Topic Outlines in Microbiology:' an Instructotes Guilae

Home , Carnivore, Herbivore, Inoculation needle

DOCUMENT RESUME ED 205 403 SE 035 468,,

° TITLE, Topic Outlines in Microbiology:' An InstruCtotes Guilae

p -forJuniorAnd. CommUnity Colleges. _ . , , INSTITUTION -American Tociety for Microbiology, Washington, D.C. . SPONS AGENCY National Science Foundation-, Washington, D.C.

PUB DATE 80 .- GRANT NSF-SED-77-18459 NOTE 517p.: Paces 111 and 115 of Section I/and pages 11,19,21,41,43, 49,55,63,65, and 67 of Section IV removed due to copyright restrictions. EDPS PRICE. MF02/PC21 Plus Postage. DESCRIPTORS *College Science: Community Colleges: Course Content: Higher Education: *Instructional Materials: *Microbiology: *Resource Materials: *Science Curriculum: Science Education: *Teaching Guides: Two Year Colleges ABSTRACT This resource' guide presents subject, matter organized,- in .outline'form for four topical areas: introductory microbiology: medical microbiology: microbial genetics: and microbial phySiology. The first two units comprise the two most frequently taught microbiology courses in community and lunior colleges. The outlines- for *microbial genetics and microbial physiology present the, instructor with more detailed information' in basic subject 'areas that are reportedly more difficult to teach..The micrcibial genetics:and physiology units are cited--extensiiely throughout the introductory and' medical-units to assist the instructor with the presentation of these,sublects. The outlines are not intended for student use but' as background information ,for instructors and 'as a guide for lei/eloping courses of instruction. The.format of each unit contains essential information, enriching information serving as.ilIustrative materials to facilitate learning, and praCtical activities including illustrative. materials. (Authon/JN)

*********************************************************m********** Reproductions supplied by EDPT are the best that can be made from the original document: *******************4************************************************ Zz

-CD CNI Topic Outlines In Microbiology

AN INSTRUCTOR'S GUIDE FOR JUNIOR AND COMMUNITY LOLL- EaES

"PERMISSIO0iTO REPRODUCE THIS MATERIAL HAS BEEN GRANTED BY ePochricio- etabb American Society for Microbiology

TO THE EDUCATIONAL RESOURCES Board of Education and Training INFORMATION CENTER (ERIC)" r°

U 5 DEPARTMENT OF HEALTH. EDUCATION 6 WELFARE NATIONAL INSTITUTE OF EDUCATION THIS DOCUMENT HAS BEEN REPRO- DUCED EXACTLY AS RECEIVED FROM THE PERSON OR ORGANIZATION ORIGIN- ATING IT POINTS OF VIEW OR OPINIONS c-1 STATED DO NOT NECESSARILY REPRE- SENT OF Po( iAL NATIONAL INSTITUTE OF EDUCATION POSITION OR POLICY PUBLISHED BY The American Society for Microbiology Washington, D.C.

1/4

1/4 ContribLiting 'Authors -,1 INTRODUCTORY MICRO- MICROBIAL GENETICS BIOLOGY 2 Eugene W. 'Nester, Chairperson George A.WistreichiiChair Person University of Washington East ,rps Angeles c011ege Daniel D. Burke David Campbell Mercer University 0i. Louis Community College at M'eramac Patricia A. Lorenz koliy T. Crabtree l Penn Valley Cornimunity College University of Wisconsin' Center Marathon County; Eleanor K. Mary Paul Edmonds 444," Dutchess COmmunity College University of Wisconsin at Oshkosh Barbara Pogosian Gary E. Kaiser Golden West College Catonsville Community College MEDICAL kICROBIOLOGY MICROBIAL .PHYSIOLOGY Nicholas M. Burdas,Chairperson Carol.Feist, Chairperson Medical' University of South Carolina University*,of Alaska Norma B rratt Joseph T. McGrellis. King Fai al University Atlantic Conimunity'College , a John P. anos Cynthia F. Norton Medical University of South Carolina, -University-of Maine at Augusta Joseph J. Previte Patricia,R. Starr FraMingham State College Mt.'Hood Community College Joseph B. Wilson MiChael L. Rickard , Macomb County Community College r UniVersity of Wisconsin School of Medicine Barbara Starks Oscar.Roselunior College

5 4 Preface ti

- In .1975, the American 'Society for Microbiology surveyed the needs of community and junior college teachers of microbiology, The study revealed that instructional materials specifically designed forthese instructors were inadequate. As a result, American Society for Microbiology, thtouglor its Board of Education and Training, applied for and received a grant from*the National Science Foundation to produce Tepie Outlines in Microbiology. This,publication is the result:Its objective is to organize bodies of scientific knowledge so that community and junior college instructors, with diffefing aims and training, may extract portions according to their instructional needs. The Material was prepared with the support of National Science Foundation grant no. SE1577-1a459. Howiever, any opinions, findings, conclusions, or recommendation's expressed herein are those of the author(s) and do not necessarily reflect the yiews of National Science Foundation. . .

1 STEERING COMMITTEE = L. Joe Berry, Qairman, Board of Education and Training, University of Texas, Austin, Texas. Helen L. Bishop, Prolec:tPirector, American Society for Microbiology, Washington, D.C. Joseph J. Previte, Chairman, Steering pComMitteeFramingham State College, Framingham, Mas- sachus etts. ), Nicholas M. Burdash, Medical Microbiology Study Group, Medical University of South Carolina, Charleston, South Carolina. Eugene W. Nester, Microbial Genetics Study Group,' University of Washington., Seattle, Washing- ton. .. . , / George A. Wistreich, Introductory Microbiology Study Gr ast Los Angeles College, Monterey . , Park, California. , . Joseph B. Wilson, Microbial Physiology Study Group, University of Wisconsin School of Medicine, Madison, Wisconsin. . Lois M. Bergquist, Community College ReOresentative, Los Angeles Valley College, Van Nuys, California. Robert J. Chinnis, Educational Constiltant, The Ameiican University, Washington, D.C. qs W.I. Kessel, Format Copsukant, University of Maryland School of Medicine, Baltimore, Maryland.Maryland. lcknowledgments I' Many _individuals and InstitutiOns have helped in the production of this book. The Natiory Science Foundation provided the funding. The American Society for Microbiology made it,lea'clejtrarters and staff -availably at all stages, and the American Association of Corm unity and Junior Colley;providevalbable consultant help. Special thanks are dire to those .institutions who ga so generously of the time of their faculty. Without this cooperation the authors' work would not have beer% possibl Appreciation is due to all those members of the scientific community who served as formal or informal evaluators of the various'drafts of this doerlent. Finally, thanks should be given to all contributing authors. They have given of their time in the belief that the resulting publication will fill a gap in the instructional materials currently available' to community and junior college instructors.

# Now to Use This Book

This book prilsents subjectmatter organized in outline form, The material isintended for two purposes: (I) as background information for instructors, and (2) as a guide for developing coursesof instruction, ylie user of this material must understand that the Outlines are nintended for use by stildents, nor should they be used in their entirety.- Rather, the instructor should solefrom,the material presented herto meet his or her particular

Li needs. , The Topic Outline is organized in four sectins: Introductory-Microbiology, Medical Microbiology, Microbial Genetitt, and Microbial Physiology. The first two units comprise the t io most frequently taught microbiology courses in community and junior colleges. The outlines for Microbial Genetics andMicrobial Physiology were prepared to present the instructor with mere detailed information in these basic subject areasthat are reportedly difficult to teach. The Microbial Genetics and Physiology units are cited extensively throughoutthe Introductory and Medical units to assist the instructor with the presentation of these,sq.ibjects.

kach unit is organized by the following format: ESSENTIAL, INFORMATION -A body of knowledge that the authors believe is essential subject matter. ENRICHING INFORMATION Information that the' authors believewill serve`as illustrative Material and which will fkilitate learning. PRACTICAL ACTIVITIES Activities 'allowing the instructor to enhance the use of the essential and enriching information.Illustrative materials are also included. Fundamental to this approach is the concept that even. though the:authors have organized thematerials in one fashion, the instructor has the option of reorganizing it in another. What may be deemedenriching at one stage of a student's progress may become essential knowledge at alater date. Similarly, information that is essential to a student with one educational aim may be enriching to another whose educational aiin is different.

1() 11- A

Resources- .d4 .. The user of this book may find a need foradditional sources of information concerningmicroorganisms and the -4fmicrobial world. 'Many are available includingtextbOOks, laboratory Manuals, magazine and journal'articles, and collectfons.. 'For ftirther informationP you may wish to consult alocal college or university, healthdepirtment, manufacturing industry), as well as audiovisual or apublic or special library. Scientific supply atd equipment firms (the and publishing distributors, are often useful - sourcesfor information. Titles of textbooks may be found in Books inPrint. Reviews of textbooks periodically appearin such publications as Quarterly. Review ASM News, The American Scientist, Bioscience, Journal ofJunior College Science Teaching, and The of Biology.

13 Table of Contents General Characteristics of Fungi Section I. Introductory. Microbiology 7.0 8Th General Characteristics of Viruses 1.0General Introdution 9.0General Characteristics of Protozoa and 2.0Survey of the Microbial World Heilminths 3.0Midioscopy and Staining 10.0Inhibition and Killing,of Microorganisms by .4.0"Microbial Cultivatidn . Physical Agents World 5.9Systematic Study olthe Microbial 11. Inhibition and Killing of Microorganisms by 6.0Bacterial Structure and Function Chemical Agents 7.0Viruses 12.0 8.0Fungi 13.0,'AntimicrobialAgentsHost-Parasite Relationships 9.0Algae 14.0 Immunology 10.0Protozoa Medical Bacteriology 11.0Microbial Ecology 16.0Medical Mycology 12.0Biological MoleculeS 1,7.0Medical Virology 13.0Microbial Growth 18.0Medical Parasitology 14.0EnzymeS 19.0Diagnostic Microbiology 15.0Metabolism 20.0Diagnostic Immunology 16.0Introduction to Genetics 21.0Epidemiology Genetically Important Macromolecules. 17.0 Section III. Microbial Physiology 18.0Microbial Genetics 19.0 'Chemical Methods ofMicrobial Control 1.0Biochemicals 20.0Physical Methods of Mirobial Control 2.0Nutrition 21.0Antimicrobial ChemOtherapeutic Agents 3.0Growth 22.0Introduction to Immunity (Resistance) 4.0Enzymes`, 23.0States of Immunity and DiagnosticImmunology 5.0Metabolism 24.0Epidemiology and Infectious Diseasds- 6.0Membrane Phenomena 25.0Envininmental Microbiology Section IV. Microbial Genetics 26.0Industrial Microbiology 1.0/ 'General Introduction 27.0The Future of Microbiology 2.dGenetically Important MacrOmolecules .Section II. Medical Microbiology 3.0Organization of Genetic Material 1.0Survey ofe Biological World 4.0Mutation 2.0Micro.or anisms 5.0Recombination 3.0Microscopy and Stains 6.0Regulatory Mechanisms 4.0Metabolism and Growth 5.0Genetics 6.0General Characteristics of Bacteria 4 5 Section I:Introductory 'Microbiology

TABLE Ok CONTENTS

1.0 GENERAL INTRODUC6TION 5 11.0 MICROBIAL ECOLOGY 53 1.1The Nature of Microorganisms 5 11.1Concepts of Ecology 53 1.2 Concept of the Cell 5 11.2 Symbiotic Relationships 55 1.3 Eucaryotic and Procaryotic Microorganisms 5 e11.3 Microorganisms and Biogeochemical Cycles 57 2.S SURVEY OF THE MICROBIAL WORLD 9 12.0 BIOLOGICAL MOLECULES 59

1 Classification .12.1Proteins 59

2 The Five-Kingdom Approach of Whittaker II 12.2 Carbohydrates - 59 30 MICROSCOPY AND STAINING 13 12.3 Lipids (Fats ancl Relateci`Canpounds) 67 31 Mic ruscopes 13 12.4 Nucleic,,VF1,s 07 3 2 Staining Techniques 17 12.5 Cellular i:omposition, 07 4.0 MICROBIAL CULTIVATION I0 13.0 MICROBIAL GROWTH

4 1 The Nature of Media 19 13.1 Measurement of Growth 69 4 2 Media Usage 2I 13 2 Factors Influencing Growth 71 5 0 SYSTEMATIC STUDY OF THEMICROBIAL WORLD 2I 13.3 Closed-System Growth 75 5 1 actena 21 13 4 Open- System Growth 77 5 2 f- 23 14.0 ENZYMES 7° 5 3. Protozoan.; 14 1Definitions 79 5 4 Algae 27 14.2 Mechanism of Enzyme' Action 8I 5 5 Viruses 27 14.3 Nomenclature 81 6.0 BACTERIAL STRUCTURE AND FUNCTION 29 14.4 Factors Affecting Rate of Enzyme Reactions 6 ICell Shape 29 14.5 Enzymes and Control of Microbial Metabolism 81 o 2 Surface-Assoc iated Structures 31 15.0 METABOLISM 87 6 3 Cell Wall 33 15.1 Cellular Chemical Reactions 87 4 Internal Structure 33' 15 2 Energy 87 6 5 Lndospores 37 15 3 Catabolism 89 70 VIRUSES 37 15.4 Degradative tar Catabolic Pathways 9I 71 Characteristics 37 15.5 Anabolism 103 72Vinon Structure 39 16.0 INTRODUCTION TO GENETICS 105 73Viral Replication 16.1 Definition of genetics 105 80FUNGI .17.0 GENETICALLY IMPORTANT MACROMOLECULES 107 81C haractenstic s 17 IDeox-yiribonucleic Acid, Ribonucleic Acid, and Protein 107 n2Re prod uc tion 1 7 2 Deoxyribonucleiccid (DNA) 107 83Nutrition and Environments 45 17 3 Ribonucleic Acid ( NA) 109 0ALGAE 47 17 4 Protein Structure 113 1 Characteristic s 47 17.5 Protein Function 113 u2Reproduction 47 17 6 Protein Synthesis, Transcription 13 0 3Physiology and C ultivation 4° 17 7 Protein Synthesis. translation 13 100PROTOZOA 4° 18.0 MICROBIAL .GENETICS 17

10 1 C hard( tenstic s 4° 18.1 Procaryotic Chromosome 17 102Reproduction 51 18 2 Plasmid 103Life Cycles 51 18.3 Eucaryotic Chromosome 117 104Nutrition and Cultivation 53 18 4 Eucaryotic Organelle,. 11°

1J 1G I..

119 18.5.'Procaryotic DNA Transfer 26.0 INDUSTRIAL MICROBIOLOGY , . 167

18.6 _Mutation 121 26.1 Food.Microi)iology : , 167 18.7 Selection 125 26.2 Dai icrobichogy 169 18.8 DNA Repair 125 26.3 Ind s rial Application of Mjcroorganistris 171 19.0 CHEMICAL N1ETHbDS OPMICROBI/L CONTROL 125 27.0. THE UTURE OF MICROBIOLOGY I 0'71 19.1 Modes of Kaion 127 27.1 Genetic Engineering 171 19.2 Factors Affecting the Action of Chemical Agents 27.2 Factors Affecting the Incidence of'Infectious Diseases -k173 20.0 PHYSICAL METHODS OF MICROBIAL CONTROL 129 27.3 Microbial Applications to Future World Problems -173 0.1 Modes of Actiork, 12 27.4 Evolution of Microorganisms 175 20.2 Factors Affecting Modes of Action of Physicll Agents 27.5 Microbiology Specialties 175' 21.0 ANTIMICROBIAL CHEMOTHERAPEUTIC AGENTS )133e 21.1 Modes of Action of Chemotherapeutic Agents 133 22.0 INTRODUCTION TO"IMMUNITY (RESISTANCE) 135 22.1 The Nature of Immunity 135 TABLE OF ILLUSTRATIONS 22.2 Antigens (Irnmunogen1) 137 22.3 immunoglobulins (Antibodies) t39 FIG.1, The General Elements of the Nitrogcn Cycle. 61 22.4 Host-Parasite Relationships 1741 FIG. 2.They 6erreral,Elementsof the Carbon Cycle erk 22.5 Microbial Virulence Factors ' 143 23.0 STATES OF IMMUNITY AND DIAGNOSTIC IMMUNOLOGY 043 FIG,3. The Sulfur Cycle. . 65 FIG. 4.['hales of Microbial Growth in Closed Culture. 75 23.1 Cell-lt40 iated Immunity 143 FIG. 5.Complementary Structure of Active Site and Substrate 83 23.2 Humoral-immunity 145 FIG.6. The Allosteric Site'and Binding of PqatiKantt Negative Effectors 83 23.3 General States of Immunity 147 Glycolysis. 95 23.4 Immune Disorders and Hypersensitivity 149 FIG. 8.Examples of f;ermentative Pathways. 99 231 Diagnostic Testing 151 FIG.9 A Summaryof the Tricarboxylic Acid Cycle. 101 24.0 EPIDEMIOLOGY AND INFECTIOUS DISEASES 153 FIG. NI DNA Replication 111 24.1 Principles of ERiderisiology 153 . . FIG. 11..The General Aspects of Protein Synthesis. 115 24.2 Mode of Transmission 153 24.3 Representative (Infectious) Microbial Agents 157 24.4 Control Measures 159 25.0 ENVIRONMENTAL MICROBIOLOGY 161 TABLES 25.1 Sanitation Microbiology 161 25.2 Aquatic Microbiology 163. is . 23.3 Soil Microbiology 165 TikBLEIIFeatures,Distinguishing Procaryobc and Eucaryoa :

Introductory Microbiology 13

a i , TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION, B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES

6 .1b0 General Introductihn - 1.1 The -Nature of Microi- Microorganisms are microscopic and sub- The smallest organism that can be seen Most microorganiitns cam be fixed (im- ganisms microscopic' forms having characteristics ofwith the naked eye would be approxithaeelymobilized) on slides andltained yvith various living systems. 0.1mm. The smallest organism that can be xlyTs to denionstrate specific strbc- seen using a light microscope would be ap-'tures, and a other distinguishing ,prokrties. prVmately 0.2 lan (1 im = 10-6 ni). Compound' lightmicroscopes are used to As a rule, viruses are the smallest, fol-observe most micrdorganisms07- various lowed by bacteria, fungi, and certain algae. preparations. Viruses, bacteria (including the rickettsias MicroorgaRisins have a wider rang) of Fi and chlamydiasC most fun), protooans, physiologicalTpotentialities than all other pr- and certain algae represente five general ganisms combined. - forms of microorganisms. All microorganisms have some means of reproducing or iacreasng their,numbers.° Microorganisms play a significant role in most areas of human activity:

1.2 Concept of the Cell With the exception of viruses (which are.- Cells are co posed of-protoplasm (a col- Staining procedures may be used to dem- acellular), the cell is the basic structural unit loidal ofgapi ornplex coOisting largely ofonstrate basic structural units ofjife; such as of life. proteins, s,landr, ucleic acids)sur- the cell wall and cell membrane alone, pro- rounded ,4r embranes and con-'toplasm; and nuclear material (chromo- , - taining us,,Or' nucleoid. Many cells somes)'. ,havelp uilitidition to Uniting mem- b *14 "1- e typical strucpites (cell askinmembrane, 'cytoplasm, n metabolic processes nor- *thliving cells. -`1,1-: ):(7; Most microorganismse united, plar7drf osalst2-n5:CIF; aniams are biologically and ganisrns. chemically -independent cells, a'ild they ;-.1/ reproduce independently in a suitable environment. A single-celled orgkpism is able to carry out all the processes associated with life within one dell; thus, microorga- tAms are quite complex physiologicjilly de- spite their small size. Unicellular organisms care aggtegate to form multicellular units, e.g., chains or filaments of bacteria.

1.3 Eucarlyotic and Procar- Two major types of cellular organization, Procaryotic microorganisms lack a de- yotic Microorganisms procaryotic and eucaryotic, are recognized. fined nucleus with typical nuclear membranes. Although the nuclear rtiaterial is separated from the cytoplasm, it may not be .% visible with the light microscope even when busing special stains. Introduhiory, Microbiology 5 1J 29 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT,INFORMATION.. C. PRACIICAL ACTIVITIES Eucaryotic organisms. possess a defined membrane-bound nucleus, which may be %) .visible with the light microscope in either stained or unstained preparations. -

Comparisonof Cell Based on the detailed organization of their Distinguishing procaryotic and eucaryotic With the exceptions of the nucleus and Structures cellular structures, eucaryotic microorga-features are summarized, in Table 1. the chlcrroplast, most eucaryotic organelles nisms are more complex. Many of the functions performed by var-are not visible with light microscopy, -but ious membrane,bound organelles in eucar-they are visible with electron microscopy. yotic cells are associated with the cytoplasmic membrane in procaryotes. Bacteria (including the blue-greens or cy- Most bacteria, including cyanobacteria Prepared niicroscope slides maybe used' anobacteria) are procaryotic, whereas fungi, (formerly known as blue-green algae), are.to show size relationships among various protozoans, and algae are eucaryotic. unicellular and lack an organized nucleus. microorganisms. Cyanobacteria contain photosynthetic ap-', paratus .(thylakoid) with chlorophylla. Other pfiotosynthetid"bacteria lack chlorophyll a, but contatih a, b, c, and d. Viruses are neither procaryoticor eucar- Viruses replicate only in living cells. ,.Typical electron micrographs may be ex- yotic, since they lack a true cellular organi- Viral groups may be differentiated on ttieamined to show that viruses lack a cell wall, zation.. basis of the type of nucleic acid core: Virusescell membrane, and cytoplasm and contain contain DNA or RNA, lipt never both. a nucleic acid core.

A- TABLE 1. Features distinguishing procaryotic and eucaryotic cells Feature Procaryotic Eucaryotic

Nuclear body , . Not bounded bytnuclear membrane Bounded by a nuclear membrane One circular chromosome One or more linear chromosomes No mitotic or meiotic division nitotic and meiotic division Nucleolus abint Nucleolus present Cytoplasmic nature and structures Cytdplasmic streaming Absent Present Pinocytosis Absent Present Internal membranetound ofganelles Absent Present Mitochondria Absent Present Golgi apparatus (dicytosomes) Absent Present Endoplasmic .reticulum Absent Present Chloroplasts t Absent \\May be present, depending on type of organism True vacuoles Absent . Present Cell wall Presence of peptidoglycan Absence of peptidoglycan Locomotor organelles Simple and noncontractile Multifibrilled and contractile Introductory Microbiology-7 21 . . IP TOPICS AND SUBTOPICS A. ESSENTIAL tpIFORMATION , B.ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES *It 2.0 Survey of the Micro- . bial World / Classificatif Biological_ classification (taxonomy) the Oneof the primary criteria used in clas- Bacteria can be classified on the basis of systematic arrangement of organis insification is the mode of obtaining carbonseveral demonstrabletharacteristics, groups or categories (tax9. (autotrophic or. heterotrophic). Autotrophs cell shape (coccus, rod, sp)rillum), Gram 'and heterotrophs can be further divided into &stain reactions, photosynthesizing -capabil- groups according to their source of energy. ity, and the ability to grow in different me- Additional classification criteria include: dia. ability to grow in the presence of free oxygen I (aerobic and anaeri:5bic), guanine-cytosine (G-C) ratio, and in some instances chemical composition of the cell wall and means of motility. In the case of bacteria, serological (im-, munologikalr classificationcan be used where niM'phology, physiology, and other et properties of two bacteria are similar. Orgar nisms identified by serological means are I referred to as serotypes. 4 ?.11 Nomenclature Nomenclature refers to the terms used in the systematic and scientific naming of organisms.

2:12 BinoMial System vAll microorganisms exceptviruses have at The naming of bacteria, for example, is least two names: genus and species (binomialgoverned by an internationally accepted set syst4n of nomenclature). of rules (International Code of Nomencla- ture of Bacteria). A genus represents a group of very closely related species. A species represents one kind of microorganism capable of in Feeding with one of its own kind o descendants from a single cell line. The names of both genus and spe ies are Latinized and are descriptive of an orga- ci nism's prorierties, e.g.,_Micrococcus (a mall grain), albus (whitell therefore, a small, spherical organism which produces white colonies. The first name of an ormaism is the genus; the second is the species. The Firstletterof the genus iscapitalized, whereas the first letter of the species is not. Both designations are italicized or under-

+ lined. IntroduPory Microbiology-9 C. PRACTICAL ACTIVITIES A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION TOPICS AND SUBTOPICS Microscope slides can be used to demon- Based on their characteristics, organisms The five kingdoms are differentiated pri- 2.2 The .Five-Kingdom strate the different organisms found in Pro- Approach of Whit- are grouped into the following fiveking-marily on the basis of the mode of obtaining energy, morphology (simple orcomplex), tista and Monera. taker (Note: other ap- doms: Animal, Plant, Fungi, Protista, and Waterbury,-J. B., and R. Y. Stanier. proaches exist.) Monera. and motility. r The Plant and Animal Kingdoms are dif- 1978. Patterns of growth and development ferentiated by the presence or absence ofin pleurocapsalean cyanobacteria. Micro- photosynthetic pigments, i.e., chlorophyllsbiol. Rev. 42:2-44. a, b, and c. Most animals are motile and lack cell walls, whereas most higher plants are non- 4 motile and possess cell walls containing cellulose and fungi and lack chlorophylls. Some Protista (algae/are more closely related to plants, whereas certain other Protista (protozoans) are more 'closely related to the an =- imals. The tissues of higher animals are grouped Most animals may be shown to respond to 2.21 The Animal Kingdom The Animal Kingdom includes organisms that are multicellular, eucaryotic, and non- into organs and systems. Most higher ani-?sternal stimuli (irritability). photosynthetic; that lack cell walls; and thatmals reproduce primarily by sexual means: Examination of either living or preserved Most animals usually ingest solid food (ho-specimens may show that most animals pos- have tissue differentiation and holozoic nu- 4, trition (capable of ingesting entire particleslozoic), which is broken down into smallersess a digestive cavity. or whole organisms). components by enzymatic activity in the digestive tract.

Tissue differentiation in higher plants be- Examination of either living or preserved 2.22 The Plant Kingdom The Plant Kingdom includes organisms specimens will show that most higher plants that are multicellular, eucaryotic, and pho- gins with parenchymal cells, which may give are differentiated into tissues and trueroots, tosynthetic and have tissue differentiationrise to supporting and conducting tissues, stems, and leaves which are composedof and holophytic (absorptive) nutrition. i.e., tracheary elements. Some plant cells are nonliving at maturityspecialized cells and tissues; lower plants but still carry out specific functions, e.g.,lack true roots, stems, and leaves, but may vascular and supportive plant tissue. be multi-cellular with little differentiation in cell types.

Most fungi are chemoorganotrophic (het- Medi ia such as5abouraud's dextrose agar 2.23 The Fungi Fungi are achlorophyllous, unicellular, acido- exhibit holophyticerotrophic) and aerobic saprophytes; how-can lie used to show that fungi are and/or multicellular and philic) i.e., grow better in an environment nutrition. ever, some are parasitic and pathogenic. All fungal class members except the Deu-with /a low pH. Prepared microscope slides teromycetes (Fungi Imperfecti) can repro-may be used to show cellularfeatures of duce sexually as well as asexually. fungi. The hyphae of some fungi are nonseptate, whereas the majority are septate. Aggre- gates of hyphae are referred to as mycelia. Diseases produced by fungi may be di- Introductory Microbiology-11 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL IMDRMATION B. ENRICBMEIF INFORMATION vided into two types, superficial mycOses and systemic mycoses (Medical Microbiol- ogy, Topic 16)P Some fungi produce powerful toxins (my- cotoxina, e.g., aflatoxin) (Medical Microbi- 1 ology, Subtopic 7.82). The algae excluded from the Protista are Living material or prepared microscope 2.24 The Protista The Protista consist of eucaryotic, predominately unicellular organs that includemulticellular and display cellular differentia-slides may be used to demonstrate that the Protista contain the two groups, the micro- alginilnd protozoans. tion with complex morphology, i.e., holdfast; stipe, and blade (which have some resew-scopic algae, and the protozoans'. . bIankes to root, stem, and leaves). Laboratory cultures may be used to dem- Some protozoans have either ingestive oronstrate the growth and reproductive prop- phagotrophic nutrition.All are hetero-erties of Protista. trophic and most are motile in at least one stage of their life cycle. Most Protista are predominately aquatic in habitat:but some are able, to grow and multiply in moist soil. Some Protista possess characteristics of algae and protozoans; e.g., Euglena and slime molds, and generally lack rigid cell walls. Autotrophic Monera may be divided into Autotrophic members of the Monera may 2.25 The Monera The Monera consist of unicellular procaryotic organisms, namely all bacteria whichphotoautotrophs and chemoautotrophs. only be grown in inorganic media, whereas heterotrophs must have organic substances include the blue-greens or cyanobacteria. Photoautotrophs are divided into two groups: those able to carry outanoxygenicfor cu4iVation. photOsynthesis (not producing oxygen) and Microscope slides prepared from living those able 'to carry out oxygenic photosyn-cultures or photomicrographs may be used thesis (producing oxygen). to show Various properties of theMonera. Most chemoautotrophs (lithOtrophs) are able to obtain energy from the oxidation of H2, NO2, NH4+, H2S. Many heterotrophic. Monera utilize organic substances as a source of energy as well as a source of carbon (Microbial. Phys- iology, Subtopics 2.1, 2,21, and 5.23); AD, Some heterotrophs are obligately parasitic and pathogenic (Medical Microbiology, Topic 15).

3.0 Microscopy and - Staining' Resolving power, or the ability to show Diagrams maybe used to show that 3.1 Microscopes Microscopes are of two categories, light Introductory Microbiology-13'" ow a 28 RIP

C. PRACTICAL ACTIVITIES TOPICS, AND- SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION and electron, depending upon the principledetail, is the true measuryment of any mi- bright-field or lightlnicroscOpes used to ob- on which the magnification is based. croscope. It is inversely proportional to thenerve biological 'corms are composed, of a wavelength of the illumination source used.,0'series of lenses and are called compound Since electrons have a much shorter' wave-microscopes and .thaethere are two types of length than° visible light, electron micro-electron microscopes, transmission (TEM) scopes are capable of much greater resolu- and -scartniqg (SEM)., Both electron micro- r tion. scopes use electrons rather than visible light Afunctionalbright-fieldmicroscope as the source of illumination. should have the capability- of.resolving objects that are 0.2 inn in diameter. Electron microscopes differ in several re-, spects from light microscopes. A series of electromagnetic lenses is usedis a condenser system to concentrate the source of illumi- 0, nation, electrons, which have the wavelength of 0.05 A (10- sun). Transmissionelectronmictitbscopes (TEM) not only are capable of better resolving power, but are also capable of producing useful magnification ranging from 100,000 to over 500,00Q. An SEM can resolve objects 0.01 to 0.02 tun in diameter and has approximately five times the depth of field obtainable with a TEM. The SEM can be used to determine size, shape, and surface features of specimens.

3,11 Choice of Microscope The choice of the microscope to use de- The bright-field or light microscope usu- pends on the nature of the specimen and theally is used for the examination of living and information required. stained preparations, e.g., Gram stains, capsule stains, and spore stains, etc.

° Dark-field microscopyciffrbe employed for the detection of microbial motility and K2, The presence or microorganisms in various specimens. Biological preparations (generally non- conductors) studied by electron microscopy must be treated in a manner to preserve structure and to distinguish them from their I respective background surfaces. Intracellular submicroscopic organelles can only be seen with the TEM andrequiren----- specially prepared specimens. 30 29 Introductory Microbiology-15 TOPICS.AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES Preparation techniques for electron microscopy inelude shadow-casting, freeze- etching, surface impressions or replicas, elec . tron staining, and ultrathin sectioning (slic- ing).

3.12 "Advantages of Specific each type of microscopy and each method Dark-field microscopy is useful in observ- Preparations of certain bfeteria which are , Types of Microscopes of specimen preparation offer some advan-ing very fragile, difficult to stain organisms,difficult to see with ordinary light micros- tage ilti the demonstration of microscopic(such as spirochetes), as well as certain se- copy may be treated with fluorescent dyes structures and other features of Microorga-lected cellular activities. and exposed to ultraviolet light to make nisms.. . . Fluorescent microscopy has been used ex- them visible. tensively for purposes of disease diagnosis and detection of proteins such Is immunoglobulins. Fluorescent dyes are attached (coupled) to antibodies or inununoglobulins which combine only with the specific antigens, or substances that caused their information. Resulting colors will vary depending on the type of fluorescent dye used and the structures stained.

Bright-Field Microscopy The bright-field compound microscope is Most bright-field microscopes can be cal- Many microorganisms may be seen with one of the most commonly used instrumentsibrated with stage and ocular microineters. the bright-field compound microscope. in the microbiology laboratory. Phase-contrast can be obtained with a special optical device to enhance differences a in/ refractive index and density of the specimen. 3.2 Staining Techniques A variety of colored organic compounds . Most dyes are basic and, therefore, have *Bacteria may be stained by techniques (dyes) and staining procedures are used ina strong affinity for the acidic componentswith singlb dyes known as simple staining the study and categorilation of microorga- of cells, e.g., nucleic acids. Some dyes, whenprocedures or techniques with combinations nisms. used in combination with other reagents,of dyes and with or without the use. of de- may fornia complex and may be retained bycolorizing agents, which are referred to as the bacterial cell. differential staining procedures. Many dyes also may be used to inhibit certain microorganisms. Some of these dyes may be added to media (selective media) or used for the-treatment of certain diseases. Major Differential The use of staining procedures enables Differential procedures may be used to Staining. Procedures the individual to observe cells and their re- separatemicroorganismsintospecific spective parts more distinctly and to distin- groups, e.g., Gram stain and acid-fast; to guish among certain cells and/or their as- promote visibility of structures such as sociated structures. spores, flagella, and capsules; and to aid in microbial identification. Introductory Microbiology 17 C) ...Ior 0 Toplcs HUBTOPICS A, ESSUNTIAL INFORMATION. B. IINRICIIMIONT INFORMATION C. PRACTICAL ACTIVITIR8 3,22 ThbOrt pi Stain On the basis of OVAM ataining restate, all Cram-negative organisms may exhibit a bacteria may be divided into two groups,:grampealtivo reaction only with improper gram positive ancl,gram negative, staining. Various factors may cause a gram-positive 'the aciVant stain is designed to detect organism to stain gram negatively; those in-. niicroorganisma possessing aubatanees (hy- elude aging, injury to the cell wail, and cul-drophobic ) on the coil wall, o.g., Mycobac- tivation in an acidic medium. terium tuberculosis.

3.23 Other gifferential Stahl- Other differential staining procedurps for i.iclosporesgenerallyresistordinary., ing Prop, es such structures as spores, flagella, and-cap-staining procedures. Application of heat dur- Is sules can be used in the identification ofing spore staining is necessary for stain pen- bacteria. etration to occur. Since bacterial flagella are so thin (0.02 to 0.028 am) theyrt!llh be demonstrated only by the depositing. of dye particles onto their surfaces,. With capsule stains using India ink, it is the background surrounding the structure that is stained rather than the capsule itself. 4.0 Microbial Cultivation 4.1 The Nature of Media The media used in the growth of micro- All complex media are not applicable for organisms can be subdivided into twothe cultivation of all types of microorga- groups: complex and synthetic. Complexnisms. Natural media such as milk and se- media are those for which the chemical com-rum can be sterilized by special techniques p. position is not defined, e.g., potato, blood;and apparatus. Filtration is example of a milk, meat extract, etc. Media must fulfillprocedure used for the sterilization of natu- the nutritional requirements of microorga- ral media as well as heat-labile material. `1iSM13. Microorganisms requiring complex or special sources of growth factors are referred to as nutxitionallS, fastidious organisms, e.g., parasitic and certain pathogenic organisms. The selection of a medium depends on the needs of the organism tribe cultured. An energy source is needed for all media xcept those preparations used by photo- aotrophs. The nitrogen source may be inorganic (N114+, N031 or organic (proteirel and related compounds) or both, depending on the orgtgusni. The carbon source may be inorganic HCO3) or organic compounds such as lipids, carbohydrates, or proteins. 3 Introductory Microbiology-19 3'1 TOPIC8 AND,f11111T01)108 A, Itl'8FlIIIIVTIA1, INFOIIMATION 11, 101 1001 IMIINT ltEFORMATION A0111V1'1'110i Homo organisms may ntliko itirganie Au', whor044 (owls rs(ilre monis 14u1Nr such as tollitir contaiing anilno acids (mu. thionitio, cynteino, eystino), 2 Media Usage Media may be used for a variety of lair- Various (Iwo, antibiotics, and Wiley ma. MO(1111 IIPPii0110011ti inuludo 80144(111vo mo poses, inchuling bioassay, identification and Oriel are incorporated into media to inhibitdill for the isolation of specifie organisms, 1. isolation of particular organisms, and anti- Or favor the growth of certain miernorga-u.g., tiabotiritturs agar, tomato lido), 0181 4 biotic snecoptibility. testing. antiblotie.containing preparationa; differen- pH indicators aro added to media to detect tialnasits to distinguish among similar or. acid or alkaline status. onions, e.g blood agar; selective nod Mr. Eh indicators are utilizial to detect aerobicforential media .proparations having the or anaerobic Conditions. cobinod properties of differential and se. O A variety of specific compounds (sub-loctive media, e.g., eosin methylene blue autos) may be added to media to detectagar; enrichment inedia to promote the enzymatic activities and patterns of micro- growth of tvle type of organism over others, organisms. e.g., prepakationa for various autotrophs; Media may be liquid (broth), semisolid, orand all-purpose media which support the solid. growth of a wide variety of organisms, 9.g.; nutrient agar.

5.0 Systematic Study of the Microbial World Bacteria 6.1 The bacteria are procaryotic, unicellular Bacterial structures such as endospores, Bacteria may be cultivated in the labora- organisms. They are structurally simple, andcapsules, and flagella are not found with alltory on various kinds of media or in an on the basis of cell shapes at least three kinds of bacteria. Endospores are structuresappropriate kind of tissue culture system. broad groups are recognized: bacilli (rods),that enable cells to withstand adverse con-The size, shape, and cellular arrangement of cocci (spherical and ovoid), and spirilla (spi- ditions, capsules are structures that enablevarious kinds of bacteria may be determined ral and corkscrews). some cells to avoid phagocytosis, and flagellawhen wet mounts and/or heat-fixed smears are organelles of locomotion. are observed under a compound microscope. 5.11 Gram Reactions Bacteria can be divid into two categories on the basis of teir reaction to the Gram stain proceduregram - positive and gram-negative.

5.12 Cell Structure Bacterial cells do 'not contain discrgte Bacterial enzymes that generate energy TEMs of bacterial cells may be used to membrane-bo,und structures. are located on membranes that are not or-demonstrate a grarualated cytoplasm and ganized into discrete organelles. dense areas (called avnuclear region), and Photosynthetic ba-cleria contain pigmentthe conspicuous absence of membrane. centered on inner membranes that absorbbound structures. light energy of a different wavelength than the photoreceptors of eucaryotic plants. IntroduCtory Microbiology 21 36 . 14. Tol'l11Pi AND ollwroslicti 144411ITIAI, HN11101IM11NT INI4)10144Sult1y ACTIVITI101 h,13 Chromosono titruenurs llootstia hit01 a sinitia, circular chromo, tntarnal mambrIOAtruatttrtia, 'moao, ThiMa of a .hteltiiikitil--oliromosoitio may ha and Function sonnet Fatah haatorial 40410004W contains aontati, Ara botioVtal4) AillotiOrl during evil uatal to jthow th/.t isairoularlooped ii1 © 044100, 044111 of which offildo for entiretliyiktiort through the replication of the FAO, atrtiotura lintlaytol 1 that is nphouch., protein aninmita, 44181 vitromotionto, coil wall formation, and clad by a itiolitimmo, Iha.ayntti lia of corrals protalna and (Aar voutpoundit

,Method ofIttiproiliio, I lacteria do not carry out the procoasoa of 0011 in kinds of lutotoria Pookietiii Hex pill, don itosia or moloaiti, Most latoterlit roroducestructuros that Ainction in Ono I randfor through asexual rocessos In which, the par,from donor to roolplont oolls. ant eon is (11.1/141041 squally hotwoell two row to/ coils, Ilootoriat coils contain only identical copies of omit goo aftor astoolal repro, duction. As a result Of phonoitionon, they always exist ill rho haploid Mai) ( IN).

5.2 Fungi . Fungi are organisms that utilizo, only or- Fungi contain° significantly to soil fer- Media contai ing nubatanceti aueh att- ganic substances as a carbon source (hotor-tility by roleaaing alma organic nioloculon to starch, gelatin, or lipids may be mod to show otrophic nutrition), ocooyateina from the degradations of W-that excrete a wide variety of hydro- ilim. Certain fungi degrade and obtain uti- lytic onzy MOH. lizable constituents from plaaticn, crude oil, paint% loather, etc.

5.21 Activities and A880611- These organisms lack chlorophyll and Some fungi can live optimally in extreme Field samples of fungi may used to tiona other photosynthetic pigments. environments as frog-living °nal& and ab-show that these organisms, which sap- sorb minerals and nutrients from rocks androphytes, obtain their nutrients from end other substrates that cannot be utilized by or decomposing organic matter. .31 other heterotrophs. -Certain fungi have characteristics that enable them to live mutualistically with higher plaits (mycorrhizae) or algae (lichens), Fungi that are parasitic obtain their nutrients from other living organisms:' Pathogenic fungi produce two general categories of infections in humanim those primarily associated with the skirl and its appendages (dermatomycoses); and those that inyolveyaritits organs of the body (systemic mycoses). Fungi such as Aspergillup fumigatus cause opporttinistic infections. In addition, A. llama and other organisms produce aflatoxins.

Introductory Microbiology 23 3 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 5.24 Structure and Organiza- Fungi have discrete membrane-bound or- Molds may have multinucleated hyphae TEMs may be used to show the presence tion ganelles. with cross walls (apptate) or without crossof various kinds of membrane-encircled walls (nonseptate) and produce variousstructures, and that chloroplasts are absent. kinds of spores that are characteristic of a particular taxonomic class. Yeasts are generally nonfilamentous, but some yeasts have a filamentous phase. Di- morphic fungi, when subjected to a specific environmental change, exhibit both a yeast phase and a mycelia] phase.

5.3 Protozoans Protozoans are predominately unicellular Types of organelles of locomotion are usu- Wet mounts or hanging-drop prepara- organisms that lack cell walls and exhibitally characteristic for protozoan groups.tions may be used to show that proki6ans some form of locomotion during their life- Many types of protozoans are motile onlyare able to move by various mechanisms and times. during certain stages. (Introductory Micro- organelles such as "amoeboid motion," cilia, biology, Topic 10). or whiplike flagella. Certain protozoan cells assimilate inorganic chemicals such as calcium and silicon into their outermost structure. Such substances tend to give cells rigidity. The variety of shapes exhibited by certain kinds of protozoans can be attributed to features of growth periods. Protozoans that concentrate inorganic substances in their shells become temporary reservoirs of those substances and play a role in geochemical cycles. The variety of morphological forms exhibited by a particular protozoan is often referred to as being analogous to stages of cell differentiation in higher organisms. Certain ciliates also can use their locomotion organelles as sensory organs of touch.

5.31 Organelles Protozoans contain discretemembrane- Members within one group of protozoa Transmission micrographs may be used bound organelles. (the Ciliata) possess two nuclei: a macronu-to show organelles such as nuclei, mitochon- cleus that regulates cellular functions and adria, etc. micronucleus that is associated with sexual reproduction. Macronuclei (sometimes more than one per cell) contain many copies of deoxyribonucleic acid (DNA), but do not function in mitosis or meiosis. Introductory Microbiology 25 4 TOPICS AND SUBTOPICS A, ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL. ACTIVITIES Certain protozoans exist in diploid (2N) - states during a major portion of their life cycle. ( 6.4 Algae Algae are photoautotrophic organisms The photopigment complement present Examination of living or preserved speci- that have various kinds of photosyntheticin the chloroplasts of different algae deter-Mem can be used to show the photosyn- )0gments. mines the spedific wavelength of solar radia-thetic organelles of algae. tion that can be used in photosynthesis. The biochemical reactions that occur during algal photosynthesis are similar to the kinds of reactions characteristic of plant photosynthesis.

5.41 Pigmentation Chloroplasts are present in all algal cells. The colors of different kinds of algae re- Pigments present in the chloroplasts of algaesult from the ratio of various chlorophylls include chlorophyll a, b, c, and d. Otherand other pigments present in their chloro- kinds of pigments vary considerably amongplasts. species of different groups. Bodies of water (ponds, lakes, streams, etc.) often appear greenish or red due to the abundant growth of algae. Such accumulations are called algal blooms.

5.42 Cell Structures These organisms have cell walls and var- Photoreceptors may be located in the ious discrete membrane-bound organellesmembranes of chloroplasts. typical of eucaryotic cells. Chloroplasts in algal species may differ in shape, size, and the number present in cells. Chloroplasts are self-replicating organelles. They contain their own DNA, which differs from that found in the cell's nucleus. Cell walls of algae are composed of plant- like constituents, but certain kinds of carbohydrates are unique to particular groups. Rigid cell walls are conspicuously absent in organisms classified as euglenoids. Certain algal cell walls contain silica and polysaccharides (agar, alginates, and carra- geenan).

5.5 Viruses All viruses are acellular, submicroscopic, Viruses do not contain metabolic enzymes obligate, intracellular parasites. and ribosomes with which to carry out the process of protein synthesis.

5.51 Nucleic Acid Compo- Viruses contain either ribonucleic acid Viruses are too small to be observed by nents (RNA) or DNA, but never both. light microscopy. (1 0 4 Introductory Microbiology 27 TOPICS AND SUBTOPICS . A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES Spector, D. H., and D. Baltimore. 1975. Viruses in an extracellular state (virions) The molecular biology of polio-viruses. Sci.are infectious, but incapable of self-replica- Am. 232:24-31. tion. flr The nucleic acid in the core of both DNA and RNA viruses may be either single- or do"uble-stranded. Fiddes, J. C. 1977. The nucleotide sequence of a viral DNA. Sci. Am. 237:54-67. The nucleic acid core of viruses is surrounded by a protein coat or other covering.

6.0 Bacterial Structure and Function 6.1 Cell Shape Bacterial species exhibit one of three gen- Some bacteria do not exhibit one of the Stained preparations of the three general eral forms: spherical (coccus), cylindrical three general forms, e.g., some bacteria are bacterial shapes (coccus, rod, spiral) are eas-

(rod), or spiral. stalked, some are branching, some are pleo- , ily observable with the ordinary microscope. morphic, and some exhibit characteristic extension of cell parts (prosthecate). If the cell wall of cocci, rods, or spirals is removed, they all tend to assume a spherical shape, e.g., spheroplast or protoplast.

6.11 Cellular Arrangements Depending on the species, bacteria exhibit Some bacteria may display pleomorphism Prepared microscope slides of cocci may a variety of arrangements of these shapes. (irregular shapes). be used to show morphological arrange- Some gram-positive and gram-negative ments in pairs (diplococci), chains (strepto- bacteria may lack cell walls when grown in cocci), clusters (staphylococci), fours (tet- media containing an antibiotic such 1113,pen. rads), or cuboidal arrangements of eight icillin, which blocks cell wall synthesis. (sarcinae). cells usually display spherical shapes in high-salt environments and lyse in a low-salt environment, and some may not revert to walled organisms. When such wall-less organisms occur in nature, they are referred to as L-forms. Members of the family Mycoplasmata- ceae have no cell walls. Spiral-shaped bacteria such as vibrios may appear in the form of short, incomplete spirals. Others may appear as a thick, rigid spiral (spirillum) or a thin flexible spiral (spirochete).

Introductory Microbiology 29 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 6.2 SurfaceAssociated everal microbrgimisms have a variety of Structures s ace-associated 'structures. These include capsules, slimes, flagella, and pill.

6.21 Capsules Some bacteria are surrounded by a vis- Capsules are structures with a definite Staining techniques may be used to dem- cous layer termed a capsule or a slime layerspherical shape surrounding the cell. Theonstrate bacterial capsules and/oi slimes. which may serve a variety of functions. slime layer has no definite shape. Most capsules and slime layers are composed of complex polysaccharides. Some are composed of polymers of glutamic acid, e.g., .02 Bacillus anthracis. Capsules may serve as extracellularly stored reserve food, may protect cells from dehydration and phagocytosis, are frequently related to virulence, and can be used in vaccines; some capsules are in commercially important products, e.g., the blood substitute dextran. ran The synthesis of capsules is greatly influenced by the bacterium's environment.

6.22 Flagella Some bacteria produce hair-like append- Flagella originate in the cell membrane ages dalled flagella which serve as organellesand are not visible with an ordinary micro- selocomotion (Microbial Physiology, Sub-scope without the aid of special staining topic .6.27), procedUres. Flagella may be found hi various arrangements. This property is used in bacterial classification. Bacteria displaying a single flagellum at one end of the cell are referred to as polai monotrichousr6 there are more than one at one end, itpolarly lophotri- chous; if there is one or more flagella at each end of the cell, it is amphitrichous; if flagella are ingited all around the cell, the arrangement is referred to as peritrichous. A few motile bacteria- \lack flagella and move ;nstead by a gliding or flexing motion. Motile bacteria represent the simplest ex- 0' ample of microbial behavior in nature in that they are capable of taxis (a motile response to an environmental stimulus) (Mi- crobial Physiology, Subtopic 6.223). Flagella are composed of the protein fla- gellin. They appear as semirigid, noncon- 0.0 Introductory Microbiology 31 (1 G TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES tractile, hollow fibrils and consist of a ho stricture and a long filament. The ability to produce flagella is genetically determined. Flagella remain attached to the cell if the cell wall is removed.

6.23 Pi li Certain bacteria possess filamentous sub- Pili have na. specific arrangement. Thek Electron micrographs may be used to microscopic appendages knowsas pill,generally protrude all around the cell, origi-show that not all bacteria. possess pill, pill which have several functions. nating from the cell membrane, and are com-are thinner and more numerous than fla- posed of the protein called pilin., gella, and piliated organisms tend to attach One kind of pilus, the sex pilus, serves'in to other objects such as inert surfaces, living the c-ransfer of genetic material during con-cell surfaces, and othbacteria. .jugaiion (bacterial mating). It has been sec- ulated that pilation is related to virulence, since pilated organisms are known to attach more efficiently to manunalian cell surfaces.

6.3 Cell Wall Most bacteria have rigid cell walls with a Bacterial cell walls are made of complex unique chemical composition. This chemical polymeric substances known as peptidogly- Qr composition is frequently used in the classi-cans, which are responsible for their rigid fication of bacteria. nature. The bacterial cell wall contains many different amino acids, amino sugars, carbohydrates, and lipids.Diaminopimelic aci (DAPA), muramic acid, and teichoic acid are unique to the bacterial cell wall. Pepti- doglycans are very large polymers composed of three kinds of building blocks, acetylglu- cosamine(AGA),acetylinuramicacid (AMA), and a peptide consisting of four or five amino acids of a limited variety. Amino acids are of the D configuration, which is not usually found elsewhere in nature. Gram-positive bacteria have a cell wall composed almost entirely of a thick layer of peptidoglycan to prevent osmotic lysis. Tei- choic acids are also components of such cell walls. Gram-negative bacteriaave a niultilay- ered cell wall consisting of ETthin inner layer of peptidoglycan to prevent osmotic lysis surrounded by an outer envelope of lipopolysaccharide and protein (which may help to retain certain essential enzymes and pre- Introductory Microbiology 33 S TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES vent some toxic substances from entering the bacterium). Gram-negaiive bacterial walls usually lack teichoic acid. The lipid component of the outer membrane of the gram-negative cell wall may also play a role in bacterial padrogenicity by acting as an endotmuisi.

6.4 Internal Structure Internally, microorganisms may contain several structures including the cytoplasmic membrane, mesosomes, nuclear region, and ribosomes.

6.41 Cytoplasmic Membrane Immedidgly beneath the cell wall is a The cytoplasmic membraneissemi- Electron micrographs may be used to thin semipermeable membrane,, the cyto-permeable, compo4ed of three layers, and show the cytoplasmic membrane. plasmic membrane, that regulates the pas-houses a large number of enzymes including sage of materials into and out of the cellcytochromes and permeases. The' bacterial (Microbial Physiology, Topic 6). cytoplasmic membrane, composed of pro, tains and lipids, is a typical biological membrane similar to those found in other cells. A cell may be killed by damage to the membrane from harsh treatment with physical or chemical agents. 1 The cytoplasmic membrane invaginates to form various structures, i.e., mesosomes, photosynthetic lamellae, and vesicles, and frequently forms expanded tubules.

6.42 Mesosomes Mesosomes are internal extensions of the The mesosome system is intricately as- Electro aphs may be d to cytoplasmic membrane which are associatedsociated with bacterial nuclear material and show that be lamellar mem-

with a variety of cellular processes. 1, its replication. Mesbsomes are also involved branes, expa Vii. or vesicles. with the beginning of cross wall formation.

