Microbiology by Michael Pelczar.pdf

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'j'd yj X4 /ff1 (& ? 1u a I1w/4f1if' I _P ' /V if I' For sale in India, Pakistan, Nepal, Bangladesh, Sri Lanka and Bhutan only Download more at Learnclax.com Microbiology Download more at Learnclax.com Download more at Learnclax.com Download more at Learnclax.com  Contents VII Preface PART ONE INTRODUCTION TO MICROBIOLOGY 3 Chapter 1 The Scope of Microbiology 18 Chapter 2 The History of Microbiology 37 Chapter 3 The Characterization, Classification, and Identification of Microorganisms 50 Chapter 4 The Microscopic Examination of Microorganisms PART TWO MICROORGANISMS—BACTERIA 73 Chapter 5 The Morphology and Fine Structure of Bactetia 99 Chapter 6 The Cultivation of Bacteria 115 Chapter 7 Reproduction and Growth 133 Chapter 8 Pure Cultures and Cultural Characteristics PART THREE MICROBIAL PHYSIOLOGY AND GENETICS 151 Chapter 9 Enzymes and Their Regulation 171 Chapter 10 Microbial Metabolism: Energy Production 196 Chapter 11 Microbial Metabolism: Utilization of Energy and Biosynthesis 227 Chapter 12 Bacterial Genetics PART FOUR THE WORLD OF BACTERIA Chapter 13 The World ofBacteria I: "Ordinary" Gram-Negative Bacteria Z61 Chapter 14 The World of Bacteria II: "Ordinary" Gram-Positive Bacteria 285 300 Chapter IS The World of Bacteria rn Bacteria with Unusual Properties Chapter 16 The World of Bacteria IV: Granf-Positive, Filamentous Bacteria of 320 Complex Morphology PART FIVE MICROORGANISMS—FUNGI, ALGAE, PROTOZOA, AND VIRUSES 333 Chapter 17 FungI—Molds and Yeasts 365 Chapter 18 Algae V Download more at Learnclax.com  vi CONTENTS Chapter 19 Protozoa 389 Chapter 20 Viruses of Bacteria 415 Chapter 21 Viruses of Animals and Plants 435 PART SIX CONTROL OF MICROORGANISMS Chapter 22 Control of Microorganisms by Physical Agents 469 Chapter 23 Control of Microorganisms by Chemical Agents 488 Chapter 24 Antibiotics and Other Chemotherapeutic Agents 510 PART SEVEN ENVIRONMENTAL AND INDUSTRIAL MICROBIOLOGY Chapter 25 Microbiology of Soil 543 Chapter 26 Aquatic Microbiology 569 Chapter 27 Microbiology of Domestic Water and Wastewater 593 Chapter 28 Microbiology of Foods 618 Chapter 29 Industrial Microbiology 643 PART LIGHT MICROORGANISMS AND DISEASE Chapter 30 Microbial Flora of the Healthy Human Host 673 Chapter 81 Host-Microbe Interactions: The Process of Infection 687 Chapter 32 Natural Resistance and Nonspecific Defense Mechanisms 703 Chapter 33 Basic and Theoretical Aspects of the linniune Response 718 Chapter 34 Assays and Applications of the Immune Response 741 Chapter 35 Epidemiology of Infectious Diseases 764 Chapter 36 Microbial Agents of Disease: Bacteria 788 Chapter 37 Microbial Agents of Disease: Viruses 824 Chapter 38 Microbial Agents of Disease: Fungi and Protozoa 850 Glossary 877 Indexes 903 Name Index Organism Index Subject Index 0 Download more at Learnclax.com  Preface "Messieurs, c'est lea Microbes qul auront Is denier mot?" Pasteur For the major partôf the twentieth century the physical sciences have dominated science and engineerin.ThiiIltuation was due to a large degree to the devel- opment of the atomic bcthb3and the achievements of the Soviet Union in outer space. The su ccessful launching of-the first satellite into space (Sputnik) in 1957 by the Soviet Union accelerated; phyM&J science research and development programs in the United States -by the government, by universities, and by-in- dustry. We became engaged-in a race 'us leadership in science and technology. We are now experiencing a rapid shift of national priorities In research and -development. As we approach the twenty-first century, we see biology emerging as one of the top priorities in the field of science, and among the biological sciences microbiology has gained new stature. Microorganisms and their activ- ities are increasingly central to many of the concerns of society both nationally and internationally. The problems of the global environment, the recognition of the need to recycle natural resources, the discovery of recombinant DNA and the resulting high technology of genetic engineering—these and other develop- ments have placed microbiology in the limelight. Microbiology is emerging as the key biological science. Microorganisms pro- vide the models used in molecular biology for research. This research at the molecular level has provided, and continues-to provide, the answers to numer- ous fundamental questions in , genetics, metabolism, and cell forms and func- tions. Microorganisms also provisleulodel systems fèrstudying the relationships between species in mixed populations. There is growing recognition of the potential of microorganisms in many applied areas The abilit y -of microorganiMns to decompose materials such as herbicides pestjcides and oils -ut-gil spills,ihe, potwtial of microorganisms as food activity t&ptoduce energy such as methane gas for rSrt coneumptton. and the potfltlai'of-new therapeutic substaioes and other -use* of microorgan- isms are beconilnginsrsaingjy amMlvq- Recombinant DNA technologybotnmay exqted to as genetic engineering is one of the principa&*rusts of the emeginjuigb technologies in the biological sciences. RecombintDNA teèhnoiogy males it-feasible to consider genetically maniputated ( en gineered) microorganisms for commercial production of new and valuable products for a variety of purposes, e.g., mediaipaj s, fuel, and food. This fifth edition of MICROBIOLOGY retains many of the features that have proved, successful In the first four editions, particularly the balance between fundamental or basic microbiology and applied microbiology. This approáh emphasizes-the importance of integrating new knowledge gainedThrough basic research with applied research and development programs. Astrong continuum VII - Download more at Learnclax.com rms l/ of research and development, from the basic to the applied, facilitates the development of benefits for society. One of the new features of this edition is a presentation of the classification of bacteria in a totally new format following the scheme introduced ill the first volume of the recently published Bergey's Manual of Systematic Bacteriology. (One of us, Noel R. Krieg, served as editor of the first volume.) We have also expanded and revised the material on metabolism, bacterial genetics, and genetic engineering and reorganized the section on microorgan- isms and disease. Careful attention has been given to updating of information in all aspects of the discipline. Many new summary tables have been developed, and new Illustrations selected. New review questions, and updated references, follow each chapter. The subject material is precented in eight parts. As a new feature, each part now opens with an essay providing added insight into the material that follows. an introduction. Many chapters Each chapter begins with a chapter outline and now contain boxed essays highlighting important discoveries and developments In microbiology. As in the past, the order of arrangement of chapters lends itself to adjustments in any sequence desired by the instructor. A considerable amount of the artwork has been drawn by Dr. Erwin F. Lessel (a microbiologist in his own right). We have found this to be a distinct asset in terms of improving the pedagogical value of illustrations. Three valuable supplementary publications are available to accompany this new edition: an INSTRUCTOR'S MANUAL, a STUDENT'S GUIDE, and LABO- RATORY EXPERIMENTS IN MICROBIOLOGY. Each has been revised to conform with the subject matter in the fifth edition of MICROBIOLOGY. We have pro- tdsd extensive cross-referencing among these four publications. The INSTRUC- TOR'S MANUAL includes suggested lecture and laboratory schedules, chapter summaries, sources of audiovisual aids, sources of laboratory equipment and reagents, as well as sample test questions. The STUDENT'S GUIDE has been developed to assist the student in his or her efforts to comprehend the subject matter. It provides for each chapter a concise statement of the content (an overview), a comprehensive topical outline, and a series of self-study questions of several types. The writing of a textbook on a subject as comprehensive as microbiology requires considerable assistance from a large number of