes Opfer,
wenn du mir die Schulden gezahlt hättest: In ein paar Jahren hätte
ich dir alles zurückgezahlt! — Das ist ja reizend! höhnte Herr Sintrup,
wohl dann, wenn die Riesengehälter eintreffen? Vorläufig bist und
bleibst du nichts weiter als ein dummer Junge, der vom Gelde seiner
Eltern lebt! Fox wurde rot und sagte: Ich verbitte mir das, ich bin
nachgerade alt genug mich nicht mehr als Kind behandeln zu lassen,
ich könnte selbst schon Kinder haben! Graf Zitzewitz zum Beispiel —
— Komm du mir nicht mehr mit dem alten albernen Gewäsch! rief
Herr Sintrup in so befehlendem Ton, daß Fox unwillkürlich wieder
sich ganz klein fühlte. Herr Sintrup ging aufgeregt im Zimmer auf
und ab, es folgte ein langes Schweigen, dann stellte er sich vor Fox
auf und durchbohrte ihn, den Mund zu einer Schlußrede öffnend, mit
den Augen. Fox wollte diesen Blick aushalten und versuchte
ebenfalls durchbohrend auszusehen, beider Augenpaare begegneten
sich, es war wie eine stumme Kraftprobe, wer von beiden es länger
aushielt, dann siegte Herr Sintrup.
Mein Entschluß steht fest! sagte er: Du bist durchs Examen
gefallen, nachdem du jahrelang gebummelt hast; ich gebe dir ein
neues, letztes Jahr zur Vorbereitung, und fällst du ein zweites Mal
durch, dann ist es aus zwischen dir und mir, dann erhältst du keinen
Pfennig Geld mehr von meiner Seite und magst meinetwegen Kellner
werden, das ist mir dann egal. Nun weißt du’s. Deine Gläubiger
werden jetzt befriedigt, sie schicken ihre Rechnungen an mich, und
ich warne sie dir ferner etwas zu borgen, da ich für nichts in Zukunft
aufkomme. Und somit adieu!
Fox begleitete ihn wortlos zum Vorplatz hinaus, und wie sich Herr
Sintrup draußen vor der Tür noch einmal umdrehte, da es seinem im
Grunde weichen Vaterherzen widerstrebte, seinen Sohn so ohne
jedes wärmere Wort zu verlassen, und er ihn halb strafend noch,
halb ermuntern wollend ansah, kam das Fräulein gerade die Treppe
herauf. Herr Sintrup sah sie nicht, sie aber ahnte sogleich, daß dies
Fox’ Vater sei, und ohne eine Stufe weiter emporzusteigen drehte sie
sofort wieder um und nahm sich vor, zu gelegenerer Zeit
wiederzukommen. —
Fox blieb in dumpfem Brüten zurück. Wieder kam er sich schlecht
behandelt vom Schicksal vor. Daß seine Schulden bezahlt wurden,
war selbstverständlich; daß sein Vater ganz brutal gesagt hatte, nun
würde nichts wieder bezahlt, das war herzlos, niederträchtig. Daß er
jetzt aber seine Lieferanten vor ihm geradezu warnen wollte, —
dafür fand er überhaupt gar keine Bezeichnung, das war
unqualifizierbar! Wenn sie auch Vater und Sohn waren, so standen
sie sich doch auch gesellschaftlich gegenüber; Herr Sintrup konnte
Gott danken, daß er Fox’ Vater war! Unter andern Umständen hätte
er ihn gefordert, — einfach gefordert!! —
Also nun hieß es arbeiten und sparen!