6.43 Nuclear Region and Ri- The cytoplasm is cpntained within the The 'CA Oplasmic area is granular in ap- Electron micrographs can be used to show bosomes cytoplasmic membrane. Components found pearance and rich in RNA. The nuclear areathat the cytoplasm contains all cellular in the cytoplasm include the nuclear region, (nucleoid) is rich in 15NA. The fluid containsstructures and materials, including the riu- which contains DNA or the cell's geneticdissolved nutrients. cleoid. material, and ribosomes, which are involved The nuclear region is not surrounded by with protein synthesis. a nuclear membrane and consists simply of a single, long, circular molecule of DNA (which codes for protein synthesis). High magnifiCation with the electron microscope may reveal the nuclear substance to be a Introductory Microbiology 35 50 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION csB. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES delicate fibrillar structure (bacterial chro- ome). 'RNAin combination with protein forms submicroscopic bodies, ribosomes, which are .6ivally de;ii.y packed througliout the cy- llismic a. Aggregates of ribosomes are knownas- polysomes and are intimately associated with rirotein synthesis. Other inclusions are photosynthetic la- a mellae, vesicles, lipids, PHB granules (poly- B-hydroxybutyrate), glycogen, and meta- chromatic granules (reserve food material).

6.5 Endospores Certain gram-positive bacteria produce Endospores are surrounded by many Laboratory experiments may be used to highly resistant and refractile thick-walledlaters of protective material, e.g., exospo-show that endospores are resistant to a va- oval bodies termed endospores. rium, ore Coat, spore cortex, etc. Theseriety of chemical and physical treatments. protecti e layers are apparently responsible for res. tant to chemical and physical treat- f ment. Endospores may germinate to become vegetative cells under suitable conditions. Heating endospores to near the boiling temperature for a few minutes may enhance germination (heat shock).

6:51 Heat Resistance The endospore wall contains a unique chemical known as dipicolinic acid (DPA) which is lacking in vegetative cells. A large amount of calcium is also present and is believed to form a calcium DPA-peptidoglycan complex.

6.52 Sporeforming Orga- Common sporeforming organisms include Endospores of Clostridium species caus- nisms Bacillus (aerobic), Clostridium, (anaero-ing tetanus and gas gangrene may germinate . bic), and various genera of the cyanobac-in deep puncture wounds (anaerobic condi- teria. tions) leading to disease produttion. The resulting vegetative bacteria produce the specific exotoxins responsible for the disease state.

7.0 Viruses 7.1 Characteristics Viruses are submicroscopic (may not usu- The smallest virus which has been ob- Electron micrographs may be used to ally be seen with the light microscope). served with the electron microscope is aboutshow the submicroscopic, intracellular na- S Introductory Microbiology 37 5 I TICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 0.01 inn in diameter (foot-and-mouth diseaseture of viruses. The filterability of viruses virus). can be demonstrated with the use of special Larger ones include vaccinia (cowpox)filters. and variola (smallpox) (0.2 to 0.3 nm). Host cell nucleic acids and organelles are directed by the viral genome to synthesize new viral components. Such activity eventually may resultin,the death of the host cell. Depending on the. virus and host cell system, virus particle release may be abrupt or gradual.

7.11 The Virion The mature transmissible virus particle is Bacterial virions may be divided into two known as the virion. groups, lytic virions and temperate virions. Lyric bacterial virions generally destroy the host cell after the Icompletion of the replication of new virions. Temperate bacterial viruses, on the otter hand, do not replicate themselves but integrate into the host cell genome and remain integrated for an indef- inite time.

7.2 Viiion Structure Mature transmissible virus particles or Most viruses have capsids. Proteins of the Electron micrographs may be used to virions consist of a protein toat, called acapsid confer specificity to the virus. show that the capsid is made up of protein capsid, which surrounds the nucleic acid of Some viruses, such as bacterial viruses,subunits called capsomers and that certain the particle. shed the capsid outside of the cell after hostvirions, such as the herpesviruses, also may penetration, whereds in other viruses thebe covered by an additional component, the uncoating occurs within the host celL envelope or peplos, which may he derived from host cell membranes.

7.21 Nucleocapsid The structure composed of nucleic acid General shape& of nucleocapsids include Electron micrographs ay be used to surrounded by the capsid is the nucleocap-helical, polyhedral; filamentous, or binal,show the variety of shapes, sizes, and orga- aid. which is a combination of helical and poly-nization of 'nucleocapsids. hedral shapes. Certain nucleocapsids are quite complex. \Some bacterialviruses,the bacterio- phages, are composed of head, neck, collar, sheath, base plate spikes, core, tail fibers, and pins.

7.22 Nucleic Acid Content Individual virus particles contain DNA or The structure of nucleic acid in different RNA but never both. The nucleic acid com-viruses may be either linear or circular. ponent of virus particles is used as one cri- Viruses are classified on the basis of terion for classification. whether they have double-stranded or single-stranded DNA or RNA. Introductory Microbiology-39 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 7.23 Envelope Some viruses have an irregular outer Many mammajian viruses have an enve- Electron micrographs may be used to lipid-containing envelope, which encloseslope (mantle). Such viruses are referred toshow the properties of envelopes such as the viral capsid if present and the viral nu-as enveloped or mantle viruses. Envelopesattachment spikes or projections and indi- cleic acid core. The presence of an envelopeappear to be derived from the host cell cy-vidual units known as peplomers. is one of several criteria used. for classifica- toplasmic membrane or nuclear membrane tion. (depending on kind of virus and cell). Membranes contain lipids or lipoproteins 41' and are composed of structural units sometimes called peplomers. Such viruses are sensitive to lipid solvents and are sometimes referred to as lipoviruses.

7.3 Viral Replication All viruses are obligate intracellular par- All viruses have a certain host range (host Laboratory experiments and electron mi- asites. specificity). crographs may be used to show viral repli- Some viruses can penetrate and replicatecation, e.g., plaque formation and pock for- in one type or kind of cell. Such viruses are mation in the chicken embryo. referred to as monovalent viruses. If viruses are capable of infecting and replicating in two different types of cells, they are known as divalent, and if they penetrate and replicate in many kinds and types of cells, they are referred to as poly- valent viruses.

7.31 Effects of Viral Replica- Certain viruses gaining entrance into sus- Cytopathic effects range from no observ- Laboratory experiments and micrographs tion : ceptible cells may bring about the formationable physical change to complete cellularmay be used to show the range of destiuctive (replication) of new virus particles, whichdestruction. cellular effects caused by viruses, which are may or may not be accompanied by imme- Virus replication differs with the type ofcalled cytopathic effects (CPE). diate cell death. -; virus and type of host cell involved. For example, one form of animal virus replication may start with adsorption, penetration, particle uncoating before penetration into the nucleus, DNA incorporation and transcription, RNA transport, synthesis of structural protein (capsid) and nonstructural protein (DNA synthetase), transport of structural protein and DNA synthetase into the nucleus, DNA synthesis, virus assembly inside the nucleus, envelope attachment as virus particles leave the nucleus (the nuclear membrane may become the envelope), and release. With certain viruses, cell lysis results in the concomitant release of new viral parti- 5 Introductory Microbiology 41 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B, ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES cies. With other viruses, viral particle Mat- uration and release are relatively slow. 7.32 , Provirus Viral DNA within the cytoplasm or in the A provirus remains as part of the host nucleus may integrate into the host DNADNA replicon indefinitely, or it may be and become a part of host DNA. Such aspontaneously or intentionally induced to segment of DNA is referred to as a provirus. enter the lytic cycle. New .virions derived The presence of a provirus may change thefrom such a cell can invade a new host cell host cell characteristics. which does not contain the same virus (host cell immune response). However, a host cell containing another type of provirus may be infected, establishing double lysogeny. Invaded host cells may display cytopathic effects (CPE) and may produce chemical compounds which interfere with viral replication (interferon). Various physical and chemical agents may induce viral replication and consequent CPE. The exposure of seemingly uninfected cells to irradiation may bring about the activation of previously undetected viruses.

8.0 Fungi Laboratory cultures and experiments may 8.1 Characteristics Fungi include molds and yeasts. Some molds produce useful ,antibiotics; others are parasitic and pathogenic. be used to show that molds are filamentous Certain molds under various environmen- or thread-like, achlorophyllousmycelium- tal conditions exhibit different characteristicproducing microorganisms and that repro- shapes (dimorphism). duction can be asexual and/or sexual. Yeasts Most yeasts can grow aerobically,.or an-are primarily unicellular and generally re- aerobically, or both. produce asexually by budding. There are Hartwell, L. H. 1974. Saccharomycesfilamentous yeasts. cerevisiae cell cycle. Bacteriol. Rev. 38:164- 198. Certain yeasts may be pathogenic. Yeasts are used industrially for several processes including the manufacture of oils and fats, feed supplements, and alcohol and vitamin production.

8.11 Multiple Forms The fungi include unicellular and multi- Several fungi produce laige fruitingstruc- cellular forms, tures, e.g., mushrooms, bracket fungi, etc. Some fruiting structures are as large as 50 cm in diameter (puffballs), and are made of a mass of mycelia. Introductory Microbiology 43 f

TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES . Some fungi are edible, whereas others are a:A otremely poisonous.

§4 Hyphae TfuK structural units of most mature Most members of the class Phycomycetes Microscopic examination of laboratory molds are the hyphae which may or may(bread molds, water molds, etc.) lack crosscultures may be used to demonstrate the not have cross walls (septated or nonsep- walls. pres6nce or absence of septa. tated). Filamentous fungi in the classes Asco- mycetes and asidiomycetes are septate. The members of these classes include some unicellular forms (yeasts) as well as molds.

8.2 Reproduction All fungi except those of the class Deuter- Asexual reproduction may involve binary Asexual and sexual reproduction may be omycetes (Fungi Imperfecti) can multiplyfission, budding, spore forMation, and thedemonstrated by growing a "+" and a "" both asexually and sexually. Members offragmentation of vegetative hyphae. strain of Rhizopus on the same plate. this group lack sexual fruiting bodies. The Yeast form buds on the 'surface of the reproductive pattern. of the fungi form themain or mother cell. Upon maturity buds basis of their classification. are called daughter cells and have genetic constituents identical to the mother cell. Buds break off from the mother cell at maturity.Undersuitableconditions,the mother cell can reproduce as many as 18 daughter cells by budding. In asexual reproduction spores 'may be zoospores, which become vegetative cells. Many fungi produce nonmotile vegetative spores. Spores may be borne on the tip of modified hyphae (sterigma) or enclosed in a sac (sporangium). Fungi can carry out meiois (chromosome reduction division, 2N 1N). In some groups sexual reproduction occurs by the fusion of fertile hyphae from + andstrains of the organitims. Zygospores (2N) or zygotes (2N) result. Some sexual reproduction occurs by the fusion of two isogametes (gamete morphology is indistinguishable, e.g., slime mold) or by heterogametes (zoospore and egg). Sexual spores may be enclosed in a structure. The type of enclosure is used in the differentiation of fungi. f 8.3 Nutrition and Environ- The fun': heterotrophic. Fungi are extremely versatile organisms ments and are able to decompose a variety of coin- Introductory Microbiology 45

5J A': 60 op

C, PRACTICAL ACTIVITIES TOPICS ANDSCIITOIlfCS A. ESSENTIAL INFORMATION 13, ENRICHMENT INFO IMATION plsx materials, thereby atoning as indis- pensable decomposers in our ecosystems. 6.1 Bauehop, T. 1970. Rumen anaerobic fungi. Appl, Environ, Microbiol. 38:148-158, Laboratory experiments may bo used to 8,31 Growth EiwirdnMents Most fungi are aerobic, osmophilio, and Fangi use molecular oxygen as a final acidophilio. electran acceptor in their respiration. show that many fungi can tolerate or prefer Funiii are important In' the food, industrya high-sugar or high-salt environment. because of spoilage effects with syrup, mo- lasses (high-sugar products), sauerkraut, or- anges, and lemons. Oungi rarely spoil canned goods because r of the anaerobic nature of such processed foods, but they are capable of forming toxic products in stored'grains. Maggon, K. K., S. K. Gupta; and T. A. Venkitaaubramatdan. 1977. Biosynthesis of aflatoxins. Bacteriol. Rev. 41:822-855.

9.0 Algae Laboratory experiments may be used to 9.1 Characteristics The algae are photosynthetic unicellular All algae contain discrete cbroplasts or multicellular organisms. Some are fila--w can contain chlorophyll a and somedemonstrate algal motility. mentous or appear in colonial arrangements. of er chlorophylls (b, or p,. pfd, etc.). Pigments and morphology are used in algal Many algae possess accessory pigments, classification. - e.carotenoid (yellow-orange), fucoxanthin (brown), and phycoerythrin (red). Multicellular algae exhibit a variety of Algae share many properties with green Examination of field or preserved speci- shapes, ranging from single filaments to di-plants; these include cell walls containingmens may be used to show that many algae, chotomously branching large structures. cellulose, chlorophylls a and b, and otherespecially certain marine species, are not pigments confined to chloroplasts and theonly multicellular but morphologically dif- production of starch in photosynthesis. ferentiated into holdfasts, stipes, and cysts. Examinationof prepared microscope 9.2 Reproduction Algae reproduce by"both asexual andliac- Unicellular algae can reproduce asexually ual means. by fission. fides may be used to show that most uni- Multicellular algae may reproduce asex-c llular algae reproduce,by transverse bi- ually by zoospores or fragmentation. Sexual fission; some reproduce by longitudinal reproduction may be by fusion of isoga- fission. metes. Multicellular algae may be hetero- gamous (eggs and sperm). Certain green algae (Chlamydomonas)k can reproduce by fusion of two zoospores to produce a 2N zygote. Some multicellular algae, e.g., aggregates A of similar cells, can reproduce sexually by Introductory Microbiology 47 IIP

TOPICS ANI) A It114814141AL INFOIIMAtION II. IdNll!(11IMI(N'1` INFORMNIION (1, I. VITIKH corthigation. Zygospores are produced upon fusion of two gametes In recipient tiont algae exhibit alternation of genera- In most algae, the completions (visible) lions in their life cycle. Such algae exist Ingeneration is the vegetative Amami) ( both haploid (IN) and diploid (2N) status. Some zygotes may &ya thick walls (cysts) to become metabolically dormant cells. Cysts may germinate when the environment Waimea favorable.

9.3 PhYiklology and Cultiva- Algae are photoautotrophs (Microbial Photosynthesis requires light as an energy tlon Physiology, Subtopics 5.4 and 5.51). source, chlorophylls, water, and carbon dioxide. The end products formed are sugar and free oxygen. Sugar may be polymerized and stored as polysaccharides, e.g., starch. Some algae may store lipids. Chloroplasts, like mitochohdria, are capable of self-replication because they contain functional DNA and RNA. Some algae possess structures known as pyrenoida which may be involved in polymerization of glucose to starch. Most media for algal cultivation are com- Laboratory experiments may be used to posed of inorganic compounds containingshow that algae are phototrophic an most bulk elements (C, N, P, S) and some trace do not' require organic compounds iniedia. elements including Fe, Mg, Zn, Cl, K, Mo, and 'B in addition to water. Algae can be grown on solid or liquid media. 10.0 Protozoa 10.1 Characteristics The protozoans are unicellular, nonpho- The cytoplasm of most unicellular proto- Examination of living and/or preserved tosynthetic eucaryotes. zoans is divided into two regions: the innerspecimens may be used to show that all fluid zone (endoplasm) and the outer or protozoans lack rigid cell walls, some contain peripheral zone (ectoplasm). pellicles or periplasts equivalent to a cell Some protozoanslayeim external cover-wall, and others have a cell membraneas ing known as a pellicle which may be com- the outermost structure. posed of lipoprotein.

10.11 Means of Motility Protozoans use a variety of means for Most protozoans are motile during at least which serve as a basis for theirone stage in their life cycles. classification. The protozoans can be sepa- Some protozoans move by means of flag- rated on the basis of motility into fourellar motion, whereas others move by means groups: Sarcodina (pseudopodia), Ciliate of the sweeping motion of cilia. Both flagella (cilia), Mastigophora (flagella), and Sporo-and cilia consist of complex inner fibrillar zoa (some movement by pseudopodia onlystructures not seen in procaryotic flagella. Introductory Microbiology-49 C .1 TOPI Cti A N till LIT 0111011 A, 10110INTIACINFORMATION 0, INFORMATION , PR ACTICAL ACTIVITIEH In immature titagoti, mule gametes Are flag, Flagella and villaappear to originate in basal ullatud), grannies. hi the ectoplasm,

10,2 Iteproduct iota PrtOZO114 reproduce by both gamma Protozoans van multiply asexually by bi- Pexamination of living or 11111111111110r0, and sexual t toa, Thu mothoila of re re- nary fission, Budding may produce individ-scope slides may be used to show itsexaahl (Notion and associated structures are el a- male oxogenounly or endogenously, Multiple reprodmetion, which includes binary fission, sification criteria, fission ()coma from the formation of multi- multiple fission, budding, and plaamotoy. nucleate organisms, Hubsuquaint cleavage re- Sexual reproduction can occur through eon- sults in the formation of annoy. uninuelonto jugatinfi, my. miaow, and irtao

Clenutie recombination can result from the modal union of two cells (conjugation). 10,21 Macro- and Microaclei , Some protozoans are multinucleated. Ciliates contain both macro, and micro. Examination of living cultures, prepared nucluL cronuclei play a-role in minim-microscope slides, or electron micrographs list and developiputa. Those colts with onlymay be used to show that ciliates have at maconuclel are unable to conjugate, Cellsleast one macrontwleus. with micronuclei can conjugate. Diploid micronuclei undergo meiosis and mitosis, resulting in two haploid nuclei (conjugal pair). One of these is exchanged daring conjugation, forming a hew diploid nucleus, which undergoes a complex series of nuclear divisions. 10.3 Life Cycles Protozoans can be free-living or parasitic. With a few exceptions, most members 'of -Sarcodina, Ciliata, and Mastigophora are free-living, saprophytic scavengers in their respective habitats. All members of thei)o- rozoa are parasitic. Many protozoans have stages, namely trophozoite (active feeding stage) and cyst. Cysts are surrounded by- thick walls and are dormant. Encystment occurs in an unfavorable environment and ,excystment occurs when the environment becomes favorable. Many protozoan parasites have t different types ofsts: definitive, or fial, and intermediate. e latter provides t environment for the immature or larval stage. Some protozoans produce serious and dis- abling diseases (malaria, African sleeping sickness, amoebic dysentery, vaginitis, gas- Introductory Microbiology-51 TOl'ICS AND SUBTOPICS / A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES tro-intestinal disturbances, and en- cephalomeningitis [Naegleria infection]). Hawking, F. 1970. The clock of the ma- larial parasite. Sci. Am. 222:123-131.

10.4 Nutrition and Cultiva- All protozoans are heterotrophs. Some In some species (ciliates), food particles tion ingest particulate material (holozoic); othersmay pass through the peristome(oral absorb dissolved nutrients. groove) to the cytOstome (mouth) and then into the cytopharynx (gullet). Ingested solid food particles are enclosed in food vacuoles. L,11,e:vlot; Wastes are eliminated via the cytopyge (anal ()titer protozoa ingest food by phagocytosis or pinocytosis.

10.41 Techniques A variety of techniques and media are Nutrients used are similar to those for utilized for protozoan cultivation. cultpring of procaryotes. d, Dyer, C. L., and G. L. Butler. 1980. A Sources for organic particVs can be living culture method for freshwater microinver-(algae, bacteria, and other protozoans) or tebrates. Am. Biol. Teach. 42:52. non-living (crushed or ground matter such as rice, wheat, etc.). 11.0 Microbial Ecology

11.1 , Concepts of Ecology Microorganisms are ubiquitous in nature . Subdivisions of habitats are called mi- Laboratory culture of environmental sam- and are often associated with the other livingttoenvironments. Microenvironments serve pies may be used to show the major habitats forms in their natural habitats. The majoras integral parts of all habitats in ecosys-of microorganisms within the biosphere. natural habitats within the biosphere are tems. aquatic, terrestrial, and biological (in or on Imshenetsky, A. A., S. V. Lysenko, living systems) (Introductory Microbiology, and G. A. Kazakoy, 1978. Upper boundary Topic 25) of the biosphere. Appl. Environ. Microbiol. 35:1-5. Within microenvironments, microorganisms occupy specific niches. The niche of an organism is determined by genetic and physiological adaptation of the organism to either fixed or variable environments. Examples of adaptations to microenvironments include organisms that are halophilic (well adapted to a high-salt environment) and osmophilic (adapted to a high-sugar environment). Some organisms are psychrophiles (adapted to cold temperatures), some are mesophiles (adapted to moderate tempere- Introductor,/ Microbiology - TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES

° tures), and others may be thermophiles (adapted to high temperatures). Certain organisms preferentially thrive in acidic environments, whereas some thrive in neutral environments and others thrive in alkaline environments.

11.11 Components of Ecosys- Microorganisms comprise an integral part Biotic components in ecosystems are di- terns of the biotic community, which togetherverse and range from the simplest (cells) to with the abiotic (nonliving) componentsthe most complex (community). form ecosystems. Microorganisms that serve as primary producers (blue-green bacteria) represent the first step' in the pyramid of food levels supporting all other living things. Microorganisms function as important decomposers. Abiotic components in ecosystems tend to function as selective factors by making ecosystems conducive for certain kinds of organisms and undesirable for others. The acid pH of fruits in, the presence of air tends to be highly selective for Acetobac- ter species. The temperature of water in hot springs in Yellowstone National Park is highly ,elective for thermophilic bacteria, e.g., Thermus aquaticus.

11.2 Symbiotic Relationships Symbiosis refers to the living together or Many fungi are ectosymbionts of plants. Laboratory examination of field samples to the association of two dissimilar orga-Although they grow around the root systemsuch as lichens and leguminous plants may nisms with a' certain degree of constancy.with little penetration into the host tissue,be used to show symbiotic associations in- Symbiosis may be divided into two generalthe association is beneficial to both fungi volving microorganisms. types: ectosymbiosis, in which one organismand plants, i.e., increased mineral uptake by lives on the external surface of another or- plants (mycorrhizal association). ganism, and endosymbiosis, in which one Certain bacteria, e.g., members of the ge- organism lives inside the cells, tissues, ornus Rhizobium, establish endosymbiosis organs of another form of life. with leguminous. plants. The bacteria infect the roots of legumes and establish themselves intracellulaEly in the plant tissue, producing root nodules. The bacteria are capable of fixing gaseoup nitrogen, and the fixed nitrogen is provided to the plant. The association is beneficial to both plant and bacteria (mutualism). Endosymbionts (bacteria or algae) of cer- Introductory Microbiology 55 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION 13. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES

tain Paramecium species are examples of A symbiotic associations between two dissimilar kinds of microorganisms.

1.4 11.21 Patterns of Association Symbiotic relationships among living or- Endosymbionts (microorganisms)that ganisms can be categorized into three pat-liy'e in the stomach of ruminants represent terns of association. If an association of two examples of a mutalistic association between or more dissimilar organisms or populationsunicellular organisms and higher animals. is beneficial td both, it is termed mutualism. Lichens represent a unique biological en- If the association is beneficial to one without tity that is formed from the mutualistic as- effecting the other, it is termed commensal- sociation between an alga and a fungus. ism. If the association is harmful to one and beneficial to the other, the associatior is called parasitism. Among the various kinds of symbiotic Commensalism may he ectocommerisal- associations that are found in nature, some ism (attachment of organisms to the host do not require cell-to-cell contact between body)or endocommensalism (organisms partners. which live in the lumen of the alimentary tract).

11.22 Facultat lye Associations A particular kind of symbiotic association Endomycorrhizae of orchids represent a may be facultative (nonobligatory I or oblig- highly specific symbiotic association be- atory. tween certain kinds of fungi and a flowering plant. Here the relationship is obligatory for the orchid and facultative for the fungi. Bac- teria that are classified in certain genera (Caedobacter, Lyticum, and Tectobacter) are obligate endosymbionts of Paramecium aurelia. Preer, J. R., Jr., L. B. Preer, and A. Jurand. 1974. Kappa and other endosymbionts in Paramecium aurelia. Bacteriol. Rev. 38:113-163.

11.3 Microorganisms and Biogeochemical cycles are those processes Molecular nitrogen (N2) represents ap- Biogeochemical Cycles through which chemical elements are trans- proximately 79% of atmospheric gases. The formed within ecosystems, often through the element nitrogen is an essential constituent metabolic activities of microorganisms. for living cells (nitrogen cycle); microorganisms are responsible for all biological nitrogen fixation. Some microbes are capable of utilizing molecular nitrogen as a sole source of nitrogen (nitrogen fixers), whereas others are capable of oxidizing ammonia to nitrite or nitrate (nitrifiers) and others are able to Introductory Microbiol,gy 57 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES reduce, nitrate to nitrogen (denitrifiers). Ni- t° trosomonas species utilize ammonia as an energy source, and release nitrite as an end product. Nitrobacter species utilize nitrite as an energy source and release nitrate as an end product (Fig. 1). Brill, W. J. 1979. Nitrogen-fixation basic to applied. Am. Sci. 67:458-466. Carbon is present in rocks and fossil material and is also an essential constituent of all living cells (carbon cycle) (Fig. 2). In environments where elemental sulfur is abundant and the pH is between 2 and 4, Thiobacillus thiooxidans oxidizes elemental sulfur. Sulfur may exist in various sulfates which may be reduced to H2S by certain anaerobic bacteria. H2S in turn may be oxidized to S or sulfate by lithotrophic bacteria (Fig. 3).

11.31 Types of Biogeochemi- Geochemical cycles include carbon, nitro- cal Cycles ggn, phosphorous, and sulfur cycles. Certain microorganisms are highly specific in terms of a specific chemical structure (substrate) that they can metabolize. Chemical elements released to the environment as a result of microbial metabolism may selectively function as enrichment or inhibitory agents for other microorganisms. Jannasch, H. W. 1979. Microbial turnover of organic matter in the deep sea. BioScience 29:228-232.

12.0 Biological Molecules 12.1 Proteins Proteins are very large complex organic There are about 20 different amino acids. compounds composed of numerous smallHundreds or thousands of amino acid mol- molecules called amino acids. They are im-ecules combine to make proteins. portant as enzymes and as structural elements of cells.

12.2 Carbohydrates Carbohydrates contain only carbon, hy- (Microbial Physiology, Subtopics1.11, drogen, and oxygen. They may be small 1.21 and 1.33.) molecules (monosaccharides) used as struc- In troductory Microbiology 59 rM N3inthe a atmaipherb Plant \protein

. Carnivore ...".1P"protein Electrochemical Herbivore , fixation protein

- N2 fixing

bacteria ? N2-fixing Assimilationof Decay and NO2 by plants Denitrifying Fish excretion bacteria Plants t INO3.NO Microorganisms Nitrate Loss to (NO, I deep sediment .------"" f s Decay and Nitrate bacteria I excretion Deposit in (NO2 ---. NO3 ) sediments f Nitrite bacteria INH.1-- NO2) Events in shallow soil

Ammonifying bacteria INH2 P NH3)

iAmino acids, urea, uric acid, organic residues

FigIThe general elements of the nitrogen cycle.

taJ R2 .11

CO, Combustion of fossil fuels

Volcanoes

Industry and agriculture

Aquatic Phytoplankton photosynthesis) Environments Death, decay

Bicarbonates CO2 in H2O Carbonificatton Respiration / CHO Animals.V Plants tJ Microorganisms Death, decay ti

Carbonates

Fig. 2.The general elements of the carbon cycle.

76 Introductory Microbiology-63 Organic compounds (protein)

plant. bacterial Microbial assimilation decomposition

Sulfatereducuun

SO1 nesuifocihrto I-1,S sulfate Hy-drogen

Microbial oxidation

Microbial Microbial oxidation oxidation

Sulfur Autotrophic bacteria l'hotosyndietic bacteria

I Thtobatllus

Fig. 3.The sulfur cycle

Introductory Microbiology 65 73 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES tural elements of the cell or as a stored energy source. i2.3 Lipids (Fats and Related Lipids are composed of carbon, hydrogen, (Microbial Physiology, Subtopics 1.13, Compounds) and oxygen, but also may contain other ele- 1.24, 1.31, and .1.34.) ments such as phosphorus and nitrogen. They are insoluble in water and are important in intermediary metabolism and in structural elements such as the cell membrane.

12.4 Nucleic Acids Nucleic acids play major roles in the (MicrobialPhysioligy,Subtopics 1.14 and transmission of hereditary traits and , the1.23 and Microbial Genetics, Subtopics 2.1 control of cell functions and protein synthe- and 2.2.) sis.

12.5 Cellular Composition, Microbial cells are composed of proteins, Composition of bacterial cells will vary Wet weights and dry weights of a mass' of carbohydrates, nucleic acids, lipids, phos-depending upon the kind of bacteria and the concentrated bacterial cells can be deter- phates, and other materials. From 75 to 90% Qowth medium. General approximations ofmined to show the water content of the cell. of the weight of the microbial cell ik35,iater. ccinposition are as follows. Component % Dry wt

Water 75-90 Protein 35-50 Carbohydrate 2-25 Nucleic acids RNA 6-25 DNA 1-4 Peptidoglycan Gram-positive cells 15-20 Gram-negative cells 0.1-55 Lipopolysaccharide Gram-negative cells 5 7 Inorganics (phosphates) 0.9

13.0 Microbial Growth Growth is the orderly increase in cellular In organisms that r u by binary constituents. Normal growth leads to cell fission, growth produces an incre in num- reproduction. Growth of microorganismsbers. Coenocytic organisms increasenly in leads to an increase in population. individual cell size rather that) in ce umbers. Single cells may continue to reprodu e and form visible accumulations such as a colony (bacteria) and mycelium (fungus). Introductory Microbiology 67 SO o it- TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION. B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 13:1 Measurement of Growth It is possible to measure various aspects of microbial growth. One chooses the grov413 measurement technique needed depending on whether the emphasis is on total ,numbers, viable organisms, or metabolic-rate of the population. Usually several techniques are used, and the results are compared.

A sample of washed cells may be dried in 13.11 Increase in' Mass Measurements of increase in cell mass Nephelometry and spectrophotometry are emphasize growth rather than reproduction. the most convenient and generally usedan oven, and the dry weight can bertletec- They may not distinguish living from dead growth measuring techniques for bacteria. mined. _ The increase in turbidity of the cul measured. _.. Spectrophotometers measure the propor- , A sample of washedcells maytettn: alyzed to determint'qhe totld nitrogen, oinotal pro- tion of light which is transmitted through a _.ic filled cuvefte placed in t1ie light' beam. tein. Nephelometers (more 'sensitive) measura." ,,,Cellsin suspension scatter light;the the proportion ot light deflected .under theamount of light scattered is proportional to same conditions." the mass of cells per milliliter of fluid. A spectrophotometer or nephelometer can be t. used to measure scattered light./

13.12 Increase in Cell numbers can be measured by direct Direct counts may be done to assist in The diriCt observation of a known volume microscopic counting. Culture techniquesstandardizing other mlods. They are alsoof a specimen spread over a known area on "which` test the ability, of each organism pres- useful for quantitating unusual organisms inslide can berilsed to calculalel:umbers of ent to grow to a visible mass or to metabolize natural samples or hard-to-cultivate species. organisms pmilliliter . The Peroff-Hauser and produce a detectable product are also Automated electronic particle counters maycounting chamber is used for direct Counts' used (viable counts). These methods are u4-be used. i.f bacteria andreher small cellular orgatt ful,,only for unicellular organisms. Viable counts are much used in. studies of .nisms. The courxt reflects bct living and , V water Samples, urinalysis, and food quality.'dead, microorganisms. determination. A diluted sample is spread- The plate count isused to determir0 the. on a solid 0i mixed. intro a tnelted dgar ine- number of CFUs in a sample. ,, dium If this is done with medium in giletri plate, it is called a,plate count. Each 'viable .organism may grow into a visible mass of cells (a colony). The number of colonies after incubation of this medium represents the number of viable bacteria in the diluted sample. Because itisdifficult to know whether ,one bacterium gave rise to one colony, the number is usually given as colonV- forming units (tU). Introductory Microbiology -6l sir

C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION . B.. ENRICHMENT INFORMATION When microOrganiains are not growing be- 13.2 Factors influencing Microorganisms are heteronii eous in Growth, their- illysical and chemical i4 uirements.',P01 "lase of inadequate conditions, they are not Optimum growth occurs only 'when all re(necessarily dead. Long perioOf metabolic quirements are met. r, inactivity may be toleratedlayouta loss of

13.21 Nutrition Growth patterns differ depending gee Microbial PhySiology, Sub -Topic 3.30 whether nutrient supply is continuous (open oor closed system information and'Sub- system). Most natural situations are open; .Topic .3;4 for open system information. most laboratory conditions are closed. Microorganisms contain regulatory mech- In natural situations such as bodies of anisms that allow them to modulate growth , water, organisms receive a continuous if flue- for survival under changing nutrient availa- Writing supply of nutrients. Wastes are also bility. removed. Growth speeds up and slows down, but rarely stops. Under in vitro conditions, when nutrients are consumed, growth stops.

13.22 Physical and Chemical Microbial growth is affected by various Influences on Growth physical and chemical factol's including the ,gaseous atmosphere, pH, temperature, and osmotic pressure. Obligate anaerobes may be recovered Aerobic and facultative anaerobes. must 13.221 Gaseous Atmosphere Microbial growth is affected by the concentrations of 02, CO2, and. other gases infrom :dons irrnature which appear tohave an 02 source for best growth. In liquid the surrounding medium. Most .gher formsbe robie-, These organisms grow in mi.culture best yields are obtained with small of life require Lk many microorga isms aocroenvironmenta thatare anaerobic. Bacte- volumes oyluicrin large vessels on shakers. not. Obligate aerobes require 02. fors growth.Tides, an obligate anaerobe, can be isolatedAnaerobic growth conditions can be pro- Their metabolism is respiratory. Facultative1,rein the mouth. It inhabits anaerobic'crev-vided in sealed jars with catalytic oxygen anaerobes grow either with 02 orwithotitit.inces around the teeth. Many anaerobes areremoval or in anaerobic incubators. Thiog&- Growth is usually better in the presence ofkilled by oxygen collate, chopped meat medium, and media O. These organisms are usually ferments- covered with mineral oil may be used to . provide anaerobic conditions. A high -0O2- tive in the absence of 02. Obligate anaerobe§ do not grow in the presence of oxygen. .Mi- low-02 atmosphere can be provided in a '" 4Froaerophiles require reduced 02 concentra- candle jar by combustion or in specially designed incubatora.1;i:. , tion and may re-quire increased CO2 concen- iR Aration. Aerotolerant anaerobes are orga- . tennette, E. Balows, W: J. nisms living totally by ferm9tation in the gander, Jr., and J. P. Truant (ed.). 1980. presence of air. TheY are not kensitive to 02. Manual of clinical rnicrobiolOgy, 3rd ed., American Society for Microbiology, Wash- ington, D.C. Detection of a pH change in media IA p.22 pH r-most organisms the optimum pH for Extreme pH occurs in some natural envi- h flea between 6.0 and 7.5. Some highlyronments. Drainage from mining operations,usually done with pH indicators,fie.g., phenol

Introductory Microbiology 71 . C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A: ESSENTIAL INFORMATION , B. ENRICHMENT INFORMATION 4)) specialized organisms can tolerate a pH a(:high mineral area, and certain fermentationred4js yellow below pH 6.8 and red above,' low as 1.0,Or as higlyas 10.4. The internalconditions yield high-acidic. environments.6.8; methyl red is red below pH 4.5 pad. pH of most:midroorganisms is close tO'neu- The inland salt or alkali lakes may have pAllo?y abovp 4.5. Electronic pH meters ,-trality regardless of the pH of the medium. pH as high as 11. lie tped,for exact readings in liquid me Many.important biochemical testa used in SeleCtive media for isolation of fungi u identification of bacteria depend on pki insfl.11y.,have. a pH below 6.0, wherea -m dicators to detect prOduction of acidic orgenerld-purpose bacteriological me bask end products., a pH -Of 6.8 to 7.4.

,microtitganisms. Soil or water samples may be plateand 13.223 Temperature A giveh strain 9f microdiganism will have ..over their entire anoptimum' temperatureforgrowth. grAth.'teitiPeraCtire At suboptimalincubated at different temperatures to dem- Growth will occur between its maximum and teinperattire4:: their,; g don' 'time 4s ex-onstrate presence of all three types of orga- minimum temperature. These poirits are ge-tended from minutes :lardtt 'Above thenisms. Many psychrophiles are killed by ex- netically determined. optinium, growth tilow4--aistiie Microorgan-posure to room temperature, so precautions The effect of temperature on enzyme ac-ism diverts ever-increasing;ainounta of Cel- must be taken to keep samples and media cold. tivity (Microbial Physiology, Subtopic 4.53) lular energy to repair heat-itidtleed damage. . tt tjt is important in, determining the range over which an organism will grow. There are three groups of microorganisms with different ranges of growth temperatures:lisychro- philes, mesophiles, and therinophiles. One commonly used definition of psychrophiles is that they have a growth range somewhere between 5° and 20°C. Meso- philes gow between 20 and 45°C. Thermo- t ' ' ' philesgrowbetween 45 and 90°C. '.*, . Mannitol salt agar may be used for the 443.24.-Osmotic Pressure Osmotic pressure is determined by the All types of dissolved colloidal particles concentration of dissolved particles in water. contribute to the osmotic pressure of a so-selective isolation of Staphylococcus aureus Microorganisms are found in environmentslution. Cytoplasm has a relatively high os-from human clinical samples because its of varying osmotic pressures. Most micro-motic pressure. The cell can vary this within high (7.5%) NaCI concentration inhibits al- organisms, with the exception of protozoans, limits by concentrating or excreting K.most all other nasopharyngeal organisms. The osmotic pressure of media for isola- -, are protected fromlysis in dilute environ-When the external environment contains ments by a rigid cell wall, more dissolved material than the cytoplasm,tion of cell walkdeficient organisms, Exposure to high osmoticessure causes,it is hypertonic, and the cell will tend to lose Mycopldsma, must be carefully adjusted. loss of water from cells. Thi.''ests growth.water to the environment. This drying effect The halophilic organisms live in highly sa-arrests metaboli most organisms; the line environments (15 to- 34, dissolvedeffect explainstO ative value of salt,' salts). They have a specialized membraneing or sugaring f flute environnients and salt-tolerant enzymes. pose a challenge toAerial survival. When . . the exterior environment is hypotonic, . 'I tends to enter the cell. Expansion and death are prevented by the rigid cell wall.Cell 'wall-deficient forms of bacteria survive only telk' oor Introductory Microbiology-73 C. PRACTICAL ACTIVITIES A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION in isotonic environments. Examples arothe L-forms and mycoplasmas: All laboratory cultivation of microorga- 13.3 Closed-System Growth, _,Within a closed system, the amounts of 'nutrients,; and their energy contents are nisms except- continuous culture follows this model.'The phases of growth may be dem- fix; , Growth produces waste products which accumulate and may he toxic. (Fig. imstrated by inoculating fresh, prewarmed medium with an inoculum of log-phase cells and following the increase in medium turbidity, Any other accurate measurement of cell components may be used.BY,;-galdng simultaneous samples for plate e?MitS and direct counts, a good approximation Of ac-

1.og 4)11 tual numbers may be obtained for each read- 111 i V1111 ing.

Time Fig.4itases of microbial growth in closed culture. The duration of lag phase is extended if Introduction of a microorganism that di- Each cell in the inoculum must carry out 13.31 Lag Phase internal regulatory shifts to start growth.the inoculum is from an old culture, if the vides by binary fission into a sterile closed chemical system such as a flask of nutrient brothAccumulation of cytoplasmic .componentsnew medium is quite different in and increase in cell size begin. Duringlagcomposition from the old, or if it is at refrig- starts growth. No appreciable increase in erator temperature. During lag phase, mea- numbers occurs for a short time. phase, new proteins may need to be made. A complete round of DNA eplication will surement of cell constituents may increase. precede the first and every ubsequt cell division,

D g log-phase growth,:readingsshould, 13.32 Log -Phil "se (Exponential Once growth begins, increase in numbers th) 'tee- is exponential for a period of time. The1.be,cnni e frequently becauSe turbidity can. culture will commence division at the max- in as little as 20 to 30 min forsome

rbadtira. Growth curve data may be plotted imum rate possible given the genetic paten. r.1 tial of the "culture and thesuitability,40Onlitiear graph paper with time 'as,theab- or the log medium. scigsit and the log of the turbidity of 'the niiiiiber of organisms as the ordinate. 7.

All populations grow or decrease exponen- During exponential growth ther 13.321 lahationo For most microorganisms one cell divides straight-line%lationship between th4 neutial Growth iitto tw?. Thus, during each generationtimetially if eaclichangeireflects addition or sub- hef d ;dation doubles. Starfiitg, with tonetraction of some cefriatant percentage of theCell number versus time. xou can illustrate'exponentialtotal individuals present tit that time. growth with7the,geometric progression: 1, 2, If exponential growth proceeded for 48 h, 4, 8, 16, 32 ... or t, 21,22, 23, 24, 25 one bacterium that divkled every20 min, Introductory Micbiology-75 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION ENRICHMENT INFORMATION yield a bacterial mass weighing 4,000 tbtites the weight of the earth.

neration times for some bacteria may Calculate generation time as follows: 13,322 Generation'Pime The generation time observed is the av- ( short as ,12 min. Thus, bacteria can erage time needed for one cell to complete be a G = or a round of division into two cells. In this1=14 ly exti'emely rapidly under optimum time the population in the closed system willcoalitions. Generation times for other bac- 4**" tl to double. The total amount of each cell con-teria and many eucaryotic microorganisms 3.3 logiobi stituent will double. may be as long as several days. to = time at first measurement ti = time at second measurement bo = number of cells at to f bi = number of cells at t1 G = doubling time or generation;time n = number of generations

Nutrient limitation is the major restric- The stationary phase may persist for a 13;33 Stationary Phase Environmental limitations put an end to exponential growth. The growth curve tion for most aerobic organisms. Accumula- long period. proaches a horizontal line. During thea-tion of toxic wastes is the most frequent tionary phase some cells grow and dide. cause of growth arrest in anaerobic culture. Some are active, others die, and the population number is unchanged.

The death phase results in a reduction in The decline is usually exponential. Small 13.34. Weath Phase A decline in number of viable cells constitutes the death phase. viable cell numbers. This phase may or maynumbers of viable cells may persist indefi- not.,afigv corresponding decline in totalnitel3at the end of the death phase... oell numbers.

ability without cell lysis can 13.341 Mechanism An acute shortage of nutrients or high The loss of concentrations of toxic waste products mayittsually be attribud to an inability-to sup- trigger activity tolytic enzymes causing ply the energy necessary to repair key gez cell lysis. ietic structures. Wh'eri autolysis occurs, it iaL:'°- usually related to low cellular- ATP lbvels, signaling activation. 4 intracelltilar4xgo-4 zyme. Clearing of.x.the/cultuie may bequite rapid if autorYtk Mechanisms are set in tion. dt'

13.4 Open-System Growth Open-System growth occurs when constant environtnental conditions., are maintained,Nutrients are continuously provided, ti and Wastes are removed. The number of cells per'unit volume is kept constant. .C9 ' Introductory Microbiokytt-272?- 90 r) 11. ENI1101 'WNW INFORMATION C. PRACTICAL ACTIVITIES TOPIC'S ANI) SIII1TO1'ICS A. ESSENTIAL INFORMATION laboratory devices for producing open- The growth rate of the bacteria in the 13.41 Conditions nutrients' , system growth provide a source of.cells invonael adjusts to the rate at which the log phase of growth. They allow theare provided, After a periodof adjustment, study of a culture under optimum physiolog-the rate of increase df(tho cells through lea! conditions. growth will just equal the rate of cell loss. The cells in a continuous culture apparatus are in log phase. ThedotplitiTti One is determined by the growth c tiditionni. If all else is constant, growth re depot-yin directly on the concentratioo arn lung nutritional factor. ) In a chernostat, the flow rate is set at a These are two types of continuous culture Continuous cultur toms provideaion- 13.42 Contitauttils Culture venient, constant so rce of log-phase cellscertain value. The rate of growth ofthe Devices devices, chemostats and turbidostats. They culture adjusts to the rate at which nutrients of log-for study. They are used for resew,11 on are, used for study of all activities are added. In a turbidostat,un electronic h as e cells. mechanisms of regulation, concentr9 ion of nutrients, selection of growth rate-n6trients, device Monitors turbidity, i.e., c 41 mass, of, and interactions among species in mixed cul- the id electronically signalsth'addition e desired tures under conditions that simulatenaturalof fres,medittin 6..maintain t environments. population d nsity.

14.0 Enzymes 14.1 Definitions Catalysts are used in a number of reac- Starch + water Glucose. This reaction 14.11 Catalyst A catalyst is a substance that affects the will proceed at a very low rate in worn being tions of everyday 'practical interest and in- , rate of a chemical reaction without water. Upon the addition of a smallquantity permanently altered itself. duptrial importance. For example, platinum catalytically enhances the -oxidation of un-of amylase, accelerated glucose production burned hydrocarbons in automotive ex-(and starch hydrolysis) is noted. After a hausts (catalytic converters). Also, the in- tittle, the enzyme can be isolated fromthe dustrial extraction of apple juice from apple system with its original activityessentially pulp is aided by treatment with pectin-hy-undiminished. drolyzing enzymes (pectinases).

dOfo The fact that enzymes are protein, can be 14.12 Enzymes Enzymes are highly specified catalysts demonstrated by their reaction with protein- produced by living cells. Enzymes have a protein component and', may or may not detecting re sits sticks, Milloifteagent or have other components such as metal ions, the Folin phenol reagent. Enzyme solutions will also exhibit maximum ultraviolet, ab- vitamin, or carbohydrate molecules. sorbance at 280 nm which is characteri of protein solutions.

14.13 Substrate A substrate is a substance which is altered in an enzyme-catalyzed reaction. Introductory Microbiology-79 TOPICS AND $1111Toplom A. 111181INTIAli INFORMATION I,' FNIIICk1110INT-INVORM ATKIN (1, PIRACTICi I, Will/111RM 14.14 Active Hite Anactive site in 11 su dl ti con on tho Thtttolye Hill) NM he pichirod an a atria!, zynaiso auvl'nce which (Maio in a highly spe- tutu which matches up with a opecitiosittli cific manner to the taihnt rote, It is obi lkol plementory conformal ion Iii the subtit riao.i hi the catalytic Site. a "lock-and-key" sort of arrangement (Fig, .1: 5), . .1'

14,15 Alloateric Site An nilosteric site is a region ontii Thu allonteric site con be pictured on Nuance, mairt from the acivii$iitii, atiewn in Fig. 0, Note that the active site will regulatory substance may that and "fit" the' substrate hi (notthe alloslarie the affinity of the enzyme for the aubstrate.effector. The effector, if hound, results in a modificationofthe.th Nettlitnellaionnl structure. of the apoenzYne. This, in turn, moth- 1 lies the structureof the active site.

'14.2. Mechanism of Enzyme Enzymes work by lowering the required Action activation eittirgy, thus facilitating Rai reaction (Microbial Physiology, Subtopic 4.3).

14.3 Nomenclature EnzyMes are named according to the type The six basic/ classes of enzymes are oxi- of reactitin catalyzed. They usually have the doeeductases, transferases, hydrolases, suffix -age, e.g., gelatinase, lipase, etc. lyases, imome)'aseki, and ligases (Microbial Physiology, subtopics 4,41 to 4.461. 14.4 Factors Affecting- Rate . The most impor an rs affecting the of Enzyme Reactions rated enzyiri react ons are enzyme concentration, substrate concentration, temperature, and pH (Microbial Physiology, Sub- S tonic 4.6).

. 14.5 ,Enzymes and Control of Sint @ allIiioChernical reactions are cata- Only a small number of the possible en- .robial Metabolism lyzed by specific enzymes, cells can control zympttic reactions occur in any cell at a given their function (metabolism) by changing thetip* Some enzymes are only synthesized activity or amount of specific enzymes. 70er particular condieris, and others are synthesized but inhibited from acting.

14.51 Control of Enzyme Syn- Certain potential food molecules, can trig- Escherichia coli does not syrdhesize the .thesis, Induction ger/the microbial synthesis of,enzymes nec- enzymes for lactose metabolism unless lac- essary for their breakdown, This type ofttose is present in its immediate environment reaction is called enzyme induction. (Microbial Genetics, Subtopic 6.12).

- V 14.52 Control of Enzyme Syn- The addition of a compound that is the End product repression is one of two feed- . thesis, Repression end prodbet of a biosynthetic pathway to a back-regulating mechanisms forf=.bio'syr.;,. growth medium causes an arrest in the synthetic pathways. It regulates enzyme ajinthiy,-?,!- s .7 thesis of the specific enzymes of the path-sis. End product repression is conipie" Introductory Microldiolcigy-81

93 rt °T.,i4 .1

Active-Site Potential Substrates

Enzyme A Will not bind Will Bind

Fig. Complementary structure of active site and substrate.

Allostcric Site

Active Site

Alternate Enzyme Conformations

I MA I I Iam B Substrate Effector A Effector B

If effector A is present, enzyme will be in a favorable conformationforinteraction with the substrate. If effector B is present, substrate binding will be reduced because this effectiw stabilizes conformation B which cannot bind substrate. 1 Fig. 6.The allosteric site and binding Of positive and negative effectors.

Introductory'Microbiology-83 e / . . - TOPICS' D IUBTOPICS / A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES way. This type of situation is called repres-tary to end product inhibition (Introductory sion. Microbiology, Subtopic 14.53). \. Both end product, repression and end product inhibition serve to modulate the L overall cellular metabolic activity to Maxi- mize the rate of growth through the efficient conversion of nutrients into cellular materials (Microbial Genetics, Subtopic 6.11).

14.53 Contro4 of enzymeAc- The activity of most enzymes is influenced Thg relative concentrations of ATP, ADP, ivity by a variety of small molecules. Some. ofand AMP determine the ratio of catabolic to these socalled modulators, enzyme biosynthetic activity in a cell at a given time. activity, and some inhibit such reactions. ADP and AMP tend to stimulate catabolic enzymes; ATP tends to inhibit catabolic and 1 stimulate biosynthetk enzymes.

14.54 End Product Inhibition en the end pr9duct of a metabolic In the foll'oting reactbn, an excess of an pathway is present i*excess, it may inhibit inhibitor (F) interacts With the allosteric site the action of one of the early enzymes in the of the enzyme catalyzijig the conversion of pathway. This inhibition involves the allo-A to B. Since production of B is blocked, C steric site so the inhibitor does not compete will not be produced. with the substrate for the active site.

inhibitor Consider the following structures: 14.55 Competitive Inhibitors which are structurally similar COOH SO2 H2 to the substrate compete with thelsubstrate molecules for enzyme active sites. As the ratio of inhibitor to substrate is increased, a concomitant decrease in product formation occurs. Competitive inhibition can be re- NH, NH, versed by increasing the ratio of substrate to (PABA) Sulfanilamide inhibitor. 0 para- -, 47 aminobenzoic acid Some microorganisms utilizeTABA as a substrate in the synthesis of cblic acid, a material required for metabOlism...The en- 0 zyme which normally binds ,PAB.A atlas active sitewillalso bind sulfani1A. Therefore, if the ratio of sulfanilde PABA is high, the probability of the eiyme--. 96 Introductory Microbiology -88\ .TOPICS AND SUBTOPICS , A. ESSENTIAL INFORMATION 13.1NRICHMENT INFORMAbON C. PRACTICAL ACTIVITIES ' reacting with sulfanilamide rather than with its normal substrate is also high. The result is a decrease of folic acid synthesis and suppression of metabolism. Such inhibition is based upon the similarity in structure of normal substrate and inhibitor. Sulfanil- amide does not effect the supply of folic acid to mammalian cells because they cannot convert PABA to folic acid and thus require folic acid preformed. 15.0 Metabolism 15.1 Cellular Chemical Re- , 'Metabolism is the sum of all the chemical In general; microbial metabolism is quite if actions reactions in the cell. similar to the chemiciii reactions that occur in higher animals and plants. However, upeni''' examination of individual microorganillms, it there is a marked diversity of metabolic pathways and end produdts. This diversity is useful in identifying microorganisms such as bacteria and fungi. Many metabolic ways known to be similar in 'all organisms were first worked out 'by investigators with microorganisms.Escher'ichia coliand"Sae- charomyces cerevisiaehave been particularly useful in metabolic research.

15.2 Energy nergy is the ability to do work. Microbial movement, growth, and reproduction occur only if there is an available w. energy source. The ultimate source of most

!*; biologicenergy is the sun. A possible ex- caption this is the deep sea volcanic gases systemhere the driving nergy is the oxidation o H2S by bacteria. Ballard, R, D., and J. F. Grassle. 19" Aeturn to oases of the deep. Natl. Getogr. 156:689-705.

15.21. Forms of Energy Energy can exist as light' or other kinds of , The form tof energy)equired/directly for radiation energy, chemicaPenergy, mechan- microbial movement, growth, and reproduc- ical eneigy, or heat. tion is chemical energy. `i 15.22 Chemical Bond Energy The energy of a molecule can roughly be If a carbon-containing substance is burned represented as the sum of the forces heilding (totally oxidized to CO2), the heat produced its atoms together. is a n1easure of the potentially useful chemical energy of that substance. Introductory Microbiology-87 03 09 - TOPICS AND SUBTOPICS ESSII1NTIAL, INFORMATION B. ENRICHMENT. INFORMATION C. PRACTICAL ACTIVITIES 15.23 . Ent li;igonicand Exer- Certain bond formations between atoms A typical'endergonic reaction may be rep': 1 ` gonic Reactions' require energy input (endergonic reaction);,resented as follows; however, such energy can later be releaSed A + B Energy --. AR. by ,I4eaking the bonds (exergonic reaction). A typical exergonic reaction may be represented as follows: 'BC + C +Energy.