professional colleagues. Among these, we wish to acknowledge the following persons who were generous in their assistance, particularly in commenting upon drafts of various chapters: Phillip M. Acttey, University of Florida; Ronald L. Crawford, University of Minnesota; Loretta C. Ellias, Florida State University; Louis R. Fina, Kansas State University; Thomas R. Jewell, University of Wisconsin-Eau Claire; Ted R. Johnson, St. Olaf College; Robert J. Janssen, University of Arizona; David lcaf- kewltz, Rutgers University; Joseph S. Layne, Memphis State University; Haideh Lightfoot, Eastern Washington University; David Prasner; Rutgers University; Rgmondj. Seidler, Oregon State University; Robert Todd, South Dakota State University; Anne H. Williams, Evergreen Valley College; and Fred D. Williams, Iowa State University. Special thanks are due Malcolm C. Baines, McGill University; John 0. Corliss, University of Maryland; A. C. Dornbush, Medical Research Division, American Download more at Learnclax.com PREFACE Cyanamid Co.; Jerome J. Motta, University of Maryland; and Robert C. Bates, Virginia Polytechnic Institute and State University, who provided extra help with certain chapters. We are grateful to our colleagues at the McGraw-Hill Book Company, Kathleen Civetta, Editor; James W. Bradley, Editing Supervisor; and Charles Hess, Pro- duction Supervisor, for their pleasant cooperation and assistance in the task of preparing and publishing this book. Thanks are due also to Karen Jacques and Edna Khalil for their skillful assistance in the preparation of manuscript. In the writing of this text, each chapter has been the primary responsibility of one author. However, each of us has read and critiqued all the chapters. As previously mentioned, we have had the benefit of reviews of each chapter from several of our professional associates. In the end we take collective responsi- bility for the complete content of this text. Michael J. Pelczar, Jr. E. C. S. Chan Noel K. Krieg Download more at Learnclax.com PART ONE INTRODUCTION TO MICROBIOLOGY Download more at Learnclax.com  agar for pure slices of sterile potatoes as the solid surtace upon wntcn about as investigators developed microscopes and used to -grow colonies of bacteria. This proved unsatisfactory them to examine droplets of natural fluids. Menageries for a variety of reasons. He tried gelatin as a solidifying of microbes were revealed from a variety of specimens. agent. This had the desirable feature of being a trans- Initially these observations were a source of great cu- parent gel, but it had the serious disadvantage of be- riosity. During the period from 1600 to 1800, consid- coming liquid above 25°C, which is below the optimum erable information accumulated about the occurrence temperature for the growth of human disease-producing of these microscopic forms of life. Great debates bacteria. emerged as to the origin of these microbes. The contro- The solution to this problem was provided in -1883 versy centered on the question of whether the y arose by a German housewife, Fannie E. Hesse, who spent from nonliving materials, i.e., spontaneous generation. part of her time working in the laboratory of her hus- Simultaneous with the development of evidence over band, Walther Hesse. Hesse, a physician, was a former hundreds of years to disprove spontaneous generation student of Robert Koch- Frau Hesse suggested to her was the growing acceptance of the concept that these husband that he use agar, a polysaccharide of algal microorganisms were the cause of many conditions that origin, as a substitute for gelatin in microbiological occur in everyday life, ranging from food spoilage to media. She had gained experience with the chth-acter- diseases of humans, other animals, and plants. In the istics of agar in the process of making jelly; the agar latter part of the nineteenth century a major problem increased the consistency. His experiments with agar confronted investigators searching for evidence to as a substitute for gelatin were dramatically successful. prove that a specific 'kind of microorganim was re- This observation was of such significance that Hesse sponsible for a disease or spoilage. How cpuld they promptly reported the experiments with agar to Robert isolate in pure culture the microorganism suspected of Koch. Koch immediately recognized the great value of causing the change and prove that it was the causal agar as a solidifying agent for microbiological media agent? What the researchers needed was a solid nutrient and adopted its use. Agar goes into solution (1.5 per- substance upon which a specimen could be spread so cent) at ioo°C and solidifies at 45°C. Upon jelling at that individual microbial cells would he distant from 45°C, if remains solid at elevated temperatures—tem- each other. Upon incubation, each cell would repro- peratures , just below joOt. This feature makes it pos- duce, resulting in a mass of identical cells (a colony). sible to incubate the inoculated media at almost any A small portion of the colony could then be transferred disired temperature and still have the medium remain to a fresh medium and be maintained as a pure culture solid. for subsequent experiments, i.e., the pure culture tech- It is a remarkable fact that agar, introduced for use as nique. a solidifying agent in microbiological media just about Robert Koch (1843-1885) was particularly concerned 100 years ago, has not been replaced. Agar is as impor- with the need to develop a technique for the isolation tant now as it was then. The manlier in which it was of microorganisms in pure culture in order to establish discovered adds credence to one of Louis Pasteur's ob- the causative agent of a disease. He experimented with servations: 'Chance favors the prepared mind." Preceding page. A compound microscope made by John Marshall of London about 1700 after a design by Robert Hooke. A condensing lens on a jointed arm allowed the instrument to be tilted on a ball-and-socket Joint. (Courtesy of the Armed Forces Institute of Pathology, Washington, D.C.) Download more at Learnclax.com Chapter 1 The Scope of Microbiology OUTLINE Microbiology as a Field of Biology The Place of Microorganisms in the Living World Haeckel's Kingdom Protista Procaryotic and Eucaryotic Protists Whittaker's Five-Kingdom Concept. Kingdom Procazyotae after Bergey's Manual of Systematic Bacteriology Groups of Microorganisms Distribution of Microorganisms in Nature Applied Areas of Microbiology Micrçblology and the Origin of Life Microbiology is the study of living organisms of microscopic size, which include bacteria, fungi, algae, protozoa; and the infectious agents at the borderline of life that are called viruses. It is concerned with their form, structure reproduc- tion, physiology, metabolism, and classification. it includes the study of their distribution in nature, their relationship to each other and to other living organ- isms, their effects on human beings and on other animals and plants, their abilities to make physical and chemical changes in our environment, and their reactions In physical and chemical agents. Microorganisms are closely associated with the health and welfare of human beings; some microorganisms are beneficial and others are detrimental. For example, microorganisms are involved in the making of yogurt, cheese, and 'vine; in the production of penicillin, interferon, and alcohol; and in the pro- cessing of domestic and industrial wastes. Microorganisms can cause disease, spoil food, and deteriorate materials like iron pipes, glass lenses, and wood pilings. Most microorganisms are unicellular. In unicellular organisms all the life processes are performed by a single cell. In the so-called higher forms of life, organisms are composed of many cells that are arranged in tissues and organs to perform specific functions. Regardless of the complexity of an organism, the cell is the basic structural unit of life. All living cells are fundamentally similar. The word cell was first used morethan two centuries ago by an Englishman, Robert Hooke (1635-1703). in his descriptions (1665) of the fine structure of cork and other plant materials. The honeycomblike structure he observed in a thin slice of cork (see Fig. 1-1) was due to the cell