Vor allem kaufte er sich noch einmal die herrlichsten Dinge
zusammen, zu einer Art von Henkersmahlzeit; die vertilgte er, und
wie er satt war, glaubte er, es werde ihm nun leicht werden, in
Zukunft auf all das Schöne zu verzichten. Bei einer ausgezeichneten
Zigarre schien es ihm leicht, sich die ausgezeichneten Zigarren
abzugewöhnen. Auch den teuren Wein mußte er in Zukunft
entbehren. Das schien noch leichter, denn es befand sich noch ein
kleiner Vorrat in dem Keller. Den trank er nun in kurzer Zeit aus, um
mit ihm aufzuräumen, um reine Bahn zu machen für die Zukunft.
Und dann war der Moment da, wo diese Zukunft wirklich beginnen
sollte: Mit dem Gefühl des Märtyrers kaufte er sich eine ganze Kiste
der billigsten Zigarren, entzündete sich eine und sah die glühende
Spitze voll unverhohlener Bitterkeit an: Das schmeckt ja abscheulich,
— einfach abscheulich! sagte er laut, mit kurzen, hochfliegenden
Endsilben, wie wenn ihn gerade jemand beleidigt hätte. Er ließ die
Kiste stehen. — Eine einzige, gute Zigarre, eine wirkliche Importe,
wird mich auch nicht ärmer machen! Und diese Kiste hier bleibt auf
dem Schreibtisch, für den Hausgebrauch. — Der ersten Importe
folgte bald eine zweite, eine dritte, nur mit dem Unterschied gegen
früher, daß er nicht mehr ganze Kisten kaufte, sondern Stück für
Stück. Und wie der Wein zu Ende war, kaufte er ihn flaschenweise.
Die einzige, die wirklich etwas von Ersparnissen empfand, war das
Fräulein. Sie bekam keinen Wein mehr, sondern nur noch Tee; und
die Zuschüsse für die Toiletten hörten gänzlich auf. Theaterbilletts
sah sie auch nicht mehr. Fox setzte ihr auseinander, sein Vater habe
geschäftliches Unglück gehabt; er hoffe aber, die Zeit der
Einschränkung werde vorübergehend sein. Auf diese Wandlung war
sie nicht vorbereitet. Fox hatte sie zwar gelegentlich um kleine
Geldbeträge angegangen, die sie ihm auch bereitwillig gab, aber das
war doch nur geschehen, wenn es sich um Kleinigkeiten handelte
und er gerade nur lauter Hundertmarkscheine bei sich hatte. — Jetzt
merkte sie nun, wie die Sache stand, und eines Abends sagte sie
ihm in aller Ruhe, sie möchte nun nichts mehr mit ihm zu tun haben.
Er machte ihr Vorwürfe, sagte, die wahre Liebe überwände alles,
aber sie sagte, nein, das könne sie nicht überwinden, und zeigte sich
gegen seine Auseinandersetzungen sehr störrisch und verärgert. Und
als er sagte: Na also, auf Wiedersehen, nächsten Freitag! schwieg
sie brummig. — Am nächsten Freitag blieb sie auch aus. Bon! dachte
Fox, wenn sie nicht mehr will, ist es ihre Sache. Die nächste wird von
allem Anfang an etwas knapper gehalten. — Es kam nun ein anderes
Fräulein, nicht ganz so hübsch wie das erste, aber viel lebendiger; ja
eigentlich viel zu lebendig. Zu Anfang schwieg er und dachte: Es
wird sich wohl legen; aber es legte sich nicht. Fox liebte die
Lebendigkeit nicht sehr. Das erste Fräulein hatte doch auch viel mehr
Gemüt gehabt! Er schrieb diesem ersten einen sentimentalen Brief,
während er sich unter der Hand nach einem dritten Fräulein umsah
und es dem Zufall überlassen wollte, welches von den dreien nun in
Zukunft bei ihm fußen würde. In der nächsten Woche war die erste
Freundin wirklich wieder da, und als sie ihr Geld erhielt für die
„Toiletten“, sagte sie, das Vergangene sollte begraben und vergessen