15.24 Coupled Reactions Biological transformations of chemical en- A counted reaction is the snm of the fol- ergy can occur because exergonic (energy- lowing events: yielding) reactions are linked to endergonic Endergonic: A + B + Energy AB . (energy-requiring) reactions by common in- Exergonic: BC B + C + Energy termediates. Sum: A + BC + C Coupled reactions mayalso be writHei: A B ndergonic , exergonic BC - The reactions will proceed in thedirection of the arrows as long as the energy released by the exergonic reaction exceeds that required by the endergonic reaction.

-15.25 Nucleoside piphos- These compounds are the mot usesil in: The conversion of adenosine diphosphate phates and Triptios- , termediates between exergoqic and ender- (4..DP) to adenosine triphesphate (AlTP4rbr phates: ADP, GDP, gonic reactions. ) example, provides an efficient short -term en- UDP, CDP, ATP, GTP, ergy storage and transfer ineChanism in bi- UTP, CTP ological systems. The addition of a third

S phosphate to a nucleoside diphosphate is an endergonic reaction and can occur only when this reaction is linked to an exergonic reaction that releases sufficient energy. En- zyme,catalyzed renwval o tiraisai phosphate is exergonic; thus, it can be used to drive cellular energy-requiring, reactions.

15.3 Catabolism Catabolism is the sum of cellular reactions , resulting in the conversion of the, energy from various organic -molecules to energy usable for cellular work. ATP is the most ':useful" cellular energy form available.

Introductory Microbiology-89 1u1 1'3 iJ A TOPICS ANI) A, ESSENTIAL INFORMATION II, ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 5,31 Oxidation-Ittltiction Oxidation. is removal of-tilectrotia and le, In biologfcal aysten oxygin is ono of the An oxidation-reduction reaction can l') lioct ions ten 'manna (IV) from a niolettle: The Wee- most avid available acceptoni -of electrons represented as (rons removed must -be donated to Another (mosk.electroncgative). Thus, an oxidation A11 Molecule. This, recipient is said to be, rein which oxygdn ends up with the tram-

duced. Thus, an oxidation reaction nitaia. furred electrons releases the intlat energy, 11 ways be accompanirl by a reduction read- - electron Lion. Oxidation-reduction reactions result in II 'proton a transfer of energy; Much of the energy A o electron dolma involved in the transfa may he lot to- the , -II electron acceptor system as neat, or if the reaction proceeds in a stepwise fashidn, some of the energy -may be conserved by coupled reactions: e ..c, , 15.32 NADs as Electron Car- Nicotinamide adenine dinucleotides 'l'he'use of NAD' and NADP''it'S electron For directions foryerforMing a lactic' de- riers (NAD') are the most conunon immediate acceptors' in catabolic protlisses is very im- hydrogenase enzymegssay which wil dein- electron acceptors of organic molecules:Re- portant to the conservation of energy re-.onstrate use ofNADas an electro accep,tork duced formg of these molecules (NADH and leased by oxidatior(NADH and NADPH dO consult: PH) act as electron donors in other not donate electrons to oxygen ditectly chid Bergnrioyer, H. U. 1974 Methils of en- reac ions. are relatively stable in the absbnCe of )enzymatic analysis, vol.1, P. 080. Academic NAD' is made from niacin and is a good zymes" specific forrinsfer of the electrons 'Press, Inc.., New, Yorit. example of the place of vitamins in metab- to a given substrate. This enables the orga- olisni. nism. to maintain control of its oxidation- N. reduction reactions.

15.4 .Degradative or Cata- Cells break down, organic molecules by a bolic Pathways series of enzyme-catalyzed reactions. A par'. ticular series of such reactions is called a metabolic pathway.

15.41 Fetmentation and Res- Fermentation and respiration are the two 'The type of catabolism associated with Simple laboratory tests can be used to piration basic catabolic schemes. bacterial species is important to their char- determine whether a particular organism is In 'fermentative. pathways, the organic acterization. Strict aerobes carry out respi- obtaining energy from fermentation or res- molecules being utilized as a source of en- ratory (catabolism only. Facultative anaer- piration. One such test depends on the abil- ergy are incompletely oxidized. No'external obes can utilize either fermentation or,res- ity or inability of an isolate to utili e a par- inorganic electron acceptor is necessary (no piratiok Aerotolerant anaerobes use fer-,-tieular organic energy source i.a sealed 02 is required): ATP is produced only by mentative pathways but are not harmed by tube. Information on *dation-fe.entation substrate level photphorylatiop, (Microbial 0.2. Strict anaerobes'use fermentative path- (OF) test iirtedia can be found. in: Phyiology, Topic 5.25). The number of ATP ways and are harnied by 02. A few are able Lennette, A. Balows, W J. molecules produced' per molecule of sub- to respire by using alternate electron accep: Hausler, Jr., and J. P. Truant (ed.): 1979.' strate catabolized is small. tors'(Microbial Physiology, Topic 5.2432). Manual of clinical microbiology; 3rd ed. Inrespiratorycatabolism(sometimes Tw_b indtistrial processes with yeast can American Society for Microbiology, Wash-

called oxidative catabolism), the organic .be used to demonstrate differencesbetween, ington, D.C. molecules being utilized are usually oxidized fermentation and respiration. When yeast is completely to CO2. The electron transport used for alcohol production, the vats are Introductory Micrbbiology-91 iO3 P.

TOPICH AND HIIIITOPIPti A, l4l-it.11NTIAlt INFORMATION 16111\11111111MFNT INFORMATION PlIAOTIOAI, ACTIVI'11101 spit out M ierobla IPyniollogy,Nuhlopic kepi HI Hedy anaerobic, The edulnol is li tierves Iit transfer eleetrons to on product of ferntenIntion, When yenott in pro, guide electron noceplor, Mont of the ATP InilnowKftir igle an baker& yennt, it In grown produced by oxidative pIttriphorylnI ion co nt. under highly nerobiil cotolitionn, beenntioI ho pled to the electron trallt111011: ardent, The..radial into added eon only he obtained, if I he yield of ATP per notleculp of growth sub- ,yeast in. living by respiration. strate metahOliZt)(1 IN 1111101 greater than the yield of ATP in fertnentatio. I

Ifi.42 Wye(!yids (in Islew (Ilycolysis is it pathway for I he degrada- Glycolysis is essentially the Home in nit- Meyerluil Pathway) tion of glucose found hl 00)81111111.001.1411- 1111111iti, HMI animals,It in atj\ 1118111H, IVO Dille 11111011)8 11100(11)(1 10 eytophounie cellular proem, skild thy en- (11 (3)11V4311 glucose to two yrtivate molecules. zymes are not mendwane-lithind. The intermediate substrates aro all phomhorylated (Ilucono 2Pyrovnle The pathway can be divided into three 2 NAI)6 2 NADI! asum. The preparative phase converts glucose, to two three-carbon phosphorylated 2 ADP 2 ATI' molecules. This conversion requires two 2 P molecules of ATP. There is one oxidative step in which The oxidative phase converts the three - A NAD' accepts a pair of electrons from aetirbon molecules to the highly reactive 1,3- throb-carbon intermediate. diphosphoglyceric acid (3-phosphoglyceroyl The ATP molecules are produced by sub- phosphate). This requires two molecules of strate level,phosphorylation. NAD+ and generates 2 NADH. The pyruvate and NADH generated have The energy transfer phase results in sub- 7 a different fate in fermentative organismsstrate- ovel phosphorylntion of 4 ADP to 4 than in respiratory organisms. ATP. Two reactions occur whereby energy and phosphakare transferred directly from the three-carrOn intermediates to ADP (Fig. 7.). t

15.43 Other Pathways ,.Other routes exist by which sugars can be A unique characteristic of procaryotic The pathway by which glucose is catabo- catabolized. cells is the diversity of catabolic pathwaysJized may be an important taxonomic char- that may be found in different species. acteristic of some microorganisms. Most members of the genus Pseudomcmas char- acteristicallyusetheEntner-Doudoroff pathway.

15.44 Fermentation Fermenting microorganisms are unable to Because the energy yield per molecule of It is possible to identify certain taxonomic carry out the reactions of electron transbortsubstrate utilized is low, a fermenting micro-groups of bacteria by their fermentation either because they lack a terminal acceptororganism must catabolize more molecules ofproducts. or because they lack the necessary enzjrmes.substrate than a respiring microorganism for The Voges-Proskauer test for the pres-

Introductory Microbiology-93 1J5 II

COH Dihydroxyacetotie phosphate

Preparative Phase: POC n COP COH

C'=-0

COP

Glucose OH OH OH Fructose-11,6 diphosphate 2 C=0

('OH Oxidative Phase Ke (' 21)

I'OH ( dvceraltlehydeI phosphate 2N A OH, 1-1* ('01'

t PhosphogIvcerovl Phosphate

, ,11 =1M =. emI . alMO 2AOP 21-1,4/ ('H 2ADP 2ATI' ( ( H EnergyI ranslet Phase // r 2 Reactions /P ('-()

=11 I React Ion r ( O 2A'I'P t.01' PhosOpenolpyrus.ale 'N ru' ale PhosphogIvceru acid

Fig H/vco/vsts

G lotroduct M IcrolmologN TOPICS AND SUBTOPICS A. BSSENTIAL INFORMATION B. ENRICHMENT INFORMATION C: PRACTICAL ACTIVITIES or cofactors. They can grow and reproduce.the same yield of growth and reproduction. ence of acetoin is frequently used in identi- on the ATP produced by glycolysis or other In fermentations .there is no exogenousfication of enteric bacteria. anaerobic pathways, but because NAD+ iselectron acceptor, so the average oxidation Two fermentative pathways are illus- in very limited supply they must be able tolevel of the fermentation products musttrated in Fig. 8. reoxidize the NADH produced to permit theequal that of the substrates utilized. This anaerobic energy-yielding pathway to con-requirement and the fact that no oxygen is tinue. Fermentative microorganisms utilize available to carry out oxidative bond cleav- pyruvate or other products directly or indi-age limit the lange of substrates that can be rectlyasrecipientsforelectrons fromfermented. NADH. Thus, much of the pyruvate carbon can ,be found in the reduced organic mole- culd4 (fermentation products) excreted by fermenting organisms.

15.45 Tricarboxylic Acid Cy- Pyruvate is converted to acetyl coenzyme The tricarboxylic acid cycle has more than cle (Krebs' Cycle, Citric A (CoA) and CO2. The further oxidation ofjust a catabolic function. Many of the inter- Acid Cycle) acetyl CoA is dependent on enzymes of themediates are starting material for synthesis' tricarboxylic acid cycle. These enzymes areof important cell components: Anaerobes associated With the cytoplasmic membranethat do not use the cycle fof respiratory 4 of procarytotic cells and are in the mitochon- metabolisM may still have many of the cycle

, dria of eucaryotic cells. For each acetyl COAenzymes. These have biosynthetic function. oxidized to CO2, 3 NADH and 1 FADH areWhen tricarboxylic acid intermediates are produced. These molecules donate electrons removed for biosynthesis, the cycle can be to the electron transport chain. kept in operation bAynthesizing new oxaloacetate (Fig. 9).

15.46 Electron Transport and Electron transport is a cellular mechanism The electron transport system consists of Oxidative Phosphoryla- for conserving the energy released duringa series of molecules (flavoproteins, qui- ,tion oxidation of organic molecules. Catabolicnones, cytochromes) that are embedded in processes involving participation of the elec- a cellular membrane. This series of mole- tron transport chain are called respirationcules is referred to as the respiratory chain. (Microbial Physiology, Topic 6.0). These molecules have the ability to be both good electron acceptors and good electron donors. When NADH provides an electron pair, the electron transport molecules are alternately reduced and reoxidized in a particular order as the electrons pass from one to the next. As these stepwise oxidation- reduction reactions occur, energy is released, and this energy can be conserved by oxidative phosphorylation. Fog the continuation of electron flow through this pathway, there must be some exogenous compound that will

Introductory M icrobiology 97 J.

a a `.

The ;educed organic molecules produced by fermenting microorganiShiS are of great commercial and diagnostic ingiMiance. Ethanol is produced mainly as a product of .yeast fermentation.

Substrate Glucose (6 ('artions) 2 Ethanol. Fermentation l'rodm;t (2 x 2 Cart

2NA1) 1(tOXI(14111011 of NAI)1.1.1r

Re I ease 1 hnergv

- 2NA1)11 11

2 l'NniNali 2 A, et .MI4. hale

211)12 , itrl)mis)

4 harlJr err III n III toeliti I ,./ (hi mill, tens) tuba, ill' utul .Pptwiwiis imp", I n 111.1011111Il 4,16e.se Wu' mgarl F

Fernier1777w Prothil t Is1111,s1 ;1 t. 1,.( 12 k h = 12 ( arbons) . 4 1.11, 11, Ar it 1 ('iirlmits) NAII

Relearn. .1 Energy RetlxIdal

4 NA1)11.1k.

III II% MI

In the brill iii bfl'a/(1/,, hen Penal (..utate., mat he identiped 0 iii, ha.11, mentutuin and prrahrrl.

FIg Examples of fermentative pathways In the clinical /chart tore. bacterial isolates may he identified on the basis of ferment(' lion substrates and products

Introductory Microbiology-99 IUD '21

(2 Carbons) Acetyl-CoA CoA

Citric Acid (6 Carbons) (4 Carbone) OxAloacetic Acid

\ 1 FADH2 rFig. 9. A summary ofthe tricarboxyli acid cycle

Introductory Microbiology -101 111 TOPICS AND SUBTOPICS A.., ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES accept electrons from, and thus reoxidize; a the lastcytochrome in the chain. N77'- 1.5.47 Aerobic Respiration Most microorganisms utilizing electron Obligate aerobes are 'microorganisms tha Nitrate reduction can be observed in the transpoi-t require oxygen to accept electronscannot grow and reriroduce in the, absencelaboratory by culturing denitrifying bacteria from the fmal reduced cytochrome. of molecular oxygen. They require oxygenunder anaerobic conditions with NO3- added as a terminal electron acceptor. to the medium. 1%102- may be produced, e.g., Escherichia coli. Denitrifiers reduce NO3- I to N2 or other gaseous products, e.g., Pseu- domonas aeruginosa.' .0 15.48 Anaerobic Respirtion A few types of bacteria can use oxidized Bacteria that use NO3 as a terminal elec- Winogradsky columns (Microbial Physi- inorganic ions as electron acceptors in thetron acceptor (denitrifiers) will use roxygen ology, Subtopic 2.12) usually develop a good absence pf oxygen. NO, (nitrate) and SO; preferentially. The conversion of soil nitrate population of sulfate-reducing bacteria. It is (sulfatof are the most common alternate(NO, ) to gaseous nitrogen products by de- possible to smell the H2S produced in these electron acceptors. nitrifying bacteria is one way that soils !Ilsecylinders or test foritwith filter paper nitrogen. The iise of SOIas a terminal soaked in lead acetate. electron acceptor is restricted to a specialized group of anaerobic organism's. 1125 is produced. Water-logged sediments with an C abundance of organic material and SO; ( tideflats, for example) are noted for their H $-producing Desulfouibrio populations.

15.49 Pathways for Degrada- Microorganisms can use a wide range of Some synthetic molecules are very resist- Microorganisms vary greatly in their abil- tion of Other Substances carbon compounds as sources of carbon andant to microbial degradation. DDT is one ofity to degrade Carbon compounds. Such sub- energy. Series of degradation reactions, of-these recalcitrant molecules. stances need not be water soluble, e.g., crude ten catalyzed by inducible enzymes (Micro- oil. Microorganisms able to degrade large/ bial Genetics, Subtopic 6.1), result in prod- Approx. molecules often excrete the degradative en- Festicide chemical zymes into the medium containing the sub- ucts that can enter the glycolytic or vicar- (yrl boxylic acid cycle pathways. strate. These excreted enzymes are called Chlordane 2-4 exoenzymes and can be detected by growing. DDT 3-10 Dieldrin 4-7 the microorganisms on solid medium con- aThlor 7-12 taining the substrate and obserl4ing substrate' disappearance around the areas of microbial growth.

15.5 Anabolism Anabolism is the sum of the cellular chemical reactions that produce the organic mol- - ecules necessary for maintenance, growth, and reproduction of the organism.

CO2 FixationinAuto- The conversion of CO2 Into the carbon The primary reaction in autotrophic CO2 CO2 fixation (the Calvin cycle) was first trophs compounds of cell material requires energy is the binding of CO2 to the five-carbon sugar investigated in green algae. The path of in and reducing power. .ribulose 1,5-diphoSphate. This results in pro- corporated "C-labeled CO2 was followed in Introductop Microbiology-103

1 1 i) .

TOPICS AND VEITQPICS A. ESSENTIAL INFORV.O.PN4 B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES '1, , ducticin -of two phosphOglyceric acid 'mole- photosynthesizing organisms. It was later cules (each three carboris). These can bediscovered that this pathway is used by al- used asotarting material to synthesiie the most all autolroli+hs. carbon compounds needed by the autotroph. .1 qt:' ...4=-4- 15162 Relationship Bet.W66n Catabolic metabollam provioes'sources of Anaboliggi an& Catabb- energy (ATP), reducing power (NADH and lism t NADPH), and carbOn skeletons for bio- syntheses.Withdrawal of intermediates from catabolic pathways diminishes he net ATP yield. Control mechanisms fundtion so that anabolic processes (biosynthedc Path- ways) are maximized when the ATP supply is high and catabolic processes predominate when ATP supply'is low.

-16.6 Introduction To Ge- Reties 16.1 Definition of Genetics Genetics is the study of the mechanisms Generation times can be estimated by de- N by which the information of cells is stored, termining the increase in the number of 1 expressed, and- modified,and how this infor- colonies arising from samples of the growing mation is transmitted to other cells in thepopulation taken at various time intervals. population and to future generations of or- For microorganisms reproducing by binary ganisms. fission, the following formula can be applied:

G= T/3.3 log ( blB ), Uses of Microorganisms Microorganisms are useful tools in genetic stiles for the following reasons: (i) microorganisms have short generation times; In)where G = generation time; T = time of cell very large populations of almost identicalgrowth; B = number of cells in original pop- cells ciln he produced asexually from a single ulation; h = number of cells in final pdpula- cell in a short dine; (iii) large populations tion. can he grown in a small volume; (iv) micro- Race bacteria divide by binary fission, a organisms are usually haploid. and genesingle cell will give rise to over 1 million cells expression is immediate; (v) and a variety ofin 20 generations and to over 1 billion cells mutants can be isolated. in 30 generations. Riley, M., and A. Anilionis. 1978. Evo- Cell populations of bacteria approach 10 lution of the bacterial genome. Annu. Rev.cells per ml in liquid cultures. Denser pop- Microbiol. 32:519-560. ulations can be grown on the surface of solid media. Since microorganisms are haploid, a mu- Inoculating "needles" may he used to tation will not be masked by its dominanttranIfer colonies in their exact position from counterpart. an enriched medium to a variety of media Genetic recombination in eucaryotic mi-lacking growth factors. Nutritionally defi- 111 Introductory Microbiology-1 115 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES croorganismssually involves fusion of nu-dent mutants, auxotrophs, can be identified clei from two individuals to form a diploidby their failure to grow on one or more n6clens. This is followed by meiosis. Haploiddeficient media. individuals develop chaiacteristic of both parents: ° 17.0 Genetically Impor- tant Macromolecules C 17.1 Deoxyribonucleic Acid, The major molecules important in ge- The genetic information coded in DIVA is Ribonucleic Acid, andnetics are deoxyribonucleic acid (D A), r transcribed into mRNA which is then trans- Protein bonucleic acid (RNA), and protein lated into protein. This flow of information Cohen, S.1975. The manipu ation ofconstitutes the central dogma of molecular genes. Sci. Am. July 233(:24-33. genetics. A diagramatic form of this dogma would be DNA -- RNA protein. Messen- ger RNA, transfer RNA, and ribosomal RNA are involved in protein synthesis. In RNA viruses, reverse transcriptase is the enzyme which produces a DNA copy of the RNA genome of the virus.

17.2- DeoxyribonucleicAcid DNA is a double-stranded polymer of nu- In procaryotes, DNA is a small circular The nuclear region of procaryotes can be (DNA) cleotides. Each nucleotide consists of a phos- molecule without a surrounding membrane.demonstrated by the Feulgen staining pro- phate group, deoxyribose sugar, and one ofProcaryotes do not have histones. cedure. Staining of eucaryotic cell nuclei or four bases: adenine, guanine) thymine, or Eucaryotic DNA is divided into chromo-\chromosomes is achieved by use of basic cytosine. The twd strands are held-togethersomes. The DNA is combined with histone ;dyes. by hydrogen bonding 'between the comple- and nonhistone proteins to form a complex mentary base pairs adenine and thyminestructure. The number of chromosomes var- and guanine and cytosine. ies among different eucaryotic organisms, and these chromosomes are located in the nucleus, a membrane-bounded structure. Mitochondria' and chloroplast DNA are ,small circular molecules located in these membraneous organelles.

17.21 Functions of I)NA I)NA has two major functions: to replicate In biosynthetic pathways which require One evidence of DNA as the genetic in- itself and to code for the synthesis of 11 otein.numerous enzyrilatic steps, the genes coding formation carrier comes from the experi- A gene is the sequence of nucleotides whichfor thejarious enzymes may be linked into ments 43.1' Hershey and Chase (1952), who codes for a single protein. The entire com-a unit called the operon. An operator genedemonstrated that only the DNA of the plement of genes in an organism comprisesand a regulator gene control the operon'svirus entered a bacterial cell,. its genome. functioning. Mirsky, A. 1968. The discovery of DNA. Sci. Am. June 218:78-88.

17.22 'Replication The two DNA strands separate a spe- Replication is catalyze'd,:by DNA polyrn- In Oductory Microbiology-107 113 IP

TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION' C. PRACTICAL`ACTIVITIES cific pOint called the origin: Eniymes cata-erases which require a single-stranded tem- lyze the Inthesis of tw new stiands com-plate; the mechanism by which the double- 'plementary to each of the original twostranded molecule septliates is unknown. strands.' The two new DNA molecules DNA replicates itself in a serniconserva- formed are identical to the original molecule.tive process as described by Meselson and Stahl in 1957. The two strands of the double- stranded DNA molecule are complementary in base sequence. Each strand serves as a template to which a new strand is formed, resulting in two DNA molecules having one strand from the original,IDNA molecule ilnd one strand wwly synthelized. The process also requires a primer segment of polyribonucleotide, 50 to 100 nucleotides long, produced by a DNA-dependent RNA polymerase (probably different from that involved in RNA synthesis). Replication is discontinuous and occurs in a 5' to 3' direction on both strands of the open region of DNA as demonstrated by Okazaki in 1968 (Fig. 10). The RNA-DNA fragments found in the initiation processes of replication are termed Okazaki fragments.

17.3 Ribonucleic Acid (RNA) RNA is a single-stranded polymer of nu- The three types of RNA are coded in the cleotides. The nucleotide subunit of RNAspecific base sequences on the DNA mole- differs from that of DNA in that ribosecules. The maturation process of the three replaces deoxyribose and uracil replaces thy-kinds of RNA differs and results in specific mine. There are three major types of RNA: spatial configurations for each RNA type. messenger RNA (mRNA), transfer RNA rRNA constitutes up to 65% of the ribo- (tRNA), and 'ribosomal RNA (rRNA). somal mass. Procaryotic ribosomes have Rich, A., ;and H. K. Sung. 1978. Thethree characteristic forms of rRNA; eucar- three-dimensibnalstructureoftransferyotic ribosomes are larger and have four RNA. Sci. Am. January 238:52-62. types of rRNA.

17.31 Functions of RNA The three types of RNA function at var- mRNA is involved in both transcription ious stages in protein synthesis. and translation/processes of protein synthesis. A specific mRNA is a transitory mole- -. cule. The tRNA is folded in a "cloverleaf" structure. Each of the 20 amino adds found in proteins has at least one corresponding tRNA. Some amino acids are carried by more than one tRNA. The mRNA codOns\ 113 Introductory Microbiology-109

a IIP TOPICS AID SUBTOPICS AI ESSENTIAL INFORMATION UPB. EIICRMENT INFORMATION C. PRACTICAL ACTIVITIES and the corresponding' amino acids in the genetic code are known.

17.4 Protein Structure A protein is a linear polymer of amino Furthermore, secondary and tertiary con- acids joined together by peptide bonds. Thefigurations of a protein are determined by protein has a unique three-dimensionalthe primary structure. Proteins can associ- structure determined by its amino acid se-ate noncovalently with other proteins in spe- quence. cific arrangements, resulting in a quaternary configuration.

17.5 Protein Function Proteins functionlas enzymes and as struc- As enzymes, the protein has specific sites tural elements. at which a substrate molecule is changed to the product of the enzyme's action. This site is called the active or catalytic site. As structural elements, proteins consti- tute at least 50 to 60% of the cell membrane.

17.6 Protein Synthesis, Tran- In transcription the rnRNA molecule is The DNA-dependent RNA polymerase scription synthesized complementary to a gene on one binds to specific initiation sites on the DNA strand of DNA. The mRNA bases form theto begin transcription of a specific RNA. pattern to be translated into the specific The initiation site-binding complex results amino acid sequence of a protein. A tripletin localized strand separation of the DNA of three nucleotides is called a codon andand the beginning of transcription (Fig. 11). will be translated into one specific amino acid of the protein.

17.7 Protein Synthesis, In translation the mRNA molecule is de- Translation of mRNA involves four major Translation coded on the ribosome by the tRNA mole- steps. cules carrying amino acids. Each tRNA at- (i) Activation of tRNA. This involves the taches to a specific codon of the mRNA. Thebinding of an amino acid to tRNA. ribosome moves along the mRNA, resulting (ii) Initiation of translation. The first ini- in the sequential polymerization of aminotiating aminoacyl-tRNA binds to the small acids to form a protein (Fig. 11). subunit of the ribosome with the involvement of specific proteins. The large ribosomal subunit attaches to this complex, thus -forming a functional ribosome, ready for the next step. (iii) Combination of amino acids into an elongating protein chain. The polymerization of amino acids is accomplished by the sequential transfer of amino acids from the tRNA molecules which attach to the mRNA at the ribosomal positions, peptidyl site (P) and aminoacyl site (A). After formation of Introducto/ry Microbiology-113 121 a

PAGE 115 REMOVED DUE TO COPYRIGHT RESTRICTIONS.

"PC) TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION t C. PRACTICAL ACTIVITIES the peptide bond between the two amino 4 acids attached to the P and A sites on the ribosome, the ribosome moves one codon further along the mRNA chain: the-peptidyl site contains the growing polypeptide site, whereas the aminoacyl site accepts a new aminoacyl-tRNA which carries the anticodon corresponding to the new codon on the mRNA molecule. In this stepwise fashion, each succeeding codon is translated into' an amino acid and eventually into the protein chain. (iv) Termination. The protein chain is completed when the appropriate termination codons are reached on the mRNA molecule. The protein product is released from the ribosomes. 18.0 Microbial Genetics 18.1 1 rocaryotic Chromo- There is one closed circular chromosome The sequence of bases in the DNA may The bacterial chromosome may be visu- so DNA molecule per cell which attaches to be used as a measure of the relationship ofalized by autoradiography. the cell membrane at a specific attachmentdifferent species. The closer the sequence, The -bacterial chromosome attached to site. No structural proteins are associatedthe closer the relationship. the bacterial membrane may be isolated by with the, DNA molecule. detergent lysis of the cell followed by centrifugation.

18.2 Plasmid A plasmid is an extrachromosomal, closed Plasmid replication may be under its own Plasmids may be phy 'cally mapped by circular molecule of DNA which varies fromcontrol or that of the chromosome. cleavage into fragments y enzymes (restric- 0.1 to 10% the size of the chromosome. Cells Plasmids must contain genes for their owntion enzymes) which cut at specific sites. may contain multiple copies. The' plasmid isreplication and may contain genes for trans- Each plasmid yields a distinct and reproduc- not necessary for essential normt cellularfer and for the synthesis of other products. ible fragment pattern. activities. Some plasmids may integrate into, Plasmids can be divided into various the bacterial chromosome. classes depending on the products for which Clowes, R. C. 1973. The molecule of in-they code. These include drug resistance, fectious drug resistance. Sci. Am. April 228: bacteriocin, and toxin synthesis. 18-27. 4Ir Plasmids in an integrated state may mo- bilize transfer of a chromosome to another 'cell. A plasmiday carry a chromosome fragment. In a state of this type it can transfer bacterial genes.

18.3 Eucaryotic Chromo- The genetic material of eucaryotic micro- Eucaryotes contain several basic proteins The chromosome number may be deter- some organisms is located mainly in the form of(histones bound to their DNA). These helpmined in some cases by staining the chro- Introductory Microbiology-117 123 1' I .111 i TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION , C. PRACTICAL ACTIVITIES multiple DNA molecules called--chromo-determine the chromosome structure. Theymosomes and counting and can often' be somes, which also contain bound structuralalso have acid proteins bound to the DNAestimated by linkage analysiti.. and regulatory proteins. The ,cell can bewhich'. .are inVolved in regulation of gone haploid or diploid. expression.

18.4 Eucaryotic Organelles Both mitochondria and chloroplasts eon- Organelle DNA contains genes for ribo. Purified DNA from organelles is generally tain small procaryote-like chromosomes. somal RNA, ribosomal protein and some,isolated by first purifying the organelle. Goodenough, U.i:and R. P, Levine. but not ally transfer-RNAs. (T7-' 1970. The genetic activity of riglochondtia Organelle DNA 'contains some genes for ' and chloroplasts. Sci. Am. November. 223:enzymes such as cytochrome oxidase in mi- 22-29. tochondria.

18.5 Procaryotic DNA DNA is transferred from donor to recipi- Transfer is recognized if the donor DNA Transfer ent cells by three different mechanisms:differs from the recipient DNA, and the transformation, conjugation, and transduc-mixture of cells is plated on a medium on I. tion. Once inside the recipient cell, the donorwhich only recombinants will be able to z/- DNA rimy recombine with the recipientgrovOricf form colonies. chromosome. Plasmids also may be trans- Transfer has been recognized in only a ferred by transformation, conjugation, andsmallininority of all bacteria. However, this transduction. may be because the proper conditions have -not been used to demonstrate transfer. 18.51 Transformation In DNA-mediated transformation, DNA Addition of deoxyribonuclease to the fragmeneleased from lysed cells are taken lysed donor cells prevents transfer by de- up by r cipient cells. grading the donor DNA. The recipient cells must be grown in a certain way to gain the. ability to take up such a large molecule as DNA. Such recipient cells are termed competent.

18.52 Co jugation In conjugation the donor DNA is trans- Chromosome transfer is detected if the ferred during the time that the donor anddonor and recipient cells are plated on a recipient cells are held in contact, by a sexmedium on which neither will grow, but on pilus of the donor. (F+) bacterium. which cells with a combination of the donor Wollman, E., and F. Jacob. 1956. Sex- and recipient cells' properties will grow. uality in bacteria. Sci. Am. July. 195:109- Most cells attach to one another by means 116. of pili. DNA is transferred only as long as the cells remain in contact. 18.53 Transduction In transduction the donor DNA is trans- The bacterial DNA takes the place of ferred from the donor to recipient cells by aphage DNA inside the protein coat of the bacteriophage (phage). phage; thus, the phage is "defective" since it Zinder, N. 1958. Transduction in bacte- doe§ not have all of the genetic information ria. Sci. Am. November. 199:38-43. required for its own replication. Introductory Microbiology-119 1"G 1,"

TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION 0, PRAgPIOAI ACTIVITIES The recipient 'cells art) plated on a me- diuln on which .tVey will not grow unless they are proyided 'with additional genetic information. Only recipient cella that are not lysed by the phage can be transduced.

7 18.6 Mutation A mutation is an inheritableAhange in Mutations can be either spontaneous or .Phenopic expressions of iiititations may e base sequence of DNA. A mutation may induced. be detect d by directobservation of colony ange any characteristic of a cell or a virus. Mutations may or may not cause an ob- color, size, texture,' or other colonial prop- servable change in phenotype dependingerty; use of differential media; solation of upon the extent or change in an enzyme.nutriti?nal mutants by replictqlating; and Examples of types of mutation are as fol- the use of penicillin selection (bacteria) or lows. filtration technique (molds) tolricti for Structural. Loss of ability to produce a mutants. capsule; loss of ability to produce flagella Exposure to ultraviolet (UV) ligJt and X (nonmotile mutant); change in colonial mor-rays may increase the yield of mutant cells. phology. Examples of mutants include pigntation Nutritional. Loss of ability to synthesizemutants as colonies on solid media phage- a biosynthetic enzyme, thereby producing aresistant mutants identifiedascolonies requirement for a particular nutrient orforming on agar plates, which hn e been growth factor not required by the parentsprayed with phage; anlibiotic-resit ant mu- cell; parent is the prototroph and the nutri-tants isolated on medit oontainin tIr anti-- tiinal mutant derived from it called an biotic; and techni' 'using auxotroph. a velveteen pad as ti-pulat vice to A drug- or virus-resistant mutant cantransfer colonies in the i4 act p from grow in the presence of a drug or virus whicha plate containing an enriched me to,... a kills or inhibits the growth of the parent. variety of media lacking,articularowth Loss of degradative enzymes is manifestedfactors. by loss of ability to use a given compound as a carbon source or energy source. Temperature .sensitive mutants grow at 'one temperature but not at another. This is an example of a conditional lethal mutant, that is, an organism that grows only under conditions not required for growth of parent.

1P} 18.61 Base Sequence Changes The base sequence of DNA may be Mutational events involving a change in changed by substitution of a base; additiona single base or base pair are called point or deletion of a single base; or deletion ormutations. Ultimately, all mutations occur addition of a segment of DNA. because of change in the base pair sequence in DNA synthesis. ( I 18.62 Mutation Frequency The rate or frequency with which muta- The mutation rate refers to "sponta- Introductory Microbiology-121 . *. v sr

hi TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION A. ENIIICIIMENT INFORMATION (r PRACTICAL AgTIVITIES Lions occur at specific sites on the DNA nootui" mutations, i.e., those awing with no molecule differs for different genes. apparent intervention on dui part of the investigator. The mutation of one gone is indeponllent of mutation in other genes.

18.83 Dintagenie Agents Chemical and physical agents which in- Mutqgonic agents are used experimen- Comparisons of irradiated and non-irra- crease the rate of mutation by interactingtally to?increase the frequency of mutation;dieted cultures of Serratia Inurement; show with and modifying the DNA are called mu- the dose and/or time of treatment is°sondifferences, in the frequency of mutation tagonic agents. - to cause approximatillY 90%"1 illinifor with regard to lethal mutations, pigmenta- No mutagonic agents are known whic I aretion, and colony morphology. specific for causing the mutation of a particular gene. General categories of mutagenic agents include the following. Substitution of DNA base analogs for normal basen results in an increased incidence of incooration of "ong" bases, Examples of such' analogs 6-bromouracil and 2- aminopurine. its Chemicals which react with DNA bases in such a way an to change their chemical structure result in a change in hydrogen bonding properties and consequent changes in base pairing, addition, or deletion of bases. Examples of such chemicals include nitrous acid, hydroxylamine, monofunctional alkylating agent (ethyl methane sulfonate), and biftinctiOnalalkylatingagents(nitrogen mustards, mitomycin, and nitrosoguani- dine). This group causes cross-linking be- tweeen the two DNA strands, prevents un- 4 winding, and may result in deletion of a segment of DNA. Acridines are inserted between bases in DNA. This causes the addition or deletion if a single base, producing frame shift mu- tions. EJV radiation exposure causes the forma- bon of covalent bonds between two adjacent thymines on the same strand of DNA (thymine dimer), producing changes in base pairing. Ionizing radiation expogure prochtaes multiple effects on the DNA molecule.

Introductory Microbiology--123 139 p

1-08 AND IlTOPICA A. liliMINTIA14 INF AlMATION mucus/11,1NT INFoRmArtilow 0. PRACTICAL AOTIVITItilil Se loot kin Clumps in the enViro en!, may give A popilliltkin funds to remain genetically Two mylloolo ()t coo he used for the. mutant an advantage so'thrIt will gI stable in 1.le absence of an environmental loolotion of eotibiotie.emiannt mot onOi ore faster than the parent andriliplesel'he change seleut Ivo for mutants; each Iiiiitantis gradient agar plates and selective moth. change environment does mit cause or Inequilibrium at a 6'oquency In the total Induce mutations HOlilelo' for preexisting population proportional to its mutation rate, udants.\ llaci:orlit are more adaptable to new un- vironments than etwaryotie cells because their haploid nature makes possible the (in- inedipte expression of mutations, In midi- tion, lbacterhil growth rates are faster; and high population density increasee the probability of obtaining a wider variety of mu- r tants. Moreover, every coil cite potentially give rise to a new strain. Some mutations are "selective" and confer an advantage on the organism dander certain environmental conditions; otliks are "unselktive" and confer neither an advantage' nor disadvantage on the mutant cell

compared to the parent. . Selective mutants are easier to detect and isolate, and direCt methods May be used; indirect methods- for examination of very large numbers of cells is required to detect and isolate unselective mutants. Principles of natural selection as it occurs in the environment are used in developing methods ,of direct laboratory selection...

I DNA Repair Microorganisms have enzymatic mecha- Photorepair or 11 air mechanisms Expose UV-irradiated cells to visible light nisms to repair DNA. require the presence oisible light. Thisand then compare frequency of mutation mechanism for repair of UV-induced dam-with cells kept in the dark after UV irradia- age involves breakage of the covalent bondstion. joining the thymine dimers; it requires a specific eRynne system. Dark repair mechanisms not,require visible light. Specific cellular enzymes excise damaged portions of a single DNA strand and other enzymes and ,then synthesize a segment of DNA complementary to the normal DNA strand.

Chemical Methods of a Microbial Control Introductory Microbiology-125 131 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION Laboratory experiments may be used to 19.1 Modes of Action Microbicides are substances that kill mi- For all practical purposes, microbicides croorganisms. and disinfectants are synonyms for agentsshow that most microbicidea are chemical that are commonly used,eor killing of all agents that kill growing microorganisms, but kinds of microorganisms. However, their usenot necessarily heat-resistant spores, and is generally confined to disinfection of inan- that many solutions of heavy salts kill a imate objects. variety of microorganisms with which they come in contact. Chemicals commonly used as inicrobio- 19.11 Growth Inhibition Microbiostatic agents are substances that inhibit the growth of microorganisms. static agents include aniline dyes, such as stains, e.g., crystal violet, which may react with cells (especially gram-positive cells) and prevent them from growing. However, cells can be quickly reactivated by washing off the dye. Other frequentlyusedmicrobiostatic agents include synthetic detergents, such as quaternary ammonia compounds. Experiments may be used to show thatI. 19.12 Lethal Effects Microorganisms may be killed by various Solutions of heavy metals, strong acids, means, including protein denaturation, cell or halogens may precipitate protoplasmiccells subjected to certain concentrations of lysis, alterations in cell membrane permea-protein (coagulation or denaturation). heavy metal solutions, phenol and phenolic bility, and interference with cellular metab- Coagulation of cellular protein is pre- solutions, high osmotic pressure-exerting so- olism and/or reproduction. ceded by breakage of hydrogen or disulfidelutions, synthetic detergents, and soap may bonds in the secondary and tertiary struc-no longer be able to produce colonies on ture of proteins (partial denaturation). Dam-suitable media. age is irreversible. Damage to cell walls, especially those of gram-positive bacteria, may be caused by the enzyme lysozyme (found in tears, leukocytes, etc.). Disintegration of the cell wall is followed by cell lysis. Phenolic compounds, synthetic detergents, soaps, and other related Substances tend to destroy the selective permeability of membranes of the cell and permit leakage of cellular constituents, especially N and P, all of which result in cell death. Interference with reproduction may be accomplished by inhibitingnucleicacid ( DNA or RNA) synthesis. Such agents may inhibit nucleotide formation or interfere withpolymerization of nucleotides into nucleic acid, e.g., 5-bromouracil, an antimetabolite for thymine. Introductory Microbiology-127 1:;3 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION 13. ENRICHMENT INFORMATION C. PRA TICAL ACTIVITIES 19.2 Factors Affecting the Microbicide and microbiostatic agent ef- As a rule, the longer the contact time with Actionof Chemicalfectiveness is influenced by factors such asthe chemical agent, the greater is the inhi- Agents contact time, concentration, pH, tempera-bition and destruction of cells. ture, nature of microorganisms, and varying Generally, the higher the concentration susceptibility of the agent within the targetof chemical agents, with some exceptions, population. e.g., ethyl alcohol (ETOH is most effective at a 70 to 75% concentration), the more effective is the killing or inhibition. As a rule, the lethal or toxic action of an agent is enhanced by increasing the concentration of H' or 01-1- ions. The warmer the agent, the more effective itis because chemical reactions in general are more rapid at higher temperatures. The types, age, and numbers of microorganisms are important factors to consider. Generally, younger cells are more susceptible than older cells. Growing cells are subject to interference with metabolism, whereby nongrowing cells would not be affected. The membranes of older cells tend to he less permeable to chemical agents than the membranes of young, dividing cells. Vegetative cells of spore-forming Orga- nisms are more susceptible than dormant spore stages. In a mixed pop ation some species are more susceptible to ertain agents than others, i,e., lysozyme tends to attack gram-positive cell walls. Generally, human pathogens are more sensitive to certain agents than the normal flora of the human body. Each agent behaves differently under different circumstances.

20.0 Physical Methods of Microbial Control 20.1 Modes of Action Physical agents may inhibit or kill micro- Intense moist heat coagulates protein, Experiments may be used to show that organisms in some or the following ways:and dry heat oxidizes protein. Damage is high and low temperatures, incineration, ra- coagulation of protein, enzyme oxidation, irreversible. diation, and ultrasonic waves may slow depression of metabolism, cell hydration, Moist heat is more efficient than dry heat. growth, inhibit, or kill microorganisms. and DNA injury and destruction. Moist heat at 120 to 121°C for 15 minutes (only obtained by autoclaving) kills micro- Introductory MicrobiologyI29 1;;L)L)

0 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES organisms, including spores, but dry, heat requires a much higher temperature for a longer period (160°C for abodt 2 hours to sterilize laboratory glassware). Most cell enzymes are denatured at boiling temperatures. Enzymes in spores are protected from heat because of the structures of the extensive outer coverings or reduced cell water content (thick spore cortex). Temperatures below optimum for growth depress the rate of metabolism, and if the temperature Is sufficiently low, growth and metabolism cease. Dessication is a process by which cell water is removed. Excessive dehydration causes cessation of metabolic actiVity. Loss of cell water may occur if the cells are placed in a highly osmotic solution. In such a solution cells tend to collapse (plas- molysis) due to excessive loss of cell water to the solution outside. However, freeze- drying (lyophilization) of the cell may remove water but the process does not cause injury to the cell; hence, the cell may be viable for many years. Various types of radiation are used in the control of microorganisms. UV light (260 Om)isused inkilling microorganisms. Shorter waves (185 nm) are more rapidly lethal but are difficult to control and apply. NRadiation such as UV, X rays, and gamma rola, tends to damage nucleic acidif such treatments either produce thymine dieters or break H bonds of nucleic acid, resulting in either the killing, injuring, or mutating of the cells. As a rule, the shorter the wavelength, the more damage to the integrity of the cells.

20.2 Factors Affecting Modes Factors affecting the modes of action of The concentration, dosages, etc., of the of Action of Physical physical agents include the nature and in- physical agent have an influence on its effec- Agents tensity of the physical agent, exposure time,tiveness in the killing or inhibition of micro- the type of microorganism, the physiologicalorganisms; for example, moist heat is more Introductory Microbiology-131 TOPICS AND SUBTOPICS A.:ESSIOITIAL INFORMATION B. ENRICHMENT INFORMATION C, PRACTICAL ACTIVITIES. state of the organism, and the surroundingefficient than dry heat and gamma rays are etwironment.: more lethal than X rays. Contact or exposure time is inversely proportional to the intensity of the agent applied, e.g., the higher the temperature, the lesstime,required; the shorter the wavelength, such as gamma raysothe less contact time required.

TY- If cells are surrounded by efficient heat- tranSferring material, they are easily inactivated or destroyed. If they are surrounded by poor heat conductors, such as soil, they tend to survive correspondingly longer.

21.0 Antimicrobial Chem- otherapeutic Agents 21.1 ModesofActionof Antimicrobial chemotherapeutic agents Arsenical compounds, quinine, sulfona- Laboratory experiments may be used to Chemotherapeutic are chemicql substances used in the treat-mides, and antibiotics are used as chemo-demonstrate the sensitivity of microorga- Agents ment ai1d control of various infectious dis-therapeutic agents, i.e., substances used innisms to chemotherapeutic antimicrobial eases. treatment of diseases by destroying or pre- agents. venting the activity of parasitic organisms without major injury to the cells of the host. Historically, the chemotherapeutic agents arsphenamine and rieoarsphenaming were used in control of syphiliit,' ,bilighey have been replaced by penicillin. Quinine and its derivatives from the bark of the chinchona tree are useful in the treatment of malaria. Antibiotics such as tetracycline anitichlor- amphenicol are broad-spectrum antibibtics, that is, they are able to inhibit many gram- positive and gram-negative organisms. Synthetiq chemotherapeutic agents are those compounds which have been found to have antimicrobial activity, for example, ni- 4. trofurans. Such agents do not occur naturally but have a broad spectrum of action. Isonicotinic acid hydrazide has been used for treatment of tuberculosis, and nalidixic acid has been used for treatment of urinary tract infections caused! by gram-negative bacteria. 1 4 0 introductory MicrobiologyI33 1 3 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES LET Definition Ingeneral,naturallyoccurringsub- Many microorganisms produce antibiot- stances, as distinguished from syntheticallyics which provide protection against other produced compounds, are referred to as an-microorganisms, i.e., they excrete .a specific tibiotics. substance that interferes with the metabo-. lism of other microorganisms, thereby-preventing their growth or killing them. Many antibiotics (naturally produced by microorganisms) are not produced synthetically on a commercial scale.

21.12 Static and Cidal Effects Chemotherapeutic agents can either, in- The actions of antibiotics include the inhibit the growth of or kill microorganisms.hibition or interference with cell wall syn- Those inhibiting growth are called micro- thesis, DNA synthesis, or injury to cell mem- biostatic agents and those killing are called branes. microbiocidal agents. Penicillin, produced by Penicillium chry- sogenum is active primarily against gram- positive bacteria and inhibits their cell wall synthesis. Tetracycline is produced by Streptomyces aureofaciens and is a broad-spectrum antibiotic and interferes with protein synthesis. Novobiocin, produced by Streptomyces griseus, attacks primarily gram-positive bacteria and inhibits DNA polymerization. Griseofulvin produced by S. griseus attacks pathogenic fungi and interferes with fungal cell wall and nucleic acid synthesis. Other microbiostatic agents include sul- fonamide drugs which act as structural analogs of paraaminobenzoic acid. Many different cellular enzymes (enzymes in the glycolytic system, citric acid cycle, and cytochrome system) are potential targets for an inhibitor. For example, cy- anamide inhibits glycolysis; trivalent arsenic compounds may block the tricarboxylic acid cycle; and many halogens (CI, B, I) and other oxidizing agents may alter orinacti- vatethe enzymes.

22.0 IntroductiontoIrni, munity (Resistance) 22.1 The Nature of Immu',, Immunity refers to the ability of an indi- nity vidual organism to resist and/or overcome Introductory Microbiology-135 11° 1 1 1 . C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION ° B. ENRICHMENT INFORMATION a disease agent to which most or many ofits species are"susceptible.

22.11 The Immune Response The immune response is a specific adap- tive response to foreign substances and materials which include infectious disease agents, certain chemicals, and cells. There are two types of immunity, cellular and hti- moral, which may aid in eliminating foreign material or rendering it harmless.

22.12 Lymphocytes Immunity depends upon specifically reactive white blood cells called lymphocytes. Most lymphocytes are found in the spleen, lymph nodes, and Peyer's patches.

Lymphocytes, which are responsible for A diagram showing the origins of T and 22.13 B- and T-Type Lymph° B cells and their functional interactions may cytes humoral immunity (the production of antibodies), are referred to as bone marrow or B be used. Scanning micrographs of these cells cells. Those cells responsible for cellular im- also may be helpful. munity and influenced by the thymus gland are called T cells (Medical Microbiology, glik7ic 14.14).

!a 22.2 Antigens (Immunogens) Antigens are substances which, when in- trodueed into a host, will provoke a detectable immune response. C

22.21 General Properties Antigens are perceived as foreign to a host's immune system. The host is normally tolerant (nonreactive) to its own tissues and does not consider them as foreign.

22.22 Chemical Composition Some antigens are proteins, whereas others may be polysaccharides. Nucleic'''Et cids and certain lipids also are antigenic in some cases(MedicalMicrobiology,Subtopic 14.22).

22.23 Antigenic Determinants Antigenic determinants are those parts of . the antigenmolecule that are actually involved in binding with antibody (Medical Microbiology, Subtopic 14.234. Introductory Microbiology-137 1 1 I

1 -4A 3 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION . B. ENRICHMENT INFORMATION 22.24 Haptens A hapten is generally a small molecule which is incapable of initiating an immune response. Such molecules Can become antigenic through linkage with large carrier molecules (Medical Microbiology, Subtopic 16.24).

22.25 Examples of Antigenic , Bacteria (or their parts) or virus particles Materials may serve as antigens. Most exotoxins are proteins. Their toxoids (altered toxins), whiCh have little or no toxic properties but retain antigenicity and antigenic determinants, upon injection bring about the formation of antitoxins. These molecules serve to protect against the effects of toxins. Microbial antigens, such as whole cells (killed or attenuated) or viruses, when injected into animals including humans, induce a response resulting in the formation of specific immunoglobulins.

22.3 Immunoglobulins (Anti- Antibodies or immunoglobulins represent bodies) a specific group of heterogeneous proteins which are formed in responie to antigenic stimulation and react specifically with the antigen that provoked their formation. lin- munoglobulins are found in body fluids such as blood, lymph, and tissue fluids.

22.31 Classes of Immunoglob- Five major classes of human immuno- Wins globulins are recognized, each with unique chemical properties and functions. The five classes'are termed immunoglobulin G (IgG), IgA, IgM, IgD, and IgE (Medical Microbi- ology, Subtopics 14.34-14.367). Most classes of immunoglobulins have been further divided into subclasses.

22.32 Mechanism of Action Antibodies may interfere with or inhibit the activities of foreign cells and substances through various mechanisms, including increased opsonization, which facilitates the phagocytosis of microorganisms and other materials; cellular destruction (cytolysis) of Introductory Microbiology-139 1 15 C. PRACTICAL ACTIVITIES TOPlegyID SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION certain microorganisms and other cells in the presence of complement (a system of serum protein's which is activated by certain antigen and antibody reactions); neutraliza- -e- a tion of toxins; and prevention of the attachment of microorganisms such as viruses to susceptible host cells.

22.4 Host-Parasite Relation Oiganisms live in various relationships to ships each other in nature. In the human body, host-parasite relationships may exist in several body sites. One of the undesirable rela-, tionships is parasitism. This is the association in which one organism lives either on or in another form of life (the host) atthe expense of the host. Natural resistance of a host depends on a 22.41 Host Resistance The ability of a host to prevent or overcome the effects of a disease- large number of factors, including the host's microorganism (pathogen) is relatto angenetic constitution, general health, nutri- individual's immunity. Such an ahy totion, and environment and social conditions. resist pathogens may be attributed to two Acquired resistance is usually due to the factors: natural resistance (factors inhexentindividual's experiences of infections and im- in the body) or resistance acquired duringmunizations(IntroductoryMicrobiology, Jlife (acquired immunity). Topic 23.0, and Medical Microbiology, Sub- tppic 14.5).

Body defense mechanisms include physi- 22.43 HostDefense Mecha- Invading pathogens must be capable of nisms overcoming various anatomical and physio-cal barriers (intact skin), cellular factors, or logical barriers of the host's body beforebiochemical factors (antigen-antibody reac- they can establish an infectious process. tions)(MedicalMicrobiology, Subtopic 13.5). Physical resistance mechanisms include intact skin, mucous membranes, and all cases of flushing or Blearing of theinvading microorganisms from the anatomical site. Phagocytosis by macrophages incorporates both physical and biochemical mechanisms. Biochemical factors include reactions involving various enzymes, body fluids,ter- feron and antibodies with invading mi o- organisms, or those reactions between microbial toxins and antitoxins. The normal flora, of the human body, S Introductory Microbiology-141 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION which is found a the skin and mucous membranes and other locations without causing disease, also contributes-ko protecting the host against pathogens (Medical Mi- crobiology, Subtopic 13.3).

Virulence The disease-producing capacity or viru- Pathogens which fully express virulence 22.5 Microbial increase Factors lence of organisms is determined by geneticmay be able to grow and multiply, properties which may be fully expressed un-in local lesions, or spread throughout the der certain environmental conditions. This host, lie able to tolerate phagocytic action of virulence may be due to various factors, leukocytes, be able to prevent production of including an organism's ability to produceantibodies, and be able to produce sub- toxic substances (toxigenicity), invasiveness,stances injurious to host tissues (Medical and ability to survive within a host. Microbiology, Subtopics 13.65-13.67). Toxins interfere with normal cellular ac- 22.51 Microbial Toxins Microbial toxins aritpoisonous substances produced by various microorganisms,....Ex- tivities. amples of such toxins include exotoxins, enterotoxins,endotoxins,andmycotoxins (Medical Microbiology, Subtop* 13.63- 13.634).

2272 Invasiveness Invasiveness is the ability of an organism to spread among the tissues of a host. This virulence factor involves not only counter- ' actiug normal host defenses but bepg able to spread from the original site of penetration.