walls of cells that were once Download more at Learnclax.com  WTROOUCTION TO MICROBIOLOGY Figure 1-1, Robert Hooke's drawing of a thin slice of cork as he observed it under the microscope. This draw- ing was included in a report made to the Royal Society (London) in 1665. He is credited as being the first person to use the word cell. (Courtesy of Notional Li- brow of Medicine,) living. But the concept of the cell as the structural unit of life—the cell theory— is credited to two Germans, Matthias Schleiden and Theodor Schwann, who in 1838-1839 described cells as the basic structural and functional units of all organisms. Schleiden and Schwann recognized that all cells, no matter what the organism, are vcry similar in structure. As the concept of the cell as the basic unit of life gained acceptance, investigators speculated on the nature of the substance contained within the cell. Protoplasm (Greek prate, first"; plasm, "formed substance", introduced to characterize the living material of a cell), is a colloidal organic complex consisting largely of protein, lipids, and nucleic acids. These substances are enclosed by membranes or cell walls; and the protoplasm always contains nuclei or an equivalent nuclear substance (see Fig. 1-2). Developments in electron-microscope techniques have made it possible to reveal the complex intricacies of intracellular organization (see Fig. 1-3). All biological systems have the following characteristics in common: (1) the ability to reproduce; (2) the ability to ingest or assimilate food substances and metabolize them for energy and growth; (3) the ability to excrete waste products; (4) the ability to react to changes in their environment—sometimes called irrit- ability; and (5) susceptibility to mutation. In the study of microbiology, we encounter organisms" which may represent the borderline of life. These are the viruses, which are simpler in structure and composition than single cells. Viruses provide an exciting challenge and an opportunity to gain a better un. Download more at Learnclax.com Nucleus Endoplàsmic reticulum Colt wall Endaplosmic Vacuole reliculu \ Cell membrane - Zz.á Cell membrane Nudeclus Tonoplosl Nuclear membrane Nucleus Vacuole p:, Galgi complex 5 ó;..;ut. q /L r_"; - -. Chromatin Chloroplast II $x/f :: A.. B Mptochondnon ( Nucleo membrare Mitochondrion C Starch cram Microlubules Golgi complex rial *p& c!0,cm:1 Nuclear material JDNA) Figure 1-3. (A) Electron micrograph of the alga Chlainydomonos reinhardii (X15000), a eucaryotic cell. (Courtesy of George E. Palade, The Rockefeller Uni- versity, by permission of Holt, New York, publishers of Ariel C. Leowy and Philip Seikovitz, Cell Structure and Function, 1969.) (B) Schematic representation of (A). (Erwin F. Leflel; illustrator.) 5 Download more at Learnclax.com  6 INTRODUCTION TO MICROBIOLOGY derstanding of the nature of complex organic substances that may bridge the gap between the living and the nonliving worlds. Viruses are obligate puasites; that is, they are obligated to grow within an appropriate host cell—plant, animal, or microbe. They cannot multiply outside a host cell However, when a virus enters an appropriate living cell, it is able to direct the s y nthesis of hundreds of identical virues, using the cell's energy and biochemical machinery. A virus is made up of substances unique to life: nucleic acids (chemicals that make up genetic material) and proteins (complex nitrogenous substances found in various forms in animals and plants). Biologists are known for their differing opinions as to how the huge field of MICROBIOLOGY biology can best be subdivided. Historically, the divisions followed the major AS A FIELD OF BIOLOGY groups of life, as in zoology (animals). botany (plants), entomology (insects), and microbiology. (microorganisms). Another manner of subdividing the subject matter of biology is based on the level at which the study is conducted: for example, studies at the level of molecular constituents of the cell (molecular biology); studies at the level of the cell (cell biology); studies at the level of the intact organism (organismal biology); and studies of groups of organisms (pop- ulation biology). Still another approach is to establish divisionron the basis of form and function, as in morphology or anatomy, physiology, metabolism, and genetics. In some colleges and universities, the study of microbiology is carried out in a department of biology; in others, it is in a department of microbiology -. or a department of molecular biology. Irrespective of where microbiology is placed in the broad field of biology, microorganisms have some characteristics which make them ideal specimens for the study of numerous fundamental life processes. This is possible because, at the cellular level, many life processes are performed in the same manner whether they be in microbe, mouse, or human. Microorganisms are exceptionally attractive models for studying fundamental life processes. They can be grown conveniently in test tubes or flasks, thus requiring less space and maintenance than lager plants and animals. They grow rapidly and reproduce at an unusually high rate; some species of bacteria un- dergo almost ioo generations in a 24-h period. The metabolic processes of microorganisms follow patterns that occur among higher plants and animals. For example, yeasts utilize glucose in essentially the same manner as cells of mammalian tissue; the same system of enzymes is present in these diverse organisms. The energy liberated during the breakdown of glucose is"trapped" and made available for the work to be performed by the cells, whether they be bacteria, yeasts, protozoa, or muscle cells. In fact, the mechanism by which organisms (or their cells) utilize energy is fundamentally the same throughout the biological world. The source of energy does, of course, vary among organ- isms. Plants are characterized by their ability to use radiant energy, whereas animals require chemical substances as their fuel. In this respect some micro- organisms are like plants others like animals; and some have the unique ability of using either radiant energy or chemical energy and thus are like both plants and animals. Furthermore, some microorganisms, the bacteria in particular, are able to utilize a great variety of chemical substances as their energy source— ranging from simple inorganic substances to complex organic substances. Download more at Learnclax.com  7 The Scope of Microbiology In microbiology we can study organisms in great detail and observe their life processes while they are actively metabolizing, growing, reproducing, aging, and dying. By modifying their environment we can alter metabolic activities. regulate growth, and even change some details of their genetic pattern—all without destroying the organisms. For example, bacteriophages, which are viruses that infect and reproduce in bacteria, demonstrate the complete sequence of host-parasite reactions and pro- vide a model by which'virus-host cell reactions can be postulated for infections in higher plants and animals. Bacteriophages have been of inestimable value in elucidating many biological phenomena, including those concerned with ge- netics. Microorganisms have a wider range j of physiological and biochemical poten- tialities than all other organisms combined. For example, some bacteria are able to utilize atmospheric nitrogen for the synthesis of proteins and other complex organic nitrogenous compounds. Other species require inorganic or organic nitrogen compounds as the initial building blocks for their nitrogenous constit- uents. Some microorganisms synthesize all their vitamins, while others need to be furnished vitamins. By reviewing the nutritional requirements of a large collection of microorganisms, it is possible to arrange them from those with the simplest to those with the most complex requirements. The increasing com- plexity of nutritional requirements in such an arrangement is also a reflection of the decreasing synthetic capacity of the organisms so arranged. In addition, this kind of arrangement provides information about the steps in the synthesis of various metabolites, e.g., from atmospheric oxygen to inorganic nitrogen salts to amino acids. The biochemist has used