sein. Das andere Fräulein verließ den Schauplatz so plötzlich, wie es
ihn betreten hatte, unter Mitnahme mehrerer wertvoller
Gegenstände, aber ohne Zeichen einer Kränkung. Das erste,
eigentliche Fräulein fand von ihrer Nebenbuhlerin noch ein paar
Haarnadeln, sagte sich sofort, daß es natürlich sei, daß Fox
inzwischen einen Ersatz gesucht habe, und steckte sie in ihr eigenes
Haar. —
Fox lebte nun fast wieder wie zuvor. Er mied die früheren
Gläubiger und fand neue, von Arbeit war nicht viel die Rede, beinah
nur in Briefen, die er nach Hause schrieb. Wieder begann sich das
Unwetter über seinem Kopfe zusammenzuziehen; aber auch diesmal
wurde er gerettet; durch ein an sich trauriges Ereignis: Frau Sintrup
starb plötzlich, und hinterließ die Verfügung, daß ihren Söhnen ein
Teil des mütterlichen Vermögens, das sie einmal zu erwarten hatten,
schon jetzt ausgezahlt würde. Herr Sintrup erzählte, seine Frau sei
plötzlich am Schlaganfall gestorben. Die näheren Einzelheiten waren
traurig: Eines Nachmittags, nach einem schweren Herrendiner, saß
Frau Sintrup schlafend im Sofa. Nach kurzer Zeit erwachte sie, fühlte
sich sehr flau und erinnerte sich, daß noch sehr viel
Hummermayonnaise da sei. Die aß sie, ihr Appetit wurde angeregt,
und sie erinnerte sich weiter, daß noch eine kleine halbe
Marzipantorte da sei. Die aß sie auch, und jetzt wurde ihr fast
nüchtern zumute. Es fiel ihr nun ein, daß heute gerade frisches
Schwarzbrot gebacken wurde. Sie ließ sich einen kleinen Laib
kommen, bestrich ihn dick mit Butter, und verzehrte ihn ebenfalls,
obgleich sie eigentlich fühlte, daß sie nicht mehr konnte. Dann kam
der erste Schlaganfall, dem sehr bald ein zweiter folgte. Sie ahnte,
daß es mit ihr zu Ende ging, und traf jene letztwillige Verfügung,
über die Herr Sintrup unglücklich war, denn er sah nur Unheil für
seine Söhne vor Augen.
Fox war aufrichtig betrübt, und als er später jenes Testament
erfuhr, erschüttert über soviel Güte. Große Tränen traten ihm ins
Auge. Zwar fuhr es ihm für einen Moment durch den Kopf, daß
vielleicht gar nicht soviel Güte dabei war — er hätte das Geld ja
später sowieso geerbt — aber er verbannte diesen Gedanken sofort
und dachte: Nein nein, dies ist wirklich groß! Es war ihre letzte große
Handlung, mit der sie aus dem Leben schied! Und er feierte das
Andenken seiner Mutter für sich allein, ganz allein, in einer
Weinrestauration. Er ließ sich ein kleines Separatzimmer geben,
bestellte eine Flasche Sekt und zwei Gläser, schloß dann die Tür ab,
füllte beide Gläser, sah sie lange gedankenvoll an und sprach
endlich: Auf dein Andenken, Mutter! Dann trank er sein Glas aus,
wußte nicht, was nun mit dem anderen werden sollte, und trank es
ebenfalls aus. Dann seufzte er tief und dachte: das ist nun alles,
alles dahin! Welch eine Fülle von Liebe habe ich genossen! Wenn
mich jetzt jemand hier sitzen sähe, ganz allein, den Sohn, der das
Andenken seiner Mutter feiert, die ihn verlassen hat! Die Tränen
traten ihm darüber in die Augen. —
Ernst, ohne nach rechts und links zu sehen, kam er endlich wieder
aus seinem Separatzimmer heraus, dessen Schlüssel nachdrücklich
und schwer sich im Schlosse gedreht hatte, und die Kellner, die
nichts von allem begriffen, sahen ihm nach als wenn er verrückt
geworden wäre.