23.0 States Of Immunity And Diagnostic Im- munology 23.1 Cell-Mediated Immu- Cell-mediated immunity is regulatedy nity T-type lymphocytes and is dependent on tlhe presence of the thymus gland at birth. Im- mune responses mediated by T-type lym- phocyteslire important in combating fungal parasites, chronic bacterial infections, and certain viral infections and are responsible for many accelerated rejections of allografts (grafted cells or tissues from genetically different individuals of the same species) (Med- ical Microbiology, Subtopic 14.4). Introductory Microbiology-143 150 1 3 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 23.11 'reel' Sensitization A T cell which comes Into contact with Sensitized T cells provide protection to an antigen for which it is specific is-said tothe host through cellular mechanisms such be committed or sensitized. Sensitized Tas promoting phagocytosis (by producing cells multiply after exposure to such anti-lymphokines such as chemotactic factor and gens and give rise to clones of cells, whichmacrophage-activating factor) preventing produce a variety of biological effects. viral multiplication via interferon, and con- tracting and destrOying tumor cells and other foreign cells, including virus-infected . cells. In cell-mediated immunity, the antigen may combine with particular lymphocytes (activated), and such cells enlarge, divide, and provide mediators (lymphokines) which induce local inflanuruition and participate in ( the elimination of the foreign material. A classical example of this response is the immunity to tubercle bacilli (Medical Micro- biology, Subtopic 14.43).

23.2 Humoral Immunity Humoral immunity is mediated by anti- Individuals who have recovered from bodies. B-type lymphocytes are responsiblemeasles or mumps or who have been im- for antibody production. munized with diphtheria, pertussis, and tetanus (DPT) antigens are protected from these diseases because they-have produced specific antibodies against each of the causative agents and/or toxoids. Certain disease states are diagn sed in the laboratory1/4by demonstrating di -,presence of specific antibodies in a patient's serum, e.g., syphilis and infectious mononucleosis. trr 23.21 Primary Response The immune response that occurs after. initial exposure to a given antigen is called the primary response. With humoral ,irn- munity, IgM is usually...44e4100iiiiibody produced and is detectable in alitut 1 week.

23.22 Secondary Response Upon reexposure to the same antigen used in an initial exposure, The body produces a greater antibOdy response in a shorter period of time, known as the secondary or anamnestic: response. IgG, which is producein this resperise, is detectable for Introductory Microbiology-145 r . TOPICS' ANII 131.440PICS A, ESSENTIAL INFORMATION ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES _ long periods and is associated with a long- .11 .r. lasting protection (Medical Microbiology, . .. Subtopics 14,512-14.514). --1 /, .. z'. , 23.S "GeneralStates of Im- Native and acqUired immunities are rec- ' :rnunity ognized as general states of immunity. .' .;*( '..' - 23.31 Native Irhmunity Native immunity refers various factors Resistance *to infection varies with the involved with species istance, racial re-species of animal or plant; e.g., infectious sistance, and individuresistance. diseases of cold - blooded animals, with a few exceptionsA,rarely occur in warm-blooded animals. racial resistance refers to genetic factors which influence disease resistance or susceptibility and is an inherited trait, e.g., Native Americans are less resistant to tuberculosis than Caucasians. The principle of specie's resistance is extensively used in agriculturethe breeding of resistant plants is essential in crop production.

23.32 Acquired Immunity Immunity resulting from an exposure to antigen is said to ,be acquired. Such immunity to a specific antigen may be actively or passively acquired.

23.321 Active vs. Passive Forms Active immunity may develop from an infection or artificially by an immunization procedure such as vaccination. The immune response here is produced by the host. In passively acquired immunity, the immune response is not produced by the host. Preformed antibodies may be acquired naturally from other sources (Medical Micro- biology, Subtopics 14.5-14.52).

23.33 Types of Acquired Im- Acquired immunity may be of four basic Example' of the four basic types of immunity types: naturally acquired active, naturallymunity include the following. Naturally ac- acquired passive, artificially acquired active, quired active: natural exposure to an infec- and artificially acquired passive. tion. Naturally acquired passive: placental transfer of IgG. IgA in the cOlostrm of -mother's milk. Artificially acquired active: injection or other artificial applica)tion of attenuated microorganisms, or toxoids. Ar- Introductory Microbiology-147 3 :J A

t! Topics AND SUBTOPICS I A. 'ESSENTIAL INFORMATION B, ENRICHMENT INFORMATION U, PRACTICAL ACTIVITIES 1 tificially acquired passive: injection of antibody-containing serum from another individual orinnintl. , I

23,4 ImmuneDisorilers and Immune disorders result from immuno- Certain individuals are unable to combat Hypersensitivity tleflciencieS such as lack of functional B-typecertain infectious agents because they lack lymphocytes, or 'P -type lymphocytes, oreither humoral immunity and/or cell -medi- Y both. ated immunity. ) Examples of primary immunodeficiency include the following: Lack of functional B lymphocytes results ;j in poor ,..mmoral immunity but normal cellular finittainity; Lack of functional T lymphocytes results in poor cellular immunity and some decrease in humoral immunity (where T- lymphocyte cooperation is involved in antigen recognition); Lack of functional T lymphocytes and B lymphocytes results in little or no humoral immunity or cellular immunity. As a rule, the immune system may de- N, crease in function with advanced age.

23.41 States of rsensitiv- Hypersensitivity or allergy may result Hypersensitivities can be experime tally ity from adverse effects of humoral immunityshown in laboratory animals, e.g., anap axis (immediate1` hypersensitivity) or cell-medi- and graft rejection. ated immunity (delayed hypersensitivity). In cases of immediate hypersensitivity or Clinically,,the states of hypersensitivity areallergy, the production of antibodies against organized into type I (anaphylaxis), type IIantigens causes more damage to the host (cytotoxic reaction), type III (immune com- than good. Reactions of this type can be plex reaction), and type IV (delayed hyper-observed within minutes to hours after ex- sensitivity) (Medical Microbiology, Subtop- posure to allergens. icS 14.61-16.64). There are three mechanisms of immediate hypersensitivity: (i) IgE mediated (anaphylactic type of type I), (ii) antibody-dependent cytotoxicity (type II), (iii) immune complex mediated (type III). In typeI,antigen-antibody reactions bring about the massive release of histamine and related substances which trigger inflammation as well as cause the constriction of smooth muscles in veins, arteries, and bron- chioles; hence, immediate injections of anti- Introductory Microbiology-149 TOPICS AND SUBTOPICS A. R8814NTIAI, INFORMATION R, NNIIICIIMENT INFORMATION 0, PRACTICAL, AOTIVIIIEH histamines may be necessary to alleviate.the symptoms of the hypersensitive reaction, Type I hypersensitivity reactions may be inducud.by the inhalation of ragweed pollen or the iogention of certain foods, These substances/react with IgE molecules attached to mqiit collo in the tissues or to ban° hila in the bloodstream, and therebybringabout the symptotns of allergy, "Allergy shots" are administered to allergic individuals to stimulate production of specific antibody (IgG) in their serum. IgG antibody neutralizes the allergen and is given little or no opportunity for contact with the mast cell-attached IgE. Diagnostic skin teats such as those used as a presumptive test, for tuberculosis are based on delayed hypersensitivity. Delayed hypersensitivity (type IV) does not involve circulating antibodies (the same mechanism ascellularimmunity).Re- sponses are usually seen within hours to days after exposure to allergens.

23.5 Diagnostic Testing Various diagnostic procedures are used widely to demonstrate the presence of both antigefts and antibodies in body fluids and/ or tissues. The results of such tests are of medical (clinical) importance to the identification of infectious agents, some chemicals, and various cellular antigens or their corresponding antibodies. In all diagnostic immunological tests, the identity of one component, antigen or antibody, is known.

23.51 Identificationof Anti- An unknown antigen can be identified or gens typed by means of known antibody.

23.52 Identificationof Anti- The presence or absence ofantibody and body the titer (level) of antibody can be determined by employing a known antigen-antibody reaction. (Medical Microbiology, Sub- topics 20.2-20.23). Introductory. Microbiology-151 1::3 TPI(.H ANI) H11111101010H A, ERHIIINTIAli INFORMATION II, 11111t1(111MHNT INFOIIMATION (I,

113,13 inuilimologival Tests A W111 1.1111$41 Of WAN 81111 1411111114of thesetests exist to tieleet, sptiellie antigens or antibodies in opeolinetts, Theme include agglutination, precipitation, inuntinothioren, centantlisly techniques, ratiloinuoutions nay (RIM, enzymelinited inmmiloasorbent assay (RURA), and skin tests, Mi- crobiology, Huhtopics 20,31-20,41,

24,1,0 Epidemiology and In- feetioun Diseases 24.1 Principles of Epidemiol- Epidenli010gy is a branch of Klemm that Factors that influence the distribution of gy deals witli the Vill1H011of disease awl with the disease-producing agents (pathogentiLwithin factors that influence the distribution of dis-a population include changes in the immu- ease-producing agents within populations. nity status, age profile, and vector characteristics of the host population. Epidemiological studies associated with infectious diseases include portal of entry and exit, survival rate outside of host, carrier state,microbial _population invasiveness, toxin production,phismid transfer, and host population. The interaction of host and parasite populations, geographical distribution, prevalence of disease, seasonal distribution, etc., also are significant. The suspected causative agent of an infectious disease must be isolated and identified by laboratory tests or incriminated 14% strongly by specific symptoms of infected individuals. The presence of appropriate vehicles such as fomites and/or -vectors in the environment will enhance the distribution of pathogens. Three interconnected factors, host, agent, environment determine the severity of epidemics caused by microorganisms. A successful pathogen may produce disease, but usually does not kill the host.

24.2 Mode of4Transtnission For a pathogen to cause a disease, it must Infected animals, humans, and contami- travel from habitats in a given reservoir, benated objects may serve as reservoirs of transported to a susceptible host, and colo- disease-causing agents. Introductory Microbiology 153 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES nize or exhibit its virulence within a new Infectious agents may be transmitted to host. susceptible bosts by direct contact or by vectors such as arthropods. . Cysts and spores of microbial pathogens enable infectious' agents to survive adverse conditions that.would be destructive to vegetative cells during dispersal from reservoir to susceptible host. Bacterial endospores are more resistant to deOruction by antimicrobial agents than are vegetative cells. Protozoan cysts, such as those of Giardia lamblia, are not destroyed by chlorine at concentrations used in municipal water pu- rification facilities. The environmental conditions associated with a susceptible host influence the wanner in which the disease agent interac with host tissues.

24.21 Direct and Indirect Con- Processes that involve the transmission of Direct transmission involves events such tact microorganisms include two broad groups ofas biting, licking, kissing, and those which mechanisms, namely, direct contact and in- involve an exchange of mucus secretions be- direct contact. twe6n an infected person or animal and a suscePtitire host. Infectious agents are transmitted to susceptible individuals by mechanical and biological means. Mechanical means include contact with contaminated inanimate objects (fomites), nuclei of mucus and saliva, water, air, soil, food, dairy products, sewage, etc. (indirect transmission). A nosocomial infection (hospital-acquired infection) is a good example of contact transmission of a pathogenic agent. Here as well in other situations, the agent may be acquired from air, food, or contaminated apparatus or directly from infected individuals. Biological means of disease transmission involve living vectors that transmit pathogens directly from one susceptible host to another. Certain infectious diseases primarily as- 1 13 Introductory M icrobiology 155

11i1.) .i, C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION sociated with lower animals may be trans- 1 mitted to humans by a variety of vectors (mosquitoes, ticks, fleas, etc.). Such diseases are examples of zoonoses. A portal of entry to the body will often determine the nature and severity of a disease. Syphilis may be acquired through sexual contact witPan asymptomatic individual having a symptomatic or asymtomatic form of the disease (direct contact transmission). Diphtheria may be acquired by inhaling droplets (nuclei of mucous secretion droplets containing virulent infectious agents) expelled into the air, through sneezing or coughing, etc. (indirect contact transmission). Rabies is most frequently spread to humans and other mammals by the bite or the saliva of an infected animal.

24.3 Representative lnfec- Pathogenic agents have .varying abilities Virulence factors include: adherence to tious) Microbial Agents to injure body tissues and/or alter body host cells; tissue affinity (certain microor- functions by a variety of mechanisms. ganisms have a particular affinity for certain cells and tissues which they may injure or destroy); toxic factors; the production of exo- or endotoxins or both (exotoxins as a rule are more detrimental to the host than endotoxins); enzymic and other factors contrib.- uting to virulence (e.g., production of hyaluronidase [spreading factor],lecithinase [alpha-toxin, which destroysredblood cells], collagenase [destroys collagen]; coagulase [clotting factor]; fibrinolysin [stiepto- kinase, dissolves human fibrin]; leukocidin [kills leukocytes in vitro]; hemolysin [liber- ates hemoglobin from red blood cells]); and capsular material (encapsulated strains of certain bacteria are pathogenic, whereas noncapsulated stcains are nonenpathogenic, but there are exct6ptions).

24.31 Infectious States and In- Infectious diseases are caused by patho- Many pathogens have a rather specific toxications gens from the following microbial groups:host range. lnt roductory Microbiology-157 1'' 3 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION bacteria, fungi, protozoans, viruses, and the Certain bacteria, fungi, and viruses may multicellular worms or helminths. Some mi- be capable of infecting more than one kind crobes such as algae and certain bacteriaof animal host. cause noninfectious diseases statessuch as As a rule, most plaht pathogens are non- poisonings (intoxications). pathogenic to animals and vice versa. Bacteria, algae, andlungi share a similar molecular basis of disease, i.e., produce toxins and enzymes causing injury to the cells or tissues. Gonorrhoea caused by Neisseria gonorrhoeae and syphilis (Treponema pallidum) are examples of bacterialdiseases. Histoplasmosis (caused by Histoplasma capsulatum), and coccidiomycosis (Cocci- dioides immitis) are examples of systemic mycoses or deep-seated fungaldiseases. Protozoans may invade the body cells and tissues, multiply within them, and cause destruction of cells and tissues, which may result in a variety of diseases. Malaria (caused by Plasmodium vivax) and amoebic dysentery (Entamoeba lusty- lytica infection) are examples of protozoan diseases. Hepatitis (caused by type A and type B viruses) and German measles (caused by rubella virus) are examples of viral diseases. Trichinosis or pork roundworm (caused by Trichinella spiralis) and infection with tapeworms (Taenia saginata, beef tapeworm; T. solium, pork tapeworm) are examples of helminth infections. Prophylactic measures may involve im- 24.4 Control Measures Infectious diseases are controlled by processes that involve one or a combinationofmunization or the administration of chemo- the following: chemotherapy, prophylactictherapeutic agents in certain extreme cases. immunization (passive measures), and envi- Many viral diseases are controlled by ex- ronmental sanitation. tensive immunization programs (poliomyelitis, influenza, measles, mumps). Smallpox has been eradicated by means of a massive immunization campaign. DPT vaccines are administered to children to protect them from diphtheria, whooping cough, andtetanus. Introductory Microbiology 159 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES Those infectious diseases requiring a spe7 ci0c vector may be controlled by the eradi- cation of the vector (yellow fever, malaria, plague, etc.). Antiseptics are, used to reduce the number of pathogens on host tissues, and disinfectants are used to remove and destroy pathogens on inanimate materials. Some diseases are controlled by chemotherapy and the detection and treatment of carriers since immunization against such diseases is ineffective, e.g., syphilis and gonorrhoea. Certain viral diseases are controlled by the elimination of carriers and prophylactic immunization (rabies), whereas some animal diseases are controlled by the elimination of diseased individuals as well as carriers (foot and mouth disease). Typhoid fever, for example, is largely controlled by good environmental sanitation, whereas poliomyelitis is controlled by immunization and environmental sanitation. Diphtheria may be controlled by prophylactic immunization and improved environmental sanitation. Postinfection measures may involve different forms of immunizatiqfi naterials, the use of chemotherapeutic agents, and im- provements in environmental sanitation.

25.0 Environmental Microbiology 25.1 Sanitation Microbiology Microorganisms are an integral part of Most airborne bacteria are transient, i.e., Laboratory examination of a variety of air, water, and solid waste pollution. Micro- no bacteria use air as a habitat. Likewise,samples may be used to show the presence organisms are ubiquitous. Polluted air, wa- solid waste may contain a heterogeneousof microorganisms. ter, and land almost always contain numer- group of organisms, whereas water or waste- ous types of organisms. Some of these orga- water may contain indigenous as well as nisms are pathogenic to plants and to ani-transient populations of organisms. mals, whereas others are saprophytes in- Increasedorganicloadingoflakes, volved in decomposition of pollutants. streams, and estuaries has taxed the natural Taber, W. A. 1976. Wastewater micro-system to recycle the waste material, result- biology. Annu. Rev. Microbiol. 30:263-277. ing in pollution. Water pollution can be detected by indicator species (i.e., coliforms) Introductory Microbiology-161 1 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES and other biological and chemical tests, i.e., biocemical oxygen demand (BOD) and chemical oxygen demand (COD). a Open dumping of solid waste in a community is an open invitation to the spread of disease by flies and rats. Solid waste, which may be constituted of fibers, metals, and animal and human wastes, may contain a number of pathogens. Improperly disposed f liquid or solid wastes may produce not only a health hazard but undesirable odors from decomposition as well. Decomposition of buried waste (sanitary landfill) involVes aft - aerobic microbial activity. Properly buried material is slowly decomposed to various 0 compounds -or elements and recycled into the environment without causing undue harm.

25.11 DecompositionActivi Microorganisms serve a vital function in Microorganisms play an indispensible role ties the elimination of some environmental pol- in the decomposition of organic materials lutants from air, water, and land. found in Source water (water for drinking Crites, R. W., and C. G. Pound. 1976.purposes). Pathogenic bacteria are elimi- Land treatment of municipal wastewater.nated by chlorination or ozonation after Environ. Sci. Technol. 10:548-551. physical-chemical treatment.

25.2 Aquatic Microbiology Microorganisms are present at all depths Microorganisms are principal agents in in the aquatic environment. No naturalbiological wastewater treatment (trickling aquatic environment is devoid of optenisms. filters, activated sludge processes). The or- Many such microbes are introduced by air ganisms stabilize noxious material as well as or soil, from surrounding environment, or by material which may fertilize receiving bodies human activity. of water. Pathogenic organisms are destroyed before water is discharged into receiving bodies by various disinfection processes, e.g., chlorination, ozonation, etc. Microorganisms may be distributed in all parts of a lake. The littoral zone (along the shore with good light penetration to the bottom) har- 0' bors more bacteria per milliliter than other parts of the lake because the nutrient concentration is higher. I riThe tic zone (open area: depth an rbidity determine the amount of light .1;,J ory Microbiology TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION . C. PRACTICAL ACTIVITIES penetration), the largest population Of microorganisms is found in the top centimeters, and it supports both autotrophic anci'l*ter- otrophic organisms. In the profunal zone4,(deeper regions of open water) the population of microorganisms is smallest. The benthic zone (bottom area: little or no light, a zone of decomposition) harbors large numbers of microflora and -fauna. Many are anaerobic', active decomposers of the sedimented organic material. Aquatic microorganisms may be divided into three groups: (i) Planktonic (floating and drifting organisms, mostly algae [phytoplanktor] and protozoans [zooplankton]). (ii) Nektonic (swimming organisms). (iii) Benthic (bottom swelters). Indigenous microorganisms may be photosynthetic, chemoautotrophic, or chemoorganotrophic. Hot springs may contain thermophilic organisms able to tolerate a temperature as high as 90°C.

25.21 Food Chains Micoorganisms form the basis of activities Photosyntheticmicroorganisms(blue- such as food chains and decomposition cy- green bacteria and others) may function as cies within aquatic environments. primaryproducersinthefoodchain, whereas other microorganisms serve as decomposers within the aquatic environment. The components of a pond food chain include: primary producer (plankton algae) primary consumer species (minnows or small fry of larger fish) -- secondary con- , sumer species (bluegill or catfish) + tertiary consumer ikpecies (bass or pike) dead plants and animals attacked by decomposers (bacteria and aquatic fungi).

25.3 Soil Microbiology The soil serves as a major habitat for The soil contains water and minerals, in Laboratory techniques may be used to many autotrophic and heterotrophic micro-addition to organic material from the re-determine microbial numbers within soil.

Introductory Microbiology I 65 1.y a

TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES organisms. Soils are, formed as a result ofmains of dead plants,: animals, insects, andTopsoil may harbor a billion organisms per geological and biological processes. . humus. It is a dynamic habitat, a constantlygraM. changing microbial population. Distribution and function of soil microorganisms: (i) Bacteria and other microorganisms degrade complex organic compounds in the rhizosphere. (ii) Fungi serve asctive decomposers, major "crumb" formers. (iii) Algae fix carbon dioxide and contribute to soil fertility. (iv) Protozoans are the prime predators. The soil harbors many different nutritional types of microorganisms. Some are photoautotrophs, some arechemolitho- trophs, and the great majority are chemoorganotrophic saprophytes. Some exist sym- biotically, whereas others exist in a free-liv- ink state.

25.31 Microbial Activities Soil microorganisms play an essential role Both autotrophs and heterotrophs in soil in the recycling of nutrients. serve as biogeochemical agents for the min- eralization of organic C, N, S, P, and other compoun4 Metaboltc activities microorganisms include the following. Transformations of nitrogen: organic nitrogen s ammonia -- nitrites nitrates. Transformations of sulfur sulfates hydrogen sulfide elemental sulfur sulfates --- organic sulfur --. hoydrogen sulfide. Transformations of carbon: carbon dioxide -- organic carbon carbon dioxide.

26.0 Industrial Microbiol- ogy Various foods may be used to demon- 26.1 Food Microbiology Microorganisms are used in the manufac- A special strain of yeast is used in prepa- ture of various foods, vitamins, drugs, andration of dough to produce CO2, whichstrate microbial processes. Microorganisms chemicals. causes the dough to rise. are used in bakerieS and other food indus- Ryther, J. H., and J. C. Goldman. 1975. Sauerkraut, pickles, sausage, and othertries for the preparation of sausage, sauer- Microbes as food in maricultures. Annu.fermented foods are produced by special kraut, and pickles. Rev. Microbiol. 29:429-443. kinds of bacteria which are able to tolerate high osmotic environments and to produce .)' Introductory Microbiology-167 TOPICSAND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES large amounts of lactic acid and other flavor - enhancing products. Lactic acid produced , a by the organisms serves as a food preservative. Single-cell microorganisms are used as sources of protein supplementi-in fcthd for domestic animals. The raw materials used for microbial cultivation are generally waste products such as spentpapermill water (sulfite liquor) or whey, blanching Water, etc. These microorganisms decoMpose and stabilize wastes and serve as a source of protein.

26.11 Food Texture and Fla- icroorganisms are used in the food in- Microorganisms impart specific or Unique Aror and taste of -freshly balked bread 4 >I vor dus to enhance the taste, flavor, texture, taste, flavor, and texture to food, but they are attributed to the activities of yeast cells. an duiritive valuekif foods. 4 also increase its digestability and nutritivtaiThe flavor of fermented folds is dice to the value. products of bacterial metabolism. A gram of yeast cells may provide not -4' only protein but almost all essential vitamins and growth factors for animals, inclu ti humans. V.re, 26.12 Spoilage-Orianisras_ Microorganismsalso are capable of food In food microbiology, time, money, and Perishable foods such as fresh fruit, veg- Sfi-t*e. effort are spent in protecting food from mi-etablts, fish, poultry, ands meat eventually decompose when they are left at room tem- crobial spoilage and elimination-, of pathogens. perature. Such foods can be shown to con- Some of the methods used-in protecting,tain a wide variety and Beige numbers of (Sod fronn,spoilage(include: aseptic handlingbacteria. of food, Pasteurization, boiling and -Steam sterilization; low temperature (refrigeration, freezing), dehydration, increasing osmotic pressure (concentrated sugar or salt), presence of pieservative chemicals (organic acids produced fro .fermentation and itariOus, products formed dining smoking), and radia-

6 a tion (ultraviolet and ionizing). Because foods are handled by many people, they are subject to contamination with a variety of pathogens and frequently cause outbreaks of food poisoning and food asso- a ciated infections.

c;og, 26.2 Dairy Microbiology The preparation ofutter, cheeses, and Bacteria which' preduce largetamounts of fermentedillra4pind cts such as buttermilk lactic acid (strains of Lactobocillits and 1 Introductory Microbiology-169

F. C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRJCHMENT INFORMATION and yogurt depend on the metabolic acti Streptoco4s) are used as starter cultures ties of particular microorganisms. 4. for many;fimented milk products. Cheeses, a8er cream, buttermilk, acidophilus milk, yagurt, kefir, and kumiss are produced

.by ferniaptation of milk. Special bacteria and fuOgi:,?Sre ,uSed in the manufacture of milk antidairY products. Ripiiing of some cheeses depends on spe- - cific kinds of bacteria, whereas other cheeses require 'Molds, e.g., bleu cheese, roquefort, etc. Fermented milk and milk products have a longei keeping quality, than raw milk, since th0 contain a variety of organic acids pro- , guced by bacteria. :Pasteurization is used to protect the public from milk-borne diseases (similar to foodborne), such as salmonellosis, tuberculosis, brucellosis, and Q-fever. .

26.3 Industrial Application of Mi&Ocirganisms are utilized in the pro- Microorganisms used industrially include molds, yeasts, and bacteria. _Microorganisms ductionof alcohols, amino acids, antibiotics, enzymes, hormones, vitamins, vaccines, and Pathogenic microorganisms and their other commercially important products. products are usektO,prepare vaccines. Kleyn, J., and J. Hough. 1971. The Most process Mosttig raw materials and microbiology of brewing. Annu. Rev. Micro-microorganisms are carried out by using the bial..25:583-608. -continuous culture method (chemostat) rather that( batch method (Microbial Phys= iolpgy, &biopic 3.4). MicroOrganisms employed in the manufacture of products are constantly improved by various genetic manipulations. Some bacteria are used in steroid conversions. Many industrial processes involving microorganisms utilize waste materials (whey, corn steep liquor) to manufacture useful and essential product&

27.0 The Future of Micro- hiol9gy The ly to produce certain desired . 27.1 Genetic Engineering The m ation of genetic material to obtain results is referred to as ge- produc antibiotics, organic acids, interferon, insulin, alcohols, etc.), the ability to 1 ,1wi netic en g. Genetic engineering can 1. Introductory Microbiology-171 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION , 13, ENRICHMENT INFORMATION be a powerful tool to construct organismsciecomitise certain materials, or the ability that may have agricultural, industrial, orto prodUte toxic substances is controlled by

medical importance. . genes which may be manipulated. '

. . . 27.11 The Use of Microorga tiicially .created...reconibinaot DNA

nisms , molejJeainliepbcAted inliacteria such as ichiao1iCenes of higher tirga- niazi*'7044,4f*:.insulin, interferon, and other useful prOduCts can be inserted to plasmida which, *when introduce-A-into mi- croorganismi; replicate andptoduCe functional products.

27.12 Scientific Concerns Some scientists are concerned that recombinant DNA technology may result in un-

expected biological hazards. to Grobatein, C. 1977. The recombinant DNA debate. Sci. Am. July. 237:22-33.

27.2 Factors Affecting the In- Infectious diseases have become less Conjugal transfer of functional cidenceofInfectious ':'prominent causes of death and disability in.plasmid(s). such as toxic factors (either exo- .,..,t, Diseases regions of improved sanitation and adequateor endotoxins or both) and enzymic factors sources of antibiotics. However, the misuse(R-factor), has created considerable prob- t! of antibiotics has resulted in an increase in lems in chemotherapy and control of gon- the incidence ofiqtibiotic-resistant micro-orrhea and other assorted diseases. organisms. '/ Acquisition of genes for pili (structures that facilitate adhesion to target tissue) or

J . the ability to produce capsules may enhance the virulence of such organisms.

obialApplications Microorganisms have played and will con- Microorganisms may be used as protein e -World Prob-tinue to play important roles in resolvingsupplements, to control pests in crop pro- world 'problems including environmentalduction, to reduce or eliminate famines, to pollution and food shortages. . control water ana soil pollution, and to re- Kihlberg, R. 1972. The micro,as acover, valuable products from wastes for resource of food. Annu. Rev. Micro 26: cycling to conserve -raw material. 427-466. Single-cell microbes, yeast cells can be harvestefromc6 w. om h#me and industry. Waste cell hay be solu- bilized by an enzyme produced by organisms which will convert it to sugar. Sugar can then be used to grow yeast, which can be consumed by humans directly or indirectly 1 Introductory Microbiology-173 9'' r!, .. . . A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES as a source of protein and vitamins or added to animal feed as a supplement. Microorganisms, e.g., Bacillus thuringi- nesis and some viruses, rather than biologically recalcitrant chemicals such as. DDT, have been used to control Clop-damaging insects. This keeps the environment free of insecticides. Solid wastes may be composted to fertil- izer or soil conditioner or fermented to re-

Pt. coyer fuel (CH4, CH3OH, C2H5OH) for home amtindustrial

27,4 Evolution of Microorga- Genetic chanties occur among members'of The ecosystem (biotic and abiotic com- nisms a species through modification of DNA (mu- ponents) provides internal as well as exter- tation). Mutants, either spontaneous or in- nal selective pressures. duced, are subject to the same selective pres- Most ecosystems are variable. Variation sure as nonmutants. Mutation results in in- may be natural, induced, or brought about creased physiological- versatility when mu- by internal or external forces. These include tants thrive bettq or can survive better afrequent changes in temperature, pH, avail- particular enVirodinent. able water and nutrients, amount of radia-' tion received, antibiotics produced, or toxic substances introduced by humans or by flora and fauna within and external to the given environment. Such variables may be selective forces. Examples would include the following: bacteria endowed with resistance (R) plas-- mids are more apt to survive in the environment containing antibiotics; lowering the pH tends to favor acid:tolerant organisms:

27.41 Causes Genotypic changes in cells also may result Alteratio#. in Aiomes may be mediated Changes; Iri pr9eesses such -as conjugation, trans- by lyk: genic,Virnaea. format'i and transduction., Mateyi bacteria are capable of conjugally transferring functiorial plaSmida such as R factor tti members Of, their genus` and to members of other genera,

Opportunit at'various levels exist for-, Microbiologists are emplpyeefin_ the fol- individuals educ *Ad well-trained in thelowing specialty categories: basic sciences t e application of micro- Agricultural microbiology:cause-effect biological principles. and techniques. control of plant pathogens, improvement of Introductory Microbiolcigy-175 1P32.

-s J

4 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENR1C MENT INFORMATION C. PRACTICAL ACTIVITIES. soil for ility, nit ogen fixation, ecto- and en-. dosymbi etc Aquatic n marine microbiology: microbial ecolog -relationship of organisms to other organ's or flora and fauna and to ablotic ,componts (eneficial-detrimental effect), and 139 u on control. Fowl mickbiol gy: preservation of food (prevent spoilage); storage; improvement in nutritional value, aroma, flavor, taste, and 1"' texture; quality control; food sanitation. Dairymicrobiology:manufactureof cheeses and other fermented milk products, quality control, epidemiology of certain microbial diseases of cows. Sanitary or public health microbiology: the study of epidemiology of food- and water-borne infections or diseases; maintain bacriological safety of potable water and swming pools; and examination of effluent from wastewater treatment. Medicalmicrobiology:epidemiology, pathogenesis, immunology, chemotherapy, and diagnostic microbiology. Industrial microbiology: search for and development of antimicrobial agents, production of bioche'cals, and vaccines, and recombinant 'DNA, 'app 'cations.

27.51 Factors Affecting Em- Salaries and employment, as in other ployment professions, are dependent on the level and extent of education, training, andexperi- ence. ,

s,,` ,1'

Introductory Microbiology-177 Section IMedicitl Microbiology

TABLE OF CONTENTS

1.0 SURVEY OF THE BIOLOGICAL WORLD 5 7.1 Fungi 37 L 1 Taxonomy 5 7.2 Classification 37 1.2 Classification 5 7.3 Reproduction 39 1.3 Nomenclature 5 7.4 YeaV Morphology and Reproduction 39 1.4 The Five-Kingdom Approach to Classification (Other Approaches Ex isti 5 7.5 Clas.4ification of Yeasts 41 1.5 Viruses 7 7.6 Dimorphism l';': 41 2.0 MICROORGANISMS -7 7.7 Cultural Characteristics of the Fungi 41 2.1 The Nature of Microoryani ms 7 7.8 Fungal By-Products ' 43 2.2 Concept of tlie'CeU....%"'' 9 8.0 GENERAL CHARACTERISTICS OF VIRUSES 43 2.3 Single-Cell Organisms 9 8.1 Physical Characteristics 43 2.4 Procaryotic and Eucaryo Microorganisms 9 8.2 Host Specificity , 45 2.5Viru. 9 8.3 Bacteriophage 45 3.0 CROSC S AINS 9 8.4 Reactions to Chemical and Physical Agents 45 3.1 Microscope 9 8.5 Attenuation of Viruses tsi, .47,. 3.2 Types of Microscopes 9 8.6 Classification ""47-. 3.3 Stains Used with Bright-Field Microscopes 13 8.7 Replication .47 3.4 Clinical Application 13 8.8 Cultivation 49 4.0 METABOLISM AND GROWTH 15 9.0 GENERAL CHARACTERISTICS OF PROTOZOA AND HELMINTHS 51 4.1 Cellular Composition 15 9.1 Protozoa 51

4.2 Metabolism ..... 15 9.2 Trophozoite 51

4.3 Heterotrophs , 17' '9.3 Cyst 51

4.4 Enzymes . . 17 9.4 Life Cycle 51 4.5 Small Molecule'S 17 9.5 Locomotion 51 4.6 Metabolic Pathways 19. 9.6 Classification 53 4.7 Fermentation and Respiration 19 9.7 Physiology 53 4.8 Quantitation of Microorganisms 19 9.8 Helminths 53 fp 4.9 Media. ' 21 10.0 INHIBITION AND KILLING OF MICROORGANISMS BY PHYSICAL 5.0 GENETICS 23 AGENTS 57 5.1 Genetics 23 10.1 'Heat 57 52 Genetically Important Molecules 23 10.2 Drying .59 5.3 DNA. 23 10.3 Freezing ) 59 , A7 .5.4 RNA ,.e840'ec 7, 27 10.4 Ultraviolet Radiation (UV Light) 59 5.5 Protein. Synthesis 27 10.iltration '59 .1 5.6 Mutation 27 10.6 Physical Disintegration 5.7 Phenotypic Expression 29 11,.0 INHIBITIONAND KILLING OF MitiOORGANISMS BYChEMICAV9" Transfer of ONA Between Procaryotic Cells 29 , AGENTS 59 Plasmid 31 11.1 Steidiation 59 si 6.0 GENERAL CHARACTERISTICS. OF BACTERIA 31 RI Disinfection 61 6.15hape 31 11.3 Antiseptics 61 6.2 Surface-Associated Structures 33 11.4 Representative Chemicals Used for.Disinfection and Sterilization 61 6.3rissification 35 12.0 ANTIMICROBIAL AGENTS ,..., , 63

ENERAL CHARACTERISTICS OF FUNGI, 37 12.1 Antibacterial Agents . . . 63 1v5 IIP TABLE OF CONTENTS-Continued 12.2 Antiftingal Agents 10.6 Qpporttnilstic Mycoses, 13'/ 12.3&Uproa/tun Agents -r 67 17.0 MIDICAI, VIROLOGYl 139 12,4 Antiviral Agents n7 17.1 Epidemiology 139 .

12.5 Susceptibility Versus Resistance of Microorganisms 07. 17.2 Disease States , . 12.6 Drug Resistance 07 17.3 Retiptrwie of the Host to Viral Infection 12.7 UseorAntimicrobial Drugs 17.4 Antiviral Agents 14 I

110 HOST-PARASITE RELATIONS, IIP5 71,1 17.5 limmutoprophylaiOs 143 13.1 Host-Parasite 10Iationships 17.0 Diagnostic Virology 1.13 13.2 Pathogen C,!, 73 17.7 RNA Viruses 145 13.3 Normal Flora 73 17.8 DNA Viruses 149 13.4 Disease States 73 17.9 Other Less-Well-Defined VirtikS- 151 13.5 Host Resistance Factors 77 18.0 MEDICAL. PARASITOLOGY 153 13.6 Factors that Enable Microorganisms to Cause Disease 79 18.1 General Features of Protozoan Infections 153 14,q IMMUNOLOGY 83 18.2 Medical Protozoology 153 14.1 ImMunity 83 18.3 General Features of Flehninth Infection 159 14.2 Antigen (Immunogen) 83 18.4 Medical Helminthology 161 14.3 Humoral Immunity 85 19.0 DIAGNOSTICt MICROBIOLOGY 167 14.4 Cell-Mediated Immunity 91 19.1 Diagnostic Bacteriology 167 14.5 Acquired Immunity 93 19.2 Transport 167 97 iWi 14.0 Immune Mechanisms in Tissue Damage 19.3 Sterile Body Sites 167 14.7 Autoimmunity 99 19.4 Normal Flora 169 14.8 Immunodeficiency 99 19.5 Isolation 169 14.9 Lymphocyte and Plasma Cell Disease 101 19.6 Antibiotic Susceptibility 169 15.0 MEDICAL BACTERIOLOGY 101 '19.7 Parasite Identification 169 15.1 Disease-Producing Bacteria 101 19.8 Fungus Identification 169 .0'- 15.2 Pyogepic Cocci 101 19.9 Virus Identification 171 15.3 Rods 107 20.0 DIAGNOSTIC IMMUNOLOGY 171 15.4 Actinomycetes and Related Organisms 109 20.1 Serology 171 15.$ .Gram-Negatixe Ji2ods 115 20.2 Antigen-Antibody Reactions 171 15.40'he Spirochete: 123 20.3 Serological Tests 173 15.7 Spiral and Curved Bacteria 125 20.4 Skirt Tests Involving Immediate Hypersenpivity 177 15.8 The Mysoplasmas 127 20.5 Skin Tests Involving Delayed Hypersensitivity 177 15.9 The Rickettsias 127 20:6 Detection of Cells in the Immune Response and Their Function 177 -16.0 MEDICAL MYCOLOGY 131 21.0 EPIDEMIOLOGY 177 16.1 General Consideration 131 21.1 EpitfrilitibloiY 177 16.2 Superficial. Mycoses 131 21.2 Transmission of Disease 179 16.3 CutaneoUS Mycoses (Dermatophytes) 131 21.3 Nosocomial Infectiods (Hospital Acquired) 183 16.4 Subcutaneous Mycoses 133 21.4 Quarantine 183 16.5 Systemic. Mycoses 135 21.5 Immunization 185

( Medical Microbiology-3

1Lj .2 IP a TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFO \ATION C. PRACTICAL ACTIVI 1.0 Survey of the Biolog- . ical V 1,1 xonomy Biological classification or taxonomy is the systematic arrangement of organisms into groups or categories called taxa according to a definite scheme.

L2 Cl'dficationification Organisms may be classified on the basis of several characteristics, including morphology, staining reactions, physiological properties, chemical structure, and antigenicity.

1.3 Nomenclature Nomenclature entails the systematio and scientific naming of organisms.

1.31 Genus and Species The scientific name of an organism consists of genus and species (binomial system of nomenclature). A genus represents a group of closely related species, but a species represents one kinthf organism.

1.32 Names of Organisms The first name of an organism is the ge- Both names of an organism frequently are nus, the second is the species. The first letterdescriptive, i.e., Micrococcus (a small grain) of the genus is capitalized, whereas the firstalbus (white), a spherically shaped bacte- letter of the species is not. rium which produces white colonies. -The scientific name of an organism is printed in italics or is underlined.

1.4 The Five-Kingdom Ap- Based on their characteristics, organisms Since viruses are not cellular, they are not proach to Classificationmay be grouped into one of the followingincluded in this classification scheme. (Other Approaches Ex-five kingdoms: Animal, Plant, Fungi, Pro- ist) tista, and Monera. The five kingdoms are differentiated primarily on the basis of morphology, motility, and mode of obtaining I energy. . 1.41 Animals The animal kingdom includes organisms Most animals are motile, lack cell walls, that are multicellular, eucaryotic, and non-'possess a mouth and a digestive cavity, and photosynthetic; lack cell walls; have tissueutilize -organic materials for food and energy. differentiation; and ingest their nutrients.

1.42 Plants The plant kingdom includes organisms Most higher plants are non-motile, pos- that are multicpllular, eucaryotic, photosyn-sess a cell wall, and are differentiated into thetic, and have tissue differentiation. tissues, roots, stems, and leaves. All plants Medical Microbiology-5. TOPICS AND SUBTOPICS AIESSE IAL INFORMATION B., ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES absorb their nutrients from their surround- . itms.

1.43 Fungi Fungiare *min-photosyntheticunicel- Most fungi are heterotrophic (use organic lular or multicellular organisms which ob-comppunds as sources of carbon) and are tain their nutrients by absorption. aerObic saprophytes..

1.44 Prod - The proti§ta consist of eucaryotic, pre- dowinantly unicellular organisms that include algae and protozoa.

1.45 Monera The,monera consist of unicellular, procar/ yotic. organisms (all bacteria)._ .

1.5 'Viruses Viruses are submicmscopic obligate intracellular parasites.Sincethey ,do .,n9t have cellular orgrn, they 'are not considered true ce

2.0 Microorganisms 2.1 The Nature of Microor- Microorganisms are microscopic or sub- The smallest organism that can be seen J ganisms microscopic forms.of life .and Can be fixed with thee naked eye is approximately 0.1 mm. (immobilized) on glass slides and stainedThe smallest organism that can he seen with with various aniline dyes to demonstratea light microscope is approximitely ,um. specific distinguishing structures and prop-Bacteria areusually iri the range of 1 p.m. erties. Compound light microscopes are used (One micrometer Dun] equals 101 meters.h' to observe most microorganisms in living a and stained prepar'ations.

2.12 Microbial Organ'tr Viruses, bacteria, most fungi, protozoa, Microorganisms have a wider range of Microscope and projection slides showing and certain algae reprisent the five generalphysiological and biochemical plotentialitiescharacteristic examples of the five forms of forms of microorganisms. Since algae arethan all other organisms combined. Theremicroorganisms may be studied. seldom of medical importance, they are notare few fields of human endeavor where considered in medical microbiology. microorganisms do not play an important role. Only a small percentage of rnicroorga: nisms are capable of growing or surviving on or'in the human body.

2.13 Reproduction All microorganisms except viruses are ca- Asexual reptoductiori involves replication pable of reproduction eithet asexually and/of 'chromosomaldelnyribonucleicacid or sexually. Viruses and certain bacteria(DNA) before division into two cells. Sexual (rickettsia and chltunydia) must grow insidereproduction of microorganisms requires f living cells to reproduce. twister of genetic material from one cell to another. r4 111 Medical Microbiology-7 7

TOPICS ANL? 'SUBTOPICS A, ESSENTIAL INFORMAT'ION B. -ENRICHMENTINFORMATION C. PRACTICAL ACTIVITIES 2.2 Concept of the Cell Truvell is the basic structural unit of life' Diagrams showing structural units of mi- and iacomposed of: cell wall (if present), cells croorganisms may be studied. membrane, cytoplasm, and nuclear material (chromosomes). r 2.3 Single-Cell Organisms Most microorganisms are unicellulgior- A single7celled organism is capable of Projection slides of bacteria and proto- ganisms which are capable of inde endent carrying out all the processes associated with zoans prepared for light and/or electron mi- existence. life Wtthin one ce11; these microorganismsi anis croscopy may be\studied to show'basic struc- are quite complex physiologically despitetures. their small size:

. I 2.4 Procaryotic and Eucar- There are pwo types of cellular organiza- yotic Microorganism tion: procaryotic and eucaryotic; (Introduc- tory Microbiology, Table 1).

2.41 Procaryotes Among other characteristics, procaryotic, Diagrams of bacteria and protoibans may microorganisms lack a nuclear membrane '° be studied. to demonstrate cell structure. Td and membrane-bound. organelles. Bacteria areprocaryotic,whereas fungi and protozoa are eucaryotic. O

2.42 Eucaryotes Among other characteristics, eucaryotic Eiicaryotic microorganisms. are structur- organisms defined membrane-ally, more complex than procaryotic orga- bound nucleus an ther organelles which nisms. niay be visible with a light microscope in stained or unstained preparations.

2.5 Viruses ,Viruses are not ;cells since they do not t, possess a cell membrane or, nuclear material capable of self replication. Viruses are, therefore, neither eucaryotic nor procaryotic a (Medical Microbiology, Topic 8). ,

3.0 Microscopy and Stains -4 --- 3.1 Midroscope A microscope is En trument consisting The ability to see microorganisms with A chart comparing relative sizes of objects o ,a4combination of lens to make small the ,aid of a microscope paved the way forafter magnification may be sfildied. bjects such as microorganisms look largerthe establishfnent of the germ theory of in order that they may be seen and studied.disease. %, The ability to distinguish two distinct points as separate entities is the resolving power of a microscope.

3.2 Types of Microscopes There are many types of microscopes. The 1J1 Medical Microbiology-9 1

C. PRACTICAL ACTIVITIES TOPICS ANDSUBTOPICS- A. ESSENTINL INFORMATION' I1, ENRICHMENT INFORMATION, choice of microscope depends on the nature of thelPecinien find the information re- ' redi, qu 1 I N V. A bright-field microscope may be studied ;3.21 Bright-Field Microscope' The commonly used microscope to The parts of a bright-field microscope in; obserye microorganisms is a bright-field mi- cludd the ocular (top lona system),objeptiveand used by all students. crosCope ip which stained organisms appear.(lower lens system), mechanical staget con- aome objects such-es stained yeast cells dark against a bright background, denser, and light source. may be observed at all the available magni- Bright-field microscopes generally mag- The magnifiCation of p light microscope fications. nify up to 1,000 times. The resolving power may be determined by multiplying the mag- a of a ilight-fieldlnicroscope is 0.2 AM. nification of the ocular by that of the objective. Viruses and certain bacteria cannot be seen with the aid of a arightjield microscope because they are smaller than the resolving power of the microscope. t` A diagram showing the principles of dark- 3.22 Dark-Field /Microscope In'dark-field microscopy, incoming light is 'Ttvonemapallidum, the causative agent directed so that the organisms appeat light of syphilis, is usually examined under dark-field microscopy and microscope slides of in a dark background. Dark-field micro)copyfield illumination. bacteria observed with a dark-field micro-- is often used to. observe viable organisms scope may be studied. and organisms slightly below the resolving power of a bright-field microscope. No stains p.. are required, , Phase - Contrast' Micro- Phase differences occur between lightal- The principles of phase microscopy 'dic- Microscope slides showing internal struc- scope tered 4n object andkhe unaltered back-,tate that when light passes through 0...leo-tures of eucaryotic microorganisms may be observed by phase-contrast microscopy. ground light. Differences in the density &organisms, it emerges in different patterns f. chemical composition of the organisms aredepending on the different properties of the contrasted by phase variation against the material through Which it passes. Igiierrial backgrovd. No stains are required. structures of living microorganisms are sd- ied in this mariner. . Projection slides showing fluorescing bac- 24 Fluorescence Micro- The second emission of visible light by an*4 The' identification -of certain pathogenic scope object skch as a bacterium stainbd -with amicroorganisms or the presence of specific'tens identified by specific fluorescent anti- fluorescent dye and illuminated with lighttifantibodies is often' determined with the aidbody may be shown. short:wavelength sudi as ultra let is fluo- of fluorescence microscopy and specific an- rescence. Fluorescence microsco tibody coupled to a fluorescent dye. For special optics and tight sources, example, rabies virus may be identified in a rabid animal'a brain tissue by use of the fluorescent microscope. 0 .° 4 e 'Electron MicroscOpe Electrons produc horter. wavelengths Electron microscopes aid in the study of A cliagrani showing the components of an than light. Instead of light, an, electron mi-viruses and have greatly added to our un-electron microscope along with electron mi- . Medicail Microbiology-11

fi 1JG 0 4,

TOPICS ANI)StIliTOPICS A. FtilinNTIAL INFORMATION cir-11, Hilitcglimim INFORMATION C, PRACTICAL ACTIVITIPIR oroscope uses bonnie of °Natrona projectedderntaliding of the morphology of utioroor.orographa of microorganisms may he stint. 4. through n vaguely And footuied by °learn- KO niMiliii, kid, magnets for Mowing on a fluorescent screen. The electron microscope can resolve particles 0.001 pin apart.

3.3 Stains Used with Bright: Most microorganisms are transparent and Many bacteriological dyes are coal tar do- The value of staining may be demon- Field Microscopes must be stained with dyes before they canrivativeli known-as aniline dyes. strated by exainining stained and unstained be aeon under the microscope. bacterial smears. IN Grain and acid-fast staining should be per- 3.31 Differential Stains Certain staining procedures such as the Differentiation occurs because of differ- Gram stain and acid-fast stain separate or- ences in the chemical contents of the various formed if possible. Microscope slides or pro / ganisms into categories, i.e., :gram-positive typps of cells. jection slides of stained organisms may els or gram-negative, acid-fast or no-acid-fast. be studied. This is often the first step in identification of bacteria.

3.311 Gram Stain The Gram stain is the most prevalently Several mechanisms for the Gram reac- used stain for bacteria. Gram-positive bac-tion have been proposed; however, it is still 'teriaretain -crystalvioletuponde- poorly understood. colorization and appear purple. Gram -negative bacteria do not retain crystal violet upon dekolorization. They are then stained red by a safranin counterstain.

3.312 Acid-Fast Stain The acid-fast stain is primarily used to The agent of tuberculosis, Mycobacterium detect mycobacteria which retain a red dyetuberculosis is a classic example of an acid- 9omplex due to their high lipid content evenfast microorganism. after decolorizan with acid alcohol. Non-acid anisms do not retain the complex but may bevisualizedafter counterstaining. Microscope slides of bacteria stained with 3.313 Special Stains The staining of spores,ilagella, and capsules is performed to promote visibility of special stains are available commercially the respective structures and to aid in cate- and should be studied. r gorization and identification. Specimens from patients are often Gram 3.4 . Clinical Application or acid-fast stained and observed under the brightfield microscope to provide the clinician. with information regarding the presence of microorganisms, their .relative concentrations, and their staining reactions. - :11a Medical Microbiology-13 IP tr,

ANI ) A, INFORMATION II, ItINI11011MEINT INFORMATION ACTIVITI101 This information in lielpt\ii regarding Omit, inept and management of the piktionts, MotabOlant tirowlit Cellular Composition Allliving things, Including microorganism, are composed of protoplasm, Th., (..common ellonfical, elements carbon (C), oxygen' (01, lohlogen (I 1), 11114I nitrogen (N), compote) about 09% Of protoplasm. These chemies1 elements are arranged into complex organic Molecules such as proteins, carbohydrates, lipids, and nucleic acids, which are necessary- for the microorganism to live and reproduce. A basic knowledge of cellular composition and metabolism is necessary to understand the concepts of medical microbiology

4.2 Metabolism Metabolism is the sum of all( the chemical The breakdown of molecule" does not pro- reactions occurring within an organism.duce energy but transforms it from one form /They involve a breakdown of large organicto another. molecules into simpler ones (catabolism) with the liberation of energy necessary for ...,-) the organiim's activities and the build-up of simp/ Imolecules tocomplexmplex ones (anabo- lismm necessary for the organism to carry out these activities.

4.21 Organic Molecules ,All living materialicontains organic molecules. All organic molecules contain carbon (Microbial Physiology, Topic 1).

4.211 Proteins Proteins are very large complex organic There are about 20 different amino acids. compounds composed of numerous small Thousands of amino acid molecules may be - molecules called amino acids. Proteins arecombined to make proteins. important as enzymes and as structural elements of cells.

4.212 Carbohydrates (Sugars), Carbohydrates are compounds that contain only carbon, hydrogen, and oxygen. They may be small molecules (monosaccharides) or large complex molecules (polysaccharides) used as structural elements of the cell or as a stored energy source. ..t Medical Microbiology-15 2 1..) TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 4.213 Lipids (Fats and Related are composed of carbon, hydrogen, Compounds) and oxygen but also may contain other elements such as phosphorus and nitrogen. They are insoluble in water and are important in intermediary metabolism and in structural elements such as the cell membrane.

4.214'1"'Nucleic Acids Nucleic acids are compounds chat play a major role in the transmission of hereditary traits, in the control of cell functions, and in the synthesis Of proteins.

4.3 l-Ieterotrophs Heterotrophic organisms obtain their carbon requirements from organic molecules. Heterotrophs use organic molecules both as a source of carbon and as a source of energy. Pathogenicmicroorganismsarehetero- trophs as are most animals. Many nonpathogenic microorganisms obtain their carbon from carbon dioxide (CO2). These organisms are autotrophic as are plants.

4.4 Enzymes The transformation of energy necessary in Enzymes and substrates are highly spe- catabolism and anabolism is carried out incific for each other. Enzymes are named part by enzymes. Enzymes are organicaccording to the type of reaction catalyzed catalysts, i.e., substances which in minute(e.g., hydrolase) or the substrate acted upon amounts promote chemical changes without(e.g., protease). Cettain bacteria produce en- being used up in the reactions. A substrate zymea which relate directly or indirectly to is the substance, e.g., an organic molecule,pathogenesis. Lecithinase, the alpha toxin of which is permanently altered in an enzyme- Clostridium perfringens causes lysis of the catalyzed reaction (Microbial Physiology, host cell membranes. Topic 4).