microorganisms having varying degrees of synthetic ability to investigate pathways of synthesis. In his presidential address to the Society of American Bacteriologists (now The American Society for Microbiology) in 1942, the late Selman A. Waksman (Fig. 1-4) observed: Figure 1-4. Selman A. Walcs man (1888-1973), world's foremost authority on soil microbiology and codiscov- erer of the antibiotic strep- tomycin. Download more at Learnclax.com  8 IKTRODUCTIO% TO MICROBIOLOGY There is no field of human endeavor, whether it be in industry or agriculture, or in the preparation of food or in connection with problems of shelter or clothing, or in the conservation of human or animal health and the combating of disease, where the microbe does not play an important and often dominant role. Waksman, longtime professor of microbiology at Rutgers University, in 1952 was awarded the Nobel prize in physiology or medicine for the part he played in the discovery of the antibiotic streptomycin, which is produced by a soil bacterium. THE PLACE OF in biology as in any other field, classification means the orderly arrangement of MICROORGANISMS IN units under study into groups of larger units. Present-day classification in bi- THE LIVING WORLD ology was established by the work of Carolus Linnaeus (1707-1778), a Swedish botanist. His books on the classification of plants and animals are considered to be the beginning of modern botanical and zoological nomenclature, a system of naming plants and animals. Nomenclature in microbiology, which came much later, was based on the principles established for the plant and animal kingdoms. Until the eighteenth century, the classification of living organisms placed all organisms into one of two kingdoms, plant and animal. As previously stated, in microbiology we study some organisms that are predominantly plantlike, others that are animall Ike, and some that share characteristics common to both plants and animals. Since there are organisms that do not fall naturally into either the plant or the animal kingdom, it was proposed that new kingdoms be established to include those organisms which typically are neither plants nor animals. !Iaeckel's Kingdom One of the earliest of these proposals was made in 1866 by a German zoologist, Protista E. H. I-Iaeckel. He suggested that a third kingdom, Protista, be formed to include those unicellular microorganisms that are typically neither plants nor animals. These organisms, the protists, include bacteria, algae, fungi, and protozoa. (Vi- ruses are not cellular organisms and therefore are not classified as protists.) Bacteria are referred to as lower protists; the others—algae, fungi, and protozoa— are called higher protists. Procaryotic and Haeckel's kingdom Protisto left some questions unanswered. For example, what Eucaryotic Prolists criteria could be used to distinguish a bacterium from a yeast or certain micro- scopic algae? Satisfactory criteria were unavailable until late in the 1940s when more definitive observation of internal cell structure was made possible with the aid of the powerful magnification provided by electron microscopy. It was discovered that in some cells, for example typical bacteria, the nuclear substance was not enclosed by a nuclear membrane.. In other cells, such as typical algae and fungi, the nucleus was enclosed in a membrane. This discovery—the ab- sence of membrane-bound internal structures in one group of protists (bacteria) and the presence of membrane-bound structures in all the others (fungi, algae, and protozoa)—was a discovery of fundamental significance. Further research has revealed additional differences in the internal structure of these cells. These two cell types, as characterized in Table 1-1, have been designated Download more at Learnclax.com  g The Scope of Microbiology Table 1-1. Features Distin- Feature l'rocarvotic Cells Itucarvolu. Cell,, guishing Procaryotic from Groups where found as unit Bacteria Algae, fungi. protozoa, Eucaryotic Cells of structure plants, and animals Size range of organism 1-2 by 1-4 pan or less Greater than 5 m in width or diameter Genetic system Location Nucleold, chromatin body, Nucleus, mitochondria, or nuclear material chloroplasts Structure of nucleus Not bounded by nuclear Bounded by nuclear mem- membrane; one circular brane; more than one chro- chromosome mosome Chromosome does not con- Chromosomes have-histones; tain histones; no mitotic di- mitotic nuclear division vision Nucleolus absent; function- Nucleolus present; function- ally related genes may be ally related genes not clus- clustered tered Sexuality Zygote nature is merozygotic Zygote is diploid (partial diploid) Cytoplasmic nature and structures Cytoplasmic streaming Absent Present Pinocytosis Absent Present Gas vacuoles Can be present Absent Mesosome Present Absent Ribosomes 70S , * distributed in the cy- SOS arrayed on membranes toplasm as in endoplasmic reticu- lum; 70S in mitochondria and chloroplasts Mitochondria Absent Present Chloroplasts Absent May be present Golgi structures Absent Present Endoplasmic reticulum Absent Present Membrane-bound (true) Absent Present vacuoles Outer cell structures Cytoplasmic membranes Generally do not contain Sterols present; do not carry sterols; contain part of respi- out respiration and photo- ratory and, in some, photo- synthesis synthetic machinery Cell 'call Peptidoglycan (murein or Absence of peptidoglycan mucopeptide) as component Locomotor organelles Simple fibril Multifibrilled with "9 + 2" microtubules Download more at Learnclax.com  10 Il¼TRODUCTIO7 TO MICROBIOLOGY Table 1-1. (continued) Foal tire Procarvolic Gills Eucarvolic Cells Pseudopodia AbaSit Present in some Metabolic mechanisms Wide variety, particularly Clycolysis is pathway for that of anaerobic energy- anaerobic energy-yielding yielding reactions; some fix mechanism nitrogen gas; Some accumu- late poly--hydroxybutyrate as reserve material DNA base ratios as moles % 28 to 73 About 40 of guanine + cytosine (G -f C %) S refers to the Svedberg unit, the sedimentation coefficient of a particle in the ultracentrifuge. Note: Definitions of technical words are provided in the glossary at the back of the book. pi'oc:ar y ulu: and i,ui:arvotii.: organisms of each cell type are called procaryotes and eucaryotes, respectively, l3acteNa are procaryotic microorganisms. The oucaryotic microorganisms in- Figure 1-5. The bacterium Escherichio coil, a typical procaryotic cell. Note the absence of any discrete in- tracellular organelle struc- tures. The light area repre- sents nuclear material; the dark area is ribosomal mate- rial. jCourtesy I. D. J. But- deft and B. C. B. Murray, I. Bacterial 119:1039, 1974.) Inset: B. call as seen by light microscopy. Download more at Learnclax.com  11 The Scope of Microbiology Kingdom Fungi Kingdom Plcntoe Nutneni Nutrie/ PhothetFc uptake by I uptake b, ingestion ngest Kingdomoosto— / — - L—— Figure 1-6. A simplified schematic representa- Kingdom Loner0 tion of Whittaker's five-kingdom system. (Erwin F. Lease], illustrator.) dude the protozoa, fungi, and algae. (Plant and animal cells are also eucaryotic.) Viruses are left out of this scheme of classification. Examples of t ypical pro- caryotic and eucaryotic cells are shown in Figs. 1-2, 1-3, and 1-5. Whittakcr's Five- A more recent and comprehensive system of classification, the five-kingdom Kingdom Concept system, was proposed by R. H. Whittaker (1969). This system of classification, shown in Fig. 1-6, is based on three levels of cellular organization which evolved to accommodate three principal modes of nutrition: photosynthesis, absorption, and ingestion. The procaryotes are included in the kingdom Monera; they lack the ingestive mode of nutrition. Unicellular eucaryotic microorganisms are placed in the kingdom Protista; all three nutritional types are represented here. In fact, as shown in Fig. 1-6, the nutritional modes are continuous: the mode of nutrition of the microalgae is photosynthetic; the mode of nutrition of the protozoa is ingestive; and the mode of nutrition in some other protists is ab- sorptive, with some overlap to the photosynthetic and ingestive modes. The tnutticellular and multinucleate eucaryotic organisms are found in the king- doms Plantoe (muhicellular