An das Examen wurde nun überhaupt nicht mehr gedacht, Herr
Sintrup war verzweifelt, aber Fox schrieb ihm ganz gelassen, es
könne ihm doch ganz egal sein, was er tue, denn er lebe ja nun
nicht mehr von seinem Gelde; er berief sich außerdem auf Pitt, der
ja ebenfalls jetzt sein Leben genieße, seinen Referendar an den
Nagel gehängt habe und auf Reisen gegangen sei.
Traurig und bekümmert war Herr Sintrup: was sollte nur aus
seinen Söhnen werden! Und er selber arbeitete sich für diese Söhne
ab, für nichts und wieder nichts! Er war doch auch noch nicht mit
dem Leben fertig! Es regte sich in ihm eine Bitterkeit gegen das
Schicksal. Seine Geschäftsreisen wurden häufiger und endeten
immer häufiger mit ungeschäftlichen Abschlüssen. Sollte er am Ende
noch einmal heiraten? Mausi hatte ihm selbst gesagt: Traure mir nur
nicht nach, das hat gar keinen Zweck und ist kindisch. Der Mensch
lebt und stirbt, und was liegt denn schließlich an einem Menschen?
Ehe er da war, hat ja doch niemand an ihn gedacht, also was kann
da so Besondres dran sein! — Ja, das hatte Mausi gesagt und ihn
darauf hingewiesen, daß er ihr ja auch während ihres langen
Zusammenlebens nicht immer treu war. Sollte er wieder heiraten? —
Vorläufig nahm er eine Hausdame, und alsbald gingen die
bedenklichsten Munkeleien über ihn und diese Dame um, die von
auswärts kam, voll und beinah üppig, und deren musternde Augen
mehr als Hausfrauentugenden spiegelten. Man begann sich leise von
diesem Hause zurückzuziehen, und nur einige Junggesellenfreunde
frequentierten es seither mehr, interessiert den neuen Zustand
prüfend.
Fox erfuhr von diesen Dingen durch alte Schulfreunde, die es für
ihre moralische Verpflichtung hielten ihm alles mitzuteilen. Aber er
berührte diese Fragen niemals, schon deshalb nicht, weil er jetzt so
gut wie ganz außer Korrespondenz mit seinem Vater war, und dann:
Männer haben Männern in solchen Dingen nicht dreinzureden.
Pitt war auf Reisen, Fox fand, er müsse auch Reisen machen. Aber
während Pitt vernünftig Geld ausgab, berechnend, wie lange er mit
ihm reichen werde, gab Fox es in unsinniger Weise aus, zumal er
nicht allein reiste. Das Fräulein hatte erst gedacht es käme mit, aber
Fox setzte ihr auseinander, daß da noch andere warteten, und daß
sie noch längst nicht an die Reihe käme. — Da sagte sie, sie könne
sich nur dann dazu verstehen, die Beziehungen mit ihm
weiterzuführen, wenn es in der Zwischenzeit genau so wäre, als
wenn Fox am Orte bliebe, das heißt, wenn sie ihre Unterstützung
weiter von ihm empfinge. Das fand er selbstverständlich, nachdem
er im ersten Gefühle dagegen opponieren wollte. —
Nach ein paar Monaten war er schon wieder da: Die Menschen
sind verschieden, sagte er zum Fräulein, mein Bruder gondelt weiter
in der Welt herum; i c h sage mir, zu Hause ist es doch am besten;
man hat sein festes Heim und — also ich habe mich wirklich nach dir
gesehnt — direkt nach dir gesehnt! So etwas im besten Sinne
Anspruchloses wie dich gibt es doch nicht wieder! Ich bin dir immer
treu geblieben, seelisch treu geblieben, von Anfang bis zu Ende,
wahrhaftig’n Gott! Jetzt leben w i r aber mal schön fidel zusammen,
was? Er faßte das Fräulein um die Taille, und sie sagte: Drücke mich
doch nicht so, Robert. — Von seinem Vornamen Fox wußte sie gar
nichts. —