4.41 Exoenzymes Many microorganisms secrete enzymes Exoenzymes hydrolyze starch to glucose into the external environment (exoenzymes) and proteins to amino acids. Determination that split large molecules into smaller onesand identification of exoenzymes often aid which can enter the cell. inthe identification of microorganisms. Many bacterial virulence factors, including toxins, are exoenzymes.

4.5 Small Molecules Small molecules for anabolism are obtained from catabolism and from outside the cell. Small molecules outside the cell may be 2. Medical Microbiology-17 'TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION 4 B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES , passively (diffusion) or actively.transported inside the cell by enzyme-catalyzed reactions.

4.6 Metabolic Pathways A particular series of stepwise chemical reactions resulting in a given end product or products is called a metabolic pathway. Glu- cose is a simple 6 carbon sugar used as ant energy source by many microorganisms, since it is widely distributed in nature and is a part of many larger molecules. The breakdown of glucose into smaller molecules by various metabolic pathways is a major energy source and produces smaller molecules as by-products (Microbial Physiology, Sub- topics 5.2 and 5.3).

4.61 ATP Adenosine triphosphate (ATP) is a mole- A- cule which is important in energy transfer. % ATP is formed from adenosine diphosphate (ADP) as a result of catabolic reactions. Energy is released by breaking the high energy bond of ATP (ATP --* ADP + Pi). Such energy is available for biosynthesis and other energy - requiring reactions.

4.7 Fermentation and Res- Fermentation and respiration are the two Organisms which use molecular oxygen in piration basic catabolic schemes found in microor-respiration are more energy efficient and ganisms. Fermentation involves energy pro-produce more molecules of ATP per mole- duction without the use of mole ular oxygen, cule of glucose catabolized. Therefore, they whereas respiration requires it Fermentersusually have shorter generation times. The are divided into organisms w"--ch cannotdetermination of an organism's catabolic tolerate the presence of molecular oxygenscheme and the end products of the scheme (anaerobes) and those which can (facultative are used in the identification of microorga- anaerobes) (Microbial Physiology, Subtop- nisms. ics 5.2 and 5.21).

4.8 Quantitation of Micro- In the clinical, laboratory, it is often im- Abnormal amounts or increased percent- organisms portant to w the amount (number ofages of an organism may indicate a disease organisms pmilliliter) of a given organism state, e.g., greater than 10' bacteria per ml or the percen ge of a given organism in a "of urine generally indicates disease. mixed population. Microorganisms may be quantitated by several means; however, in the clinical bacteriology laboratory, count- 2 Medical Microbiology-19 TOPICS AD SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT, INFORMATION C. PRACTICAL ACTIVITIES mg, which etermmes, the number i,viable bacteria, is generally used.

4.81 Generation Time 'Although 'growth refers to an increase in The ability of pathogenic microorganisms the volume of protoplasm, with niicroorga:to rapidly ieproduce enables them to colo- nisms.it generally refers to an increase innize whost and produce disease in relatively cell numbers (reproduction). The averageshort periods of time. time needed for one microorganismdivide In nature] growth of microorganisms is into two is its generation time, e.g., Esche-slowed due to the accumulation of their own , richia coli =417 minutes, enabling large pop- waste materials, .depletion of nutrients, and ulations of cells to be produced in a 'shorttheir inability to adjust:to changes in environment. The growth of pathogenic micro- time (Microbial Physiology, Topic 3 ands. Microbial Genetics, Subtopic 1.2). organisms in a host is also slowed by the host's defense mechanisms. 1 4.9 Media To study Microorganisms such as bacteria The ideal medium and environment in the laboratory, nutrients and environmentshould duplicate those conditions in nature must be provided which are conducive to inwhicka given inkcroorganism thrives.. Me 1r7 growth and reproduction. "Medium" is a be sterile or free from microorgal A generid term used to denote nutrient mate-nisms whose development might influen& O rials used for the culture of microorganisms. or prevent the normal growth and metabo- The proper temperature, pH, oxygen ten-lism of the inoculated type. Sterilization of sion, and other environmental factors-mustmedia is usually by means of autoclaving or also be provided to insure growth. in certain cases by filtration.

4.91 Uses of Media Media may be used for isolation, identification, and mainkefiance of microorganisms. Media may be liquid, solid, or semi-solid. e

4.92 Agar Agar, used most frequently as a solidifiing Agar plates, agar medium' in a petri Agar plates and slants may -15e prepared as agent at 1.5 to 2.0% for media, is a paiyiac-dish, provide idea] media. for isolation ofa laboratory exercise. Plates and slants may charide obtained from seaweed: It producesbacterial colonies and for study of colonialthen be inoculated with a microorganism a substance the consistency of gelatin. It ischaracteristics, such as color, shape, size, such as E. coh. f not metabolized by most bacteria, liquefies and consistency. at 100°C, and solidifies at 45°C. Nutrient material necessary for metabolism is incorporated in the agar.

4.93 Complex and Synthetic Complex media are those for which the Growth factors are small compounds that Media chemical composition is not defined, e.g.,"i.ertainbacteria cannot synthesize and blood and milk. Media in which all the com-therefore must be added to the media. ponents are known, in kind and amount, are synthetic. Medical Microbiology-21 TOPICS AND SUBTOPICS A. ESSENTIAL INFO!MATION B. ENRICHMENT FORMATION C. PRACTICAL ACTIVITIES k 4.94 Differential Media Differential media are used to more easily A differential Medi is one which will dett colonies of gticteria which have dis-cause the colonies of ular organism tinctive operties such as hennolysis ofto have a distincti rere.g., E. coli blood; gproduction, or acid production. has a characteristiCilividcent flheen on EMB (eosin-methyl niblu ) agar. O 4.95 Selective Media /An ideal selective medium is one that In clinical labor tones, media used are, inhibits the growth of all microorganisths inoften selective and differential. a specimen and supports the growth of a I desired species or other defined group. Se- lective media are formulated by: inclusion of inhibitorysubstances;establishingex- tremes, e.g., pH and salt concentration; or 1. 1 2 the presence or absence of growth factors. 4.96 Enrichment Media An enrichment medium is one to which Many bacte is isolatOd from clinical spec- special nutrients have' been added to en-imens require ,blood of blood products and hance the growth of certain microorganisms,increased CO tension. These conditions are e.g., blood agar. more reflecti e of the natural environment. C 5.0 Genetics 5.1 Genetics Genetics is the study of the mechanisms by which the infotmation of cells is stored, expressed, and modified and of how this information is transmitted to future generations of organisms and to other cells in the population.

5.2 Genetically Important The major molecules important in the Molecule§ study of genetics are deoic'bonucleic acid (DNA), ribonucleic aci. NA), and protein (Microbial Genetics, Vs pic 2).

5.3 DNA DNA is a double-stranded molecule in the A nucleotideconsistsofdeoxyribose- form of a double helix, each strand of whichsugar, a phosphate group, and one of four t is a polymer of nucleotides. DNA is thebases: adenine, guanine, thymine, or cyto- reservoir of genetic information in a cellsine. rrr (Microbial Genetics, Subtopib 2.1).

Electron micrographs may be used toil- 5.31 DNA of Procaryotes DNA of procaryotes is a circular molecule A fully extended bacterial chromosome is located in the cytoplasm and is not sur- abqut 1 mm long. lustrate the circular nature of DNA. rounded by a membrane. This molecule is called a chromosqme and contains segments Medical M icrobiology 23 2 , 'U

,. \' 4 B. ENRICHMENT INFORMATION T PIcS AND SUBPICS . A. ESSEgriAtrFORMATION calledgeffet lines-determine the charac\- teristi .and act as,,the control units\ of all celln tivity. 17,3 ".."\ -tv -6.32 DNA of Eucaryotes 131114 eucaryotes is distributed into \The number of chromosomes found in the chrtAisnmes. A eucaryotic chromosome isnucleus varies among eucaryotic organisms. , ,' cOnitorig'ed of one very large DNA molecule, Human body cells contain 46 chromosomes, histoner proteins," and other proteins. Thean onion has 16, but procaryotic cells have 'chromosomes are located in the membrane- only 1. hound nucleus.

,5,33 Gene A gene is the part-of the DNA molecule Ge es code for the enzymes which regu- which carries the genetic informaticulfor th;late al the chemical reactions of the cell. synthesis of one protein.

5.34 Genome The entire complement of genes in an organism comprises its ge ome. ( 5.35 DNA Function DNA has two major fu ctions: to dupli- DNA is replicated so that each daughter cate itself and to code for synthesis of pro-cell receives an exact copy of the genetic tein. material.

5.36 DNA Replictktion The two DNA strands separate. Enzymes Each strand of the original molecule (Duplication)' catalyze the synthesis of new strands ofserves as a template resulting in two DNA DNA complementary to each of the originalmolecules having one strand from the origi- two strands. The-two new DNA moleculesnal DNA and one strand of newly synthe- formed are identical' to the original. sized DNA.

5.361 Mitosis I. Mitosis is the separation of previously rep- Mitosisoccurs -in all animal and plant Heated chromosomes of eucaryotic cells, cells.

5.362 Meiosis Eucaryotic cells, which have a sexual cycle, carry out a reduction-division process (meiosis) which results in one diploid cell becoming foul -haploid cells. Diploid cells contaia pair (2n) of each chromosome, wherea\aploid cells contain half (n) the number of chromosomes as diploid cells.

5.363 Procaryotic,ellRepli- Procaryotic cells do not undergo mitosis "4. cation or meiosis. The Circular DNA is replicated, and ssach cell produced by asexual cell division has one DNA molecule as did the parent cell. The. result is two equal daughter 21 Va. 2) MedicalMicrobiology 25 . TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION .C. PRA6TICAD-ACTIVITIES cells which are replicas o e parent (binary fission) (Microbial Genetics,ubtopic 2.2).

5.4 RNA RNA is a single-stranded polymer of nucleotides. It differs from DNA in that the sugar ribose replaces deoxyribose and the base uracil replaces thymine.

5.41 RNA Synthesis RNA molecules are enzymatically synthesized as complements to one strand of a particular region of DNA. RNA molecules are temporary and are constantly synthesized.

5.42 Types of RNA There are three' types of RNA: messenger NA (mRNA); transfer RNA (tRNA); and r4 osomal RNA (rRNA). They function at ous stages of protein synthesis.

5.5 Protein Synthesis enetic information flows from DNA to Each amino acid may be ended for by NA to prOtein. Each codon, a sequence several different codons. Rf three nucleotides, specifies a particular amino acid. Special codons specify sites of initiation Or termination of a protein (Micro- bial Genetics, Subtopic 2.3).

Transcription The mRNA molecule is synthesized as a A specific mRNA is a transitory molecule complement to a gene on one strand of involved in the synthesis of one specific pro- DNA. The sequence of the bases in mRNAtein or portion of a piptein. mRNA may be determines the amino acid sequece of acopied several times during the synthesis of protein. a protein.

5.52 Translation ryrThe mRNA molecule is decoded on the Several ribesomes may move along one ribosome by the tRNA molecules carryingmRNA at the same time. This results in a amino acids. Each tRNA attaches to a spepolysome, which appears as a \cluster of ri- cific amino acid and also recognizes one spe-bosomes on an electron micrograMi. cific codon of the mRNA. The ribosome moves along the mRNA, resulting in the sequential polymerization of amino acids to synthesize a protein.

5.6 Mutation A mutation involves an inheritable change in the base sequence of DNA. A mutation may change any characteristic of a cell or Medical Microbiology-27 2 1 0 2 .1AL TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION 9. PRACTICAL ACTIVITIES virus. There are diff,erent types of mutations (Microbial Genetics, Topic 4).

5.7 Phenotypic Expression Most mutations result in the change of a The following are examples of phenotypic protein. Mutations may or may not cause anchanges: structuralloss of ability to pro- observable change in phenotype, dependingduce a capsule; nutritionalloss Qf ability.tO upon the extent or site of change. synthesize an enzyme resulting in a requirement for a particular nutrient or growth factor; and drug or virus resistanceability to grow in the presence of a drug or virus which kills or inhibits growth of the parent.

5.71 Mutation Rate The frequency with which inutations occur at specific sites on the DNA molecule is referred to as the mutation rate. Mutation rate ekpresses the probability that a cell will undergo mutation at a particular gene.

5.72 Spontaneous Mutation A spontaneous mutation arises without apparent intervention. An average value for frequency of spontaneous mutation per gene is one mutation in 106 cells.

5.73' 1 Mutagenic Agents Agents which interact with and modify ,DNA and thereby increase the rate of mutation are mutagenic agents. Examples of mutagenic agents include irradiation, DNA base analogs, and certain chemicals which react with DNA in such a way as to change its chemical structure.

5.74 Selection Changes in the environment may give a An example of natural selection is the Antibiotic-containing media can be used mutant an advantage so that it will growincreased incidence of penicillithesistantto select antibiotic-resistant mutants in a 1. faster than the parent and replace it. TheStaphylococcus strains after initiation ofpopulation. The replica-plating technique change in environment does not cause orpenicillin therapy in the 1940s. This muta-can be used to demonstrate that the mutants induce mutation but selects for preexisting tion was not induced by penicillin, but pen-were preexistent and not induced by the mutants. icillin acted as a selective agent for preexist- antibiotic. ing mutants.

5.8 Transfer of DNA Be- DNA is transferred from donor to recipi- tween Procaryotic Cells ent cells by three different mechanisms. Once inside the recipient cell, the donor DNA may recombine with the recipient chromosome (Microbial Genetics, Subtonic 5.3). a O Medical Microbiology-29 2 3 e TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 5.81 DNA-Mediated Trans- In DNA-mediated transformation, DNA formatkin fragments released from cells that lyse are taken up by recipient cells (Microbial Ge- netics, Subtopic 5.31).

5.82 Conjugation In conjugation, the' DNA may be transferred by means of sex pili. The donor DNA fragment is transferred during the time the donor and recipient cells are in contact (Mi- crobial Genetics, Subtopic 5.32).

5.83 Transduction In transduction, the donor DNA is transferred from the donor bacterium to a recipient bacterium by a bacteriophage (virus which infects a bacterial cell) ( MiFobial Ge- netics, Subtopic 5.33).

5.9 Plasmid A plasmid is extrachromosomal circular Plaitmids may be very important in trans- DNA which may exist independently in theferring drug resistance among bacteria. cytoplasm or become integrated into the procaryotic cell chromosome (Microbial Ge- netics, Subtopic 3.12).

5.91. Plasmid Transfer An entire plasmid may be transferred by Many plasmids have wide host ranges and any of the three mechanisms for DNA trans-may be transferred between different gen- fer (Microbial Genetics, Subtopic3.34). era.

5.92 Lysogeny Some bacterial viruses integrate their nu- Corynebacterium caphtheriae strains only cleic acid into the DNA of the cells theycause diptheria when lysogenized by a spe- invade. The viral nucleic acid is replicated cific bacteriophage. Scarlet fever is caused along with the bacterial DNA (Microbialby a lysogenic strain of Streptococcus py- Genetics, Subtopic 5.12). ogenes. 6.0 General Charac- teristics of Bacteria 6.1 Shape Most bacterial species exhibit. one of three Microscopicslidescontainingstained general forms; cylindrical (rods or bacilli); preparations of bacteria having these shapes spherical (coccus); or spiral (spirillum). may be observed under the microscope.

6.11 Arrangement Although bacteria are unicellular, the in- Cellular arrangements often aid in the Typical arrangements may be observed dividual cella may not separate completelyclassification and ide tification of bacteria.under t.e. microscope using stained prepa- after cell dIvision and therefore exhibit aWhen bacteria are founds a patient's spec-rations on microscope slides. variety of cellular arrangements. Dependingimen, the Gram reaction, ce ar shape, and on the plane of division, cocci may be ar- cellular arrangement are oftereported to MedicalMicrobiology-31

21.5 216 r

TOPICS AND SUBTOPICS . A. ESSENTIAL INFORMATION. B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES ranged in pairs (diplococci), chains (strep-the clinician before the organism isidehti- tococci), fours (tetrads), cuboidal packetsfied, because they provide valuable infor- (sarcina), or clusters (staphylococci). ,Rodsmation regajding disease. may appear singly, form chains, line up in parallel (palisades), or form unusual letter patterns (X, Y, or Z). 6.12 Colony ... Bacteria grown on solid culture media Colonial morphology ,is often used as an Various species of bacteria streaked for (agar) multifvly repeatedly to form a visibleaid in identification and classification. Col-isolation on agar media may be studied for structure called a colony. A colony is theo-onies differ in many characteristics, includ-colonial morphology. retically a clone of bacteria resulting from ing size, shape, color, and texture. the multiplication of a single organism, that organism & referred to as a colony-forming unit. 6.2 Surface-Associated Structures

6.21 Capsule Some species of bacteria are surrounded Capsular antigens are also used for sero- Projection slides of organisms suchas by a viscous layer termed a capsule. Cap-logical identification (typing) of bacteria inStreptococcus pneumonia and Cryptococcus sules are important in medical microbiologythe clinical laboratory (Medical Microbiol- neoformans, specially stained to show their since they aid in protecting bacteria from ogy, Subtopic 15.212). capsules, may be shown. engulfment (phagocytosis) by host cells and thus enhance the bacteria's ability to produce disease.

6.22 Flagellum Some bacteria produce long appendages The flagellar or H antigens are often used Microscopic slides of specially prepared called flagella which serve in locomotion.for serological typing of certain bacteria. bacteria such as Proteus uulgarus with Flagella are composed of protein subunits' stained flagella may be observed under the arranged in a helix. The various arrange- microscope. ments in which flagella occur are useful in classifying certain bacteria (Microbial Phys- iology, Subtopic 6.22).

6.23 Pilus Certain bacteria, especially freshly iso- Piliated bacteria tend to attach to other Electron micrographs of organisms with laAed, gram-negative rods,poAsess submi- objects such as inert surfaces, living cellpili may be studied. croscopic appendages known as pill. Theysurfaces, and other bacteria. are thinner, shorter, and more numerous than flagella and are not involved in motility. One kind of pilus (sex piluOis involved in the transfer of genetic material from one bacterium to another during conjugation.

6.24 Cell Wall Bacteria possess a rigid cell wall which Mycoplasmatales are the only family of gives the bacteria their shape (rod, coccus,bacteria which do not possess a cell wall. Medical Microbiology-33 213 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES spiral). Gram-pesitive bacteria havea thick Wall-defective microbial forms (WDMF) cell wall composed almost entirely of a com-are bacteria which have lost some or all of plex polymer called a peptidoglycan. their cell wall. is The cell wall of gram-negative bacteria is Certain antibiotics and enzymes produce composed of peptidoglycan, lipopolysaccha-WDMFs. Protoplasts, spheropku3ts, and L ride, and protein. Lysis of gram-negative forms are specific types of WDMFs. bacteria releases the cell wall component's, which are now called endotoxin, and produce disease symptoms.

'6.26 Cytoplasmic Membrane The cytoplasmic membrane- ia a thin The cytoplasmic membrane is similar to semipermeable membrane immediately be- the eucaryotic cytoplasmic membrane and neath the cell wall. It regulates the passageis also called the plasma or cell membrane. of material between the cell and its environ- 4- ment.

6.261 Mesosomes Mesosomes are internal extensions of the yytoplasmic membrane which aret;tociated with cross-cell wall formation at time of cell division. They are unique to bacteria. 1 6.27 Cyt i plasm .The cytoplasm includes all the contents within the cell membrane. It is granular in appearance rich in RNA and ribosomes.

6.28 Nu LearRegion(Nu- The uclear region is composed primarily Many bacteria contain circular extrachro- cleo of D A and carries the complete geneticmosomal strands of DNA called plasmids. A inf'ation of the bacteria. It is equivalentplasmid may exist independently in thary- the nucleus of eucaryotic cells Nit consists,teplasm or may become integrated into the only of a single circular DNA moleculechromosome. (chromosome). of 6.29 Endospores Certain-bacteria produce highly resistant Endospores of certain bacteria such as the and refractile,thickwalled, oval bodiesclostridia are commonly found in the soil. termed endospores, which enable them toEndospores of the Clostridium spp. causing survive harsh environments. Endospores aretetanus and gas gangrene may germinate in highly resistant to physical and chemicaldirty wounds leading to disease production. treatment. When the environment becomesThe resulting vegetative bacteria produce favorable, each endospore yields a singleexotoxins responsible for the disease state. vegetative bacterium.

6.3 Classification Bacteria are divided into 19 categories The description of the 19 categories is based on readily determinable criteria. Thisgiven in Bergey Manual of Determinative 220 21J Medical Microbiology-35 TOPICS AND SUBTOPICS A. ESSENTIAL INFO MATION B. ENRICHMENT INFORMATION C, PRACTICAL ACTIVITIES division avoids confusion n assigning orga- Bacteriology (8th ed., Willinumand Wilkins nisms to discrete classes. Co., Baltimore, 1974), Which is the standard reference on classification of bacteria. 7.0 General Character's- ties of Fungi 7.1 - Fungi 41V The fungi are non-photosynthetic eucar- Hyphae grow by elongation at their tips Moldy bread or fruit, or a portion of a yotic microorganism having a cell wall andand by producing side branches. The hyphae yeast cake may be 'examined in the labora- usually growing eit er as branching tubularmay be aerial and bear sexual or asexual tory. filaments (hyphae) which form a mat (my- spores, or may be vegetative and extend into celium) or as unicellular yeasts. The cell size the medium to provide the mycelium. with is larger than that of bacteria. nutrients and water. Yeasts reproduce by budding. F1.11 Importance or Rele- Fungi may be saprophytic (living on dead Saprophytic fungi are important in break- vance organic material) or parasitic (living in or oning down organic materials in nature. Plant another organism). pathogens such as rusts and smuts are fungi. Other fungi may be parasites of animals including humans.

Molds and Yeasts Molds are fungi which produce mycelia. A Molds may produce mycelia which are Tube cultures of mycelial (Aspergillus) few fungi are unicellular, oval or spherical, compact and tough or loose and fluffy. Yeast fungal growth can be compared with that of do not form true mycelia, and are calledcolonies usually appear as thick, moist,a yeast (Saccharomyces). yeasts. pasty-looking colonies. 7.2 Classification Fungi are classified as Phycomycetes, As- In the Phycomycetes, two compatible comycetes, or Basidiomycetes on the basiscells fuse to form a zygospore. In the Asco- of their type of sexual reproduction. A fourthmycetes, sexually produced spores (asco- group, the Deuteromycetes (Fungi Imper-spores) are contained within the ascus. Sex- fecti) either have no sexual stage or theually produced spores (basidioapores) are c sexual stage has not yet been found. borne in the basidium in the Basidiomy- cetes.

7.21 Morphological Types of Molds

7.22 Molds with Nonseptate Multinucleate molds without cross-walls The nonseptate (coenocytic) molds char- Mucor or Rhizopus may easily be cul- Hyphae (Phycomy- that produce asexual spores in a sporangium atteristically have wider hyphae and growtured as examples of coenocytic molds. cetes), arej the Phycomycetes. faster than other molds.

7.23 Molds with Septate Hy- Most molds have hyphae divided by sep- The hole in the middle of the cross-wall Aspergillus and Penicillium may be cul- phae tae, or cross-walls, which extend in from theallows nutrients, nuclei, and cell organellestured as examples of molds with septate periphery of the hyphae. These include As-to pass through from one cell to another. hyphae. comycetes, Basidiomycetes, and Deutero- mycetes. Medical Microbiology-37 2 `?, 0

l'OPIOS,ANO SUBTOPICS A, ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C, PRACTICAL ACTIVITIES 7,3 Reproduction Molds reproduce naturally by spore for- While fungi Are classified on the basis of Slide Cultures may be prepared by using mation, but in addition moat parts of those their sexual spores, the sexual atagos arebits of mycelium as the inoculum, The com- fungi are capable of growth after being rm. difficult to induco, so the descriptioh of fungi mon mold Penicillium may be used. merited. is based principally upon their asexual structures.

7.31 Speres Asexual spores which vary greatly in Asexual spores (6 to 20 inn) are formed in color, size, and shape are produced on spe- lae numbers, light-weight, easily disbursed cialized hyphal stalks. Morphology andinto the, environment, and usually provide mode of origin of the asexual spores consti- the typical color of the mycelium. tute the main basis for classifying fungi that lack sexuality. Sexual spores (haploid cells from the parents) fuse to give a diploid cell. After meiosis haploid spores are produced again.

7.311 Conidiospores (Conidia) Conidiospores (one type of asexual spore) The dermatophytes Trichophyton, Mi- Conidia of Aspergillus and Penicillium which occur singly or in groups are borncrosporum, and Epidermophyton are differ- may be studied in the laboratory. externally at the tips or sides in a hyphaentiated by the presence or absence and type called a conidiophore. Some species haveof micro- and/or macroconidia (Medical two types of conidia differing in size and Microbiology, Subtopic 16.3). structure and are designated microconidia and macroconidia.

---.7.312 Thallospores Thallospores are formed by segmentation.. Coccidioides immitis produces arthro- Geotrichum produces arthrospores which of the mycelium in actively reproducingspores which may remain dormant in themay be studied in-the laboratory. cells. -These include arthrospores (oidia)arid countryside until the next rain activates which are fragmented segments of septatethem.(MedicalMicrobiology;Subtopic hyphae and blastospores which are pro- 16.51). ducal by yeast cells.

7.313 Chlamydospores Chlamydospores are thick-walled,en- Chlamydospores are formed when grow- Chlamydospore,s\can be readily seen in larged, resting spores formed within hyphae ingconditionsbecomelessfavorable, most old cultures, for example with Candida or terminal cells. Chlamydospores are morewhether cultured in the laboratory or in thealbicans. resistant to heat and to drying than otherhuman body. parts of the mycelium or other fungal spores.

7.314 Sporangiospores Sporangiospores are asexual spores borne The sac readily ruptures and releases Mucor or Rhizopus spores may lid easily internally inside a sac and are characteristicspores into the culture tube, or into the air.studied in slide culture. of the Phycomycetes. ,

7.4 Yeast Morphology and Yeasts are oval, spherical, or cylindrical Certain yeasts may also reproduce by fis- Reproduction unicellular organisms with rigid cell walls;sion to yield sexual haploid yeast cells. they normally reproduce by budding. Medical MicrObiology-39 -10 2 "£1 0. PRACTICAL ACTIVITIES TOPICS AND HUBTOPICti A, 148414NTIAL INFORMATION 11, 111411101IMPINT INFORMATION 7,41 Patsulohypitne Some yeasts and their progeny under spe- The ability to form peetslohypllite helps cial cultural conditions or in the body adhereto differentiate .iipeekiti (twat% to each other and form a chain called piani. dohyphao.

7,42 'Capsules Certain yeasts are covered with a capsule The presence of the capsule makes phag- e.g., Cryptococcus ncolormans. ocytosis of pathogenic encapsulated yeasts difficult and they are therefore more virulent.

Classification of Yeasts Although yeasts exhibit uniformity, of Yeasts may be members of the Ascomy- Projection slides of the biochemical roue- morphology, they do not form a naturalcotes, Basidiomycotes, or Deuteromycetes. tiona of various yeasts may be shown. taxonomic group and tire found in three classes of the fungi. The taxonomy of most yeasts is based upon biochemical tests.

7.6 Dimorphism Some fungi (dimorphic) can grow in either Many fungal pathogens are dimorphic and the form of molds or yeasts depending upon may be differentiated from nonpathogens by the environment. their ability to form yeasts at._375)-C and molds at room tetilperature.

7.7 Cultural Characteristics of the Fungi Plates of Sabouraud agar will readily sup- 7.71 Growth Fungi are heterotrophic and will grow on Fungi can grow on almost any medium ordinary laboratory media. and normally grow more slowly than bacte- port the growth of common laboratory fungi. ria. Sabouraud agar is the most common fungal medium.

7.72 02 Requirements Fungi are usually strict aerobes, but some Saccharomyces and other yeasts grown yeasts are facultative anaerobes. aerobically produce CO2 from glucose and -may be used in baking bread. The same 0 Yeasts grown anaerobically produce alcohol and CO2 from glucose and are used in beer and wine making.

7.73 Temperature Fungi grow overa wide temperature range Fungi grow at refrigerator temperatures from 0 to 62°C. The normal range is 20 toand may be found growing in improperly 30°C. , disinfected laboratory incubators.

7.74 Moisture Fungi require at least a slightly moist environment for growth, but the spores may survive a more arid environment. 2,,G Medical Microbiology-41 S 14" TOPIQH ANI) 81111TOPICH iivotianim, iNFouroATIoN HNICRIIIMNNT INFORMATION 0, PlIACTICAI, rnixed propitroil with difforsiil,!Fl palings 7,75 pH ungl a p11 rlkilit,) from %LI to 9.1) Willa mod io isolate 611141 1'1'0111 a moSt oroi,Y host at shout p)1 II to 7, NOUN oriomodlY p11 ,11 or lower ao that ho mos, to compiiro"iho firoWlh sliocts hnotorlawill,ho Inhibited while the moldsou it given Amps, flourish,

Fungi grow moldy on Jolly (whim and will (lrowth of fungi and bnotorin may ho con, 7,711 Ornnnphilits Nature Many fungi are osmohilic, 1.0., 111)10 l0 tOlOrali high Holt or sugar concentrations, grow on 1181115 ronoryod with parts! On 50 mid 100(if, syrup,

7,8. Fungal Ily-Products

7,t#1 Antibiotics Certain hingi produce antibiotics, e.g Ihioftil antibiotics are produced by some podoilliam and cophaloaporins. Pena:Whoa and some Cephalosporium epo- des,

7.82 Mycotoxiiis Some Nngi produce poisonous subatancos, Aflatoxin is a mycotoxin produced by As- termed mycotoxins, which are highly toxic. pergillus fhwasgrown on corn or peanuts which wore improperly harvested or stored. Mycotoxin has been shown to be" carcino- genic in laboratory animals.

8.0 . General Character's'. tics of Viruses 8.1 Physical Characteristics Viruses are the smallest known infectious The nucleic acid (DNA or RNA) of a Diagrammatic sketches of different types agents and contain either RNA or DNAgiven virus may exist as either a single-of viruses showing nucleic acid, capsid sym- (never both) as their genetic material (ge-stranded or double-stranded molecule. metry, and envelope configuration may be nome). They are noncellular chemical par- studied. Electron micrographs depicting ac- ticles and obligate intracellular parasites. tual viruses are also available.

8.11 Capsid The capsid is the protein shell which en- The capsid protects the nucleic acid and closes the nucleic acid of the virus. facilitates attachment of the virus to its host cell.

8.111 Capsomer The capsomer is the basic morphological repetitive polypeptide unit that collectively forms the capsid.

8.12 Nucleocapsid The capsid with the enclosed nucleic acid is called the nucleocapsid.

8.13 Envelope Many viruses have an external envelope Some enveloped viruses have projections surrounding the capsid. It is lipid or lipopro- on the envelope known as spikes. tein and is acquired in part from host cell membrane. Viruses that do not have envelopes are termed naked. Medical Microbiology-43 2 '1 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION 8.14 Virion The complete infective virus particle is The virion may be identical to the nucleo- called a virion. capsid or it may include a nucleocapsid plus the envelope. A diagram comparing viral size with that 8.15 Size Viruses range from 20 to 250 nanometers Viral size may be determined by filtration in size. A nanometer (nm) is 10-3 microme-through a membrane filter of known poreof other microorganisms would help in [p- ters (um). size, by ultracentrifugation, or by use of elec-predating virus size. tron micrographs in which the virus is compared with a particle of known size.

8.16 Symmetry of the Capsid The influenza virus has a helical capsid. 8.161 Helical Symmetry The capsid of some viruses is coiled like a tightly wound rope.

The cubic symmetry of animal viruses is 8.162 Cubic Symmetry Cubic symmetry occurs in viruses which have morphological units arranged in var-icosahedral (20 triangular faces). ious regular patterns.

The poxviruses show complex symmetry. 8.163 Complex Symmetry Certain viruses have a combination of symmetry types called complex symmetry.

Host specificity is reflected in part by the 8.2 Host Specificity Viruses are capable of invading plant, bacterial, or animal cells. Some viruses can pen- fact that animal and bacterial viruses must etrate and replicate in only one type of hostinteract with a specific receptor site on the cell, whereas others may infect a variety ofhost cell surface to invade the cell. cells within the particular host.

8.3 Bacteriophage Viruses which invade bacteria are called Bacteriophage are the most easily studied bacteriophage (phage). A given bacterio- of the viruses since their hosts are bacteria. phage infects only one or a few strains of aCoagulase-positive staphylococci mny be given species. The specificity of the phagesubdivided by bacteriophage typing. for its particular bacterial host cell may be used in identification of the bacterial species. This is called bacteriophage typing.

8.4 Reactions to Chemical In general, viruses are susceptible to the Some purine and pyrimiaine analogs have and Physical Agents same antiseptics and disinfectants as arebeen used tb treat herpesvirus infections of bacteria. Viruses are not susceptible to an-the eye. tibiotics. Enveloped viruses are made non- infective by treatment with ether, whereas naked viruses are not (Medical Microbiol- ogy, Subtopic 12.4). Medical Microbiology-45 TOPICS AND SUBTOPICS' A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 8.5 Attenuation of Viruses By growing viruses under varying cultural Live attenuated virus vaccines produce conditions, mutants which are capable ofbetter immunity than killed virus vaccines producing immunity but are no longer ca- because the viruses actually continue to rep- pable of producing disease may be selected. licate in the cells of the host. These viruses are said to be attenuated.

8.6 Classification Vipses have been classified according to: Other criteria used in classification of vi- Diagrams of viral classification schemes their physiochemical properties (type of nu- ruses include susceptibility to physical andmay be shown. cleic acid, symmetry [enveloped or naked],chemical agents, especially ether, immuno- and size); the type of symptoms produced logical properties, natural methods of trans- (respiratory, intestinal); and other methodsmission, and host tissue and cell tropism. of classification.

8.7 Replication All viruses require actively metabolizing Diagramatic sketches of various replica- cells in which to replicate. The host cells tive cycles of viruses may be studied. provide the necessary enzymes, small molecules, and energy for viral synthesis.

8.71 Adsorption, Penetra- Animal viruses adsorb to specific host cells Viruses attach to host cells by means of tion, and Uncoating and are engulfed into the cells by phagocy-specific attachment sites on the host cell tosis (viropexia). Once inside the cell thesurface. If the host cell does not have the nucleic acid is released from the capsid (un- specific receptor sites it cannot be infected coating). by that particular virus.

8.72 Production of Viral Virus nucleic acid and virus components During the production of virus nucleic Components are produced by the host cell by using infor- acids and virus components, the complete mation provided by the viral nucleic acid. virus cannot be identified in the cell.

8,(73 Maturation Maturation of virus particles occurs when On maturation, completed virus particles the nucleic acid is packaged within the cap - may be identified within the cell. aid.

8.74 Release of Virions

8.741 Cell Lysis Some types of viruses cause cell death and It is this cell destruction which causes the lysis of the cell with release of mature naked symptoms of the virus disease. virions.

8.742 Budding Some viruses are expelled from the host The infected host cell may die or may not cell one at a time by a type of reverse phag- be affected at all. The unaffected cell may ocytosis. The host cell is not disrupted. Thedivide and the virus can be passed on to the virus buds through the plasma membranedaughter cells. Membranes through which which has been altered to incorporate viralthe virus is extruded may be nuclear, endo- protein. This budding process results in theplasmic reticulum, or cytoplasmic. virus becoming enveloped. Medical Microbiology-47 2" f) TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 8.75 Site of Replication In general, DNA virus replication is initi- Exceptions are the poxviruses, which are ated within the nucleus of the host cell,DNA but replicate in the cytoplasm. whereas RNA viruses replicate in the cytoplas

8.76 Inclusion Bodies During virus multiplication, virus-specific Virus inclusion bodies are considered to Prepared microscope slides of various structures calledinclusion,bodies may bebe the site of development of viruses or, in types of inclusion bodies may be studied. produced. These become large enough to besome cases, masses of virus particles. Negri- seen with the light microscope after staining. bodies, found in neurons in cases of rabies, / They may be cytoplasmic, nuclear, or both.are examples of inclusion bodies.

8.8 Cultivation Viruses ,have no metabolic machinery of their own and therefore must rely on the host cell to reproduce the virus particles.

8.81 Embryonated Chicken For diagnostic purposes, for preparation After incubation of the egg the presence A diagram demonstrating egg inoculation Eggs of vaccines or for research, viruses may be of virus is suggested by curling or tucking ofmay be shown. cultured in elnbryonated eggs. The eggs are the embryo, thickening of membranes, in- inoculated in the yolk sac, the allantoic sac, crease or decrease of fluid, hemorrhage, or or into the chicken embryo, depending uponcongestion. Specific tests have to be per- a the" virus. formed to detect the presence of a virus.

8.82 Tissue Culture A tissue culture consists of individual cells Antibiotics may be added to the medium A projector slide of a tissue culture may suspended in a rich medium in a test tube or to prevent bacterial contamination since thebe shown. petri dish and supplied with proper gaseous antibiotics will not inhibit the viruses. Pres- and temperature conditions. In the classicalence of virus may be determined by plaque example the cells reproduce until the con-formation or by certain changes in the'cells tainer is covered with a single layer of cells. (cytopathogenic effects or CPE). These cells may be subcpltured to produce 40' multiplesingle-layeredcultures(mono- layers).

8.821 Types of Tissue Culture Tissue cultures may be produced from Monkey kidney or human placenta may tissue freshly removed from an animal (pri- be used for primary tissue culture. HeLa mary cell culture). An established cell line iscells are an example of an established cell a tissue culture which has mutated so thatline. These cells are available commercially. it can be transferred repeatedly without dying out.

8.83 Experimental Animals Animals such as_ mice may be inoculated Suckling animals are particularly suscep- successfully with certain viruses of human tible to virus infection. The presence of virus origin. may be noted by signs produced. 24.1 Medical Microbiolog4-49 24 TOPICS AND SUBTOPICS A: ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 8.84 Cultivation of Bacterio- bacteriophage may spread on medium in Bacteriophage which is specific for E. coli Students may heavily inoculate phage phitge +, a petri dish which has been inoculated heav-may be isolated from sewage or other natu-agar with E. coll. The bacteriophage T4 may ,4ily with, a bacterial suspension. After incu-ral sources. be added to liquefied phage agar, which is 43ation;-the bacterial growth will show areas then added to the petri dish as an overlay: (plaques)" where the virus particles invaded After overnight incubation, the plaques the bacteria and caused cell lysis. (areas where the E. coli were destroyed) may be observed. 9.0 General Characteris:; tics of Pr4tozoa and lielminths` 9.1 Protozoa' Protozoa are unicellular non-photosyn- Some protozoa live as saprophytes; others Live protozoa may be purchased from bi- thetic, eucaryotic organisms. They lack aare parasitic for all or part of their lives. ological supply houses or found in ponds or tell wall and generally are motile. streams.

9.2 Trophozoite Trbphozoites (active vegetative stage) are Prepared microsAve slides of tropho- susceptible to dehydration, acid, and various zoites and cysts of aariety of species may 'other unfavorable environmental conditions. be studied.

9.3 Cyst The vegetative or trophozoite stage of cer- In many parasitic species the more resist- fain protozoa may undergo nuclear division,ant cyst stage is necessary for survival out- store reserve' food material, and secrete a side the body during passage from one host

4.4 resistant wall forming an inactive stage (theto another.' This stage also protects the or- cyst). ganism from the destructive action of gastric 4 ' juices after the cyst is swallqwed. Therefore it is the infective stage for :'(species which develop cysts.

9.4 Life Cycle The life Cycle of protozoa may be simple Human intestinal, vaginal, and urogenital Charts which depibt the life cycles of var- and direct or complex and indirect. A simpleprotozoa have a simple life cycle. Tissue andious types of protozoa, both free living and itrld directilife cycle would involve a tropho-blood protozoa suchfcliS Leishmania andparasitic, may be studied. zoite.or OA which is infective for a new hostPlasmodium have a complex life cycle. In some timpjilter it leaves the human bodyLeishmania, humans are the definitive host and does not involve an intermediate host.(harbor the intracellular stage) and arthro- A "complex and indire6t life cycle involvespods are the intermediate host (harbor the one or more intermediate hosts which har- extracellular stage). In Plasmodium (the eti- bor the immature stage and a final hostological agent of malaria), mosquitoes are which harbors the mature stage of the par-the definitive host (sexual reproduction asites. j)epending on the organism, humanstakes place here) and humans are the inter- may be either the intermediate or the finalmediate host (only asexual reproduction oc- host. curs here).

9.5 Locomotion All protozoa are motile during at least one Locomotion may be used in obtaining food stage in their life cycle. Protozoa are classi-as well as responding to other stimuli. In Medical Microbiology-51 2"G 11111 ill

TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES fled, at least in part, on the basis of theirgeneral, parasitic protozoa are less motile locomotor organelles. These include pseu-than are free-living species. dopodia, flagella, cilia, and undulating membranes.

9.6 Classification Protozoa of medical importance may be Infectious diseases caused by .these orga- Prepared microsco0 slidea and preserved divided into four groups; Sarcodina, Masti- nisms are common in many parts of thespecimens are available for all four groups. gophora, Ciliate, and Sporozoa. world. In areas where the diseases occur, asymptomatic cases are common.

9.61 Sarcodina The Sarcodinainclude the amoebae Reproduction is by binary fissionor which move. by pseudopodia. A pseudopo- through cyst formation in which two or more dium is formed by the amoeba extendingnuclear divisions may take place. part of itself forward and then pulling the rest of the cell up into the extension.

9.62 Mastigophora In the trophozoite stage, the Mastigo- Reproduction occurs by longitudinal di- phora possess one or more flagella and, invision or by encystment with the formation some species, an undulating membrane. Theof small immature forms. Some parasitic cell membrane is differentiated into a well -forms require alternation of hosts to com- defined pellicle which gives a definite shapeplete their life cycle. to the trophozoite.

9.63 Ciliate The Ciliate possess cilia (fine h irlike ap- The Ciliate reproduce by binary fission pendages which sweep in unison) duartand by conjugation, the latter taking place ri or all of their life. cycle. They possess twoevery several hundred generations. nuclei: a vegetative macronucleus anda gen- erative micronucleus:

9.64 Sporozoa Sporozoa are parasitic, generally without Reproduction has become highly special- visible locomotor organelles, and usually ized in these protozoa and involves both form spores at some stage in their life cycle. sexual and asexual phases. Vertebrates and invertebrates serve as hosts.

9.7 Physiology Protozoa are heterotrophs requiring large The Mastigophora and most Sporozoa ob- amounts of water. Most are either strict tain dissolved organic material through their aerobes or facultative anaerobes. cytoplasmic membranes. Sarcodina obtain nutrients through phagocytosis, whereas the Ciliate have a cytostome which,. leads to a gullet through which food particles are taken in.

9.8 Helminths Helminths are worms that are considered Projection slides of eggs, larvae, and in medical microbiology because the diag- adults of helminths may be studied. The Medical Microbiology-53

2'__ C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION nosis of these infections depends principally larger helminth adults may be purchased as upon the identification by microscopic ex- preserved specimens. Charts of the life cy- amination of eggs or larvae found in feces or cles of the various helminths may be studied., other excretions. Some species of helminths are free living and others are parasitic.

9.81 Aschelminthes (Nema- Nematodes, are elongated, cylindrical, un- The roundworms are characterized by a todes or Roundworms) segmented worms tapering toward the headcuticle-covered unsegrnented body, com- and tail. They have separate sexes and aplete digestive system, with mouth and anus, well-developed digestive system. There may nervous system, excretory system, genital be finlike projections of the neck region orsystem, and separate sexes. Diagnosis is the tail which are an aid in diagnosis. made by finding eggs or larvae in feces, urine, blood, or tissue.

Nematodes with simple life cycles include 9.82 Life Cycles The life cycles of the intestinal nematodes vary from simple, in which the egg stage isEnterobius, Ascaris, and Trichuris. Nema- infective within a few hours or weeks aftertodes with complex life cycles include the it is passed in excrement; to complex, inhookworms Necator americanus and An- which the developmental cycle outside thecylostoma duodenale. host involves free-living generations with the ultimate production of an infective stage.

9.83 Platyhelminthes (Flat- In platyhelminthes the digestive tract is worms) absent or, if present, does not have an anus, with wastes regurgitated through the mouth.

9.831 Cestodes (Tapeworms) The cestodea or worms consist of a The scolex is adapted for attachment to "head" or scolex, a neck, and a series of body the intestinal mucosa. The unsegmented segments (proglottids). Cestodes are mo- neck is a budding zone that gives rise to the noecious (both sexes in the same. organism).rest of the worm. Each proglottris c pable- Worms may reach 20 feet in length. of producing eggs. 0

9.832 Life Cycle The life cycle of the Cestodp is normally Taenia soliumz may involve pigs as the indirect and includes alternation of genera-intermediate host and humans as the defin- tions between intermediate and definitive itive host, whereas Hymenolepsis nana may hosts. or may not have an intermediate host, although the cycle is rarely direct.

9.84 Trematodes (Flukes) Trematodes are elongatedandleaf- They often have a mouth surrounded by - shaped parasites usually having two suckersa muscular sucker and a second sucker for found on the ventral surface. attachment. Most trematodes are monoe- cious, but the schistosomes (blood flukes) have separate sexes. Medical Microbiology -55 2*.;.) C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION 9.85 ° Life Cycle Trematodes have complicated life cycles Mollusks involved as intermediate hosts involving one or two intermediate hosts (oneof flukes include many families of snails, of which is usually a mollusk) #nd severalboth freshwater and terrestrial. developmental stages and generations before becoming infective for the 'definitive host.

10.0 Inhibition and Killing of Microorganisms by Physical Agents The temperature that kills a 24-hour liq- 10.1 Heat Heat is an effective means of sterilizing many solids and liquids. Sterile is defined asuid culture of bacteria at pH 7 in 10 minutes absence of viable organisms. Spores areis called the thermal death point. The time more resistant to heat than are vegetativerequired to kill all bacteria in a given, sus- bacteria. Moist heat is more efficient thanpension at a given temperature is the ther- dry heat. mal death time.

10:11 Moist Heat

10.111 Boiling Boiling for 10 minutes will kill vegetative pathogens, but not necessarily endospores of pathogenic organisms.

Autoclaving is the most effective practical Heat-stable media for bacterial cultiva- Pictures of autoclaves (largefloor models 10.112 Steam Under Pressure and (table models) may be studied. Use of (Autoclaving) method for heat sterilization. Exposure totion are usually sterilized by autoclaving, steam (121°C) in an autoclave at 15 lb ofand cultures, growing, on media, are auto-the autoclave, if available, may be demon- pressure for 20 minutes will kill all forms ofclaved before being discarded. strated. life including endospores.

10.113 Pasteurization Pasteurization destroys vegetative patho- The pasteurization of milk ,involves heat- gens but does not destroy all bacteria noring at 60°C for 30 minutes /10 r 72°C for 15 does it destroy endospores. Foods commonly seconds (flash method). Ttie heat of pas- pasteurized to destroy pathogens includeteurization reduces the nu ber of bacteria milk and cheese. to levels making disease transmission im- probable. Some toxins, however, may survive pasteurization temperatures.

10.12 Dry heat Glassware, metal, and petrolatum oils are Dry heat destroys rif i.00rganisms by ox- usually sterilized in a dry heat oven at 160idation, whereas moist eat destroys micro- to 180°C for 2 hours. This procedure killsorganisms by protein lenaturation. vegetative organisms and endospores.

10.13 Incineration Incineration (burning) kills all microor- Carcasses of infectedanimals, infected Medical Microbiology-57 4 2 2 2-11 C PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION ganisms, Inoculation loops for transfer ofdressings, etc., should be incinerated. Incin- ,microorganisms are sterilized by incinera-eration of inoculation loops containing or- tion. ganisms with a Bunsen burner may cause sputtering and allow some of the microor- a ganisms to escape before they are killed. 2' . , 110 10.2 Drying Drying in air at room temperature is lethal There is much variation in.the lethal ef- to many vegetative pathogens but not nec-fects of drying. Tuberculosis organisms and essarily to endospores bacterial endospores are very resistant, whereas the gonococcus is quite sensitive. For this reason, intimate Contact is necessary for continued transfer of the gonococcus.

10.3 Freezing Freezing can be lethal to many bacteria Repeated freezing and thawing is an effec- although generally it is bacteriostatic (in-tive way of, killing many microorganisms. hibits growth). Freezing and then removing the moisture under negative pressure is a mechanism used for preserving organisms and is known as lyophilization.

'Ultraviolet Radiation UV light is often, used for reducing the UV light acts by forming pyrimidine (thy-) The effects of varying exposures of UV 10.4 light on the growth of bacteria on nutrient (UV Light) microbial population in operating rooms andmine) dimers from adjacent monomers on laboratories. The effectiveness of UV lightthe same DNA strand. In media it also pro- / agar may be shown. is limited 6y its low penetrability. duces peroxides. Both of these mechanisms are harmful to microorganisms and, depend- L ing on concentrations, may be lethaL UV light is also harmful to the eyes and skin. Pictures or exampled\pf various filtration 10.5 Filtration Heat-sensitive substances such as toxins, Cellulose acetate or cellulose nitrate mem- carbohydrates, serum, and plasm ster-branes with predetermined pore sizes aredevices may be shown or demonstrated. ilized by being passed through filters whichmost commonly used for filtration. hold back bacteria. Viruses are not retained by bacterial filters.

10.6 Physical Disintegration Grinding bacterial cells will destroy them. It is difficult to attain 100% kill by these High-frequency ultrasonic waves will alsomethods although ultrasound is more effi- disrupt bacterial cells. cient than grinding.

11.0 Inhibition and Killing - of Microorganisms by Chemical Agents 11.1 Sterilization Sterilization is a process which kills all Sterilization of materials is important in Medical Microbiology-59 2 2 :1 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION forms of life in a given preparation or envi-operating and delivery rooms* Gowns, in- ronment. struments, etc., must be sterile. In the laboratory all glassware and media must be sterile before microorganisms can be isolated and identified. A laboratory experiment which exposes 11.2 Disinfection Disinfection is the destruction of patho- Disinfection does not necessarily destroy gens on inanimate objects. It includes anyspores or certain nonpathogenic microorga-bacteria and bacterial spores to disinfectants process which kills all microorganisms whichnisms. for various lengths of time and various con- are capable of producing infectious disease centrations may be performed. (pathogens). The action of disinfectants is affected by time, temperature, acidity, and the susceptibility of the various bacteria. The presence of organic material, however, may interfere with the action of many disinfectants. An experiment comparing the growth of 11.3 Antiseptics Antisepsis is defined as destruction of pathogens in wounds or on body surfaces. bacteria in the presence and absence of com- Antiseptics may inhibit the growth of bac- mercial antiseptics may be performed. For teria without necessarily killing them. example, saturate a filter paper disk with an antiseptic, place it on a plate seeded with a bacterial culture, and incubate it overnight.

Skin flora may be cultured before and 11.4 Representative Chemi- Chemical agents inhibit or kill microor- The types of microorganisms and partic- cals Used for Disinfec-ganisms by disrupting membranes, denatur-ular situations often determine the type ofafter hand washing with an antiseptic solution and Sterilization ing proteins, or interfering with nucleic acidchemical agent used for disinfection or ster-tion to demonstrate a decrease in total bac- synthesis or functions. ilization. terial population.

11.41 Halogens (Chlorine, Io- Halogens owe Much of t eir bactericidal Chlorine is commonly used to destroy dine, Bromine, and Flu-action to their oxidizing act vity which in-pathogens in drinking water. However it also orthe) activatesenzymes. They areeffectivereacts indiscriminately with organic mate- against sporulating organic rial. Iodine -is commonly used in a dilute alcoholic solution (2.5%) as a skin antiseptic.

11.42 Alcohols Alcohols disorganize the lipid structure of Both 70% denatured ethyl or isopropyl cell membranes and also denature cellularalcohol are commonly used as antiseptics. proteins. They do not kill spores and should not be relied upon for instrument sterilization.

11.43 Phenols (Carbolic Acid) Phenols and cresols cause membrane A 5% solution of phenol kills all vegetative and Related Compoundsdamage with leakage of cell contents andorganisms and after a few hours will kill su 2 5 Medical Microbiology-61 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION ItLB. ENRICHMENT INFOR TION C. PRACTICAL, ACTIVITIES resultant lyais. They are also bactericidal bybacterial spores. Cresol is more effective and inactivating enzymes. Their action is notless toxic than phenol. inhibited by the, presence of pus, serum, or other extraneous organic matter,

11,44 Detergents (Wetting Detergents aresurfaceactiveagents There are cationic, anionic, and nonionic AgentsorSyntheticwhich act by disrupting cell membranes. detergents. Cationic are most effective. An Soaps) example of a cationic detergent is a quaternary ammonium compound which is water soluble. Detergents are often used in hospi- tals.

11.45 Heavy Metals(Silver, Metallic ions, especially of the heavy met- Silver ions combine with and inactivate- '3 Mercury, Lead) Ms, are toxic to bacteria. A solution of silver enzymes. Mercury reacts with the SH nitrate is commonly used to guard the eyesgroups of proteins and inactivates bacterial of the newborn infant at birth from infection enzymes. Organic materials reduce the effi- and possible blindness due to Neisseria gon- ciency of heavy metals. orrhoeae.

11.46 Ethylene Oxide Ethylene oxide is a gas which is strongly Ethylene oxide is very inflammable and bactericidal and virucidal provided the hu-concentrations of the gas in the air as low as midity is 20 to 40%. It readily penetrates3% make an explosive mixture. Ethylene polyethylene wrapping material and is usedoxide is made nonexplosive by4ombining it for sterilizing heat-sensitive plastics, e.g., sy-with 90% carbon dioxide. It' is ixtensiyely ringes, pipettes, needles, and petri dishes. used for disinfection of suigiiCal ins r6ents and materials in specially; desi: ed autoclaves.