green plants and higher algae), Animalio (multi- cellular animals), and Fungi (multinucleate higher fungi). Their diversified nu- tritional modes lead to a more diversified cellular organization. Microorganisms are found in three of the five kingdoms: Monem (bacteria and cyariobacteria), Protista (microalgae and protozoa), and Fungi (yeasts and molds). Download more at Learnclax.com  12 INTRODUCTION TO MICROBIOLOGY Kingdom Bergeys Manual of S ystematic Bacteriology places all bacteria in the kingdom Procarvotue after Procaryotoe which in turn is divided into 4 divisions as follows: Bergey's Manual of Division 1 Graciticutes Systematic Procaryotes with a complex cell-wall structure characteristic of Cram-neg- Bacteriology ative bacteria Division 2 Firmicutes Procaryotes with a cell-wall structure characteristic of Gram-positive bac- teria Division 3 Tenericutes Procaryotes that lack a cell wall Division 4 Mendosicutes Procaryotes that show evidence of an earlier phylogenetic origin than those bacteria included in Divisions I and 2 (above) Bergey's Manual is the international standard for bacterial taxonomy. More detailed descriptions of groups of bacteria are given in Part Four of this book. A comparable manual of classification does not exist for fungi, algae, or protozoa. There are, however, schemes of classification for each group that have wide acceptance and usage. An international system for classification and no- menclature of viruses is in the process of development. GROUPS OF The major groups of protists are briefly described below. Although viruses are MICROORGANISMS not protists or cellular organisms, they are included for two reasons: (1) the techniques used to study viruses are microbiological in nature; and (2) viruses are causative agents of diseases, hence, diagnostic procedures for their identi- fication are employed in the clinical microbiological laboratory as well as the plant pathology laboratory. AI" ac are relatively simple organisms. The most primitive types are unicel- lular. Others are aggregations of similar cells with little or no differentiation in structure or function. Still other algae, such as the large brown kelp, have a complex structure with cell types specialized for particular functions. Regard- less of size or complexity, all algal cells contain chlorophyll and are capable of photosynthesis. Algae are found most commonly in aquatic environments or in damp soil. Viruses are very small noncellular parasites or pathogens of plants, animals, and bacteria as well as other protists. They are so small that they can be visualized only by the electron microscope. Viruses can be cultivated only in living cells. Bacteria are unicellular procar y otic organisms or simple associations of sim- ilar cells. Cell multiplication is usually by binary fission. Protozoa are unicellular eucar y otic organisms. They are differentiated on the basis of morphological, nutritional, and physiological characteristics. Their role in nature is varied, but the best-known protozoa are the few that cause disease in human beings and animals. Fungi are eucaryotic lower plants devoid of chlorophyll. They are usually multicellular but are not differentiated into roots, stems, and leaves. They range in size and shape from single-celled microscopic yeasts to giant multicellular Download more at Learnclax.com  Ismi on 13 The Scope of Microbiology mushrooms and puffballs. We are particularly interested in those organisms commonly called molds, the mildews, the yeasts, and the plant pathogens Figure 1-7. Morphological known as rusts. True fungi are composed of filaments and masses of cells which features of various groups of make up the body of the organism, known as a mvcelium.Fungi reproduce by microorganisms. (Note-that fission, by budding, or by means of spores borne on fruiting structures that are this illustration is only in- quite distinctive for certain species. tended to convey the Some morphological and characteristic features of these various microbial impression of morphological groups are shown in Fig. 1-7 and Table 1-2. diversity. No size relation- Microbiologists may specialize in the study of certain groups of microorgan- ship between groups can isms. Strictly speaking, Iniuteriolug' is the study of bacteria, but the term is obtained from it. The wide range in microbial sizes does not permit both con- 'S stancy in magnification and.5 -e showing of meaningful mor- phological details at the s" same time.) (A) Escherichia cob (X1,000). (B) Tobacco F - - mosaic virus (X100,000). 'g (Hitachi, Ltd., Tokyo.) (C) - Rickettsia tsutsugamushi in C, '_5 cytoplasm of infected cell.5 (X940). (N.J. Kramis and I A - The Rocky Mountain Labo- ratory, U.S. Public Health p Service.) (D) Candida utilis (X2,000 approx.). (Courtesy of G. Svihla, f. L. Dainko, and F. Schlenk,J Bacteria], 85:399, 1963.) (E) Aspergillus sp. (Courtesy of. Douglas F. Lowson.) (F) Amoeba. (Carolina Biologi- cal Supply Co.) (C) Chlo- i-ella infusionum (X1,000). I-.. (Courtesy of Robert W. Kwuss,) C.- 04 -I r a. -t Ira:7 I Dot p t31: 6LO Ca , WI F 0 Download more at Learnclax.com  14 INtRODUCTIoN TO MICROUIOLOGY Table 1-2. Some Characteristics of Major Groups of Microorganisms (Erwin F. Lasso!, illustrator) p rslorpltologv Size Important Characteristics Practical Signiticance Bacteria , Typical: Procaryotic; unicellular, simple Some cause disease; some per- / 0.5-1.5 p.m internal structure; grow on arti- form important role in natural by fIcial laboratory media; repro- cycling of elements which con- I 1.0-3.0 p.m duction asexual,'characteristi- tributes to soil fertility; useful -.- Range: callv by simple cell division in industry for manufacture of 0.2 by 100 valuable compounds; some 5pm p.m spoil foods and some make foods Viruses - Range: Do not grow on artificial labora- Cause diseases in humans, other 0.015-0i Into tory media—require living cells animals, and plants; also infect within which they are repro- microorganisms S? duced; all are obligate parasites; electron microscopy required to see viruses loo.. c:T ) Fungi: Yeasts - Range: Eucaryotic; unicellular; labora- Production of alcoholic bever- 5.0-10.0 p.m tory cultivation much like that ages; also used as food supple- of bacteria; reproduction by ment; some cause disease asexual cell division, budding, or sexual processes sum Fungi: Molds Range: Eucaryotic; multicellular, with Responsible for decomposition 2.0-10.0 pn many distinctive structural fea- (deterioration) of many materi- by tures; cultivated in laboratory als; useful for industrial produc- several mm much like bacteria; reproduc- tion of many chemicals, includ- tion by asexual and sexual pro- ing penicillin; cause diseases of 7, cesses humans, other animals, and plants 2oprp Protozoa Range: Eucaryotic; unicellular; some Food for aquatic animals; some 2.0—ZOO p.m cultivated in laboratory much cause disease like bacteria; some are intracell- ular parasites; reproduction by asexual and sexual processes 50 pm Algae ,.. Range: Eucaryotic; unicellular and mul- Important to the production of - 1.0 Pan ticellular; most occur in aquatic food in aquatic environments; many feet environments; contain chloro- used as food supplement and in phyll and a?a photosynthetic; pharmaceutical preparations; reproduction by asexual and source of agar for microbiologi- sexual processes cal media; some produce toxic lOam substances Download more at Learnclax.com  15 The Scope of Microbiology often used as a synonym for microbiology. l'i'ulo,.inilugv is the study of protozoa; a special branch of protozoology called piintsilolir.z' deals exclusively with the parasitic or disease-producing protozoa and other parasitic micro- and macroor- ganisms. Nix cr,lmz is the study of fungi such as yeasts and molds, \ ,I, is the science that deals with viruses. ph, is the study of algae. Fuither specialization in some aspect of the biology of a particular group of organisms is not uncommon; e.g.. bacterial genetics, algal physiology, and bacterial cytol- ogy. DISTRIBUTION OF - Microorganisms occur nearly everywhere in nature. They are carried by air MICROORGANISMS IN currents from the earths surface to the upper atmosphere. Even those indigenous NATURE to the ocean may be found many miles away on