Es bildete sich jetzt ein Kreis von Existenzen um ihn, der ihm
schmeichelte und von ihm profitierte. Fox hielt lange Reden über
Studium und Bildung, und wenn er fragte: ist das nicht glänzend,
was? nickten jene mit eifrigen Worten und sahen dabei zerstreut auf
die Likörflasche, auf den Wein, auf die Zigarren, denn ihr Interesse
war durch Entbehrung noch viel mehr auf diese Genüsse gerichtet
als bei Fox, der alles aus dem vollen nahm. Sie getrauten sich
anfangs nicht immer wieder selbst von neuem zuzugreifen, verloren
aber im Laufe der Zeit alle Schüchternheit, und den Übergang zu
rücksichtslosem Sichaneignen bildeten sie, indem sie irgend einen
Satz mit augenscheinlicher Geisteskonzentration sagten und dabei
wie in Zerstreutheit mit der Hand in die Luft langten, bis sie den
ersehnten Gegenstand ergriffen fühlten. — Sie umgaben Fox wie ein
Stab, und er sagte manchmal zu dem Fräulein: Wirklich, es kommt
nicht darauf an, daß der Mensch bei Ministern verkehrt, den
eigentlichen Adel, den Geistesadel, findet man auch anderwärts! Ich
wundere mich, wo auf einmal so viele echte einfache Bescheidenheit
herkommt in meinem Bekanntenkreise, so viel neidloses Anerkennen
eines andern, der mehr bedeutet als sie selbst — ich meine mich
damit.
Fox wurde allmählich ausgesogen. Man zehrte von seiner
Freigebigkeit, ließ es nicht genug sein von den Wirkungen seines
Geldes zu leben, sondern bat ihn um direktes Geld. Er gab immer
und bekam nie etwas zurück. Oft nahm er sich vor nein zu sagen,
aber er vermochte es nicht. Dies Gefühl, in die Westentasche zu
greifen und dort vornehm mit dem losen Gold zu klimpern, es
hervorzuholen wie wenn es Pfennige wären und es von oben in eine
ausgestreckte leere Hand zu legen, war zu angenehm, es war zu
schön, zu denken, daß der andere dächte: Ja, der hat’s gut, dem
macht es nichts ein paar Goldstücke weniger zu haben. — Immerhin
konnte Fox sein gutes Leben mit diesem Gelde eine gute Weile
weiter fristen, obgleich er sehr viel ausgab, aber dann begann die
Zeit der Sorgen wieder. Er berechnete, wie lange er sich noch halten
könne; dann erhielt Pitt einen Brief: Er habe gewiß etwas Geld für
seinen Bruder übrig, und wirklich schickte ihm Pitt eine größere
Summe, obgleich er sich sagte, daß es dann mit seinem eigenen
Wohlleben schneller zu Ende gehen werde. Aber er dachte: dies Geld
würde vielleicht für ein halbes Jahr länger reichen, nehmen wir an,
es läge schon in der Vergangenheit. — Fox hielt sich noch ein paar
Monate und dann begannen die früheren Jämmerlichkeiten wieder.
Schulden hatte er schon längst wieder gemacht, zu einer Zeit noch,
wo er alles hätte bar bezahlen können, und, so wie damals,
begannen die Gläubiger jetzt sich zu regen, erst einzeln, dann immer
mehr. Es gelang ihm neue Anleihen zu machen, die er zum Teil dazu
verwendete, alte zu begleichen. Schließlich brachte er in diese ganze
Tätigkeit ein wohlüberlegtes System: Einer mußte immer den andern
substituieren. Alle bildeten ein in sich geschlossenes Ganzes, das
sich in sich selbst verschob, das leise hin und her schwankte, da Fox
den Schwerpunkt bald hier-, bald dorthin verlegte. Aber allmählich
brachen von dieser Scholle, auf der er selber trieb, einzelne Stücke
ab, sie wurde kleiner und kleiner, man weigerte sich, ihm weiter zu
borgen.