11.47 Forma lin Vapor Formalin vapor2t a concentration of 1 or 2%, is bactericidal and may be used to disinfect roops( blankets, and bedding. It is cie9ttf the humidity is over 50% and the temperature is raised to about 50°C; It is also used to disinfect breeding facilities for e 4, poultry and other livestock.

12.0 Antimicrobial Agents 12.1 Antibacterial Agents Antimicrobial agents. are chemicals which The bases of selective toxicity depend are used for the treatment and control ofupon unique structural features or metabolic infectious diseases. Desirable properties ofprocesses of the microorganism against antimicrobial agents include: (i) selective ac- which the antimicrobial agent is directed. tion against the microorganism with minimal damage to the host cells; (ii) minimum side effects; (iii) stability within the host; Medical Microbiology-63

J. 'TOPICS-AND SUBTOPICS A. ESSENTIAL INFORMATION . B, ENRICHMENT INFORMATION. C. PRACTICAL ACTIVITIES (iv) minimal development of bacterial resistance to them,

Mechanisms of Action Antimicrobial drugs act by inhibiting cell wall 9r protein synthesis, injuring the cell 24 membrane, interfering with nucleic acid synthesis, or interfering with intermediary metabolism.

12.12 Antibiotics Antibiotics are chemicals which are pro- Some antibiotics have d narrow spectrum duced by bacteria and fungi and are able to in that they are effective against either inhibit the growth of (bacteriostatic) or de--4egram-positive or gram-negative bacteria, stroy (bactericidal) other microorganisms.whereas others have a broad spectrum and The chemical structures of many antibioticsare effective against both gram-positive and have been determined and a few are nowgram-negative organisms. produced synthetically.

12.121 Cell Wall Inhibitors A number of antibiotics interfere with the Antibiotics which interfere with cell wall cell wall synthesis of bacteria. The penicil-synthesis at earlier steps in the development lins and cephalosporins, the major group ofof the cell wall include cycloserine and bac- cell wall inhibitors, interfere with the final itracin. Since animal cells do not have a cell step in cell wall synthesis. Bacteria usually wall, they are not directly affected by these do not survive without intact cell walls, thus antibiotics. cell wall inhibitors are generally bactericidal.

12.122 Interferencewiththe Certain antibiotics act as surfactants and j Polymyxins and the antifungal agent am- Cell Membrane attach to the cell membrane, thereby dis-photericin B are examples of antimicrobial rupting its integrity. The regulatory activityagents which interfere with cell membrane of the membrane is compromised, resultingintegrity.. in bacterial death.

12.123 Protein Synthesis Inhib- A large number of antibiotics interfere One important group of protein synthesis itors with protein synthesis. The inhibition ofinhibitors is the amjnoglycosides which in- protein synthesis does not result in the deathclude such antibiotics as streptomycin and of the organisms and thus this group ofgentamicin. In contrast to the other protein antibiotics is generally bacteriostatic. Ex-synthesis-inhibiting antibiotics, this group of amples are chloramphenicol, tetracyclines,antibiotics is bactericidal. and erythromycin.

12.124 Nucleic Acid Inhibitors Because of the universality of nucleic acid synthesis and function among all living forms, compounds which selectively interfere with microbial nucleic acid and not host nucleic acid synthesis and function are dif- Medical Microbiology-65 2 a 250 TOPICS ANI) 8.011T01104 . A. NSSENTIAL INFORMATION Ii,I.NItIUIIMtN'l'INPORMATION C, PRACTICAL ACTIMIN6 ficult to develop, 'Chun, only a few antibiot- ico,'Huch as griseofulvin, an antifungal agent, and nalidixic acid, used in urinary tract infections, are clinically useful.

s 1-212b_ --Antimicrobial prOga Several synthetic compounds have been that Interfere,with In- developed which show antimicrobial activ- termediarY M-i3tabolisrn ity. Since intermediary metabolism is gen- (Antimetabolite), erally similar in all forms of life,, many compounds which have antimicrobial activity are also toxic to host cells. Only a few have been found to be selective for bacteria and include the sulfa drugs, a number of anti- tuberculous drugs, and some drugs effective 'against bacteria causing urinary tract infections.

12.2 Antifungal Agents Antifungal agents are usually also toxic to Griseofulvin is used to treat dermato- host cells because fungi are metabolicallyphytes; amphotericin B is used to treat sys- similar to human cells (fungi are eucary-temic mycoses such as histoplasrnosis and otes). cryptococcosig. Mycostatin is used to treat superficial candidiasis.

12.3 Antiprotozoan Agents Arsenicals, antimony compounds, qui- Metronidazole is a common agent used to ci nines, and other chemical agents are used totreat intestinal hepatic amoebiasis and tri- ci? treat protozoan infections. chamoniasis.

12.4 Antiviral Agents Antimicrobial agents are not effective I.doxuridine (IDU) is effective against eye against viruses because viruses are not cellsinfections caused by herpes simplex virus. and do not have intrinsic metabolic capabil- Adenine arabinoside is also effective against ity. A few chemical compounds that haveherpes simplex of the eye and also herpes limited effectiveness against certain virusessimplex encephalitis. Amantadine hydro- have been developed. chloride is an effective chemoprophylactic and chemotherapeutic agent against influenza Type A.

12.5 Susceptibility Versus An organism is said to be susceptible to a The higher level required to destroy or Resistance of Microor-given antimicrobial agent if it is either killedinhibit the resistant organism may be toxic ganisms or inhibited when exposed to a concentra-to the host or may be unattainable because tion of the drug known to be attainable in of rapid excretion or metabolism by the host. the patient. A resistant organism is not killed or inhibited by attainable concentrations.

12.6 Drug Resistance Many bacteria develop resistance to an Resistance may be due to production of Medical Microbiology-67 2 IP TOPICS ANI) SUBTOPICS A. ItISHIINTIAL INPORMATICN II, 101111011MIOIT C. PRACTICAL ACTIVITIHR antibacterial agent, -.Wootton of resistantenzymes which brush down the drugs, a strains may be enhanced as a result of in-change in membrane permeability which adequate or prolongedtreatment,Re- does not allow the drug to enter the cull, an mistime° is more likely to occur with bade- alteration of one or more of the synthetic riostatic agents, Resistance to a given agentpathways, or an increase'in the production moans that, the microorganism has devel- of a compoetitive substrate. oped the rosiatnco, not the patient.

12.61 Mechanisms of Micro- Bacteria develop resistance to antimicro- IQ Resistance bial agents either by mutation or by gene transfer involving conjugation or transduction. DNA transferrod_t2 a susceptible recipient bacterium contains genes from a resistant bacterium which code for resistance factors. Ip this manner, an organism susceptible to one or several agents becomes resistant to these agents.

12.7 Use of Antimicrobial Drugs

12.71 Chemotherapy In chemotherapy or treatment of a dis- A bloodborne infection would require a ease, antimicrobial agents are used in thedifferent treatment than would an abcess minimum dose required to treat a particularwhich is walled off from the bloodstream. infection in a patient. This will vary withAn immunocompetent individual would re- the location, distribution, and metabolic ac-ceive different treatment from one whose tivity of the particular organism as well asiminunocompetence was impaired. the adsorption, distribution, and concentration of the agent in the host.

12.72 Chemoprophylaxis Chemoprophylaxis is the use of antimicro- Chemoprophylaxis might be indicated for bial drugs to prevent the establishment ofan individual with rheumatic fever and heart pathogenic microorganisMs in the body. Itvalve damage who may be given antibiotics is often used in selective cases after exposurebefore .a tooth extraction. This prevents cer- to, pathogens or following certain types oftain oral microorganisms which enter the surgical procedures. Chemoprophylaxis isblood stream from establishing themselves usually limited to the action of a specificon the dame d heart tissue. Patients un- drug against a specific microorganism. Gen-dergoing card ovascular surgery are usually erally speaking, the use of antimicrobialgiven antibio cs for a few days before and drugs for chemoprophylaxis is discouragedseveral d after surgery to prevent bacte- and used Qnly under certain specific condi-ria antation on damaged heart tissue. tions. The indiscriminate use of antibiotics for chemoprophylaxis tends to enhance the selection of resistant organisms. Medical Microbiology-69 2L 21) TOPICA ANI) A, It:SHItINTIALINVOI1MATI9N H. iiNitinimilINT iNroitmArrioN 0. PRACTICALACTIVITtleiti 127:1 Choice awl Ilse of Anti- Mont antimloroial drugs are eiicretedin huniequato ntlidotie therapyis ofien icrobialAgenta the urine and10111111111.0 II1010.1.04A(41by the more iannilli than lingapy, liver, Therefore,to maintain/aconstant An individual who takeshalf the prearil; 1 blood level, one must give an,lnicroldals dosage ('us' half the time Interval In invitin constant intervals orIna 1911g-Ileallg I.01111. the selection Of rosiatont microorganism; Once begun, the adminiattation of «Mimi- (Tubb& should he continhed long enough to eliminate the mi ifourfonism and to mini- mizeor prevent lho selection of resistant mutants. '

13.0 Host-Parablte'---Itela. ..._3 tionahlps . I lost, Parasite Relation: Organisms live in various rolationti.hips to ships each other in lature. In the human body, host-parasite ri lationshipa.may exist in the mouth, the intestinal tract, vagina, skin, and pathological Winona. Several types of relationships exiswhich account in part for different orga iisms living together in the same area.

13.12 Symbiosis Symbiosis Js the coexistence of two or more dissiliar orgaims with a certain degree of co latency.

13.13 Commensalism Commensalism is a relationship between Aerobic and anaerobic microorganisms two types of organisms in which one orga- may live in the same place. The aerobic nism benefits from the association but theorganism may create a low oxidation-reduc- 14% other is not affected. tion potential which allows the a robic organism to grow and multiply.

13.14 Mutualism Mutualism is a type of symbiosis in which Termites eat wood but are unable to di- both organisms are benefited and survival gest it. Certain flagellates in the termite's depends upon the relationship. intestinal tract digest the wood. The products of digestion are then metabolized by the termite. The flagellates are in turn unable to survive outside the termite.

13.15 Parasitism Parasitism is the state in which one organism lives on or within another living organism (the host) in any environment favorable for growth. The parasite benefits at the expense of the host. 2,J Medical Microbiology-71 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 13.2 Pathogen A pathogen is a microorganism capable of causing disease.

13.3 Normal Flora The normal flora of the human body in- The resident flora consist of those micro- The normal flora of the skin may be cub clude all the microorganisms which live onorganisms which live on or in the body fortured and identified in the laboratory. the skin and mucous membranes of humansrelatively long periods of time. The "transient without producing disease. flora consist of microorganisms that inhabit the body for perhaps hours, days, or weeks but do .. not establigh themselves permanently.

13.31 Significance of Normal The normal flora are valuable to the host Pseudomonas (a potential pathogen) may Flora in keeping the relative numbers of each typebe present in the intestinal tract. The num- of organism in balance and excluding otherbers of such organisms are kept in check by potentially pathogenic microorganisms. the normal flora so that clinical disease does not occur. ao 13.32 Opportunists Opportunists are organisms present in the Alpha-hemolytic streptococci are part of environment or as part of the normal florathe normal flora of the mouth. After tooth of the host which will produce disease under extraction however, these organisms may extraordinary circumstances. If in an indi-gain entrance into the bloodstream and pro- vidual the balance of the normal flora isduce heart damage. upset or the host defenses are compromised an opportunistic organism may produce disease.

13.33 Changes in Body Flora Antibiotic therapy, natural or therapeutic %n individual who has had extensive changes in hormone level, infectious or or-treatment with antibiotics may develop can- ganic disease, massive X-irradiation, and itp- didiasis. This fungus, which is normally pres- munosuppressive agents may cause a change ent in low numbers, flourishes after the an- in the number and kind of bacteria present tibiotic-sensitive members of the normal in a given area. bacterial flera are removed or diminished.

13.4 Disease States

111.41 Disease Disease is a detrimental change in function or structure in an individual which is discernable clinically. An acute disease has a relatively rapid onset and a short duration. i.e . influenza. Chronic diseases such as tuberculosis have a gradual onset and a long duration.

Medical M icrobiology 7:1 2: C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION 13.42 Infection Infection is the invasion and multiplica- Poliovirus may produce an infection with tion of microorganisms in tissues with oror without symptoms. without the production of disease.

13.421 Infectious Disease An infectious disease is one produced by Influenza, gonorrhea, athlete's foot and a microorganism. 4. malaria are all infectious diseases.

13.422 Communicable A communicable disease is one in which The common cold is a communicable dis- (Contagious) Disease the etiological agent is readily transmitted ease. A patient with tetanus is considered to from person to person. have a noncommunicable disease since it is not spread from person to person.

13.423 Localized Infection A localized infection is an infection re- A boil or a wart on the skin is a localized stricted to one part of a tissue or area of the infection. body.

13.424 Generalized Infection A generalized infection is an infection in- Typhoid fever is an example of a gener- volving many tissues, organs, or systems ofalized infection. the body.

13.425 Inapparent (Subclinical) An individual who had an inapparent in- Mumps virus may produce an apparent or Infection fection does not have clinical symptoms, inapparent infection, both of which produce though specific immunity may be initiated a long-lasting immunity. or bolstered. The infectious agent may or may not spread to other susceptible individuals.

13.426 Latent Infection A latent infection is a persistent inappar- Herpesvirus may produce a latent infec- ent infection which has the ability to inter- tion. The infected individual may show no mittently produce clinical signs of disease. clinical signs until after a severe sunburn or a common cold, after which fever blisters due to the viral infection appear.

13.427 Carrier. A carrier is an individual who harbors a Shigella species may be carried in the potential pathogen and is capable of spread- intestinal tract without producing symptoms ing it to the environment or to other suscep- in a given individual, but the individual may tible individuals. The carrier does not ex-be able to transmit the organism to Other hibit clinical signs or symptoms of the dis- people by means of food or water contami- ease. nated with fecal material.

13.428 Secondary Infection When one organism invades a host and, . An example of a secondary infection is a produces clinical or subclinical disease, alesion produced when a person who has second organism then establishes itself and chicken pox scratches the pox lesion, allow- produces clinical disease on the damageding dirt and bacteria to reach the damaged Medical Microbiology-75 2 u ".) TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES tissue. The second organism is usually nottissue. A purulent lesion (one containing capable of initiating an infection alone. pus) may result as a secondary infection.

13.5 Host Resistance Fac- tors

13.51 Mechanical Factors Intact skin, the mucous coating of the Burn patients develop infections rapidly upper respiratory and intestinal tract, andbecause of the loss of intact skin. Decreased the cilia of the respiratory tract are all ex- capillary action and the loss of mucous coat- amples of mechanical barriers to microor- ing of the respiratory tract in heavy cigarette $ ganisms. smokers may lead to increased respiratory infections.

13.52 Chemical Factors Chemical barriers to disease-producing Chemical barriers to disease-producing organisms include gastric juice, complement,organisms include: gastric juice, which is lysozyme, beta-lysine, and interferon. extremely acidic; complement, a normal blood protein which is active in certain antigen-antibody reactions as well as in some non-immunological ones; lysozyme, an enzyme which dissolves some bacterial cell walls and is found in tears, nasal secretions, and saliva; and interferon which acts by preventing viral replication in other healthy cells. %.

13.53 Individual Factors People differ as to their susceptibility to Debilitation, physical stress, hereditary infection. This varies with their nutritionaldifferences, and emotional factors alter an state, age, sex, and certain physiochemicalindividual's resistance to infection. factors.

113.54 Nonsusceptibility of the An organism may be pathogenic for one Non-susceptibility of a host may betee\ Species host species, but another host may be com-perature related as in the case of anthrax, a pletely resistant to infection and/or diseasebacterial disease of cattle. The chicken is by the same organism. not succeptible to anthrax unless its body temperature is lowered, in which case it then becomes susceptible. In many cases the exact cause of non-susceptibility is not known, e.g., humans get mumps but canines do not.

13.55 Environmental Factors Climate, crowding, poor sanitation, smog, Tuberculosis is an example of a disease and other factors may all act as predisposingwhich occurs more frequently in crowded factors to infectious disease. conditions such as in institutions and hous- ing developments. Medical Microbiology 77 2 u 1 2 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION 13. ENRICHMENT INFORMATIONr C. PRACTICAL ACTIVITIES 13.56 Inflammation Inflammation is the normal response of a The inflammatory response to injury may host to injury. It is characterized 14 an in or may not be associated with an infectious crease in permeability of\capillaries in theprocess. A sterile blood clot may cause an area which leads to swelling and redness. inflammatory response. On the other hand White blood cells (neiitr4hils and laterinflammation caused by a bacterium in the macrophages) invade the area and phago-lung (pneumonia) is an infectious inflam- cytize cell debris and/or foreign substances.matory process. The inflammatory response includes the release of certain compounds which increase the permeability of small bloodvessels(capillaries).These compounds are said to be vasoactive.

13.57 Phagocytosis Phagocytosis is the engulfment of,,parti- Phagocytosis is the means by which the A diagram illustrating phagocytosis may cies by a polymorphonuclear neutrophil-or,body rnoves dead host cells and debris asbe studied. macrophage. well as foreign particles.

13.58 Reticuloendothelial Sys- The reticuloendothelial system (RES) in- The spleen, liver, and lymph nodes are tem cludes the mononuclear phagocytic cells ofespecially important in filtering microorga- the body found primarily in the liver, spleen, nisms, dead cells, and debris from the blood lymph nodes, and bone marrow. These cellsand lymph. help to protect the body by phagocytizing Intracellularfluidisreturnedtothe foreign substances and so are important in bloodstream by means of the lymphatic sys- helping to rid the body of microorganisms. tem. The unidirectional transport of lymph follows a route passing through a series of tissues by means of lymphatic vessels which resemble veins. These tissues include lymph nodes and lymph nodules which Filter lymph and add cells such as lymphocytes to it.

13.59 Specific Immunity Specific immunity is directed toward a specific antigen such as a microorganism or its product and is dependent upon the immune system.

13.6 Factors that Enable Mi- croorganisms to Cause Disease

13.61 Pathogenicity Pathogenicity is the ability of an organism Mycobacterium tuberculosis and Staphy- to produce disease. It may involve penetra- lococcus aureus are considered pathogenic tion of the host by the parasite or by its45teria. toxic products.

13.62 Virulence Virulence is the degree of pathogenicity of Some strains of M. tuberculosis and S. The virulence of an organism may be Medical Microbiology-79 2'J 2'' TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORM/6'10N C. PRACTICAL ACTIVITIES a given strain of a- pathogenic organism.aureus are avirulent. That is, these particu-tested by injecting it into an appropriate Many authors use the words pathogenicitylar strains are not capable of producing dis- animal. For example, virulence of the pneu- and virulence synonomously. The virulenceease. A pathogen may be virulent for onemococcuCmay be tested, by injecting it into of an organism' may be due to toxigenicity,host and not for another. a mouse. invasiveness, or oIher unknown factors.

13.63 Microbial Toxins Microbial toxins are poisonous substances Toxins interfere with the normal activity produced by a microorganism. of a host cell.

13.631 Exotoxins Exotoxins are poisonou proteins excre d Tetanus, botulism, and gas gangrene are by certain gram -post ivand a few gram-examples of diseases caused 6y exotoxins. negative bactena.,,They are released intoSome bacteria produce exotoxins only after their immediate environment which may be lysogenic conversion. the circulatory system or tissue of the host.

13.632 E.n terot oxins Enterotoxins are exotoxins which act on Enterotoxins may be under the genetic the intestinal tract of the Most. control of a transmissible plasmid, e.g., E. coli enterotoxin. 13.633 Endotoxins Endotoxins are part of the cell wall of All endotoxins produce the same general gram-negative bacteria and are liberated symptoms in the host regardless of the spe- upon cell lysis or death. cies of the organism from which they originate.

13.634 Mycotoxins Many fungi produce toxins that cause Aflatoxins from Aspergillus [lams may 4 acute or chronic intoxication or cell damage. contaminate animal feed or human food and Some toxins are able to produce neoplasiaproduce severe disease. (tumors).

13.64 Invasiveness Invasiveness is the ability of an organism Invasiveness involves counteracting nor- to spread within a host. mal hat defenses or having attributes which help the organism spread within the host from the original-site-of the infection.

13.65 Inhibition of Phagocy- Microorganisms may decrease engulfment Encapsulated pneumococci or cryptococci tosis (phagocytosis)by leukocytes or macro-are virulent. These same strains without phages by producing toxins which destroytheir capsules are avirulent and more ame- these cells (leukocidin) or by producingcapL. nable to phagocytosis. sules which make phagocytosis difficult.

13.66 Survival Within Phago- Some microorganisms may be virulent be- Mycobacterium tuberculosis is capable of cytic Cells cause they survive or multiply within phag- multiplying within phagocytic cells. In ad- ocytic cells and are resistant to the phago- dition, the infection may be spread from one cyte's hydrolytic enzymes. part of the body to another by these parasitized phagocytic cells. Medical Mic .ology 8 I 2..c TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAJ ACTIVITIES 13.67 Extracellular Products Certain bacteria produe enzymes which Coagulase produces ciotforrnation around are involved in the infectious process. a lesion and is producedby Staphylococcus , aureus. Leukocidin; ,a substance which destroys leukocytes is also produced by S. aureus.Hyaluronidase,1 an enzyme which brealts down the ground substance in con- nective tissue, is produced by certain streptococci. Collagenase, a proteolytic enzyme which promotes the spread of organisms through tissue, is produced by Clostridium perfringes.

14.0 Immunology 14.1 Immunity Immunity is the ability of an individual to resist and/or overcome a disease to which most or many of its species are susceptible. Immunity is security against any particular disease, infection, or toxic agent.

14.12 Basis of Immunity Immunity depends upon specifically re- A diagram of the human lymphoid system active leukocytes called lymphocytes. The and a microscope slide containing stained human body contains approximately 10'2 lymphocytes may be studied. lymphocytes which comprise about 1% of total body weight. Most lymphocytes are found in the spleen, lymph nodes, and Peyer's patches.

14.13 The Immune Response The immune response is a specific adap- If an individual has had measles as a child, tive response to a foreign material such ashe will not get ill on subsequent exposure. an infectious agent. There are two types ofHe is immune. However, he may still be immunitycellular and humoralwhichsusceptible to other diseases to which he has may aid in eliminating foreign material ornever been exposed. rendering it harmless.

14.14 Stem CellDifferentia- A bone marrow stem cell is an undiffer- Stem cells may differentiate into cells of diagram depicting the origin of T and B tion entiated precursor cell. Certain stem cellsthe erythrocyte series, leukocyte series, in-cells and their functional interactions may differentiate into lymphocytes. Those which eluding granulocytes (neutrophiles, baso- be studied. are responsible for humoral immunity (an-philes, and eosinophiles) and agranulocytes tibody) are called bone marrow lymphocytes(monocytes and lymphocytes). or B cells. Lymphocytes which are influenced by the thymus are responsible for cell- mediated immunity and are called T cells.

14.2 Antigen (Immunogen) An antigen is a substance which can in- The immunizing effect of certain antigens Medical Microbiology-83 2 ' n

..1 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES duce a detectable immune response when may be enhanced by mixing the antigen with introduced into the tissues of an animal. insoluble materials (adjuvant) which keep the antigen in the tissues for long periods of time. '

14.21 Foreign to the Body Antigens are perceived as foreign to a host's immune system. The host is normally tolerant (nonreactive) to its own tissues and does not consider them as foreign.

14.22 Nature of Antigens The best antigens are large,- chemically The term antigen is sometimes used complex macramolecules such as proteinsloosely to refer to viruses, whole bacteria and certain polysaccharides. Antigenicityand other organisms and cells used to stim- however; not only depends on the molecule,ulate an immune response. These materials but on the host system and the specificactually may contain hundreds of different conditions under which the molecule is in-antigens. troduced to the host.

14.23 Antigenic Determinants Antigenic determinants are those parts of Antigenic determinants may be as few as the antigen molecule that are actually in-three amino acids of a particular protein. A volved in binding with antibody. - single protein molecule may possess many different antigenic determinants.

14.24 Haptens A hapten is generally a small molecule Some individuals are sensitive to their which is incapable of initiating an immune metal watchbands. The metal, as a small response unless it is linked to a large carriermolecule or hapten, must combine with the molecule. protein of the individual's skin to act as a carrier before the hapten can become antigenic.

14.3 Humoral Immunity Humoral immunity results from the stim- Humoral immunity is especially impor- ulation of B cells and their transformationtant in protection from bacterial and viral into plasmticells which secrete immunoglob- disease. The types of infections combatted ulins. by B cells was determined partially by studying children born without B cells.

14.31 B Lymphocytes (Cells) B cells are located in germinal centers within follicular areas of the spleen and lymph nodes, and a small number circulate through the lymphatics and bloodstream. The presence of brununoglobulins on their cell surfaces differentiate B cells from T cells. About 8 to. 10% of circulating lymphocytes are B cells. Medical Microbiology-85 C. PRACTICAL ACTIVITIES TOPICS AND.SUBTOPICS' A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION arelargeMononuclear 14.32 Sensitization of B Lym- B lymphocytes 'are able to recognize for - Macrophages phocytea eign material (antigens) her directly orphagocytic cells which appear to be involved a with' the help of T lymphocytes or macro- with processing certain antigens before they ' phages. They undergo changes as a result ofaxe Presented to the lymphocytes. the reaction and are 'said to be sensitized. The sensitized B lymphocytes differentiate into clones of memory cells for future re- sponseandintoantibody-synthesizing L plasma cells. o tcr

1'4 14.33 Immunoglo6idins (Anti- Antibodies are immunoglobulins. Immy- Older terminology for immunoglobulins !a- body) e boglobulins are a specific family of heterb-includes ganunaglobulins' singe these' geneous proteins which are formed in re-teins migrate primarily in the gammaea sponse to antigenic stimulation and reactupon electrophoresis of serum.

la specifically with the4antigen that induced ti their production. They are present in the humors, or fluids of the body such as blood: 4 lymph, and tissue fluids.

Immunoglobulins are composed of one or The polypeptide chai e, joined te A diagram depicting the structure of the 14.34 Structuref Immuno- t basic inununoglobulin unit may be studied. globulins more ,basic units or monomers. Each mon-gether by disulfide bridge , omer contains two pairsof polypeptide chains; one pair is approximately twice the molecular weight of the other pair. The chains of high molecular weight are always ID identical and are called heavy or-H chains. The, loWer molecular weight chains are also identical with each other and are terined tight or L chains. , Charts listing the ,properties. of the five r, 14.35 Classesof Inununoglob- There are five antigenically different H Most classes of immunoglobulins hitve., chains designated as alpha, gamma, mu,been further antigenjcally divided into sub- immunoglobulin classes may be studied. ulinsr' delta, and epsilon., They are the baskfor the ;classes. 'A! five classes of immunoglobulins: IgA, JgG, c IgM, IgD, and IgE. There are two antigenically different L chains designated as kappa and lambda. .L chains are not'' distinct for any class of immunoglobulin, however, on any given immunoglobulin molecule both L chains are either kappa or lambda.

14.36 V,and C Regions Each immunoglobulin chain contains an anpino terminal portion called the variable Medical Microbiology-87 2' B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES TOPICS AND 'SUBTOPICS A. ESSENTIAL INFORMATION '. or V region and a carboxy to al portion called the constant or C regik 1 Any given antibody molecule is specific 14.361 V Region The V region is very heterogeneous and', contains the portion of the moleculewhich lk_i.for a given antigen and does not react with combines with the antigen. Theantigei "-lother;antigens. The heterogeneity of the an- binding site of the immunoglobulin molectkretigerNinding site enables inununoglobulins is formed by small numbers of amino acidOil': to be specific for any given antigen.' the V region of the H and L chains. A given plasma cell is committed to making antibody of only one specificity.

14.362 C Region The C region is fairly constant as to amino acids and is therefore used to antigenically classify the H chains into the five, imfuno- globulin classes and the L chains intolappa and lambda. The C region ofbthe H Chains is that portion of the antibody molecule which binds complement after the molecule has combined with its corresponding antigen (Medical Microbiology, Subtopic 14,38).

14.363 IgG IgG is the major immunoglobulin (80%) in the serum of humans and is extremely important in providing protection fromreinfec- tion. It is the only immunoglobulin to freely pass the placental wall and is therefore important in providing immunity to the new- 4 born.

14.364 IgA IgA occurs in serum but it is most impor- IgA aids in preventing an antigen from tant in mucous secretions. Two molecules ofinvading the body and establishing infection.

iil IgA are joined by a secretory, piece and are called secretory IgA. This antibodyis a major.defense mgchanism due to its abundunce in saliva, ears colostrum and other secre- II. it. tions. IN efficiently agglut.ites cells. Trans- 14.365 IgM IgM is the 'attestimm4noglobulin and with a molecular weightfusion reactions between: :binod types A, B, of4. imately 900,000. It is predominant0, and AB are a result of IgM antibodies to ...0%etrily immune responses and is especially the different types of erythrocytes. IgM is po,rtant in protection against infection formed actively by the fetus and reaches sea by gram-negative bacleria. adult levels in the serum by 1 year after birth. Medical Microbiology-89

t B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTI,NL INFORMATION r 14.368 IgD IgD is normally present in serum in trace), amounts. Its specific, functions have yet,to 4 be established; hoWeVer, it is the predominant irrinfundglobulin` on the surface of hu- -, nin;13.1ymphoCytesi,,, ,

14.367 IgE iii6sent in even smaller amounts These sensitizing ,antibodies are responsible for type )';- hypersensitivity or aller6-suchati hayfeveriThey exist in high concen-tratiohs inallecgic individuals and are bound to mast cellii and certain leukocytes. Upon reaction with a specific antigen (aller-. gen) they trigger the release of certain substances, e.g., histamine, which are responsible for the signs and symptoms of immediate

A hypersensitivity.

14.37 Mechanismof Action of Antibodies interfere with or inhibit the Antibodies activities of foreign agents through mechanisms such as: increased opsonization which facilitates phagocytosis of microorganisms and other materials; cytolysis of certain microorganisms and other .cells in the presence of complement; neutralization of toxins; and prevAtion of the adherence of microorganisms such as viruses ?to their target host cells.

Complement is a system of serum proteins Although complement consists of m which is activated by certain antigen-anti-proteins, it is referred to as if it were one body reactions and is necessary for anti-substance. An alternate pathway in which body-mediated cell lysis. When antigen and antigen-antibody reaction'is not necessary is antibody react in vitro, complement is alsoalso khown to activate complement. Com- consumed. This is used basis of theplement is also ,important in many other complement-ationfix test h may be usedphases pf inflammation. to detect the presence of antigen or anti-. body.

Organ transplants '!(e.g., kidney, heait) areJ1.0,sigrains, projection'slides -des showing 14.4 Cell-Mediated Immu- ,Cell-mediated immunity is mediated by T cells and is dependent on the presence of therejected because the antigens of one individ-.t..lig'aritiitecturepf the thymus may be stud- thymus at birth. It is responsible for allograftual may be different from those of another. ied. rejections and delayed hypersensitivity re-.Lymphocytes of a recipient react with the Medical Microbiology-91 216 leS,A1413 SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES

. actions and is important in reCovery fromhistocompatibility antigens of the donor or- viral infections. gan and cause rejection of the graft if To donor and recipient are not compatible.

14:41 T Lymphocytes (cq T cells' are lymphocytes which have been T cells are found in the subCprtical region 'influenced by the thymus. They vary greatlyof lymph riiidea and are the only type of in the chemical nature of their surface struc-lymphoCytes found in the thymus. 'Both T tute and are therefore capable of reactingand B cells may be stimulated by the same with different antigens and causing a varietymolecule. T and B cells occupy different of effects. They do not produce antibodies,,areas within the same lymphoid tissue. Each T cell will react only with the particular antigen for which it hasyle appropriate receptor sites on its surface. Some T cells assist other cell types such as B cells and macrophages by producing molecules that regulate cell function. These T cells are 4 either helper or suppressor cells.

14.42 Sensitization of T Cells A T cell which comes in contact with an . ,.antigen for which it isspecific is said to be l'ellcommitted or sensitized. Sensitized T cells multiply after exposure to antigen and give rise to clones of cells.

14.43 Lymphokines When a sensitized T cell comes in contact There are many lymphokines. A few of with its corresponding antigen it may releasethe more investigated include: macrophage soluble factors (lymphokines) which play ainhibition factor (MIF), lymphotoxin, chem- role in cell-mediated immunity. Lympho-otactic factor, and interferon. kines are active in localizing an infection or tumor, in increasing the inflammatory response, and, ultimately, in degrading an antigen.

Immunity attained as a result qpitposure' (. to antigen is said to be acquiredlniinunity: to a specific antigen may be actively or pas,' sive cqtured.

14. Acquired Active immunity may be acquired natu- nity '" -rally by having an infection or artifi'Cially by 4i; immunization proceditre such as vacci- high.

" 1) 1. ". '14'511 Primary Resgrdnse = The immune response that occurs after /Medical Microb:alogy 93 TQPICS AND SHI)TOPICS A. ESSENTIAL INFORMATION 4 ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES initialexposure to a given antigen is called the primary response. With humoral munity, IgM is usually the first antibo produced and is detectable in about a week.

14.512 Secondary Response ' Upon reexposure to the same antigen, the Two or.more injections of the same -antibody produces a greater response in. a spaced over a period of time are given in shorter period of time duo to the increasedinimunization programs toprod1cea long- amount of memory lymphocytes formed lastingimmunity.Teststodetermine after the primary contact. The humoral sec--whether an individual has specific anti- ondary or anamnestic -response produces bodies to an.antigen are performed to deter- IgG, and thesevantibodies may be detectable mine past or present experience with a spe- for long period& of time providing long-last- cific agent (Medical Microbiology, Topic 20). ing protection-

14.513, .,jrnmunization Vaccination is the injection of substanceS The exact timing of an immunization se- tion) which stimulate an immune response thatquende depends upon the type of ,vaccine protects the host against an 'infectious dis-used, the route of inoculation, rInd the im- ease. 'The term vaccinatial was finally munological competence of the individual. used to indicateinurrization agaat vac- Vaccines are not always injected, e.g., the cinia or cowpox -kit utm,Vhich confed pro- Sabin vaccine is taken orally. tection to smallpox vitut. The injected agent is called a vaccine. 14; Types of Vaccines Products used as vaccines include: killed The Salk polio vaccine contains killed microorganisms;attenuatedmicroorga- polioviruit whereas the Sabin polio vaccine nisms, i.e., organisms which are living butconsists of live attenuated poliovirus. which have been altered so as to produce nization to tetanus and diphtheria immunity but not disease; and toxoids, i.e.,is c'erriec out with tetanus and diphtheria toxins which have been altered so as totoxoids.' produce immunity but not disease. DPT v ccine refers to diphtheria, pertus- sus, and- tetan s and is given-to infants at 2 months of age with boosters,at various time intervals. . 0 14.52 AcquiredPassive Im- In passively acquired immunity, tie im- A baby will receive passively aCqUireVin- .munity mune response is not producedby the host. munity to antigefis for which the mother hes,. Preformed antibodies may be acqued nat- eirCulatineigG'initibodies. urally,from the mother by the unborn child Tetkiiis is an example of a disease treated across the placenta, or'in the colostrum afterby immunoglobulin that has specific anti- birth. Specific antibodies may also be ob--bodies to the tetanus toxin which have been tained in'the form of onirnaglobulin or hy- formed in another individual. perimmune serum which is administered by ti injection.

Medical Microbiology1,-95

-I 2 3 1 2y') qr ' TOPICS AND tillilTOPICS A. ESHENTIA1, INFOI1MATION EN 11101 iN FORM ATiON `1.1. ACTIVIT1104

Ill1111l111l1Mechanisms In Protective Immune rosponpea to an,infec. 'Visalia Damage thaw agent, may oleo produce. aignificatit pathological effects in the holt. In fact; the inducing Agent; if any, for many. deleterious effects of the immune 'HyPteni IH unknown. Whether or not the immune responseIa beneficial or deleteriogiqr resultant tissue damage is classified four basic types: anaphylactic, cytotoxic, immune complex, and delayed hypersensitivity.

14..61 Hypersensitivity is the immune response IgE antibOdiea pre also called reagin or Type I Anaphylaxis or d Immediate HypersAiiii-, gone awry; resulting in irritation and/or tis-homocytotropic antibodiea becausetheNi tivky sue. damage: Immediate hyperseraiitivity isbind to mast cells and cause thefilease of. mediated by IgE and is manifest by reactionsvaaorictive substances such as histhnrine occurring within.miliutes afteiantigen com- when they.combine with anfigen. °Fr bines with antibody. The reaction may occur Ardrphylaxis may be generalized;resulting in any member of a species and is calledin shock and possibly death. Localized aria- ana h !axis: Atopy is also tnedirited by,IgE.sphylaxis may occur in target organs such as It i e hereditary tendencft develop im- th n, nasal mucosa, or gastAintestinal mediate hy0ersens*itystatb'suchas haytract. fever and asthma to allergens such as pollens People who are allergic to a specific sub- and insect venoms that do not ,Mduce astance, e.g., penicillin, must receive at least Dresponse in normal individuals. one sensitizing dose. Reexposure to the same Pk: substance may then elicit the anaphylactic reaction. Allergy derisensitizing treatment consists of small doses of the offending allergen (antigen), which stimulate the production of blocking Antibodies in the serum. These IgG antibodies act to block or neutralize the antigen so that it does not have an opportunity to come in contact with the cell membrane-attached IgE.

14.62 Type II CytotoxiRe- 'Cytotoxic reactions involve IgG or IgM The 'target for the cytotoxic reaction may actions antibodies which bind to antigens 'on cell b_e_either a formed blood' element (red cells, membranes. Complement is then activated, *White cells, platelets) 'area specific cell type resulting in cell lysis:: within a particular tissue.

14.63 Type III Immune Com- These reactions are a result of IgG or IgM Treatment of diphtheria and tetanus with plex Reactions immune complexes (antigen-antibody) oc-horse antiserum to the toxins of these orga- curring in tissue with resultant inflamma-nisms has resulted' in many patients devel- tion. Serum sickness is a classic' example inoping antibodies to the hors&serum proteins Medical Microbiology-97 281 2 c' Tol'ICH AND A. NMI fINTIAld INFORMATION It. NNIIICIIMHNT-INFOIIMATION C. PRACTIC41;ACTIVITIEM which signs Intl syminotisi develop about 2With riniultnut. serum sickness, 'rho isle of i weeks after injection of foreign serum, An, !ultimo sermit Or I rest Intuit of I b W,IlilltilltI11 tititm'IltailmflYitatittlexii form In the blood- 1111(4essentially eliminated thikk:Acinulary MI1111111 and are deposited at different sit es in side effect. the body. 7.t . fo, Type IV Delayed Hy- Delayed liyiiersetutithoityreactions are In tuberculosis, the response to the ortga, persetttitivity cull mediated and occur MI the result of thenliun may haul to host cult destruction and interactions between suktitized '1' cells and 115tr CAVItlit1011 in the lungs, i.e., the lin- theiecourpontling antigons (Medical Mi- I no response is the major cause of tissue crobiology, $uhtopie14.4). The reactionda Igo. takes 24 to 72 hours' to reach its peak. Voition ivy, detergents, and heavy metals flu are examples of ,substances which may induce delayed hypersensitivity. Second/ anal *sitbsetinent;exposures of the natient to the allergen may result in a reaction. Delayed hypersensitivity akin testing fle- tectii cutaneous sensitivity to an antigen. In testing for sensitivity to an infectious agent such as tuberculosis, a positive teat does not necessarily imply active infection but merely a previous exposure.

14.7 Autoimmunity '-The body is normally tolerant to its own There are many theories regarding the . tissue; autoimmunity reflects a loss of toler-methods for indUction of autoimmunity in- ance to self, It is specific, humoral or cell-volving genetics, physiology,. immunology, .s mediated, immunity to thebody's own tis-and infectious agents as factors; however, 4^'`4/ sues. the exact mechanisms remain uncl .4utoimmiine disease is a clinical disorder jssociated with the immune response to self. , Examples of autoimmune diseases-include. rheumatoid arthritis, systemic lupus ery- thematosus, and chronic thyroiditis. th,'"

14.8 Immunodeficiency Humoral immunity, cell-mediated immu- The type of infection often indicates the nity, phagocytosis, and complement ad in- type of immunodeficiency-present. Deficien= dependently or in concert with each other incies of T cells, B cells, or both are the most combatting infection and disease. Deficiencyserious disorders. Functional deficiencies ex- of one or more of these systems results inist 'in which,a cell or component is present increased susceptibility to infectious agents. but is not pelforming in an aldeitiale man- Deficiencies may be congenital or acquired ner. with symptomatology related to the degree of deficiency. 11 Medical MicroPiology-99 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACLCAL ACTIVI?IE 14.9 Lymphocytekapd Diseases which affect the leukocytes, par- Plasma Cell Disease tiCularly the lymphocytes and Plasma cells, result -in in-ununologicill abnormalities. In rmultiple myeloma for example, there is a malignant transformation of a single clone. 41 of plasma cells, resulting in the production of an abnormally high- level of a monoclonal (one .specificity) immunoglobulin. Patients with myeloma often present with recurrent infections sincethey do not have enough normal immunoglobulins to combat disease.

h 15.0 Medical Bacteriology 15.1 'Disease- Producing Bac- Although myriad numbers of bacteria in- Bacteria are ubiquitous, and most sp9cies Projection slides, motion pictures, and teria habit the earth, only a comparatively smallare not involved in disease production. Flow- stained microscope slides of medically im- number of species are capable of causingever, most treatable infectious diseases areportant bacteria are available. disease. probably caused by bacteria. Culturing and 'examination of live pathogens are not recommended for the untrained microbiologist as serious disease may occur: In many cases other badteria which may be used safely in the student laboratory are listed. k,

15.2 Pyogenic Cocci Pyogenic cocci cause a variety of infections which involve suppuration (put pro- thiction).

15.21 Gram-PositiveCocci (Bergey's #14; refers to the category of Bacteria asgiveninBergey's Manual of Determina- tive Microbiology) I

15.211 Staphylococcus Staphylococci are, arranged in grapelike Diseases produced includeboils,ab- S. epidermidis is recommended for labo- clusters and form large opaque gold to white scesses, impetigo, pneumonia, meningitis, ratory experiments involving staining, colo- colonies. S. aureus is a pathogen which is aand septicemia. Staphylococcal infectionsnial, and biochemical characteristics. transient inhabitant of the anterior nares oftend to be more localized than other py- Slides of coagulase production by S. au- an appreciable percentage of the population.ogenic infections such as streptococcal infec- reus may be projected. It produces many toxins and enzymes in- tions. cluding coagulase, leukocidin, and entero- Staphylococci are a common cause of food toxin. Staphylococci are the most common poisoning due to consumption of preformed cause of wound infections, boils and otherenterotoxin in food which has been im- Medical Microbiology-101 0 .

TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION , C. PRACTICAL ACTIVITIES - acute pyog nic (pus-producing) infections in,prOperly handled and contaminated with S.. human's. .aureus is differentiated from-aureus. 'other staphylococci oh the basis of its coag- S.. aureus, especially methicillin-resistant ( ulase production. - 8f-rains:is an important cause of nbsocomfal Coagulage-negative staphylococci such asinfections. Antibiotics can be used to .treat S. epidermidis are part of the normal skin,staphylococcal disease, but susceptibility

flora .bui may also cause disease such as-testing should be performed due to p'reva-4 subacute bacterial endocarditis. lence of plasmid-associateitantibiotic resistance. ,

5.2j2 Streptococcus (other gtreptococci are arranged in chains and Major- diagnostic tests include determi- Projection'slides showing hemolysis may than S. pneumoniae) profluce small, translucent colonies on solid nation of hemOlySis type .(alpha-hemolysis, be shown. media. Streptococci are abundant in nature,greening Of erytliiocytes; beta, clearing of and some-are often memberi of the normalcells; gainma, no change), and serotyping. flora of the moutb and intestinal tract. The streptococci maY be divided flit° three groups'according to their hemolytie activity on blood akar.plates. Hemolytic streptococci maybeserolbgically grouped (A to 0) based on the C carbohydrate of their -cell, wall (Lancefield typing). J

15.213 Streptococcus pyogenes S. pyogenes. (beta-hemolytic, group A), Diseases produced by S. pAgenes include Prepared blood agar plates may be pur- elaborates a wide variety of toxins 'and en- septic sore throat; scarlet fever, endocarditis, chased. Throat swabs inoculated onto blood zymes which are believed to be involved inurinary infections, and wound it fec-agar plates may exhibit hemolytic zones. its extreme invasiveness and'disease produc- tions. Important sequelae include rheumatic' tion. The M protein in the cell wall aids infever and glomenilonephritis. Group 5 preventing phagocytosis of the organism, streptococci are a major cause of meningitis The streptococci produce a wide varietyin infants. Alpha - hemolytic spreptococci are of acute, fulminating infections and chronicnormal inhabitants of the upper respiratory diseases. The common "strep throat" is thetract but may cause disease such as subacute best known of these. Human cases and car- bacterial endocarditis. , riers are the source of infection of S. pay- Penicillins are generally used in treatment ogenes. of streptococcal disease. Enterococci are part of the normal flora of the. gastrointes- 0. final tract. They may cause disease when displaced into other body tissues. Th&ent..,,e;/ - ococci are resistant 'to penicillin.

15.214 StreptococcuF. pneumo- The pneumococci are lancet-shaped al- S. pneumoniae are often differentiated Projection slides showing susceptibility of nide,(the Pneumococ-pha:bemolytic diplococci that sometimes oc- from other alpha-hemolytic streptococci bythis organism to optochin disks may be stud- cuS) cur in short chains. Vulent strains are en-their susceptibility to optbchin. ied. Films showing resistance of encapsu- lIeclical Microbiology -103 ° Lip up

TOPICS AND,SUBTOPIdS ;A". ESSENTIAL INFORMATION B. VNRICHMENT INFORMATION C. PRACTICAL. ACTIVITIES 4 capsulated, flail over 85 strains have been A pentlivalent 'capsular vaccine is avail-lilted organisms to phagocytosis are avail- identifkil according to capsular antigen. S.o' able for immunizittion against strains thatable. pneumoniae can be a member of the normal Cause 90 to 95% of U.S. infections. flora of the upper respiratory tract of hu- Penicillin is du? antibiotic of choice, fol- .1 lowed by erythromycin. mans, who then act as carriers.- kr. This is the most common agent of tfacte-A' rial pneumonia, especially in young anfirel- derly patients, and is still a ,major alliise of death in the United States. The virulence of the organism depends upon the presence pf the capsule which preverlts agocytnais, thus permitting multiplicatioof the organism in infect9c1 tissue.

15.22 Gram - Negative (Bergey's #10)

15.221 Neisseria This genys consists of kidney- shaped dip-''{These organisins are strict probes but the Nasal swabs may be cultured on agar lococci somewhat flattened along adjokting.pathogenic species grow best in an enhancedplates and then tested for oxidase-positive sides. Some species are normal inhabitantsCO2 environment such as a candle jar. colonies demonstrating normal flora such as of the respiratory tract and occur extracel- The pathogenic Neisseria are extremelyBraphamella (Neisseria) catarrhalis. lularly, whereas the human pathogens occurIabileynd require special media. A typical candle jar may be demonstrated. ntracellularly.

15.222 Neisseria gonorrhoeae N. gonorrhoeae, le gonococCus, 'causes Goilocpccus infection of the eye (ophthal- Microscope slides with stained smears (the Gonococcus) gonorrhea, a highly contagious; sexually chia ) is, acquired by newbornsfrom the local venereal disease clinic show- transmitted disease! The gonococci attackduring ,passage through an infected birthing gram-negative intracellular diplococci mucous membranes. of the genitourinarycanal if not,Prevented by application of sil-may be examined in the laboratory. tract "(land produce acute, pus-filled lesionsver, nitrate onto the eyes immediately after. . which may result in chronic inflammation. delivery. Penicillin G jr ampicillin is the Both women and men nay be asympto-drugof choice in treatment of N. gonpr- ,matic.carriers. The only natural host for r4oeae infections, butexficillin-resistant gonorrhoeae is the human. Gonorrhea nowstrains have recently bec me a. pro)ilem. occurs in epidemic numbers, Witt the high- &Incidence in the 20- to 24 -year group., The infection rats ,can be reduced by early ditgnosis. and treatment of Cases and sexual cointacts and by education.

15.223 Neisseriameningitidis N. menipgitidii is an obligate parasite of Microscopic observation of organisms in (the Meningococcus) humans and is found as part of the transientthe cerebrospinal fluid permits presumptive flora of the nasopharynx in up to 25% of theevidence of bacterial meningitis and indi- population. catesprothpt chemotherapy. Penicillin G is From the nasopharynx the-meningococcusthe antibiotic of choice. Medical' Microbiology -105

4 2 1/4-) 2 n 1. TOPICH AND NOIITORIQ8 , A. 1.11.111ENTIAI, INFORMATION II. PINWORM IINT INFORMATION pitAcricAll. ncluttnim \ 1 may spread to the control nervous . _ wborttlt VielsoH ITIOniottilla, Untreated cases are fatal in 70 to l00% of patients, and death may occur in 24 hoots,

15;y1 Rods (Dergey'a #15) These organisms are saprophytes or intim- I i , I tine) commensals of worth -blooded animals. The' spores may remain viable in soil for

years; to

15.31 Clostridium The clostridiA are large gram-positive, Differentiation Of species within the genus C.le' tanomotplums may be grown on- spore-forming, linaerobic bacilli. They pro- is .by colonial appearance, morphology ofplates in anaerobic jars or grown anaernbi-

' duce powerful eicotoxins and enzymes. They spores, and biochemical tests. The antigenic'catty in thioglycelate broth. Its morphology are free-living inhabitants of soil. In addition type of toxin is identified by neutralizationis similar to that of C. tetani. many are normal commensals of the intes -..with specific antiseruin tinal tracts' of humans 'and animals. These organisms, are not highly invasive but may ty produce disease by elaboration of highly in- juriqus toxins. 10, 15.311 C/ostriclitA &gut:nit/Ai The botulism bacilluva producesth'most Botulism is a neurological disease, and Projection slides or films may be shown to potent neurotoxin knoWn The toxin acts by gastrointestinal symptoms are slight or ab-illustrate the epidemiological principal of blocking transmission of nerve impulses tosent. The mortality rate is high but occur-this foodborne disease. the* muscles. rence of the disease is rare. The toxin can be Botulismds anintoxfation resulting from inactivated by heating to 100°C, for 20 min- ingestion of improperly preserved food inutes. Oich C. bottilinum has grown and produced Epidemics of botulitm occur when im- toxin. \ properly home-cannedorcomrnelically The orgiinisiiii does not have to be present canned foods are served without adequate to cause the disease. cooking. Infant botulism results frOm toxin Permed by C. 'botulinum present in'! -Iwin- testinal tract. Botulism i§ treated with specific antitoxin:

Clostridium tetani This prganisrn produces round terminal Clinical. diagnosis with bacterial confir- ProjectiOn slides showing the endospbres spores Which give the organism a charactef difficult because of the few orga-or victims of the disease may be shown. istic "drumstick" appearance. nisms resent. C. ° tetani is. not an invasivorganism. This is a preventable disease. Prevention When accidentally introduced via pener depends on active irrununization with toxoid trating wound, the rpores germinated the to stimulate antitoxin production. Diphthe- organisms multiply' tps,,tpacilli remain lo- ria toxoid, killed pertussis Organisms, and calized but produce V9 hand 'tetanus tetanus toxoid are the three antigens admin- (lockjaw) results.° Tliteto*in believed to isterein a DPT immunization. A "booster" travel by way of thiMcitOi .nerves to theinjectid r may ble given to a previously im- Medical Microbiology107 O I 2'2 )TO11118 AND N't 111'1st) 1104..., A. It It-111INTIAI, INFORMATION 11; IdNitltJlIMl INT IN VOI ATION U.PRACTICAACTIVITIgti brain and spinal cord, Hy Internally withseedbed individual who lots relatived a pen- (wimp& t1.11111111tholt011 It increases oscitAll- otrating wound, Troia Mont or tut antis in wit ity of molor peveli and rulmmo (moillon/otetanus antitoxin nod cmit;aefiitiii of voluntarymuscles.

15.313 Glum (AndeanUrge- most enemata epochal of clostridia atagrene is often pegresnive and may Projection sliden nhowing the pathology of nislnm which produce the disease gam gangrene arenecessitate amputation of a limb. Treatment thitedinetwe may be shown. C. perfriagena, C. novyi, and C. septiciatt. Inofgas i gangrene Involves surgical &bride-. gas gangrene, ainked infection is the rule. moat, antimicrobial drug therapy, and po- OM gangrene develops after 81800 con -lyvalent mahatma. ,Preventionincludes tained In soil are introducedf\ into ll(1001) early and adequate cleaning of the wound. wound in which necrotic tissue has devel- In addition to gas gangrene, C. perfringens oped. Pi the organisms multiply, carbohy- also Produces nu OlitOrotOxiti Which when drates are fornicated and gas is producedingested in food induces profuse diarrhea. which causes distension of tissue and interference with the blood supply. The organisms also secrete necrotizing toxins and hyaluronidase, which favors spread of infection. This kills tissue and results In gangrene (local dent f the tissue).