mountaintops. Microbes are found in the bottom of the ocean at its greatest depths. Fertile soil teems with them. They are carried by streams and rivers into lakes and other large bodies of water; and if human wastes containing harmful bacteria are discharged into streams, diseases may be spread from one place to another. Microorganisms occur most abundantly where they find food, moisture, and a temperature suit- able for their growth and multiplication. Si*ce the conditions that favor the survival and growth of many microorganisms are those under which people normally live, it is inevitable that we live among a multitude of microbes. They are in the air we breathe and the food we eat. They are on the surfaces of our bodies, in our alimentary tracts, and in our mouths, noses, and other body orifices. Fortunately most microorganisms are harmless to us; and we have means of resisting invasion by those that are potentially harmful. APPLIED AREASOF Microorganisms affect the well-being of people in a great many ways. As we MICROBIOLOGY have already stated, they occur in large numbers in most natural environments and bring about many changes, some desirable and others undesirable. The diversity of their activities ranges from causing diseases in humans, other ani- mals, and plants to the production and deposition of minerals, the formation of coal, and the enhancement of soil fertility. There are many more species of microorganisms that perform important roles in nature than there are disease-producing species. A summary of the major fields of applied microbiology appears in Table 1-3. MICROBIOLOGY AND Many explanations have been offered for the origin of life on earth. One of the THE ORIGIN OF LIFE more acceptable of these proposals suggests that life originated in the sea fol- lowing millions of years of a chemical evolutionary process. According to this hypothesis the inorganic compounds of the atmosphere, under the influence of ultraviolet light, electrical discharges, and/or high temperatures, interacted to form organic compounds which precipitated into the sea, where they accumu- lated. These organic compounds, subjected to additional physical effects of the environment, combined to form amino acids. The amino acids interacted to form peptides, polypeptides, and other more complex organic substances which served as the precursors of the first form of life. Download more at Learnclax.com  16 INTRODUCTION TO MICROBIOLOGY Table 1-3. Major Fields of Field Some Applied Areas Applied Microbiology Medical microbiology Causative agents of disease; diagnostic procedures; diagnostic procedures for identification of causative agents; preventive measures Aquatic microbiology Water purification; microbiological examination; biological degradation of waste; ecology Aeromicrobiolagy Contamination and spoilage; dissemination of diseases Food microbiology Food preservation and preparation; foodbome diseases and their prevention Agricultural microbiology Soil fertility; plant and animal diseases Industrial microbiology Production of medicinal products such as antibiotics and vac- cines; fermented beverages, industrial chemicals; production of proteins and hormones by genetically engineered microor- ganisms Exomicrobiology Exploration for life in outer space Ceochemical microbiology Coal, mineral and gas formation; prospecting for deposits of coal, oil, and gas; recovery of minerals from low-grade ores The time scale of chemical evolution, biological evolution, and the emergence of microbial life is shown in Fig. 1-8, Millions Geological Approximate time of yeon ago era invertebrates Proterozoic '—Eucaryotic cells 1,000 —Aerobic bocleric 1 2,000 -1Archaean I —ProcaryoIic cells 5 Orygen-evolving Anaerobic bacteria Biological evolution 3,000 - i , — Firct fossil eviden, S life 4 First living "cells" 3 Piotocell: membranes enclosing prototypes of nucleic acids 1 2I Organic Coocervate formation concentration and aggregation of molecules soup: synthesis of amino acids, sugars, and peptides Chemical evolution Figure 'i-fl. Time scale of the chemical evolution, the biological evolution, and the occurrence of microbial life, Download more at Learnclax.com  17 The Scope of Microbiology QUESTIONS 1 List the characteristics common to all biological systems. 2 Why are microorganisms useful as subjects for research in the field of biol- ogy? 3 Explain why a knowledge of microbiology is useful in understanding life processes in higher plants and animals. 4 How did the term protists arise? What organisms do we refer to by use of this term? What is the difference between lower protists and higher protists? 5 Discuss the differences between procaryotic and eucaryotic cells. 6 How do viruses differ from other microorganisms? 7 What is the basis of the five-kingdom classification scheme according to Whittaker? Give a reason why it is so widely accepted in the biological community. 8 Discuss the place of microorganisms in Whittaker's five-kingdom classifi- cation scheme. B Why is Bergey's Manual of Systematic Bacteriology so important to bacte- riologists? 10 Where are microorganisms found in nature? How may they be transferred from place to place? 11 Name several applied areas of microbiology. Describe the importance of microorganisms in each of these applied fields. REFERENCES Delauriey, A.. and H. Erni (eds.): The World of Microbes, vol. 4, Encyclopedia of the Life Sciences, Doubleday, Garden City, N.Y., 1965. A beautifully illustrated and well-summarized account of microbes in relation to human beings. It is an inspiring introductory book for the new student. Edmonds, P.: Microbiology, An Environmental Perspective, Macmillan, New York, 1978. This volume serves to introduce nonspecialists to the important functions corned out by microorganisms in nature. Jennings, R. K., and R. F. Acker: The Protistan Kingdom, Van Nostrand Reinhold, New York, 1970. This small book dealing with the protists and viruses is both informative and entertaining. It may provide the stimulus for further study by students and nonstudents alike. National Academy of Sciences: Microbial Processes: Promising Technologies for Developing Countries, National Academy of Sciences, Washington, D.C., 1981. A report of scientific and technological developments in microbiology now avail- able that might be applicable to less-developed countries for improvement of their environment and their economy. Postgate, J.: Microbes and Man, Penguin, Baltimore, 1975. A very good introduction to microbes. It gives an account of how microbes keep our terrestrial biochemistry moving and influence our food supply at every stage. Other aspects of applied microbiology are also covered. Additional topics include microbes in evolution and in the future. Rossmore, H. W: The Microbes, Our Unseen Friends, Wayne State, Detroit, 1976. An elementary discussion of the many useful activities of microorganisms. Whittaker, R. H.: New Concepts of Kingdoms of Organisms,' Science, 163:150-160, 1969. A now classic paper stating the case that evolutionary relations are better represented by new classifications than by the traditional two kingdoms of plants and animals. A new scheme of classification consists of five kingdoms. Download more at Learnclax.com Chapter 2 The History of Microbiology OUTLINE The Microscope Spontaneous Generation Versus Biogenesis Fermentation The Germ Theory of Disease Laboratory Techniques and Pure Cultures Protection Against Infection: Immunity Widening Horizons Medical Microbiology Agricultural, Industrial, and Food Microbiology Microbiology and Modem Biology: Molecular Microbiology Microbiology and Society iiorV is I h stoic of the achievements of men and women. but it records relativel y few cul.t;irrdiiig names and events Man y important (:ontrillutions IN ere 1111de b y peoph whose ilailies have hoeR iorgotten and whose accomplish- juno Is have been lost ill the I oi ,er and deeper shadows cast b y those who i:acrglii liii, fi'!k;\' of the chroniclers. It has been said that in science the credit gets to the One who convinces the world, not to the one who first hail the idea. Si,, in the ci evel opine n t of microbiology. iologv. t lie outstanding names are often of those who convinced the world----vho developed a technique, a tool. or a con- (:( , Ill that was generall y adopted. or who explained their findings so clearl y or drarniaticallv that tire science grew arid prospered. Anitoiiv van ,euweiiIiiieks lucid reports in the ubiquit y of microbes enabled Louis Pasteur 2(o) years later to discover the involvement of these creatures in jol ii en tat ion react ions Ind ill owed Robert Koch, Theohald Smith. Pasteur,r, and roan a y others to discover the association of microbes with disease. Koch is renci piheree for his isolation of the bacteria that cause anthrax and tuberculosis and for thu rigid criteria he demanded before a specific bacterium be held as the cause of a disease. His important contributions to the creation of the science Of liii cro Ii 01 og y %%-oil him (lie 1905 Nobel prize. 