Was soll nur werden! dachte er nun öfter und öfter. Seinen Vater
nochmals um Unterstützung anzugehen erschien ihm zwecklos,
zumal er nicht recht wußte, ob er mit ihm eigentlich gebrochen habe
oder nicht. Waren die Gläubiger das erstemal schon zudringlich und
dreist, so stürzten sie sich nun auf ihn wie losgelassene Hunde.
Jeder wollte derjenige sein, der aus dem allgemeinen Ruin noch sein
Teilchen Habe herausriß. Fox’ Manipulationen wurden fieberhaft und
sinnlos. Er schickte Blumensträuße, wie wenn seine Lieferanten
Primadonnen wären. Stück für Stück verkaufte er von seinen
Sachen, was nur irgend zu verkaufen war. Zuletzt wanderte seine
goldene Uhr und sein Brillantring, das alte Familienerbstück, ins
Versatzamt. Endlich entschloß er sich doch an seinen Vater zu
schreiben; das schlimmste was die Folge sein konnte, war eine
Weigerung. Er bat um weitere Unterstützung für ein Jahr und um
Begleichung seiner Schulden; dafür wollte er dann auch sicher das
Examen machen und das Geld nur als geliehen betrachten. Herrn
Sintrups Antwort war ein Wutschrei. Kaum kannte Fox diese wilde
Handschrift wieder, die sonst stets denselben kaufmännischen,
kulanten Duktus führte. Wie Ohrfeigen klatschten ihm die Worte um
den Kopf. Am Schluß des Briefes stand: Entweder ich stecke dich als
Lehrling in ein Geschäft in irgend einem Orte, wo dich und mich
niemand kennt, und dann zahle ich deine Schulden, oder du bleibst
wo du bist, gehst hin wo du magst, und dann will ich nie wieder das
geringste von dir hören.
Aus dem Gefühl der Zerschmetterung, das Fox zu Anfang
ausschließlich beherrschte, löste sich allmählich eine tiefe Entrüstung
heraus, die sich in seiner Antwort in eine kalte, höfliche Reserve
umwechselte: Auf den Lehrjungenstand verzichte er, im übrigen
erlaube er sich, über seine Pläne Stillschweigen zu bewahren, da er
bei seinem Vater kein Interesse voraussetze und, selbst wenn
solches bestände, sich nicht in der Lage sähe es zu befriedigen.
Diesen Brief schickte er eingeschrieben und nahm sich vor, eine etwa
eintreffende Antwort uneröffnet zurückzuschicken. Er wollte seinem
Vater schon zeigen was ein stilvolles Benehmen ist! Aber es kam
keine Antwort, und nun dachte er: Da steckt nur die Person dahinter,
die jetzt im Hause ist! Ohne sie wäre der Alte ganz anders! —
Was blieb nun übrig? Fliehen? Wohin? Wahnsinnig erschien ihm
dieser Ausweg. — Aber — rief er plötzlich, liegt nicht auch im
Wahnwitz oft ein Sinn, ein tiefer Sinn sogar, der sich nur nicht leicht
enthüllt? Wenn ich jetzt fortgehe von hier, ist das wahnsinnig? M u ß
nicht irgend etwas erfolgen, wo ich auch bin? Und besser anderswo,
wo man mich nicht kennt, als hier, in dem verfluchten Neste! Es fiel
ihm auch ein, daß in Romanen oft Wendepunkte eintreten, wo
niemand weiß wie es nun weiter geht, und wo dann doch etwas
passiert. War das nicht im grünen Heinrich so? Und er selbst war gar
nicht einmal mehr grün! Aber er fühlte: Er stand an einem
Wendepunkte seines Lebens, im Brennpunkte seiner Entwicklung,
das Leben selbst packte ihn nun mit seinen Klauen. Seine
„Bekenntnisse“ wollte er später schreiben; war Rousseau nicht auch
einmal Kellner gewesen oder so was? — Er wollte sich jedem Dienste
unterwerfen, die härteste Arbeit übernehmen, — immer schon mit
der Gewißheit, daß ihn dann später das Leben um so glänzender
entschädigen müsse.