15.32 Bacillus a:attracts The antax bacillus is a large aerobic Virulent strains of the organism produce B. subtilis may be used to demonstpt gram-positive spore-forming rod. Anthrax isroughcolonies(non-encapsulatedorga; some oft.the characteristics of this genus. primarilY a disease of sheep and cattle butnisma) 'on laboratory media. In the presence may be transmitted to humans as an occu-of CO2 or in the body capsules are.ftirmed. pational disease of hide and wool handlers.Ti-k virulence of the organism depends on In huMans, anthrax may produce pulmonarythe presence of the polypeptide capsule and ttr cutaneous infectionswhich may lead rap-possibly the exotoxin which is produced. idly to septicemia and death. Diagnosis depends on finding the large bacilli in blood or tissue smears. Spores are not seen. The bacilli may be identified by the fluorescent antibody technique. Penicil- lin may be used to treat anthrax. Historically this organism was used by Robert Koch in establishing his four postu- lates of the bacterial etiology of a disease.

15.4 Actinomycetes and Re- lated Qrganisms (Ber- igey'4 #17)

15.41 Actinomycetaks hese bacteria are gram-positive and may The four medically important genera are Streptomyces griseus may be used to produce branching filaments although theyActinomyces, Nocardia, Streptomyces, andstudy some of the characteristics of the Ac- cdmmonly have diphtheroid morphelogY-Actirtomadura. These bacteria are relatedtutomycetales. 2'2 k -Medical Microbiolog 109

2 (1 TtIPICH AND tit 1111'0141CH A, 1111-111111N111A14- INVOI1M It14111111 IMI+INT IN1+1111MATION a Atrl'It1A 1, ACTIVITIM Home 11111 10'111-61111 01)(1 )011110 Ao 0111101.04, It) 1 Ile coryliebileteria anti lilt' illyvoltecterte 0.:111111 Al'h/11101.1'14/(11/11(1 111'0 10111111 11111111 111111, and eimealchilly resemble 1 lit+ fungi. Home 1 110 air, or may he part of the normal liora of hooks on medical tnicroltiolottv &cuss ills r many 1111111111111 111111 111111111101, 1111'00 111011 1011 eases produced by the 'Octimonycelo/00 un- Race(' by these organisms are infrequent, der the fungus (IlitIM11014, Many antibiotics 111'1/ 141(110`1/11 by StrAptont tern spp.

4 . 15,4 1 1 iletinotri,vcon israulti This organism is non-ackl-fast and prof- Act inornyeeteti deprive host littiof, Int- own( ii).119,111111t11.016. It °count an a 11(11'111111 Irrott1 0, thereby causing tisane and !oil tie. commensal in the mouth and In considered C110110. 1(10111,111(111101 of A, isroc/ii i1 gener- relatively noninvasive, In tissue it ON11111-1 110 ally by Ilttoreacent antibody in ocodures. mycelia surrounded by suppurative inflam- Treatment consistH of prolonged adminintra- matory material which products "tadltirtitm of penicillin and surgical drainage of the granule')." lesionsnecetairy. Human actinomycosis is a chronic suppurative and .granulonurtous disease occurring. in the jaws, lungs, or ileocaecal region. Infection in the head or neck area often follows a tooth extraction or denta4surgery. 'the infection spreads by direct extension through draining tracts.

15.412 Nocardia asteraides These organisms are aerobic and many The organism has a predika Lion for the and N. brasitiensis strains are partially acid-fast. They arebrain, and. abscess formation nut ccur by found in, the soil and are not part of the spread of the organism through the blood- I- microbial flora of humans. The nocardiae, stream.Actinomycetoma mayalsobe probably enter the body through the respi- caused by these organisms. Treatment is ratory tract or through breaks in the skin. with sulfonamides. Nocardiosis is an opportunistic pulmonary disease that may spread to other parts of the body. 15.42 Mycobacterium These organist-its are acid-fast, strictly The organisms are slow growing nd re- M. smegmatis may be safely cultured to aerobic rc41s which frequently show deeplyquire special media. Identificationf myco- Show the slowly growing waxy colonies and staining granules and often occur in clumps.bacteria species is by biochemical ch racter- the acid-fast rods which tend to stick to- The mycobacteria do not stain well with theistics. gether. Gram stain. They occur as saprophytes and Prepared microscope slides showing my- pathogens of humans and animals. cobacteria stained with fluorescent dye may The mycobacteria are rich in lipids which be studied. are concentrated in the cell wall. These lipids are partly responsible for the acid-fast- ness of the organism, the host's cellular reaction to the tubercl bacillus and the tendency of the organisms to clump. 2 Medical Microbiology-111- C. PRACTICAL ACTIVITIES TOPICS .AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT, INFORMATION Projection slides of the organism and of tuber- The tuberclebacillienter the body Tuberculosis is typically a wasting disease 15.421 Mycobacterium patients with tuberculosis may be shown. culosis (I;ercle Bacil- through the lungs and are ingested by phag-and was originally called consumption. 'lus) ocytes. Intracellular multiplication' follows. -The lung becomes extensively: damaged Progressive damage to lung tissue occursbefore death. The human and bovine tuber- through a complex series of events. Duringcle bacilli (M. tuberculosis and M. .bovis, this time, delayed hypersensitivity develops respectively) are the most common causes and- the disease may becomeilarrested orof tuberculosis. There are many other spe- progressively spread through 'the lungs orcies of mycobacteria which produce disease throughout the body, depending on host fac-opportunistically in humans. tors. Prevention and control of cases are of Malnutrition, poor sanitary conditions,primary importance. These measures in- crowding, and hereditary susceptibility areclude detection by mass survey, treatment all factors which predispose the individual of cases, elimination of tuberculosis in cattle, to active tuberculosis. and pasteurization of milk. Detection is by skin test Which shows delayed hypersensitivity to a purified protein derivative (PPD) of the organism. This is followed by X-ray examination of skin test-positive individuals. BCG vaccine (an attenuated bovine organism) provides some level of immunity after its inoculatioo tuberculin-negative individuals who beCOnTe...... tuberculin positive. The vaccine is not corn- monly used in the United States. Treatment involves isoniazid, ethambutol, rifampin, and streptomycin.

15.422 Mymbaeterium leprae M leprae is the etiological agent of lep- rosy,a chronic granulomatous infection largely confined to tropical countries.

Corynebacterium sp. may be isolated from 15.43 Corynebactertan, These granular gram-positive rods are Diphtheria is characterized by the pres- club shaped and, because of incomplete fis- ence of a grayish necrotic pseudomembranethe throat or Cifterosis may be purchased sion, produce V and Z forhis. in the throat and the production of toxicand used in the student laboratory. Each This genus includes C. diphtherme, thedamage to the heart and central nervouswill show pallisades, Y and Z forms, and causative agent of diphtheria, and a number.system. Paralysis of the throat, eye muscles, clubbing of organisms. of commensal diphtheroid bacilli found inand.severe damage to the heart are frequent the upper respiratory tract or the skin. C. sequelae. Active immunization with diph- diphthertae is not invasive and grows pri-theria toxoid is important in prevention of marily in the pharynx where it produces a the disease and is administered routinely in powerful protein exotoxin which blocks pro- the United States in DPT vaccine. Diagnosis tein synthesis and causes the symptoms ofincludes demonstration of the specific toxin the disease. Only strains of C. diphthertaeproduced by the isolated organism. Treat- Medical Microbiology -11:1 6 2 "R B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION which halve undergone lysogenic conversion ment includes early antitoxin administration produce the exotoxin. Diphtheria is acquiredand penicillin. by inhaling bacilli contained in droplets ex- haled by infective cases.

15.5 Gram-Negative Rods

15.51 Gram-Negative Faculta- tively Anaerobic Rods (Bergey s #8) This bacterial family exhibits diverse met- A wide variety of biochemical tests are 15.511 Enterobacteriaceae Many of these short bacilli are part of the normal intestinal flora of humans and ani-abolic activity. All members ferment glu-performed to identify the Enterobacteria- in mals. The pathogenic mechanism of these cose. Endotoxin is a potent pyrogen(fever- ceae. A few of these may be performed organisms is uncertain but has been attrib- producing substances'. Since antibiotic sus-the laboratory as time permits. uted to include endotoxin, which all contain, ceptibilitiesoftheEnterobacteriaceae Many commercial identification systems may be used to identify these organisms. and to exotoxins, which some produce. strains vary tremendously, testing should be There are many genera and species ofperformed on all isolates. Enterobacteriaceae which are important as opportunistic pathogens; however, only a few examples will be given in this outline. E. coli is used as an indicator organism, Eosin methylene blue (EMB) plates may 15.5111 Escherechta E. colt is a normal commensal in the intestinal tract, but it may invade opportun- providing evidence of fecal contamination ofbe inoculated with E. coli to show typical istically and cause infection in many organswater supplies and prepared food. It is thecolonies and green metallic sheen. of the body such as the bladder,' appendix,most widely studied bacterium and is em- and kidney. Certain strains may produceployed in recombinant DNA research be- enterotoxins which cause traveler's diarrhea.cause its chromosome has been mapped in Some strains may also produce epidemicdetail,. diarrhea in babies and infants. K. pneumoniae is primarily parasitic but 15.5112 Klebstella Klebsiella typically produces a well-defined capsule and profuse mucoid growth on may be found as a commensal of the upper solid media. K. pneumoniae causes a de- respiratory and intestinal tracts in humans. structive type of pneumonia and is a secondary invader of the respiratory tract.This organism occasionally may produce suppurative infections in many parts of the body and may be responsible for infections of the urinary tract! Serratta strains arefound commonly in 15.5113 Serrate° The type species is S. marcescens, which May produce red pigment on cultivation. Itwater and soil. Serratta strains are often isu an opportunist, and nonpigmented strainsresistant to many antibiotics. Medical Microbiology 1 15 2' ' 3u2 ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES TOTS AND SUBTOPICS A. ESSENTIAL INFORMATION B. have been the cause of noso 'al infections. It causes urinary and respit, tory tract AN infections and septicemia. , Y These free-living organisms of soil and P. uulgaris may beown on agar p tes , 15.5114Proteus The type? species is P. vulgaris. Its cultural characteristics include a tendency towater frequently inhabit human intestinalto shqvv swarming. Mot' *ty of the organism mount observed swarm over the agar surfaces. These oppor-tracts. They are often resistant to manymaY",be detected in a w tunistic organisms may cause .$evere infdcrantibiotics. unde(f a microscope.

of tithetions urinary tract and wounds.

The salmonellae are parasites of the intes- The salmonellae'cause three main types Typing of S. gallinarium may be used as 15.5115Salmonella salmonel- tinal tracts of humans and animals, inClud,of disease, but mixed forms are frequent: a dernqnsti-ation of serotyping of ing birds. These, organisms are spread inenteric or typhoid fever, in which the sal- lae. food or water via the fecal-oral route, pro-monellae are ingested with food or drink, ducing a clinical or subclinical infection. On-enter the intestinal tract, pass through the set of symptoms coincides with liberation lymphatics to the bloodstream and are dis- of a large number, of organisMs into thetributed to many organs; septicemias, which may lead to local suppuration; gastroenteri- 1.7 bloodstream. S. typhi pioduces the most serious disease, followed by S. enteritidis andtis ("food infection"), due to S. enteritidis S. choleraesuis. serotype typhirnurium and other senitypes. Imptoperly handled raw meat, poultry,During the first 2 to 3 weeks of th)e enteric shellfish, or cracked eggs may serve asfever the patient's blood is culqvated to sources of infection. Food handlers or ex-isolate the organism. creta from infected rats or mice may also be sources of infection. S. dysenteriae causes the most serious 15.5116Shigella Although #fe shigellae are found in a few primates, humans are thought to be the sole disease and produces an exotoxin which has reservoir ofdnfection. The shigellae produce neurotoxic and enterotoxic properties. The bacillary dysentery, with infection limitedrrsulting bacillary dysentery isal acute in- almost entirely to thegastrointeattal tract.flammatory condition that leads to ulcera- The most common species are S. flexneri tion of the large intestine. No bacteremia is and S. sonnei. All species are pathogenic byproduced. way of endotoxin released uponautolysis. Diagnosis is by cultivation, with differen- Shigella infections are acquired by the oraltiation from the salmonellae. Ampicillin or route from feces of human cases or carriers.chloramphenicol may be used for treatment but the disease is usually self-limiting, and only fluid replacement may be necessary. Prepared microscope slides showing bi- Y. pestis is a small bacillus showing bipo- There are three clinical types of plague: 15.5117 Yersinia(Pasteurella) polar staining may be studied. Projection pestis lar staining and causes plague in humansbubonic, which causes enlargement of re- and animals. Some strains are highly viru-gional lymph nodes; pneumonic, which mayslides of fleas, rodents, and humans with the lent. Plague, a disease of rats and otherlead to bronchopneumonia; and septicemic. disease may be shown. rodents including the ground squirrel, isPrevention includes control of rats and their Medical Microbiology-117

3,1 3 )1.0 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT' INFORMATION transmitted by kea bite's from rodenttofleas. The most effectivedrug is'streptomy- rodent and from rodent to huma cm. Y. enterocolit iv3ible for an increasing number of casof bacillary gastroenteritis and, baciereia. Swine, dogs, a and humans may be the source of infection. Y. pseudottiberculosis produces infection in" birds, rodents, and other animals that is rarely transmitted 'to humans. Disease is often chronic and associated with nodules superficially resembling tubercules that occur in the intestine or abdominal organs.

15.512 Vibrionaceae

15.5121 Vibrio The vibrios are comma-shaped, motile In cholera, an eriterotoxin is responsible bacteria. The most important species is V.for the outpouring & fluid from the intes- cholerde, which causes choleraa diseasetinal tract. This potent protein stimulates endemic in parts of Asia and Africa. excessive formation of cyclicadenosine Cholera is a violent infection of the intes-monophosphate, causing the intestinal wall tinal tract causing loss of gallons of waterto secrete electrolytes and water into the within a day or two. A general invasion oflumen. The disease is spread by water con- the body does not occur. taminated by human feces. Effective treatment of cholera is by oral administration of a balanced salt solution containing glucose.V. parahaTolyticus causes acute enteritis after ingestion of contaminated seafood.

15.52 Bergey s"Generaof Uncertain Affiliation" Projection slides of Haemophilus spp. 15.52 aemophilus This genus consists of tiny coccobacilli Transmission is from person to person by which require one or both of the growththe respiratory tract. H. influenzae was mis-showing satellitism and need for X and/or factors known as X (heroin from blood) and named and does not cause influenza whichV factors may be shown. V (nicotinamideadeninedinucleotideis a viral disease. [NAM). CO2 enhances growth. Some of Diseases caused by other species include theie bacilli produce satellitism around S.pink eye (H. aegyptis) and chancroid, a ve- aureus or certain other bacteria which ex-nereal disease (H. ducreyi). crete large amounts of NAD. The nonencapsulated form of H. influenzae is a part of the normal respiratory flora of humans. The encapsulated form, especially type b, produces suppurative res- 3 ,4 Medical Microbiology 1 19 3 ' 3 r

C. PRACTICAL ACTIVITIE TOPICS, AN1) $UWI'OPICs A.Sq_ENT4I., INFORMATION 'ENRICHMENT INFORMATION piratoryatititifections, including middle- %... ear.'fee inifi and 'meningitis in yOung chil- clre ct arn of meningitis is highly fatal . .

ese bacilli are primarily nonenteric op- -15.53 Qiino-Negative Aerobic. 1. Bpds (Bergey's *7) portimist.Vresponsible for diverse infections. Pseudomonas infections are common in P. aeruginosa may be grown on Trypti- 11631 Pseuciornonas: The pseudomonada are plant pathogens or free-living organisms of soil and waterburn and immunosupPressed patients. P.case soy agar to show blue-green pigment. that may be pa' of the normal flora of theaeruginosa grows at room temperature andThe strictly aerobic nature of its growth may skin and iniestintsof humans. An important produces a water-soluble blue-green pig-be observed in broth or semisolid media in species is P. aeruginosa. It causes woyndment. P. aerugendsa is the prototype of aa tube where itonly grows near the surface. and urinary tract infections and is an impor- group of organisms termed nonfermenters tant cause of nosocornial infections. since they are ,generally aerobic and fail to ferment glucose. Clinical disease may be localized at the 15.532 Franciaella (Padeu- This organism produs tularemia or rab- _ rella) ta larensis bit fever, a disease of ronts transnapted by portal of entry such as tthe eye, skin, or lungs various arthropods such as ceram flies, where granulomatous lesions occur. Bacter- ticks, and lice. , emia with pneumonia and septicemia may Humans develop the infection after han-follow. Diagnosis is by serology or, acute dling, eating, or skinning infected animals orcases, repeated blood cultures may allow by drinking dater contaminated by them. cultivation of the organism. The bacilli erir the host via the arthropod Tularemia can be Prevented by taking bite or througthe respiratory or gastroin- proper precautions with wild animals and by testinal traci. adequately cooking infected meat. Control The disease i. produced by rapid invasionmeasures control of rodent popula- of the host and multiplication of the-bacillitions and surveillano of animals. in rnany tissues. . Thediiease in humans is characterized by 15.533 Bruce/in The brucellae are obligate parasites of 4nirnals which cause, undulant fever in hu-a long-continued bacteremia with a remit- mans. The infectious agents are primarilytent "undulant" fever. Organisms multiply the animal pathogens B. abortus in cows, B. inside the cells of the reticuloendothelial ;gas in pigs, B. meatensis in goats, and B. system, allowing persistence of the organism cams in dogs.' in spite of high-level chemotherapy- In animals the bacilli are localized in the DiagnOsis is by repeated cultivation using mammary glands and transmitted via milk,special media and techniques for this slowly or they are localized in the genitalia.Farm- growing organism or by serology. workes or 'veterinarians may become in- Prevention is by slaughter of infected an- fested by handling contaminated material.imals and immunization of animals with a Sensitivity to endotoxin may play a role inlive vaccine plus compulsory Pasteurization the pathogenesis of brucellosis. of milk and milk products. Medical Microbiology-12 I 3' a 3'1" TOPICS AND-SUBTOPICS At ESSENTI L INFORMATION - B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES Long-term treatment with tetracyclines or arnpicillin Inay be effective.

15.534 'Bordetella B, pertussis is the etiological agent, of Whooping cough consists of a catarrhal stage with release of a large number of or- A whooping cough. Virulent strains are encaP- . sulated.B. pertussis is a fastidious organism ganisms and a paroxysmal stage with an requiring special media for primary isola-explosive whoop on inhalation. Laboratory tion. On Bordet-Gengou agar the coloniesdiagnosis is by cultivation of nasopharyngeal are raised and have a characteristic sheen,exudate and im;nunofluorescent stai" g. like a mercury drop. The bacillipossfss Treatment includes early use of e endotoxin which is probably responsible formycin and anipicillin. r degenerative changes in the respiratory tract. Prevention is with a vaccine of killed organismsgiven especially to infants and young children (DPI).

15.54 Orem-Negative Anaero- Anaerobic, grarn-negative, non-sporulat- Elaborate procedures are required for re- Projection slides and films of methods of bic Rods (Bergey's #9) ing bacilli represent the most common an- covery of these organisms from clinical spec-cultivation may be studied. aerobes,' encountered in infections. imens as well as identification. a

15.541 Bacteroides B, fragilis is the most common anaerobe. B. fragilis causes abscesses, appendicitisj Anaerobe jars or pictures of transport an- strain isolated. It represents 95% of the bac-bacteremia, and heart valve infections. Most,aerobes may be demonstrated. teria of the intestinal tract and is responsible strains are sensitive to erythromycin and for the foul odor of feces. In anaerobic infec- clindarnYcimbut resistant to penicillin. tions, bacteroides are usually found in association with other organisms.

15.55 Grain-Negative Rods The Legionnaires bacillus was isolated (Unclassified) and identified after the publication of Ber. gey's Manual and so is not placed in one of the other categories.

15.551 Legionellapneio-im These Gram-negative, pleornorphic bacilli Legionella requires enriched media and is phlia are responsible for a very severe formofoxidase and catalase positive. Different an- pneumonia called Legionnaires disease. The tigenic strains have been found. It can be organisms can live saprophytically in stag-deffnitely identified only by the direct flu- nant water. orescent antibody test. Treatment is with erythromycin or tetracycline.

15.6 The Spirochetes(Ber- Spirochetes are thin-walled, flexible, nrio- The spirochetes are actively motile, rotat- Material scraped from around the teeth gey's #5) tile, helical rods. `These microorganisms may ing around their long axes. will reveal nonpathogenic spirochetes. be seen readily in the darkfield microscope. Electron micrographs showing the special structures of the spirochetes may be shown. Medical Microbiology-123 3-I TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. 'ENRICHMENT INFORMATION" PRACTICAL ACTIVITIES 15.61 TreponenTa T. pallidum causes syphilis, which is A pregnant syphilitiC woman can transmit Electron miceographa of the organism and transmitted by sexual or direct contact. Thisthe spirochete to the fetus through the pla-projection slides demonstrating lesions may pathogenic organism has never been cul-centa. This may result in death of the fetus be shown. tured. or a variety of birth defects. Early adequate Syphilis is a disease of varying duration.treatment prevents congenital syphilis. , The primary "hard chancre" at the site of Laboratory diagnosis includes serology us- the infection heals and may be followed bying non-treponema antigen. Control of the a secondary stage characterized by a rash.disease includes treatment of all cases, fol- The skin lesions are highly contagious andlow-up of all contacts, and education.,Sero- mimic a variety of other dermatological dis- logical tests for syphilis are usually required eases. A later or tertiary stage may involveto obtain a marriage license, on all hospital granulomatous lesions of anCYpart of the admissions, and during .early, pregnancy. T. body or cause degenerative changes in thepall/ilium is extremely susceptible to penicil- central nervous system or cardiovascularlin. system.

16.62 Leptospira L. interrogans is a tightly coiled spiral Leptospirosis (Wed's disease) produces microorganism having one or both ends benthemorrhage and necrosis in the liver and into a hook. These orga4isms can be cul-kidneys and results in dysfunction of these tured on media containing serum. organs. Diagnosis includes darkfield exami- The leptospira are primarily pathogens ofnation, culture, and serology. the urinary tract of animals such as dogs, Prevention includes avoiding ingestion of rats, and cattle which excrete the organismscontaminated water, control of rodents, and in their urine. Human infection is acquired vaccination of dogs in endemic areas. Peni- by ingestion of food or water containing thecillin and tetracyclines are helpful but do organisms or less commonly by direct pen- not eradicate the infection. etration of the skin. Many infections are mild or subclinical.

15.63 Borrelia B. recurrentis is the etiological agent of Rodents are the main reservoir fdr the relapsing fever, a tick- and louse-borne dis-infection. They transmit ehe spirochete to ease. The organism is an irregular spiral thattaks that may transmit them to humans or stains readily with bacterial stains. transovarially to other ticks. The human The disease is characterized by 3 to I()louse may transmit the disease from human bouts-of sudden fever followed by chills andto human. Prevention is by avoidance of weakness. The disease is endemic in areas inexposure to ticks. Blood smears obtained which the particular tick carriers are found. during the fever may reveal the' organisms.

`15.71 Campylobacter fetus Campylobacter fetus subspecies jejeuni is Stool specimens must be inoculated on a a motile gram-negative curved rod which ishighly selective media, incubated under re- a major cause of acute gastroenteritis. Itduced oxygen tension and incubated at 42°C causes a rapid-onset diarrheal disease which in order to isolate campylobacter. They are Medical Microbiology-125 /3 n TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES is self-limited and lasts less than a week.generally susceptible to tetracyclines, eryth- These organisms are strict microaerophilesromycin, and aminoglycosides. and grow at 42°C.

15.8 The Mycoplasmas (Ber- The mycoplasmas are small highly pleo- Mycoplasmas do not revert iiiwalled type gey's #19) morphic microorganisms which do not haveforms. These fastidious organisms can repro- a cell wall. They require special stainingduce on cell-free media. On agar, colonies of methods to be seen. MycoPlasmas have ancertain species are characteristically be- affinity for cell membranes, are found inneath the surface giving a "fried egg" ap- plants and animals, and are part of the nor-pearance. mal flora of themouth and genitourinary Penicillin and thallium acetate may be tracts ohumans. The mycoplasmas areadded to media used in the primary isolation highly host specific. of mycoplasmas to inhibit growth of other Mycoplasma pneumoniae is the only spe-bacteria. Serological tests are available. cies proven to be a human pathogen and Tetracyclines and erythromycin are used causes atypical pneumonia. for treatment. Ureaplasma urealyticum (T strain of mycoplasma) hydrolyzes urea and is thought by some to be associated with nongonococcal urethritis.

15.9 The Rickettsiae(Ber- The rickettsiae include the orders Rick- gey's #18) ettsiales and Chlarnydiales.

15.91 Rickettsia les The Rickettsia les are extremely small The Rickettsiales stain poorly with Gram Prepared microscope slides of rickettsiae gram-negative obligate, intracellular para-stain but can be seen with Giemsa stain.in chick embryo tissue smears may be stud- sites of arthropods. They are transmissibleRickettsia may-be cultivated on the yolk sacied. to humans by the arthropocp, with the ex-of embryonated eggs, in tissue cell culture, Projection slides of the vectors and pa- ception of Q fever, which does nqt requireor in experimental animals. tients with the disease may be shown. an arthropod vector. Rickettsial infections Diagnosis is by specific serological tests or are generally characterized by fever andby the Weil -Felix test in which antibodies rash. produced against rickettsias cross react with Proteus sp. Treatment is with tetracyclines and chloramphenicol.

15.911 Rickettsia R. rickettsii is the etiological agent. of Rocky Mountain spotted fever occurs in Rocky Mountain spotted fever. The bacteriadeer, rabbits, and other vertebrates which are transmitted from tick to tick (trans- live in heavily wooded areas where the ticks ovarially or from tick to humans. occur. Most Rocky Mountain spotted fever The nilettsiae grow in endothelial cellsnow occurs in the eastern and southeastern of small blood vessels causing obstruction, states primarily in the late summer or fall. thrombosis, and necrosis of the skin (pro- Prevention is by avoidance or control of Medical Microbiology-127

311 3 14. Ivo0 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES ducing a rash), the heart, and central ner-ticks by clearing heavily wooded areas. A vous system. vaccine kr the disease is available. Epidemic typhus is a disease restricted to humans. it is caused by R. prowazekii and transmitted by the human louse. It is not found in the United States at the present time. Endemic typhus, caused by R. typhi, is transmitted by the rat flea from the rat to human. It is prgalent worldwide, especially in seaports. Q fever caused by Coxiella burnetti is a disease resembling influenza, atypical pneumonia, and hepatitis. Transmission is via the air instead of through the skin. It is found worldwide.

Ch15.92 lamydiales These organisms are @mall gram-negative Subclinical infections are the rule as the r.'041 obligately intracellular bacteria which mul-para)3ite reaches a balance with the host, tiply in the cytoplasm of the hbst cell.-Al- while spread of the infection from one spe- though they are gram negative,. they arecies to another is more apt to produce dis- visualized better by Giemsa or other stains. ease. They undergo a developmental cycle from Diagnosis is by serological tests or culti- the infectious particle to the Larger initialvation of the organism in embryonated eggs body. or tissue culture cells. Treatment is with tetracyclines.

15.921 ChlamYclia The two species of gylamydia are C. psit- Psittacosis in humans varies from a se- taci and C. trachomatis. vere, even fatal pneumonia to a mild inap- C. psittaci causes ornithosis or psittacosisparent infection. Birds imported from South in IiiImrins. The disease is acqUired by in-America and the Far East are quarantined halation of dried bird excreta or handlingto prevent entry of this disease.

contaminated plumage. . The inguinal lymph nodes in lymphogran- C. trachomatis causes lymphogranidoma'uloma venereum may become inflamed, sup- yenereum, a venereal ilisease reported mostpurate, and 'discharge pus, especially in commonly from the tropics and 'subtropicsmales. Trachotha is spread from eye to eye but found worldwide. Trachoma, the world'sby fingers, fomites, and flies in areas of the leading cause of blindrieSs and inclusion con- world where poor sanitary conditions pre-: junctivitis are also caused by C2trachoma-vail. Nongonococcal urethritis may coexist tis. About 50% of the cases of nongonococcalwith other venereal diseases. urethritis are caused by C. trachomatis.

Medical Microbiology-129 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 16.0 Medical Mycology 16.1 General Consideration Moat pathogenic fungi are inhabitants of Of the thousands of species that "exist, Prepared projection slides of cultures or the soil. Invasion of the host is by directfewer than 100 are considered pathogenic. developmental stages of the pathogenic contact, resulting in akin lesions, or by in- fungi or lesions may be studied. halation of spores, resulting in respiratory or Aspergillus, the Phycomycetes, Saccha- systemic infections. Candida albicans is the rornyFes, and various other fungi found in .-Ponly pathogenic fungus which is part of the the environment may be safely cultured in normal flora. It is not an inhabitant of the the student laboratory. Cultivation of path- soil. Diseases caused by fungi are called my- ogenic fungi should not be attempted. "coses. 16.12 Diagnosis Diagnosis of the mycoses is made by Cultivation of pathogenic fungi is very means of one or more of the following meth-hazardous and should be performed only by ods: isolation of the etiological agent in cul-well-trained specialists using appropriate ture media with subsequent identification;safety cabinets. microscopic examination of exudates or skin scrapings;. microscopic examination of his- topathological slides; examination of the skin or hair for fluorescence; and aerological techniques.

16.13 Pathogenesis Pathogenic fungi generally do not produce The diseases produced in deep tissues re- toxins but induce hypersensitivity in thesemble those produced in chronic bacterial host. In the mycoses of deep, or internal,infections, especially those produced by the tissue, the tissue reaction commonly is amycobacteria, e.g., tuberculosis. chronic granuloma. Human mycotic infections are more conveniently grouped by type of infection (superficial, subcutaneous, and systemic) rather than by organism.

16.2 Superficial Mycoses These fungi invade only hair and the outer Causal organisms and disease are Clados- layer of the epidermis. Since the host doesporium werneckii, which causes tinea nigra not produce a response such as redness, itch- (palmaris), Piedraia hortai, which causes ing, or swelling, the problem is often onlyblackpiedra,Trichosporoncutaneum, cosmetic. which causes white piedra, and Malassezia furfur which causes tinea versicolor.

16.3 Cutaneous Mycoses The dermatophytes are fungi which infect A cutaneous mycosis is described clini- (Dermatophytes) skin, hair, or nails by means of direct hyphalcally by the area of the body affected; i.e., penetration. They are not dimorphic. In skintines corporis is ringworm infection of the lesions, these fungi generally look alike,body, tinea pedis is athlete's foot, and tines showing hyphae and arthrospores. Somecapitis is ringworm infection of the scalp. species are found primarily in. human skin, Therapy often consists of topical applica- some in domestic and wild animals, and ation of antifungal agents over an extended few are found as saprophytes in the soil.period of time. In widespread involvement Medical Microbiology-131 316 315 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION..., , B. ENRICHMENT INFORMATION C: PRACTICAL ACTIVITIES Human to human and animal to humanof hair or nail infection griseofulvin is the transmission is common for some diseases. drug of choice. The dermatophytes produce chronic, low- Microsporant may be found on skin and grade infections. Skin lesions show inflam-hair, Epidermophyton may be found on skin mation at the site of the actively growingand nails, and Trichophyton infects skin, hyphae found on the periphery of the le-hair, and nails. Most species are found ( °'' Mons. This gives the typical ring-type lesion worldwide, but a few are localized. There is Cringworm." some age variation as to the type of disease produced, i.e., athlete's foot is more common in adults, while scalp infectiops are more common in children.

16.4 Subcutaneous Mycosek,/ The subcutaneous mycoses are caused by certain organisms introduced via a break in the skin (splinters, thorns, etc.). Lesions involve akin, subcutaneous tissue, fascia, and bone. Once established, these fungi tend to remain localized.

16.41 Sporotrichosis Sporotrochosis is caused by Sporoihrix In infected tissue, the fungus appears as a schenckii, which is a dimorphic funguscigar-shaped, budding yeast. The disease found worldwide on plants or plant struc-may rarely progress from subcutaneous le- I tures. In sporotrichosis, a local ulceratedsions to the respiratory tract or by extension lesion develops at the site of inoculation andto bones and joints. Sporotrichosis, while may progress to the formation of multiplenot a common diseas9; occurs' mostly in gar- 7 nodules and abscesses along the superficialdeners, florists, an /others having contact draining lymphatics, usually along the armwith plant mate Treatment is oral po- or leg. tassium iodine or the subcutaneous lesions or amphotericin B for the systemic disease.

16.424. Chromomycosis Chromomycosis is caused by a group of In lesions, the fungi appear as thick-walled slowly growing, dimorphic fungi. Chromo-brown ,cells which multiply by fission. On mycosis is an infection characterized bycultivation the fungi appear as darkly pig- slowly developing cauliflower-lik lesionsmented, slowly growing mycelial colonies. along the lymphatics of the feet or 'legs andFungi isolated include: Phialophora verru. is caused by several different black molds. cosa, Fonsecaea pedrosoi, and Cladospor- ium carrionii.

16.43 Mycetoma Mycetoma is a fungus nodule or tumor : A mycetoma originates from an injury . which may be caused by filamentous fungiwith resulting inoculation of microorganisms (Eumycetoma or Maduramycosis) or certaininto the body. Identification as to fungal or branching bacteria (Actinomycetoma). Thebacterial agent is important because of the disease consists of a localized lesion, oftendifferences in treatment. Treatment in- on the foot, with draining sinus tracts whichcludes sulfonamides and penicillins for the Medical Microbiology-133 313 ,TOI!ICS AND SUBTOPICS A. RSSItINTIAli INFORMATION B, tIN11101 1M ENT INFORMATION C,PR)CTICALACTIVITIR's yield granules consisting of hypluie or frag-aotinornycotio mycetoma, There is no etitab- ments of bacterial filaments, Ished treatment except surgical removal for he fungal mycotoma.

16.5 '' Systemic Mycoseii The systemic mycoses are diseases of the In addition to the usual methods of diag- 4 internal tissues usually caused by dimorphicnosis of mycoses, histopathology is espe- fungi which grow as soil saprophytes. Infec- ciallyimportantinsystemicinfections. tion is primarily acquired by spore inhala-Treatment of all those diseases is with pro- tion, with most infections being asympto-longed administration of amphotericin B. A matic. The chronic form starts with a pul- variety of domestic and wild animals may be is monary lesion and spreads via the blood- found infected with the systemic mycoses. stream or by direct extension. A small percentage of cases are fatal. There is no human el to human or animal to human transmission.

16.51 CoCcidioidomycosis Coccidioides immitis is present in tissue In culture, the fungus is characterized by as thick-walled spherules containing up toarthrospores which alternate with clear hy- several hundred endospores. Spherules rup- phae. Infection is through inhalation of ar- ture, releasing endospores which start theirthrospores. The resulting respiratory infec- own spherules. The major endemic area intion may be asymptomatic or influenza-like the world is the southwestern United Statesor 1% may progress to the disseminated, where C. immitis grows abundantly in deserthighly fatal form. Very high percentages of areas. all of the population of the endemic areas have positive skin tests showing previous infections.

16.52 Histoplasmosis Histoplasma capsulatum grows in soil, Histoplasmosis is a disease of the lungs especially those enriched with blackbird orand reticuloendothelial system. Initial infec- chicken droppings. In tissue it is seen astion is often mild and self-limiting or may small yeasts in macrophages and cells of theproduce granulomatous lesions and a disease reticuloendothelial system. The endemicwhich may resemble tuberculosis and is a area is predominately along the Mississippimajor cause of lung calcification. Supportive and Ohio River Valleys where 90 to 95% oftherapy usually is sufficient for primary pul- the people show positive skin tests. Themonary histoplasmosis. Systemic histoplas- infectiousformproducesinicroconidiamosis is treated with amphotericin B. A which enter the body through the lungs. Inreliable skin test is available. vivo these spores change into yeast cells..

16.53 Blastomycosis Blastomyces dermatitidis appears in tis- Blastomycosis, a disease of skin, lungs, sue or pus as a thick-walled nonencapsulatedand bone, may be asymptomatic or develop a yeast with broad-based buds. Blastomycosisinto a serious pulmonary disease resembling is usually restricted to the upper Mississippituberculosis or cancer. Paracoccidioidopy- and Ohio River valleys where it is thoughtcosis, a somewhat similar disease, caused by Medical Microbiology--135 3 j 320 TOPI(18 ANI) HI 1111'OPICH A. 101),IIIINTIAL INFORMATION IL PINRICIIMIiINT INFORMATION C, PRACTICAL MITIVITIF8 to be a mil aaprophyte, 'nal Infectious phasePoravoreirliolthor brazilienWri le not found probably produces spores which enter the naturally in the I hilted Stales, body by the kings, In vivo these rpores change into yeast cells.

16.54 Cryptococcosia Coptococcus neoforninnaisthe only Cryptococcosin is found worldwide, being pathogenic fungus found as an encapsulatedmore prevalent in urban areas with pigeon yeast in tissues, This organism grows 00 soil, populations. Treatment is with 5-fluorocy- especially in the presence of pigeon cure-tosine and/or amphotericin B, Diagnosis of mut, Pulmonary disease in the most com-cryptococcal meningitis involves detection mon form and may be asymptomatic. Inof encapsulated yeast in the cerebrospinal more severe canes, the organism has a pre- fluid by microsco examination with India dilection for the brain and meninges. Thereink and/or crypt 'cal antigen detection. is little or qt) inflammiltory reaction but sometimes granulomntous lesions are found. The capsule of this yeast inhibits phagocytosis,

16.6 Opportunistic Mycoses Opportunistic mycoses are caused by Severe diabetes, malignant tumors of the fungi which are part of the normal flora oflymphoid tissue, extensive treatment with the individual or are present in the environ-broad-spectrum antimicrobial agents or with ment but do not produce disease except in agents which suppress the immune response special circumstances, such as a debilitated(corticosteroids,X-irradiation,.cytotoxic state of the host or an overwhelming expo-drugs) all predispose an individual to the sure to spores by healthy individuals. opportunistic'mycoses.

16,61 Candidiasis (Candi- Candida albicans is a budding yeast Disease may be produced on the mucous dosis) which may produce hyphae or pseudohy-membranes or the skin. Candida may be phae in tissues. It is found as part of thetransmittedbetweenindividuals,from normal flora of the mucous membranes ofmother to baby as the baby passes through the mouth, vagina, and intestinal tract in athe birth canal, or by fomitesJ Other species high percentage of the population. Diseaseof Candida may be present as membeis of may occur in many parts of the body and the normal flora and more rarely cause dis- from a variety of causes of lowered resist- ease. Candidiasis is a common cause of death ance. Diseases include thrush, vulvovagini-in cancer and transplant patients. Treat- tis, infections around nails, or systemic in-ment is with nystatin locally or amphoteri- fections in inununosuppressed individuals. cin B for systemic infections.

16.62 Aspergillosis A large number of species of the aspergilli Patients with lunglesions from a previous which are distributed widely in nature andtuberculosis infection, farmers and others include many plant pathogens produce op-handling dead and decaying vegetation are portunistic infections. Aspergillosis is usu-most apt to acquire aspergillosis. Certain ally a disease of the respiratory tract andAspergillus species prevalent on stored results in either suppurative, or granuloma- grain or peanuts produce mycotoxins, which 7 Medical Microbiology-137 . 3N1 3 "2 410/

TOPICH t41111TOPICH A, Mkt !INTIM. IN AtMATION is, fitiNiii(siiimitiNT PltACTIOAI, ITIV1111101 taus Nelms'. In tissue, Aspergillmt speckle are hiAllytow when 'tweeted and may be produce militate hyphite with dieholonaecarcinegenio, ampergilli are found branching, A, itosioittus Is the most con',()mitt only as tiontnaiminte on lahoratoy mon pathogenic epeciee. medit, repeated 4'01016mi and identifies; time may be novetestry to est ablieh Own' as etio ogical agents of disease Isamu's,

10,63 Zygomycosis (Mticor- Mew or Ithizopus are occasionally found "he lesion may he ono of septa intim- mycsis, Phycontycosis) in tissues of diabetics and hutunnologicallym lion or show little tissue response. Ding- compromised Them 'organisms itRiff is made by (lading the large non-sep- aggressively grow toward, and may produce t ite hYpIpie in tissue or by repeated (mitt- occlusions in blood vessels, talons, Metabolic acidosis is a predisposing actor for acute zygomycositt.

17.0 Medical Virology 17.1 Epidemiology

17.11 Portals of Entry The primary routes of viral entry in the Examples of specific viruses _sand their human host include the skin, the respirator routes of entry include: wartsskin; influ- and gastrointestinal tract mucosa, and, to enza respiratory mucosa; poliovinises lesser extent, the eye and genitourinar, gaFtrointestinal mucosa; adenovirusesmu- tract. Certain viruses may be transmitted tincous membranes of the eye; herpes simplex utero. type 2genitourinary tract; and rubella placenta.

17.12 Transmission Inapparent infections are the most impor Humans are the only host for many vi- tant source of virus transmission from hu-ruses such as mumps, measles, and polio, man to human. Viruses may be transmittedwhereas other viruses such as rabies are by healthy individuals harboring latent vi-transmitted to humans from other animals. ruses.

17.13 Periodicity Many viral diseases such as the common Many viruses remain endemic and others cold are seasonal whereas others such asremain sporadic in a given population. hepatitis B, herpes simplex, and rabies are not. Some seasonal viruses such as the rhi- . noviruses cause epidemics yearly, whereas measles and influenza A virus tend to cause a epidemics at 2- to 3-year intervals.

17.2 Disease States Most pathogenic viruses cause acute in- Measles is an example of an acute viral fections. Certain viruses however, may enter disease. Fever blisters are caused by a virus a host and remain latent indefinitely or may (herpes) which remains latent and is ex- become clinically expressed at intervals laterpressed clinically at infrequent intervals. Medical Microbintogy -139 34.23 3 J TOPICS AND SUBTOPICS A. ESSENTIALINFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 4 in life. Slow viruses may enter a host and Subacute sclerosing panencephalitis (SSPE) incubate for months or years before symp- is an example of a slow virus infection. toms appear.

17.21 Localized Infections Many respiratory viruses invade the mu- Examples are the rhinoviruses and other cous membrane of the respiratory tract and cold-causing viruses. replicate and produce their clinical manifes- tations at this primary site.

17.22 Systemic Infections Many viruses invadp the mucous mem- Examples of such viruses include polio- branes of the respiratory or gastrointestinal virus, measles virus, and chickenpox virus. tracts and, after replication, enter the lymphatic system where they continue to replicate. The virus is then released into the Mood resulting in a viremia. The circulatory system delivers the virus to its target organ with resultant clinical disease.

17.3 Response of the Host to Host response generally includes inflam- Interferon induces cells to form transla- Viral Infection mation involving phagocytic cells, interferon tion inhibitory protein (TIP) which prevents production, and a specific immune response. the translation of viral mRNA. Interferon is Mononuclear cells (macrophages) are the tissue and host specific but is not specific for primary phagocytic cells involved, whereas a particular virus. bacterial infections elicit a polymorphonuclear cellular response. Interferon is a protein produced by a virus-infected cell and indirectly interferes with continued virus replication in other cells. It is important in recovery from viral disease. The cell-mediated immune response (T lymphocytes) appears to be primarily responsible for host recovery from viral infection, whereas the humoral immune response (B lymphocytes) seems to be primarily involved in the prevention of reinfection.

17.4 Antiviral Agents Viruses have no inherent metaboliC capa- Idoxuridine (IDU) and adenine ara hino- bility but rely on host cellular metabolism side (are-A) are nucleic acid antagonists use- for replication. Antiviral agents are therefore ful against certain herpes simplex infections. generally directed toward inhibition of viral Amantadine is indicated for the prevention nucleic acid synthesis and/or function. and/or treatment of influenza A and the thiosemicarbazones have been successfully used against smallpox.

Medical Microbiology 141 J TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 17.5 Immunoprophylaxis Live vaccines are superior to killed vac- Killed vaccines are available for influenza cines in promoting high levels of lasting im-and rabies. Live attenuated vaccines are munity. Killed vaccines' require boosteravailable for polio, rubeola (measles), and doses to maintain adequate immunity,rubella (German measles). whereas single injections of live attenuated vaccines provide a lasting immunity. A variety of viral vaccines, both killed and live- attenuated, are commercially available.

17.6 Diagnostic Virology Viral diseases may be diagnosed in the laboratory either by virus isolation or by detecting a rise in specific antibody.

17.61 Virus Isolation The presence of virus in cell culture sys- Projection slides of CPE in cell culture tems can be detected by separation and systems and slides of positive hemadsorp- rounding of cells from a monolayer (cyto- tion in cell culture systems may be studied. pathogenic effect, or CPE), the interference of cytopathogenesis, the ability of certain virus-infected cells to adsorb erythrocytes onto their surface (hemadsorption) or the ability of virus-containing fluids from cell culture systems to hemagglutinate erythrocytes.

17.62 Virus Identification Virus isolated in cell culture systems can If measles antibody is mixed with fluids be identified by a variety of tests utilizingsuspected of containing measles virus and specific known antisera to the suspected vi-this mixture is then inoculated into a cell rus, which will inhibit the expression of theculture system the CPE, which would char- virus in the cell culture system. act,eristically be caused by the measles virus, will be prevented from occurring. Similarly, if influenza antibody is mixed with influenza virus and inoculated into a susceptible cell culture system, the virus will be incapable of growing and the adsorption of erythrocytes to the cell surface will be prevented.

17.63 Serological Diagnosis To diagnose a viral disease serologically, Techniques for the detection of specific a significant elevation in specific antibodyantibody levels in a patient's serum include level between an acute and convalescenthemagglutination inhibition, complement serum should be demonstrated. The acutefixation, immunofluorescence antibody, and serum should be collected immediately afterradioimmunoassay. the onset of the disease, and the convalescent serum, with some exceptions, should be collected 10 to 14 days later. Medical MicrobiologyI43 3 " f") 1$ ) TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 17.7 RNA Viruses

17.71 Picomaviruses Picornarviruses are small (pico) RNA viruses.

17.711 Enteroviruses Enteroviruses initially replicate in the gas- Enteroviruses when swallowed' can find jection slides of an enterovirus rash trointestinal tract and are excreted in thetheir way to the gastrointestinal tract sincemaybe shown. stool. they are acid stable and are not destroyed by the acidity of the stomach.

17.7111 Polioviruses The three antigenically distinct polio- Viral meningitis involves inflammation of viruses may spread from the gastrointestinalthe meninges (the coverings of the brain and tract via blood and infect the spinal cordspinal cord). It is usually a self-limiting dis- and/or brain, which results in limb and/orease. Other enteroviruses include the echo- respiratoryparalysis.Polioviruses mayviruses and coxsackie A and B viruses. These cause viral meningitis and upper respiratorylatter three groups may be associated with tract infections but most commonly cause a raised (papular) trash. subclinical infections. The Sabin trivalent live oral polio vaccine has virtually eliminated paralytic disease in immunized popu- a lations. The Salk vaccine uses inactivated poliovirus.

17.712 Rhinoviruses There are over a hundred antigenically In contrast to other human viruses, rhi- distinct rhinoviruses, all of which may causenoviruses propagate best at 33°C rather the common cold. than 36° to 37°C.

17.72 Togaviruses (Arbovi- Togaviruses are arthropod (vector) trans- The equine encephalitides, yellow fever, ruses) mitted and are comprised of two serologi-and dengue are some of the important vi- cally distinct groups, the alphavirus group ruses in this group. They are mosquito trans- and flavivirus group. mitted.

17.721 Rubella (German Mea- Rubella is a mild rash-associated disease WoMen of childbearing age should be sles) which belongs to the togavirus group but ischecked for antibodies to rubella. If they not vector transmitted. If the virus is con- have none they should be immunized. Preg- tracted during the first 3 months of pregnant women should not be immunized with nancy, infection of the fetus may result in the rubella vaccine. death or birth defects such as cataracts and heart abnormalities. The use of the live rubella vaccine has significantly decreased the incidence of rubella-caused birth defects.

17.73 Orthomyxoviruses Medical Microbiology-145 3r) TOPICS AND SUBTOPICS1. A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 17.131 Influ'enza A sf Influenza A is the most common cause of The antigenicity of the influenza A virus influenza in humans, occurs in epidemicchanges periodically. For instance, the Hong form,every 2 to 3 years, and is characterized Kong strain may be present for a time and by sudden onset of headache, fever, achi-then another antigenic strain, such as the ness, and minimal respiratory symptoms. Russian strain, may take over Influenza B and C are similar clinically to influenza A but epidemiologically are not as significant.

17.74 Parampro.viritses ft Projection slides of a case of mumps may 17.741 Mumps, The par3lid glands are the target organs Mumps is the most common cause of pos- of the mumps virds. Occasional complica-tinfectious encephalitis; however, the en- be shown. tions of the young adolescent include inflam- cephalitis is usually of little clinical signifi- matibn of testes and the heart muscle. Thecance. Postinfectious encephalitis is an im- live mumps vaccine given early in life hasmune type of disease, as opposed to a pri- significantly decreased the incidence of corn,mary encephalitis which results in virus in- plications. vasion of nervous tissue.

17.712 Parainfluenza Viruses there are foiir antigenically distinct parainfluenza viruses, They are responsible for croup, an asthmatic type of bronchitis (bron- cliiolitis)fpneumonia, and upper respiratory tract afecilions in infants.

17.743 Measles (Rubeola) Measles is a common disease of childhood Other complications of measles include and is associated with raised lesions (a pa-inaddle ear infection, bronchitis, and pneu- pular rash). Measles is associated with a nia. Combination livevirus vaccines postinfeCtious encephalitis which may result rubella - mumps) are avail- in permanent braih damage or death in the ble. very yourirg. The live measles vaccine given/ duringdnfancy has significantly decreased the inciderlFe of encephalitis.

17.744 Respiratory Syncytial RSV nausea pneumonia and bronchiolitis Virus (RSV) in the very young. It may cause epidemics, usually in,,,the winter.

17.75 Rabies Rabies, ar rhabdovirlis, is transmitted to A new nonallergenic vaccine prepared in humans by the bite of a rabid animal. Thehuman fibroblast cells (cell cultures) and virus contained in the saliva reaches the requiring fewer injections provides a longer- centrarnervous system via the traumatized lasting immunity. nerves. The disease is characterized by a long incubation period and is almost 100% fatal. Rabies therapy involves the use of Medical Microbiology-147 or`.1 3"? %.1 6J-A- C.' PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION antirabies serum and the daily injection of killed duck embryo vaccine (DEV) for 14 to 21 days. In the United States rabies is most commonly found in skunks and, to a lesser degree, in raccoons and other wild animals, as well as in a few domesticanimals and bat&

17.8 DNA Viruses Viruses from maternal genital warts may 17.81 Pap'ovaviruses The human papilloma virus (wart virus) causes the characteristic juvenile and/orbe acquired at the time of birth by infants plantar warts. Venereal warts are sexuallywho may later develop tumorous growth of transmitted and may undergo cancerousthe vocal cords. Treatment of warts is by change if untreated. ' surgical or chemical removal or by cauteri- zation.

A live oral vaccine incorporating types 4 17.82 Adenoviruses The 31 antigenically distinct adenoviruses cause respiratory illness and/or conjuncti-and 7 is used in military recruits where vitis. infection rates are higher than in the lay population.Some human adenoviruses cause cancer when injected intolaboratory animals.

17.83 Herpes Group of Viruses This group includes viruses which, following primary infection, may remain in the host for life. HSV-1 infections occur early in life and Projection slides showing HSV-1 and 17.831 Herpes Simplex Virus HSV-1 is the cause of the common fever Types 1 and 2 (HSV-1blister or cold sore. It may also cause infec-by adulthood 70 to 90% have type 1 anti- HSV-2 infection may be shown. and HSV-2) tions of the eye and brain. HSV-2 is sexually bodies. HSV-2 infection usually occurs after transmitted and causes genital lesions. In-puberty or is acquired during birth. fection of the fetus or newborn may be fatal. Projection slides of cases of chickenpox Varieella-Zoster The V-Z virus causes chickenpox in chil- Shingles in the adult can be the source of 17.832 and of shingles may be shown. Virus (V-Z Virus) dren and shingles in adults. The chickenpox chickenpox in children and initiate large out- virus remains latent in the host and may be breaks. Shingles may be associated with expressed as shingles in later life followingspinal cord trauma or malignancy. some predisposing factor. The EB virus was first associated with 17.833 Epstein-Barr Virus (EB The EB virus is thought to be the etiolog- Virtis) ical agent of infectious mononucleosis and isBurkitt's lymphoma, a malignant tumor of possibly associated with Hodgkin's disease. the jaw which occurs in African children and Medical Microbiology-149 3 IP

TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES nasopharyngealcarcinoma commonin Chinese males.

17.834 Cytomegalovirus CMV is endemic in humans and causes Antibody to CMV is found in 80% of in- Projection slides of congenital CMV infec- (CMV) subclirlical infection in otherwise healthy individuals over 35 years of age. The virustions may be shown. dividuals. CMV may cause congenital ab-may be excreted intermittently by a host normalities of newborns, post-transfusionwith latent infection. mononucleosis and pneumonia, eye infections, and other systemic complications in ,. patients undergoing organ transplant or on immunosuppressive medication. 4

17.84 Poxviruses The poxviruses are large, have a complex symmetry, and include agents affecting a variety of animals, including humans.