'l'lie hnildi,ig of the Panama Canal dramatized Walter Reed's studies of the e1iitleniinlogv of vehhciw fever. hut historians remember that l'heohald Smiths work oil of Texas fever pointed the wa y for Walter Rood's suhse- queni t work. 18 Download more at Learnclax.com  is The His ory of Microbiology In d' 'gi:osis b y Iihoratorv u,etlitj,ls. C. F. I. Widal and :\Ligust yea \Vasserivari p n,se,i etl th,s(? vhi, tolloweSI theill with tools and ideas with which to work. 'at,! E irtch'. ,liscovimrv old chemical compound that would destro y toe svplm- ilisspiror.hete ill Ow liminiami hod' u'itlimiimt injur y to tissue colls paved the way or lim in' (lm' y cliipi,l'ots in the use of chemicals in treating disease. Fm , il l is lie sham',,,' llie Nobel pri.'.m in JfltW with Eli,, \tetchnik,itt, ''liu discovered ,i system in th iiinl;nl bod y Iliiit combated illfcclitill. ih, fig of rehjmtj el y short ilmiration, tin' histor y of flhjcfohiolfl°' is IlUecl with thrilling di.Imi,'vnmmomts, \\: have 'viii] mi[anv battles w ith ink :rin)rgomlisnls and hay,, ''mmcd liot onlEr flake them work for 'is hut also to SfluliO (It thusi t bat p. mirk mgaiilsi mis. THE MICROSCOPE Microbiology began when people learned to grind lenses from pieces of glass and combine them to produce magnifications great enough to enable microbes to be seen. During the thirteenth century Roger Bacon (1220-1292) postulated that disease is produced by invisible living creatures. This suggestion was made again by Girolamo Fracastoro of Verona (1483-1553) and Anton von Plenciz in 1762, but these people had no proof. As early as 1658, a monk named Athanasius Kircher (1601-1680) referred to "worms"4nvisible to the naked eye in decaying bodies, meat, milk, and diarrheal secretions. Although his description lacked accuracy, Kircher was the first person to recognize the significance of bacteria and other microbes in disease. In 1665 Robert Hooke's description of cells in a piece of cork established the fact that the bodies of "animals and plants, complex as they may appear, are yet composed of a few elementary parts frequently repeated"—a quotation not from Hooke but from Aristotle's description of the cellular structure of living things back in the fourth century B.C. Although he was probably not the first to see bacteria and protozoa, Antony van Leeuwenhoek, who lived in Delft, Holland, from 1632 to 1723, was the first to report his observations with accurate descriptions and drawings (Fig.. 2-1). Leeuwenhoek had the means and opportunity to pursue his hobby of lens grinding and microscope making. During his lifetime he made more than 250 microscopes consisting of home-ground lenses mounted in brass and silver, the most powerful of which would magnify about 200 to 300 times (Fig. 2-2). These microscopes bear little resemblance to the compound light microscope of today, which is capable of magnifications of 1,000 to 3,000 times. However, the lenses of Leeuwenhoek's microscopes were well made and Leeuwenhoek had the openness of mind that is so very important in an investigator. His descriptions of protozoa were so accurate that many of the forms he described are easily recognized today. Leeuwenhoek carefully recorded his observations in a series of letters to the British Royal Society. In one of the first letters, dated September 7, 1674, ad- dressed to Henry Oldenburg, Secretary of the Royal Society, he described the 'very little animalcules" which we recognize as free-living protozoa. On October 9, 1676, he wrote: In the year 1675, 1 discovered living creatures in rain water which had stood but a few days in a new earthen pot, glazed blue within. This invited me to view this Download more at Learnclax.com  20 IWTRODUCTION TO MICROBIOLOGY Figure 2-1. Antony van Leeuwenhoek (1632-1723), a Dutch student of natural history whose hobby was making microscopes is shown here with one of the more than 250 microscopes that he made. His best lenses were capable of mag- nifications up to x 270, and he was the first person to report descriptions of mi- croorganisms in detail. (Cour- tesy of Lambert-Hudnut, Division Warner-Lambert Pharmaceutical Company.) fi;G. (,1I Figure 2-2. (A) The Leeuwenhoek microscope. Replica of a simple mi- croscope made in 1673 by Leeuwenhoek. (From the collection of the Armed Forces Institute of Pathology, Washington. D.C.) (8) Side view of a Leeuwenhoek microscope, illustrating the manner in which observa- tions of specimens were made. (C) Front and side views of Leeuwen- hoek's brass aquatic microscope. Lenses could be slotted in at the front and focused by the butterfly nut at the rear. (Erwin F. Lessel, illustrator.) (0) Leeuwenhoek's sketches of bacteria from the human mouth, from letter of September 17, 1683. Note particularly shapes of cells and rela- tive sizes. The dotted line between C and D indicates motility (move- ment) of a bacterium. (Courtesy of C. Dobeil, Antony van Leeuwenhoek and His "Little Animals," Dover, New York, 1960.) Download more at Learnclax.com  21 The Illst,ry of Microbiology water with great attention, especially those little animals appearing to me ten thousand times less than those. which may be perceived in the water with the naked eye. He described his little animals in great detail, leaving little doubt that he saw bacteria, fungi, and many forms of protozoa. For example, he reported that on June 16, 1875, while examining well water into which he had put a whole pepper the day before: I discovered, in a tiny drop of water, incredibly many very little animalcules, and these of divers sorts and sizes. They moved with bendings, as an eel always swims with its head in front, and never tail first, yet these animalcules swam as well backwards as forwards, though their motion was very slow. His enthusiastic letters were read with interest by the British scientists, but the importance of his discoveries evidently went unappreciated. The talents and astuteness of this remarkable man can best be appreciated by reading Dobell's biography of Leeuwenhoek. Before the time of Pasteur, microorganisms were studied mainly to satisfy curiosity concerning their characteristics and their relationships to higher living forms, without awareness of their importance in fermentation and disease. SPONTANEOUS The discovery of microbes spurred interest in the origin of living things, and GENERATION VERSUS argument and speculation grew. As far as human beings were concerned, the BIOGENESIS Greek explanation that the goddess Gaea was able to create people from stones and other inanimate objects had been largely discarded. But even the astute Aristotle (384-322 B.C.) taught that animals might originate spontaneously from the soil, plants, or other unlike animals, and his influence was still strongly felt in the seventeenth century. About 40 B.C., Virgil (70-19 B.C.) gave directions for the artificial propagation of bees. This was but one of many fanciful tales of a similar nature that persisted into the seventeenth century. For example, it was accepted as a fact that maggots could be produced by exposing meat to warmth and air, but Francesco Redi (1626-1697) doubted this. Proof that his skepticism was well founded came from an experiment in which he placed meat in a jar covered with gauze. Attracted by the odor of the meat, flies laid eggs on the covering, and from the eggs maggots developed. Hence the experiment estab- lished the fact that the origin of the maggots was the flies and not the meat. This experiment and others