Als das Fräulein ihn am Freitag besuchen wollte, war er nicht mehr
da; seine Hauswirtin erzählte unter Tränen, wie sie am letzten
Feiertag über Land gegangen sei, und als sie heimkam, war der
saubere Herr mit Koffern und Habseligkeiten verschwunden. Sie
habe sofort an seinen Vater einen Eilbrief geschrieben, aber Herr
Sintrup habe geantwortet, sein Sohn sei mündig und er selbst hafte
für nichts mehr was ihn beträfe. — Das Fräulein antwortete nicht viel
und ging noch einmal in sein Wohnzimmer, um nachzusehen, ob er
nichts zurückgelassen habe was sie noch irgendwie gebrauchen
könne, aber selbst die Likörflasche, die immer im Winkel neben dem
Klaviere stand, selbst die hatte Fox nicht vergessen: Dick,
leergetrunken stand sie da, und wie sie die Nase daran hielt, duftete
ihr ein recht trauriger, abgestandener Geruch entgegen.
 Intermezzo.
Fräulein Nippe saß im Stadtgarten, auf der kleinen Bank neben der
Marmorgruppe: „Venus, Amor die Flügel beschneidend.“ Auf ihrem
Schoß lag „Waldmeisters Brautfahrt“ aufgeschlagen, aber sie las
wenig darin, bei jedem fernen Schritte durchzuckte es sie unruhig,
— bald mußte er kommen! —
Wie heilig hatte sie ihre Rolle gespielt, im Schicksal Lottes und
Herrn Könneckes! Und was war nun der Dank dafür? Sie fühlte sich
abgesetzt, ihre Rolle war ausgespielt, man brauchte sie nicht mehr.
Und sie war doch innerlich noch so jung, ihr Herz verlangte noch
nach Liebe. —
Es nahten Schritte. Sie besah schnell noch einmal ihre Fingernägel
und nahm dann „Waldmeisters Brautfahrt“ mit nachlässiger Eleganz
zwischen die Finger. Ein junger Mann; sie umfaßte schnell die
Erscheinung: Soigniert, proper, adrett. — O Gott, wenn er das doch
wäre! Ob er das wohl war? Er kam näher, er schien nicht überrascht
als er sie sah, und erst, als in ihren unverwandten Blick etwas wie
eine leise Beschwörung trat, schien er zu stutzen, doch er ging
vorbei und warf nur einen flüchtigen, etwas verwunderten Blick auf
sie zurück. — Ob er es dennoch war? Hatte er vielleicht nur den Mut
nicht, sie anzureden? War er zu schüchtern? — Sie zog eine Offerte
aus der Tasche und räusperte sich laut. Aber die Gestalt verschwand
langsam in dem Grün. „Herr von mittleren Jahren“ hieß es auf dem
Papier. — Nein, ihr schnelles, impulsives Herz hatte ihr wieder einmal
einen Streich gespielt, dies war kein Herr von mittleren Jahren, oder
vielmehr: Leider war der Herr von mittleren Jahren nicht dieser
junge Mann. Und sie hatte sich in den wenigen Augenblicken schon
in diese Gestalt eingelebt, sie sah ihn schon in Gedanken auf
irgendeiner Hotelterrasse Kaffee trinkend, sich gegenübersitzen, und
hinter ihnen erhoben sich blaue Berge. —
Sie wartete.
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