involving mice and scorpions appear to have settled the matter so far as these forms of life were concerned. But microbes were another matter; surely such minute creatures needed no parents! There appeared champions for and challengers of the theory that living things can originate spontaneously, each with a new and sometimes fantastic expla- nation or bit of experimental evidence. In 1749, while experimenting with meat exposed to hot ashes. John Needham (1713-1781) observed the appearance of organisms not present at the start of the experiment and concluded that the bacteria originated from the meat. About the same time, Lazaro Spallanzani (1729-1799) boiled beef broth for an hour and then sealed theilaska. No mi- crobes appeared following incubation. But his results, confirmed in repeated Download more at Learnclax.com  22 INTRODUCTION TO MICROBIOLOGY Figure 2-3. The theory of Air ste'diz^ inhlomos spontaneous generation was disproved with the devices illustrated here, all of which £othSnIe& eliminated airborne bacteria. Schwann heat-sterilized the Leing" Overhlow air which flowed through the glass tube to his culture -- — Tube or fiIIir.g flask (A). Schroder and von cs Gooseneck I,jbe Dosch filtered the air enter- 1 F--- oirConvoluiedTubos. 601015 through thor, ing the culture flask through cotton (B). Simple goose- II necked flasks (C) were de- vised by Pasteur. Tyndall S.",!eb,c,Ih constructed a dust-free in- Slera b.oth cubation chamber (U). experiments, failed to convince Needham, who insisted that air was essential to the spontaneous production of microscopic beings and that it had been excluded from the flasks by sealing them. This argument was answered some 60 or 70 years later independently by two other investigators, Franz Schulze (1815-1873) and Theodor Schwann (1810-1882). Schulze passed air through strong acid solutions into boiled infusions, whereas Schwann passed air into his flasks through red-hot tubes (Fig. 2-3A). In neither case did microbes appear. But the die-hard advocates of spontaneous generation were still not convinced. Acid and heat altered the air so that it would not support growth, they said. About 1850, H. SchrOder and T. von Dosch performed a more convincing ex- periment by passing air through cotton into flasks containing heated broth (Fig. 2-33). Thus the microbes were filtered out of the air by the cotton fibers so that growth did not occur, and a basic technique of plugging bacterial culture tubes with cotton stoppers was initiated. The concept of spontaneous generation was revived for the last time by Felix- Archimede Pouchet (1800-1872), who published in 1859 an extensive report 'proving" its occurrence. But Pouchet reckoned without the ingenious, tireless, and stubborn Pasteur (1822-1895). Irritated by Pouchet's logic and data, Pasteur performed experiments that ended the argument for all time. He prepared a flask with a long, narrow gooseneck opening (Fig. 2-3C). The nutrient solutions were heated in the flask, and air—untreated and unfiltered—could pass in or out; but the germs settled in the gooseneck, and no microbes appeared in the solution. Pasteur reported his results with a great flourish at the Sorbonne in Paris on April 7, 1864. His flasks would yield no sign of life, he said: For I have kept from them, and am still keeping from them, that one thing which is above the power of man to make; I have kept from them the germs that float in the air. I have kept from them life, Download more at Learnclax.com  23 The History of Microbiology In his exuberance, Pasteur sent a few darts at those he disagreed with: There is no condition known today in which you can affirm that microscopic beings come into the world without germs, without parents like themselves. They who allege it have been the sport of illusions, of ill-made experiments, vitiated by errors which they have not been able to perceive and have not known how to avoid. Finally, John Tyndall (1820-1893) conducted experiments in a specially de- signed box to prove that dust carried the germs (Fig. 2-31)). He demonstrated that if no dust was present, sterile broth remained free of microbial growth for indefinite periods. Louis Pasteur (Fig. 2-4) began his brilliant career as professor of chemistry at the University of Lille, France. A principal industry of France being the man- ufacture of wines and beer, Pasteur studied the methods and processes involved in order to help his neighbors produce a consistently good product. He found that fermentation of fruits and grains, resulting in alcohol, was brought about by microbes. By examining many batches of "ferment," he found microbes of different sorts. In good lots one type predominated, and in the poor products another kind was present. By proper selection of the microbe, the manufacturer might be assured of a consistently good and uniform product. Pasteur suggested that the undesirable types of microbes might be removed by heating—not enough to hurt the flavor of the fruit juice, but enough to destroy a very high percentage of the microbial population. He found that holding the juices at a temperature of 62.8°C (145°F) for half an hour did the job. Today pasteurizatior is widely used in fermentation industries, but we are most familiar with it in the dairy industry. Figure 2-4. Louis Pasteur In his laboratory. (Courtesy of Institut Pasteur, Paris.) Download more at Learnclax.com  24 INTRODUCTION TO MICROBIOLOGY THE GERM THEORY OF Even before Pasteur had proved by experiment that bacteria are the cause of DISEASE some diseases, many observant students had expressed strong arguments for the germ theory of disease. Fracastoro of Verona suggested that diseases might be due to invisible organisms transmitted from one person to another. In 1762 von Plenciz not only stated that living agents are the cause of disease but suspected that different germs were responsible for different diseases. That the concept of parasitism was becoming quite genera] is reflected in the following bit of dog- gerel written by Jhathan Swift (1667-1745) early in the eighteenth cantor),: So naturalists observe, a flea Hath smaller fleas that on him prey; And these have smaller fleas to bit 'em; And so proceed ad infinitum. This is better known in the colloquial version: Big bugs have little bugs, Upon their backs to bit 'em; And little bugs have smaller ones, And so ad infinitum. Oliver Wendell Holmes (1809-1894). a successful physician as well as a scholar, insisted that puerperal fever, a disease of childbirth, was contagious and that it was probably caused by a germ carried from one mother to another by midwives and physicians. He wrote The Contagiousness of Puerperal Fever in 1842. At approximately the same time, the Hungarian physician Ignaz Philipp Semmelweis (1818-1865) was pioneering in the use of antiseptics in obstetrical practice. Deaths due to infections associated with childbirth were reduced in the cases handled according to hisinstructions, which minimized chances for infection. As part of his crusade he published The Cause, Concept and Prophy- laxis of Childbed Fever in 1861. Still, most physicians ignored his advice, and it was not until about 1890, when the work of Joseph Lister in England had become known, that the importance of antisepsis was fully appreciated by the medical profession. Pasteur's success in solving the problem of fermentation led the French gov- erment to request that he investigate pebrine, a silkworm disease that was ruining an important French industry. For several years Pasteur struggled with this problem, heartaches and disappointments following one after another. Even- tually he isolated the parasite causing the disease. He also showed that silkworm farmers could eliminate the disease by using only healthy, disease-free cater- pillars for breeding stock. Turning from silk to wool, Pasteur next tackled the problem of anthrax, a disease of cattle, sheep, and sometimes human beings. He grew the microbes in laboratory flasks after isolating them from the blood of animals that had died of the disease. Meanwhile Robert Koch (1843-1910) was busy with the anthrax problem in Germany. Koch, a quiet, meticulous physician, sometimes neglected his medical practice to play with the fascinating new science of ba