In 1798, Jenner was the first person to use Projector slides allowing classical small- 17.841 Variola (Smallpox) _ Smallpox is characterized by a vesicular lueruption of the skin and is associated with a immunization procedures to prevent disease.box and the various stages of smallpox im- *gh morbidity and mortality. Through theHe used cowpox to protect against smallpox.munization may be shown. efforts of the World Health Organization theA laboratory strain of cowpox (vaccinia) is world today is essentially smallpox free:4now used as the agent in smallpox immuni- Smallpox immunization is required only forzation. those returning to the United States from certain risk areas.

17.9 Other Less-Well-De- fined Viruses

17.91 Hepatitis A Virus HAV is ubiquitous in that most adults About 30 to 60% of American adults have (HAV) have had exposure to it. It is transmittedantibody to HAV and an even higher prev- primarily from person to 'person by the feCal- alence occurs in lower socioeconomic groups. oral route, although epidemics pave been associated with infected shellfish and contaminated drinking water. Clinical manifes- tations involve the liver although the disease is usually of a mild, nature.

17.92 Hepatitis B Virus HBV is transmitted by blood and blood All donated blood is screened for the HBV (HBV) by-products and can also be transmitted bysurface antigen. Most of the, hepatitis that other routes. Contaminated needles of drugresults from blood transfusions is not due to addicts are also a common method of trans- HAV-or HBV but to a norrA-nInB virus and mission. HBV clinically is similar to HAVperhaps yet unknownviruses.( although it tends to be a more serious disease. A small percentage of infected individ- Medical Microbiology-151 3 -' 3"G C. PRACTICAL ACTIVITIES 3 AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION uals may become chronic carriers and remain potentially infectious to others.

Slow Viruses Infecting Kuiu, Creutzfeldt-Jakob disease, sub- Kuru, a disease ofthe.New Guinea high- Humans acute sclerosing panencephalitis (SSPE),lands, is transmitted by ingestion of under- and progressive multifocal leukoencephalo-cooked infected human brain and is on the pathy (PML) are examples of diseasesdecline due to a decrease in cannibalism. caused by slow viruses. They produce a neurological disease which is progressively fatal. These diseases are characterized by long incubation periods (months to years).

Medical Parasitology General Features of Pro- Cozoan Infections

Transmission of Proto- The life cycle of protozoa found in the Diagrams and charts depicting.he life cycles and transmission characteristics of zoa mouth and intestinal and urogenital tracts is usually simple and direct, i.e., the protozoa medically important protozoa may be stud- are in the infective stage shortly after they ied. leave the host. Protozoa which invade blood and other tissues generally have an indirect life cycle in that certain developments of the organisms take place in intermediate hosts such as insects before the protozoan is infectious for humans.

Pathogenesis of Proto- Protoza affect the host by multiplication Many pathogenic protozoa secrete prote- zoan Diseases of the organisms, invasion of tissue, andolytic enzymes, hemolysins, cytolysins, and producti& of toxins. In certain cases, theother toxins which aid in their virulence. host deiralops a hypersensitivity to the foreign proiozoan protein, resulting in an inflammatory reaction. Intestinal parasites produce less local and systemic effects than tissue parasites.

Symptoms of Protozoan Protozoan infections may be stibclinical, More than 90% of all protozoan infections Infection chronic, or acute. Often, an acute diseaseare asymptomatic, showing mutual toler- may result id a chronic infection. ance between the host and parasite. ,

Medical Protozoology

Sarcodina, the Amoebae Most intestinal amoebae are nonpatho- Entamoeba coli and Endolimax nana are Medical MicrobiologyI 53 3 3j3 TOPICS AND SUBTOPICS A. B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES IAL I FORMATION cr genic and occur as commensals in the cecumcommensals in the large intestine of humans and colon. They must be differentiated fromand are only rarely implicated in disease. the only primary pathogenic species Enta-Their presence indicates that an individual moeba histolytica. has ingested something contaminated with feces. GA. Prepared microscope slides of tropho- 18.211 E. histolytica E. histolytica is the most important path- Important sources of infection are asymp- ogenic amoeba which infects humans. Infec- tomatic carriers. Fresh 'iegetables fertilizedmites and cysts of E. histolytica may be tion is acquired by ingestion of cysts from with human feces are sources in certain for-compared with those of Entamoeba coli and contaminated food or water. Infection ofteneign countries. Trophozoites may be seen inE. nana. results in asymptomatic carriers, but in dis- runny stools while cysts are more common ease caused by invasion of colon wall, resul-in formed stools. Extraintestinal disease may tant tissue destruction occurs accompanied be detected serologically. Metronidazole is by diarrhea or dysentery. Extraintestinalgenerally the treatment of choice for extrain- disease, while rare, may occur in the liver ortestinal disease. other organs. el 18.22 Mastigophora, the Flag- ellates Prepared microscope slides of tropho- 18.221 Giardia lamblia G. lamblia is -the most frequently recog- Diagnosis is by demonstration of cysts or nized pathogenic protozoan in waterbornetrophozoites in feces. Metronidazole is thezoites and cysts of G.-lamblia may be com- disease outbreaks ant in tourists returningtreatment of choice. pared with those 9f Chilomastix mesnili, a to the United States from abroad. Large nonpathogenic flagellate. numbers of G. lamblia attached to the wall of the small intestine may cause irritation and low-grade inflammation. Diarrhea may "occur, especially in children, but asymptomatic cases are more common. The organisms ele acquired by ingestion of cysts from contaminated food or water.

18.222 Trichomonas vaginalis Three species of trichomonads are found Prevention is by attention to personal hy- in humans. The only pathogenic species, T. giene and detection and treatment of in- vaginalis may cause vaginitis in the femalefected females and contacts. Diagnosis is and urethritis in the male. T. vaginalis ismade by microscopic examination of exu- transmitted by sexual contact. dates or urine. No cyst exists. Metronidazole is the treatment of choice.

18.223 Trypansoma Trypanosoma gambiense and T. rhode- Trypanosomes, which spend part of their siense are causes of African trypanosomiasislife cycle in Vertebrates and part in inverte- (sleeping sickness) in humans. The orga-brates, may be found in the bloodstream of nisms are transmitted by the bite of theinfected humans and other vertebrates. Di- tsetse fly. A mild systemic illness graduallyagnosis is by identification of the trypano- Medical Microbiology-155 3" j zn TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES progresses into encephalitis. As cerebralsomes in blood, lymph glands, or spinal fluid. damage increases, sleep becomes continuousTreatment varies with stage of disease and and develops into a coma and death. Chaga'sis often not successful. disease(Americantrypanosomiasis)is caused by T. cruzi and is transmitted by the treatomine (cone-nosed) bugs. It is the lead; ing cause of cardiovascular death in South America but is generally not found as far north as the United States.

18.224 Leishmania Infection by members of the genus Leish- An important example of leislunaniasis is mania results in various diseases which in-kala-azar. Diagnosis is made by finding the clude visceral and/or dermal lesions. All spe-organism in infected tissue or by serology. cies are transmitted by the bite of sand flies.Treatment is varied and not often successful. Leishmania are not normally found in the United States.

18.23 Ciliata, the Ciliates

18.231 Balantidium coli Balantidium coli is the only ciliate which The large intestine is invaded by the mul- is parasitic for humans. The incidence is low,tiplying organisms which form nests that and many of these cases are asymptomatic.prodlice ulcers and abscesses. Oxytetracy- Infection is acquired by ingestion of cystscline is the drug of choice. from contaminated food or water. Symp- toms include alternating diarrhea and con- stipation.

18.24 Sporozoa The sporozoa of major medical importance are the four plasmodial species: Plas- modium vivaxf P. ino/ariae, Pialciparum, and P. ovule, all of which cause malaria. Prepared microscope slides containing the 18.241 Plasmodia Malaria is initiated by spor zoites from In malaria, the sudden rupture of parasi- the salivary glands of infected femaletized erythrocytes with release of parasitesfour species of plasmodia may be studied. Anopheles mosquitoes. The sporozoites areis responsible for the characteristic cycles of injec'ed into the bloodstream during the biterising temperature followed, by chills and 1 of the mosquito. The organisms Undergo anor more days free from symptoms. exoerythrocytic cycle in the liver followed Pathology is caused by the destruction of by an intraerythrocytic cycle in the blood aserythrocytes, blockage of capillaries in the well as a sexual cycle which is completed inviscera, and damage to the liver due to a the mosquito. The major features of infec-lack of oxygen. Diagnosis is by microscopic tion include anemia, spleen, and liver en- examination of blood smears. The disease is largement and a classical fever pattern. P.commonly treated or prevented with chlor- falciparum is the most important species. oquin or atabrine and by other synthetic drugs. 34 Medical M icro biology157 TOPIC) AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 18.242. Toxoplasma gondii T. gondii is an obligate intracellular pars Prevention is by thorough cooking of meat Prepared microscope slides of tissue con- mite of the reticuloendothelial system. Theand careful handling of cat litter boxes, es-taining T.-gondii may be studied. parasite is coccidian, and sexual reprOduc-pecially by pregnant women. Diagnosis is don occurs in the intestine of cats. A tissueusually by serological inetthods, but micro- phase is found in many animals .(humans,scopic examination. of appropriate tissue cows, pigs, sheep', chickens, and ratsj whichmay reveal organisms. Treatmeht is with have acquired the infection from ingestingpyrimethamine and sulfonamides. fecal matter frOm cats or meat from infected animals. Toxoplasmosis in, adults may_ be asymptomatic, chronic, or more rarely acute. This organism may be transmitted from an infected mother to the fetus leading to birth defects, abortion, or stillbirth. -o 18.3 General Features of Hel- minth Infection

18.31 TranarnisSio of Hel- Transmission of helminths may be direct Many ova ale infective for a new host minths by ingestion of ova or by penetration of the,after the ova have undergone some devel- skin by larvae. In certain cases, transmissionopment outside of the body. Other ova are is by eating improperly cooked meat con- infective immediately. Historically, the reli- taining larval forms. Some larvae may also gious prohibitions-against eating pork were penetrate humans skin after undergoing alaid down because of the illnesses which developmental cycle in snails and then be- were produced after eating incompletely coming free-living larvae. cooked infected meat.

18.32 Pathogenesis of Dis- Injury may be produced by the adults in eases Produced by Intes-the intestinal tract where irritation or pen- tinal Helminths etration of the intestinal wall may take place or where attachment of the helminth may open the way for bacterial infection. Larvae may produce local or generalized reactions during their invasion and migration through the host's tissue. Localized reaction depends on the degree ofsensitization of the host to products of the parasite.

18.33 Symptoms of Intestinal Symptomatology of helminth infections Helminth Infection depends upon the species, number, and locations of these parasites, the amount of migration of the organisms through the tissues, and the degree of sensitization of the host to the parasite. Chronic and repeated helminth infestations may result in anemia, Medical Microbiology -159 3J`) .TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION R. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES intermittent abdominal symptoms, signs of pneumonia during larval migration, and lowered general health.

18.4 Medical lielminthology

18.41 NematOdes, the Round- worms

18.411 Ascaris lumbricoides The adult ascaris is the largest of the Transmission is by the fecal-oral route, human intestinal roundworms. It may reachwith infestation most prevalent in young 20 cm in length. Ascariasis is the most cdh- children. Eggs leave the body of the host in mon helminthic infestation of humans. Itfeces, develop in soil, and become infectious occurs when ingested eggs hatch in the small after 10 to 14 days. Large numbers of adult intestine, enter the bloodstream, and reachworms may cause obstruction of the small the lungs, where they are coughed up and intestine. swallowed. Adults reach maturity in the in- Diagnosis is made by finding eggs in the testinal tract. Symptoms may include ab-stool. Treatment is with piperazine citrate dominal pain and allergic reactions to theand other drugs. pulmonary cycle.

18.412 Enterobius vermicu- These small roundworms (2 to 3 mm) are Enterobiasis is extremely , prevalent in laris (Pinworms) inhabitants of the cecum. Infection is com- groups of young children and their house- mon in temperate climates and is not limitedholds. Infection is self-limiting in the ab- to low pr socioeconomic groups. At night, thesence of reinfection. Diagnosis is made by femateffierges from the anus and lays hermicroscopic examination of cellophane tape eggs in the perianal region. Itching developswhich has been pressed around the perianal around the perianal area which leads to rein- region. Treatment is with pyrivinium pa- fection. The eggs may be spread throughoutmoate, piperazine, or pyrantel pamoate. the house by contaminated bedclothes, fin-Generally an 'entire household must be gers, and other objects. treated.

18.413 Necatur americanus The adult N. americanus, the Nevi World Hookworm infection may be prevented by hookworm, 'is approximately 1 cm long. Itproper sanitary measures and by wearing attaches to the mucosa of the small intestineshoes in infested areas. Diagnosis is made by by two pairs of chitinous plates in its mouth. demonstrating eggs in stoolspecimens. Eggs leave the host in feces and develop toTreatment of hookworm infections is usually yield larvae in 24 to 48 hours. Larvae pene- not necessary. An iron-rich diet helps to trate through the skin to the blood vesselsprevent symptoms. and lunga.where they are coughed up and Ancylostoma duodenale, the Old World swallowed. Maturation takes place in the hookworm, is common in southeast Asia. A. small intestine. Infestation with a smallbraziliense and A. caninum are parasites of number of worms may be asymptomatic. In dogs and cats which occasionally infect hu- chronic infestation with a large number ofmans. Creeping eruption or cutaneous larva Medical Microbiology-161 3 G 3 TOPIC'S AND 81114TOPICS A. ESSENTIAL INFORMATION 11, NNItICIIMEINT INFORMATION C. PRACTICAL ACTIVITIKS worms, 100 to 200 unt. of blood per day may migrans caused by these animal parasites hi be removed remitting in iron-deficiency ane- produced when the larvae burrow under the mia. human skin "producing irritating tunnels, Diagnosis is made by observing the tracks in I. the skin. 18.414 Strongyloi(lesstercor- The adult worms of S. stercoralis parasi- The life cycle of Strongyloides is very ails (Ttirila(1)l1orrn) tize the duodenum or jejunum of humans. complex; differkit life cycles may occur dur- The eggs, passed by the f9rtilizod female,ing a single infestation. Strongyloidiasis is hatch in the intestinal mucosa, and the lar- characterized by watery, mucoid diarrhea. vae leave the body in the feces. Infection isTreatment is with thiabendezole. acquired by penetration of the larvae into the skin of the new host followed by passage through the bloodstream to the lungs, where they are coughed up and then swallowed. Diagnosis is made by finding the larvae in the feces.

18.415 Trichuris trickura Adult worms are found in the cecum. The Prevention is by sanitary waste disposal Prepared microscope slides and preserved (Whipworm) eggs are pasSed in the feces to the soil whereand washing of hands. Diagnosis is made by material may be studied to illustrate eggs embryonic development takes place.In- examining feces for unsegmented lemon- and adults. fected eggs are ingested by the new host and shaped eggs with plug-like ends. Trichuris develop into the adult stage in the intestines.infections may be treated with mebendazole. Trichuriasis is a common disease among children.. of tropical countries and is often asymptomatic. In heavy infections, prolapse of the rectum, loss of weight, vomiting, diarrhea, and dysentery occur due to the worms penetrating the mucosa.

18.416 Trichinella spiralis Humans become infected with T. spiralis The domestic animal cycle depends upon Prepared microscope slides of encysted by eating insufficiently cooked meat of flesh- the feeding of raw garbage containing in- larvae may be observed. eating mammals or certain domestic animalsfected table scraps to swine. Diagnosis of (pigs) which contain larvae encysted intrichinosis is by intradermal testing, muscle striated muscle. Worms from digested cystsbiopsy, or serological testing. There is no develop in the intestine and produce larvaespecific treatment, but steroids may provide which penetrate the mucosa of the small symptomatic relief. intestine and are disbursed by the blood. They invade striated muscle primarily of the diaphragm, arms, and legs. Most cases are asymptomatic; however, symptoms may occur depending on the particular tissue invaded and severity of infection. An acute inflammatory reaction may occur around parasitized muscle fibers. Medical Microbiology-163 'l'OPICH AN I)O1ITOPIC8 A. 88,PINTIAI, INFORMATION 11. ENRICHMENT INFORMATION 0, PRACTICAL ACTIVITIM 18,42 Cestodes (Tlipewornsi)

18,421 Taman solito(Pork The adult of T. ?Whi is found In the Diagnosia Is mode by finding proglottids Prepared microscope slides and preserved Tapeworm) upper part of the Jejunum of humans whore or eggs in feces or on swabs from the imaginal fecal matter containing ova may be ob- it tannn' by 111011110 of hooks on Its scolex. region, Occasionally, uneystud larvae may served,Preservedproglottidsorentire Eggs or proglottida are passed in the fecesdevelop in humans, Eggs in the focus mayadults may also be attune(I. and eaten by hogs. Thu eggs hatch in thebo swept back into the duodenum by reverab intestinal tract of the hog, then penetrateperistalsis. Thu eggs hatch and the larvae the intestinal wall and migrate to musclesmigrate to muscle tissue or brain tissue and other tissue'. Infection of humane oc-where they encyat and cause disease. curs from eking undercooked pork in which the larvae are encysd. Worms are 2 to 4 motors in length and have than 1,000 proglottids.

18.422 Taenia saginata (Beef Humans are the definitive host of T. sa- Diagnosis is made by finding eggs or prog- Prepared microscope slides and preserved Tapeworm) pinata. Intermediate hosts are herbivores, lottids in feces ok on swabs from tho perianal fecal matter containing ova may be ob- with cattle being the most important. Graz- region. Treatment is with niclosamide. If theserved.Preservedproglottidsorentire ing cattle are infected by eating ova or pro-scolox is not passed following treatment, adults may also be studied. glottids contained in contaminated feces ofmore proglottids may develop. humans. The eggs hatch in the intestine, larvae penetrate the intestinal wall and migrate to muscles, where they become encysted. Infection occurs when the cyst is ingested in undercooked meat. Worms are 4 to 10 meters in length and have approximately 2,000 proglottids. T. saginata however produces little toxic, irritative, or allergic reaction.

18.423 Diphyllobothrium la- D. latum is the largest tapeworm of hu- The fish tapeworm is found in cold, fresh- turn (Fish Tapeworm) mans reaching a length of more than 10water lakes. In areas where untreated sew- meters. There are more than 3,000 proglot-age is allowed to enter fresh water, infection tids per tapeworm and 1 million eggs mayof fish and crustaceans occurs. be excreted by the tapeworm each day. In- fection occurs fromating raw or undercooked freshwater anials. Eggs are passed A in the feces and hatch inesh water. If the intermediate host (f, crustaceans) is ingested; larvae develoto the adult stage in the ileum.

18.424 Hymenolepsis nana H. nana is the most common tapeworm Diagnosis is by finding 4 eggs LI feces. (Dwarf Tapeworm) of humans, generally occurring in children Medial Microbi logy-165 3 -I j ToilwMAND 81IIITOP (11I A, 10IHNNTIAL INFORMATION 11, IN111(IIM14N'I' INFORMATION C. PRACTICAL A ITIVITI EH lit tropleal dimities, No intermediate host Is required, Ingested eggs hal eh in the small intestine, penetrate the vllli, and litter laalc out into1110 It1111011and attach to the muca, Light iiifeetions show no symptoms, wboruns heavierinfections produce abdominal pain and diarrhea,

Liver Flukes Liver flukes are flattened and monouclous, Most easesare asyptoniatiu; however, possessoral and ventralkill01(001, Mid liveIn heavy Infection results In abdominal pain the bile ducts of the liver, Major genera and diarrhea, Diagnosis Is made by finding IncludeClotiorphis, OpiNthorchis,and Pas- eggs infeca In many Far East countries, cio/o, Life cycles involve snails es interino- raw fish are traditionally yam% and are a Mate hosts and mammals including humans common source of infection. Liver flukes as aefinitive hosts. Fish may also act as may live for 20 years." additional intermediate hosts. Flukes are endemic in the Far East where figh ponds are fertilized with human feces.

Schistosonia (Blood Schistosomiasis is a disease caused by Diagnosis is made by finding eggs in feces Flukes) members of the genus$chistosornawhich and/or urine. Schistosomiasis is found in live in the blood vessels. Eggs are passed in Africa, South Asia, and tropical America. urine and/or feces. The eggs are ingested by specific snails which serve as intermediate hosts. The free-swimming lal.val stage is infective by penetrating the human skin. Symptoms may vary from a transient rash 1.4 to acute illness and possible death.

19.0 Diagnostic. Microbiol- ogy 19.1 Diagnostic Bacteriology Diagnostic bacteriology is primarily concerned with the identification of organisms as they relate to infectious disease and to the relative resistance or susceptibility of pathogenic bacteria to antibacterial agents.

19.2 Transport Specimens should be taken from appro- Examples of specimen sources include priate body sites by using aseptic techniques throat,nasopharynx,wounds,sputum, and transported to the laboratory as quickly blood, cerebrospinal fluid, urine, feces, and as possible in appropriate transport media. tissue biopsies.

19.3 Sterile Body Sites Many body sites or tissues such as blood and cerebrospinal fluid are normally devoid Medical Microbiology-167 35: 3 , l'OPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES of microorganisms so that recovery of microorganisms from these areas is significant.

19.4 Normal Flora Many parts of the body have normal mi- Escherichia colt is normally found in the Throats may be swabbed and specimens crobial flora so that microorganisms cul-stool but is a common urinary tract patho-cultured on blood agar plates. tured from these areas must be considered gen. in this light.

19.5 Isolation To identify bacteria, it is generally neces- It is difficult and usually impossible to Pure and mixed cultures of bacteria sary to streak the specimen on appropriate/identify bacteria if they are not in pure cul-streaked for isolation should be studied. semisolid agar in a petri dish and isolateture. colony-forming units.

19.51 InitialInformation on The Gram reaction, cellular morphology Speed and accuracy of organism identifi- Colonial characteristics of bacteria grown Bacteria and arrangement, and colonial morphologycation is necessary for patient care. on semisolid media in petri dishes may be and growth on selective and differential me- studied. Gram stains should be performed. dia provide valuable information toward identification of many bacteria. Many commercially produced systems are 19.52 Bacterial Identification The ability to grow and reproduce in var- Identification of anaerobic bacteriais ious biochemical media, the end products ofaided by the use of gas-liquid chromatogra- used in clinical laboratories for the identifi- such growth, susceptibility to certain anti-phy for detection of characteristic fatty acid cation of Enterobacteriaceae, nonfermen- biotics, and reactions with spkific antibodyend products. tera, and yeasts. These systems might be are common methods in bacterial' Identifi- obtained from clinical laboratories for dem- cation. onstration.

1-9.6 Antibiotic Susceptibility When a pathogenic bacterial strain is iso- The susceptibility pattern of a bacterial An experiment showing the effects of var- lated from a patient, antibiotic susceptibility isolate is often determined and reportedious antibiotic disks dropped on a lawn of patterns of that organism are often consid-along with a brief description of the orga- bacteria growing on semisolid agar in a petri ered more important than its identification.nism (e.g., gram-negative bacillus) beforedish may be performed. A bacterial isolate is generally subjected tothe organism is identified. a panel of antibiotics to determine its susceptibility or resistance to each drug.

19.7 Parasite Identification Diagnostic laboratory parasitology pri- Some aerological procedures are available Prepared microscope slides of parasitic marily involves searching for the adult or- which aid in the diagnosis of certain parasitic protozoans may be studied. ganisms, eggs, cysts, or trophozoites in pa-diseases such as amoebiasis and trichinosis. tient specimens. Material is rarely cultured.Toxoplasmosis is primarily diagnosed by serological means.

19.8 Fungus Identification Detection and identification of fungi in Serological techniques are available as an Projection slides illustrating macroscopic the diagnostic laboratory are based primar- aid in the diagnosis of certain fungal infec-and microscopic fungal characteristics may ily on recognizable macroscopic and micro-tions. However, serological cross-reactions be studied. scopic characteristics. Growth of fungi inamong some of the fungal agents make in- Medical Microbiology-169 , 3: I TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES clinical specimens often requires manyterpretation difficult. Diagnosis of crypto- weeks. coccal meningitis is aided by the immunological detection of the cryptococcal antigen in cerebrospinal fluid.

Projection slides showing rytopathic ef- 19.9 Virus Identification Few clinical laboratories are equipped to For the most part, laboratory evidence of isolate and identify a virus. Growth mustviral infection is obtained by serologicalfects in tissue or embryonated eggs may be dike place in living tissue, and identificationmeans rather than by virus isolation. Theshown. is usually made from this tissue based ontime involved in viral isolation and identifi- specific antibody reactivity or characteristiccation can vary from a few hours (herpes cytopathogenic effects of a given virus do simplex virus) to 3 to 5 weeks (cytomegalo- specific cell cultures, embryonated eggs, orvirus). Also, serological evidence of viral in- laboratory animals. fection requires submission of an acute and convalescent blood specimen (usually 10 to 14 days apart) to demonstrate a significant rise in antibody titer.

20.0 Diagnostic Immunol- ogy 20.1 Serology Serological tests attempt to detect specific antigens or antibodies in any body fluid or tissue of a patient. The results are of clinical importance in the identification of infectious agents, self and fetal antigens and their corresponding antibodies. In all serological testing, the identity of one component, antigen or antibody, is known.

20 I I Identificationof Anti- An unknown antigen can be identified or An organism may be identified as a mem- gens typed by means of known antibody. ber of the Salmonella genus by the use of specific antiserum.

20.12 Identificationof Anti- The presence or absence of antibody and Serial dilutions of a patient's serum may body the titer (level) of antibody can be deter-be tested with known antigen. A low titer mined by employing known antigen in themay suggest that a patient has had-previous antigen-antibody reaction. infection or immunization with that organism. A rise in titer, when acute and convalescent serum samples are compared, sug- gests that active infection is or recently was present.

20.2 Antigen-Antibody Re- actions Medical Microbiology 171 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES ( 20.21 Valence of Antibody Valence is the number of antigenic deter- Electron micrographs show antibodies at- A diagram showing sites for atitlop bind- minants or sites with which one antibodytached to antigens by twq attachment sites ing in IgG and IgM molecules may be siud- molecule can combine. Immunoglobulin Gin the case of IgG and two or more for IgM. ied.< s- (IgG) antibodies are bivalent (two sites), whereas IgM has a valence of 5 to 10. A+

20.22 Valence of Antigen Antigens are multivalent, i.e., have many Electron micrographs show that a micro- antibody-combining sites. organism may have many antibodies attached to it. A diagram or drawing of the lattice effect 20.23 Lattice Effect When multivalent antigens and bivalent The lattice effect is dependent upon pH, antibodies combine, they link in a lattice-ionic strength, and temperature. may be studied. type formation.

20.3 Serological Tests Projection slides of agglutination tests andi 20.31 Agglutination Test The reaction between antibody and a par- Agglutinationreactions are dependent ticulate antigen is referred to as an aggluti-upon pH, ionic strength, and temperature. reactionsay be studied. nation reaction. The antigen may consist of suspensions of organisms, erythrocytes, or uniform particles such as latex beads which have antigen adsorbed onto them. Agglutination reactions such as those for 20.311 Basis of Test Particulate antigens mixed with specific Salmonella typing may be carried out by antibody clump due to the antibody holdingeither slide or test tube agglutination pro- the detection of rheumatoid factor are com- them together. cedures. mercially available and may be performed.

20.312 Hemagglutination Hemagglutination is the ability of certain Many other viruses are also able to agglu- substances to attach to (adsorb) the surface tinate erythrocytes. This phenomenon is not of erythrocytes resulting in a clumping ofdependent on the presence of antibody. the red cells. Influenza virus hemagglutin- ates erythrocytes and can be detected and quantitated by this method.

20.313 Hemagglutination Inhi- Specific antiserum against the influenza Hemagglutination inhibition testing may bition virus will combine with its antigen (virus)be used to identify an unknown virus or and prevent the virus from attaching to thespecific antibody to a known virus. erythrocytes and thus prevent hemagglutination. This is known as hemagglutination inhibition. Typing of students' blood may be per- 20.314 Blood Typing -`,...Blood groups are systems of different an- A person who has group A antigens on his tiOns on the surfaces of erythrocytes. Theerythrocytes (type A blood) will also have formed with commercially available typing most important are the A. B, 0, and Rhantibodies to group B antigen and con- sera. Medical Microbiology -173 3: 2 3Li 6 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFOI5MATION C. PRACTICAL ACTIVITIES systems which may be involved in transfu- vertely, type B blood contains antibodies to sion reactions and heMolytic disease of thegroup A antigens. Therefore, transfusion of newborn. blood': from a type A donor into a type B recipient or vice versa would result in a tranfusion reaction. 20.32 Precipitation The reaction between specific antibody Qualitative andluantitative tests for spe- Precipitation testing using albumin or any and soluble antigen is referred to as a pre-cific serum proteiris such as albumin areprotein and its specific antibody may be cipitation reaction. The- antigen-antibodyoften performed by precipitation testing. conducted by using capillary tubes or test complex becomes insoluble. tubes.

20.321 Gel Diffusion Gel diffusion is a precipitation reaction in Orious methods involving gel diffusion The reaction between albumin and anti- which the antigen and antibody reactantshave been developed of which double diffu- albumin may also be detected by gel diffu- are supported by an agar matrix. The reac-sion in agar is a characteristic example. sion. Filter paper disks saturated with the tants diffuse toward one another and form reactants are placed 1 cm apart on an agar a detectable insoluble complex recognized as gel surface and allowed to diffuse over night. a precipitation line.

20.33 Complement Fixation The binding (fixation) of complement occurs following the reaction between an antigen and its specific antibody. The fixation of complement can be used as a tcst to detect and semiquantitate antibodies in a patient's serum. This test is most commonly used to detect antibodies to viruses.

20.34 Immunofluorescence Antigen or antibody is coupled to a flu- The presence of certain antibodies in se- Microscopy orescent dye and then reacted with the un- rum, identification of microorganisms, and known. The reaction is generally carried outdetection of antigens in tissue sections is on a microscope slide. A specialized micro-often determined via immunofluorescence scope is used for observation. If fluorescence microscopy. is seen, a specific antigen-antibody reaction has taken place.

20.35 Radioimmunoassay Radioimmunoassays are useful in measur- Radioimmunoassay is very sensitive and (RIA) ing the serum levels of many hormones,capable of detecting substances in nano- drugs, and other biological materials. The ams per milliliter. method is most often based on competition for specific antibody between radioactively labeled known material and unlabeled unknown material.

20.36 Enzyme-Linked Immu- ELISA is analogous to radioimmunoassay ELISA testing approaches the sensitivity nosorbent Assay or immunofluorescence except that the an-of RIA without the drawback of using radio- (ELISA) tibody is conjugated to an enzyme. Detec-active materials. Medical M icrobiology 175 3 . TOPICS ANDSUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES tion of an antigen:antibody reaction is then dependent on a color reaction resulting from the reaction of the enzyme with its substrate. The substrate i,s chosen so that it is colorless initially and, on degradation by the enzyme, yields a colored product whose intensity can be measured spectrophotometrically.

20.4 Skin Tests Involving Im- In atopic allergy( testing, the patient is Genetic factors seem to predispose indi- mediate Hypersensitiv- injected intradermally with-a series of anti-viduals to atopic allergies. Approximately ity (Atopic Allergy) gens (allergens such as pollen, danders, etc.) 10% of the population suffers from asthma, to determine the ones to which he is sensi- hay fever, and other atopic allergies. tive. If specific antibody (IgE) is present, a localized reaction occurs at the injection site within minutes. 20.5 Skin Tests Involving Delayed hypersensitivity skin testing in- Delayed hypersensitivity skin testing is DelayedHypersensitiv-volves the intradermal injection of certainused to demonstrate hypersensitivity to tu- ity antigens to demonstrate cutaneous hyper- berculosis, systemic fungal diseases, and poi- sensitivity. Since these tests involve a cell- son ivy. It is also used in the assessment of mediated (T-cell) response, a localized re- immunocompetence. action at the injection site occurs within 24 to 72 hours.

20.6 Detection of Cells in the The amounts and the function of lympho- Lymphocytes which bind sheep erythro- Immune Response andcytes, granulocytes, and macrophages arecytes to form rosettes are T cells. This Their Function important in determining the immunocom- marker is used to identify and quantitate T petence of a host. There are a variety ofcells. Lymphocytes with surface immuno- methodologies by which the amounts andglobulins [B cells] can be identified and function of these cells can be determined. quantitated by immunofluorescence.

21.0 Epidemiology 21.1 Epidemiology Epidemiology is the study of the factors Knowledge of the distribution of a disease which determine the frequency and distri- within a population can be used to identify bution of infection and disease in a given the etiological agent and to design strategies population. for its prevention and control.

21.11 Sporadic Some diseases may be present in a com- Virus diseases such as mumps may occur munity with only a few cases becoming clin- sporadically in school age children. ically evident from time to time. These cases are said to occur sporadically.

21.12 Endemic A disease is said to be endemic if it is Measles is an example of an endemic dis- continuously present in a given po i ion ease in the United States. at a loevel of 'ncidence. Medical obiology-177

3' 1J-a. TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 21.13 :Epidemic An epidemic is a rapidly progressing out- No specific number of cases can be used Weekly epidemiological reports of com- break of an infectious disease through ato identify an epidemic. One hundred casesmunicable diseases within each state may be population of susceptible individuals. An en-of bacillary dysentery per 100,000 persons inobtained from the local health department. demic disease which significantly increasesthe United States would be considered an in incidence within a given period of time epidemic, whereas the same incidence in the can become epidemic. Far East might iDt be considered unusual or may even be cnsidered as endemic. The possibility of a particular organism producing an epidemic depends in part on the number of individuals in the population who have immunity to the organism either from previous infection or from immunization. The genetic background of the population, the virulence of the particular strain of the pathogen, and the intensity of exposure of a given group of individuals in the population areallfactors involvedindetermining whether a disease becomes epidemic.

21.14 Pandemic A pandemic is an epidemic of unusally Influenza is a classical example of an in- great proportions often involving more than fectious agent that has caused pandemics in one country or continent. the past.

21.15 Recognition of Epidem- A continuing survey of the morbidity rate Statistics are kept by health departments ics (number of cases per 100,000 persons) andas to the seasonal incidence and localities mortality rate (number of deaths per 1,000involved in the spread of disease. cases) of a given disease will indicate an epidemic if either rate suddenly increases in frequency.

21.16 Reservoir of Infection Reservoirs of infectiop include carriers or The reservoir of a disease-producing infec- active cases, soil and water which mighttious agent is the sum of all the potential harbor organisms, or animals which mightsources of that infectious agent. harbor an infectious agent which is transmissible directly or indirectly to humans.

21.17 Zoonotic Infections Zoonoses are infections of animals which Rabies is primarily a disease of skunks can be transmitted directly or indirectly to and bats which act as a reservoir with poten- humans. tial infection to humans.

21.2 Transmission of Disease Factors determining the method of trans- Portals of exit and entry include direct mission of disease are the means by which contact between individuals, the respiratory the microorganisms leave the body (the por-route, the fecal-oral route, and inoculation tal of exit), survival of the microorganismsby animals, insects, or instruments. Medical Microbiology-179 3' TOPICS AND SUBTOPICS A..ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES while outside the body, 'and the methOef of entering the new host (portal of entry).

21.21 Diseases Transmitted Diseases which require direct contact for The fastidious nature of these organisms by Direct Contact transmission are caused by microorganismsshow a high degree of adaptation of the that require a living host and which have aparasite to its host. low resistance to the environment outside of the host.

21.211 Sexually 'Transmitted VD are transmitted.almost solely by sex- Syphilis and gonorrhea are sexually trans- Films about venereal diseases may be Disease (STD), or Ve- ual contact or byi routes which include directmitted diseases. The necessity of a closeShown to offer an understanding of epide- nereal Diseases (VD) contact from a lesion of one person to therelationship between these parasites andmiological principles as well as information skin or mucous membranes of another. host is shown by their inability toabout the diseases. wit nd environmental conditions outside o the host.

21.22 Diseases Transmitted Airborne transmission of disease is caused The common cold and influenza are two by Air by inhalation of droplets of sputum or salivaexamples of diseases transmitted by droplets from an infected host or inhalation of dustand which use the respiratory tract as the which contains spores from an organismportal of entry and the portal of exit. growinginsoil.Air-borne(respiratory) transmission is the most common method by which infectious agents are transmitted in humans.

21.221 Dust-Borne Organisms Fungi which cause systemic infections Blastomycosis,histoplasmosis,coccidi- grow in the soil and produce spores in largeodomycosis, and cryptococcosis are exam- numbers which are inhaled with circulatingples of systemic mycoses transmitted by this dust. method.

21.23 Food-Borne and Water- Organisms which leave the body in excre- Organisms such as intestinal viruses may Food-handler laws and Environmental borne Diseases ment may fmd their way directly into wateronly survive in water, but bacteria such asProtection Agency regulations may be dis- and indirectly into food by way of foodhan-salmonellae may multiply in great numberscussed. dlers or flies. Bacteria, viruses, and manyin food such as meat and egg products. intestinal protozoa are infective if consumed by a new host. Some helminths, however, complete part of their life cycle in the water or in another organism before becoming infective for a new human host.

21.231 Food Infection Versus Disease produced by microorganisms may Boils caused by Staphylococcus aureus Food Intoxication be either infeCtious (the organisms attack,are an example of an infection, whereas food the cells of the host) or intoxications (thepoisoning caused by enterotoxins produced organisms do not have to be present but by S. aureus is an intoxication. Medical Microbiology -181 B. ENRICHMENT INFORMATION C PRACTICAL ACTIVITIES . TOPICS AND SUBTOPICS A. ESSENTIALINFORMATION toxins produced by the microorganisms may

,o cause the disease)t. Direct inoculation of microorganisms by 21.24 Diseases Transmitted Inoculation can occur by insect and ani- 7by,Inoculation mal- bites, by puncture wounds, or, morearthropods may be necessary to transmit rarely, by needle and syringe. certain organisms which have no other portal of entry to the host. Other organisms require damaged tissue to become established. 4 .

21.241 Transmission by Arthro- Many arthropOds (insects such as mos- Rock Mountain spotted fever and malaria are examples of diseases transmitted PPds quitoes, ticks, and fleas) may act as vectors by' transmitting infectious agents from oneby arthropods. Control of an` reservoir 6 host to another (animal to human or humanhosts and elimination of arthr 'vectors to humans). help control the spread of certain diseases. The rabies virus iiiitesent in the salivary 21.242 TransinAsion by Animal A small number of diseases are transmis- Bite sible to humans by animal bites. Rabies isglands as well is other tissues of,,infected the classical example. carnivores. The virus in the saliva is able to establish itself only because 'the bite produces abraided t. kr.- If an individual has been previously im- '21.243 Transmission by Pune- Gas gangrene and tetanus are caused by ture prtind. organisms which gain entrance to the bodymunized or is given a prophylatic injection 1.4., f ., through a deep wound. The diseases areof antitoxin, tetanus may be prevented. *"," caused by the exotoxins produced by these anaerobic sporeforrning.Pitreilli.

, :!=1.244 Transmission by Artifi- Although any infectious agent can -be cial Inoculation (Needletransmitted accidentally by a contaminated and Syringe) 'needle puncture, hepatitis B virus (HBV) is

the most- significant. Hospital personnel, 11. homosexuals, and drug addicts are at greatest risk.

'21:3 NosocomialInfections Hospital-acquired infections can be trans- These diseases may be especially severe because of the virulence of the orgfinisins , (Hospital Acquired) mitted to patients by direct contact- with other patients or hospital personnel, by air,and/or the lowered resistance of the patient. 60, food or water, or by contact with contami-Gram-negative rods are a frequent cause of nated forintes (inahirnate objects) such asnosocomial infections. syringes, needles, or bed clothing. Some stlites permit state health depart- 21.4 Quarantine Quarantine is the isolation of individuals with highly contagious disease to minimizements to close ithoolS, theaters, or othefq: orteliminate spread of this disease to others.large gathering.places to avoid ,epidemics.'

NieditalMicrobioloJ 1R3 C. PRACTICAL ACTIVITIES A ND SUBTOPICS A. ESSENTIAL INFORMATION A B. ENRICHMENT INFORMATION f epidemics ca4 be L5 .Immunization Prevention or contro facilitatedby active unization ofstikep- tible populations. Many infectiqus?agents capable of causing epidemics such ,afi polio and measles are now controlled by immunization programs. t e

Medical Microbiology-185 3'a' , 1*. ,

-1 '4j

Seption IlleMicrobial Physiology 14LL ..tte TABLE OF CONTENTS TABLE OF ILLUSTRATIONS ..*. 1.0 WOCHEMICA.i 3 Fig.].Phases ofMicrobial .Growth. 1,1 Small Molecules 3 ' -.inClosed Cullirre 1.2 Macromoliiiifeic;',. " 0.,Fig, 2, Plots.yrf Exponential Growth 1.3 Macromolecular Complexes 7 Fig;3,Complementary Structure of a 1.4 Cellular Composition . 4elive Site and Substrate 2.0 NUTRITION Fig4 Vric,Site and Binding of 35 2.1 Energy Requirements ' - 11 , tte arid Negative Effectors 2.2 Required Elements and Compounds 11 Fig: 5d riz lutes and 4ctivatan Energy 17 39 2.3 Media , 3.0 GROWTH 19 Fig. 6.E1zy10.CoricenitatiortandReaction Rate a 43 3.1 Measurement of Growth 19 Fig. 7. 5ubstr14 Concentration and Reaction Rite ? 43 3.2 Factors Influencing Growth 21 8._pH and;ROction Rate 45 47 3.3 Ctiosed System Growth 25 Fig. 9. edback Inhibition 0 57 3.4OpenSystem Growth 31 Fig. 10. picalElectron TransportSystem 63 4.0 ENZYMES 33 Fig. 11. Glycolysis . 4.1 Definitions 33 Fig. 12.Examples of Fermentative Pathways 65 4.2 Structure 33 Fig.13. Oxidative Decarboxylation of Pyruvate 67 71 4.3 Mechanism 37 Fig. 14. Tricarboxylic Acid Cycle Summary 4.4 Nomenclature 37 Fig. 15.Tricarboxylie Acid Cycle 73 4.5 Methods of Measurement 43 Fig. 16.Cyclic Electron- Flow in Phototrophic Bacteria 79 4.6 Factors Affecting Rate of Enzyme Reactions 43 Fig.17. Noncyclic Electron Flow in Cyanobacteria 4.7 Enzyme Inhibition 45 aridEucaryotic Photosynthesizers 81 4.8 Enzyme Synthesis 47 Fig.18.Hexose Synthesis 85 5.0 METABOLISM 47 Fig. 19. Origin of Carbons and Nitrogens in Purines ' -,.. 47 and Pyrimidines 89. j 5.1 Energy 93 51 Fig. 20.Synthesis of Polysaccharides 5.2 Catabolism 97 5.3 Inorganic CompoundS'as Energy Sources 75 ,. Fig. 21.Proton Translocation and ATP Synthesis 5.4 Light as an Energy Source 75 5.5 Anabolism 77 6.0 MEMBRANE PHENOMENA 91 6.1 Membranes 'and ,Energy 91 6.2 Biological Activities of Membranes 101

C=glirs-r.

11.

t.:1

404

C ..

ra TOPICS AND RUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES '1.0 Biochemicals To understand the nutrition and metabo- For all of this section, a, textbook of bio- For all of Section 1.0, sets of molecular lism of microorganisms, it is necessary tochemistry is advised as a reference. models may be used for three-dimensional have some basic knowledge about the chem- :1 visualization of these compounds. icals that are involved in life processes.

1.1 All living material is assembled from small A niun'Ther of texts in microbiology have organic molecules. chapters giving information about these ,11 compounds.

1.11 ' Sugars &gars have the general formula (CH20).. Sugars are carbohydrates. The name car- Various chemical testtican be used to They are water soluble. bohydrate is derived from the fact that fordetect sugars. The BenediCtilest is in many yr each carbon atom there are usually two hy- introductory biology 190:oratory manuals. drogens and one oxygen (the same ratio as in water).

1.111 Monosaccharides Some important monosaccharides are Glucose, fructose, and galactose are com- the three-carbon sugar glyceraldehydemonly occurring six-carbon sugars. Common 1(C311603), ,various five-carbon sugars (pen- five-carbon monosaccharides are ribose and toses), and a number of six-carbon sugarsdeoxyribose. The differences in the arrange- (hexoses). ment of hydrogen (H) and hydroxyl (OH) groups around carbons in the central chain account for the different six-carbon sugars.

1.112 Disaccharides Dis)'tocharidesarecomposedoftwo Sucrose is composed of one molecule of monosaccharidescovalentlybondedto- glucose and one molecule of fructose; lactose gether. Two common disaccharides are su-has one molecule of glucose and one of ga- crose and lactose. lactose.

Substituted Sugars Substituted (modified) sugars are impor- So'me of the ways that simple sugars can Muramic acid, a modified sugar found tant in biological structures. be modified are removal of hydroxyl grodps, only in procearotic cell walls, has been used e.g., deoxyribose;:addition of nitrogen-con-as a sensitivVindicator of the presence of taining groups, e.g., N-acetylglutosamine;procaryotic organisins in various natural and oxidation of one of the hydroxyl groups,samples4 e.g., gluconic acid. z

Acids Amino acids have an amino 'group (NH2) A basic formula is Some microorganisms synthe: ize all of the ahil a carboxyl (C00$) group. .1'here are amino acids they need, bu rs require p.A some preforMed amino aci feet that ' about 20 amino acids important in protein . structure. These units are building blocks of NH2 some microorganisms re' o acids' proteins. Where R is an organic radical. These -organic, forgrowth has been the bslisquantitative radicalatiVa wide variety of chemicalassays of amino acidrifith ooritasszn ,s" properties. Depending on the R-group prop- (bioassay). la: erty, an amino acid may be acidic, neutral, , or basic, and snore --6 watO soluble. 3 :3 Microbial Physiology 3 TOPICS AND SUBTOPICS A. ESSENTIAL. INFORMATION/ B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES E ninnies of amino acids are: glutamic, R C2114C00-; lysino, R'... C4FINNI-13+; gly- cine, R . CH3.

1.l3 Fatty Acids These are chains of carbon (C) with a Volatile short chain fatty acids, for ex- Growth of some fungi can be inhibited by tached hydrogen ending with a carboxy q(nple acetate (2C), butyrate (3C), and pro- fatty acids. For example, some athlate's foot group (COOH). They range in length frompionate' (4C) are produced by fermenting remedies contain fatty acids. two carbons up to 22 or more carbons. microprganisms as fermentation products. The natural fatty acids on skiii inhibit Longer-chain fatty acids are, synthesizedbacterial growth. by essentially all microorganisms for use in Volatile fatty acids produced by anaer- membrane lipids. obes are useful in identification of these organisms.

1.14 Nucleotides Nucleotides are composed of a nitroge- Nucleotides function as subunits of nu- nous base (often a purine or pyrimidine)cleic acids (Microbial Genetics, Subtopic linked to a five-caricOn sugar (ribose or deox- 2.11). Also some nucleotides, e.g., adenosine

r. yribose) which has one to three phosphatetriphosphate, function as energy carriers groups on it. (Microbial - ]physiology, Subtopic 5.16).

1.2 Macromolecules These are composed of The smallolecules are linked together covalently bonded together inn orderly by enzymaticocesses which eliminate wa- and repeating arrangement. ter (condensation o dehydration reactions). )3 Polysaccharides These are composed of monosaccharides Some biologically important polysaccha- You can demonstrate that starch is made linked together. rides are starch, cellulose, glycogen, and dex-of monosaccharides by adding some saliva transall composed of glucose units linked(which contains the enzyme amylase) to a together in various ways. Other polysaccha-starch solution and testing for monosaccharides are composed of more than one type ortrides released as the starch is broken down. monomer (Microbial Physiology, Subtopic 1.33).

1.22 Proteins These are composed of large numbers- of Peptide bonds are formed by linkage of amino acid linked together according to athe carboxyl (COOH) group of one amino geneticallyetermined pattern (Mier bialacid to the amino (NH2) 'group of4nother Genetics, Subtopic 2.31). The bond betweenamino acid. (Microbial Geneiicii,,tutttopic, 1. each amino acid in the sequence is called a 2.31).; peptide bond. q)

T1.23 Nucleic Acids These are composed of large numbers of (MicrobialGenetics, Subtopics 2.1 and Charts showing the nucleotide composi- nucleotides linked together. If the nucleotide 2.2). tion and the helical structure of nucleic sugar is ribose, the compound is ribonucleic acids, as well as molecular models, are avail- acid (RNA). -If the nucleotide sugar is deox- able. Nucleic acids are negatively charged yribose, the compound.is deoxyribonucleic and stain with basic dyes. If you look at A,acid (DNA). eA\ methylene blue-stained preparations of pro- Microbial PHiioloiy, 5 ) 3" or , up '1'01'108 AND 81111TOPICS A. P,88RIVIAL INFORMATION_ 13, ENIllellIMENT INFORMATION 0, PRACTICAL ACTIVI'I'IRS towns or algae, the nuclei stain blue be: cause of the negative property of the nucleic keid.

1.24 Lipids Lipids are a heterogeneous group of com- Fats and waxes are examples of simple pounds that are insoluble in water but are soluble in organic solvents such as ether. TriglycMdes, storage lipids,are composed PhoSpholipids are important in mem-of three fatty acid molecules linked to glyc- brane structure. erol (a three-carbon compound). Phospbolipids, essential compounds of membranes, contain a non-water-soluble portion ,(often a glycerol substituted with two lo4-chain fatty acids) and a water-soluble portion (a phosphate on the third carbon of glycerol and some charged groups attached to the phosphate). Sterols are more complex lipids found in large amounts in membranes of eucaryotic microorganisms. The procaryotic exceptions are the members of the genus Mycoplasma which incorporate sterols from the medium into their membranes.

1.3 Macromolecular Com- Biotogical structures are specific assem- plexes blagelof macromolecules.

1.31. Lipopolysaccharides These are composed of lipids linked with These compounds are structural compo- and Lipoproteins polysaccharides and lipids linked with pro-nents of bacterial cell envelopes and mem- teins. branes.

1.32 Riboaiime (Ribonucleo- Ribosomes are complexes of RNA and Ribosomes of procaryotic cells can be dif- There are antibiotics, e.g., streptomycin, proteitkomplexes) protein molecules. They are the site of pro- ferentiated from those of eucaryotic cell cy-that selectively inhibit procaryotic cells by tein synthesis. toplasm by sedimentation in an analyticalbinding to 70S ribosomes. There are other A. - centrifuge. The Svedberg unit is a measurechemicals) e.g., cycloheximide, that inhibit of sedimentation velocity. Procaryotic ribo-eucaryotic cell growth by binding specifi- somes.sediment at 70 Svedberg units (70S)cally to 8,0S ribosomes. and eucaryotic ribosofile5 sediment at 80S. Mitochondria and chloroplasts of eucaiyotic cells have 70S ribosomes.

,ro 1.33' Peptidoglycan Peptidoglycan consists of a polysaccha- Peptidoglycan is unique to procaryotic Most textbooks of microbiology have dia- ride backbone composed of two substituted cells. grams of the specific interrelationships of monosaccharides (muramic acid and N-ace- the amino sugars and amino acids under a tylglucosamine). ShOrt chains ofamino acids discussion of bacterial cell walls. There are Microbial Physiology 7

3'7 _v.2 3';'3 lf IV' 111 11041 TOPIC8 AND HIMITOP108 A.PIHNNNTIAI, INFORMATION: . 11, HNIII4111MENT INFORMATION U.1111ACTIOA connect the polysaccharide bliekbontai. to. antibiotics, 414, penicillin, that inhibit the --1-gother to give a rigid layer to the cell wall of formation of the Peptidoglycan cell wall of bacteria. procaryotes while.10,ajur;groWlait,

.;. : 1.34 Membrane Thu outer and inner membrane surfaces Cell membranes have sitesfor active .Some fungal membeaties. centahl large have the waterloluble mule of phospholipidtransport of nutrients into the 'cell. Active amounts of Sterols. Certain chemicals which molecules interapersed iirtith protein mole-transportHyatt/fit allow Ceiletaltrati011 ofnu- react with sterols destabilize the membranes . cules. The middle layer consists of the non-trients inside the cell. There are also en-and thus can be used as antifungal agents. water-soluble fatty Had chains of the phos-zymes that are active in energy metabolismThese chemicala are not active against pro- pholipids. Interposed-are some protein mol-associated with the%membrane. Invagina-caryotic pathogens because these organisms ecules that extend through the membrane. tiOns of the membrane provide more func-generally lack membrane sterols. The inhi- tional membrane surface in many photo- bition of yeast but not bacterial growth by trophic procaryotes (Microbial Physiology, a compound like Nystatin can be demon- Subtopic 6.0). strated.

1.4 Cellular Composition Microbial cells are composed of proteins, Composition of bacterial cells will vary Wet weights and dry weights of a mass of carboh9drates, nucleic acids, lipids, phos-depending upon the kind of bacteria and theconcentrated bacterial cells can be deter- phates, and other materials. Seventy five togrowth medium. Very, rough generalities ofmined to show the water content of the cell. ninety percent of the weight of the microb-composition are as follows: ial cell is vater. Water 75 to 90% Dry weight of: Protein 35 to 50% Carbohydrate 2 to 25% Nucleic acids RNA 6 to 25% DNA 1 to 4% Peptidoglycan Gram positive cells 15 to 20% 46At Gram negative, cells 0.1 to 5% Lipopolysaccharide Gram negative cells 5% Inorganic (Phosphates) 0.9%

2.0 Nutrition Nutrients are those materials taken from' Materials from the environment must be,,, Various media may be, made to demon- the environment from which cells draw en-able to enter the cell to be used as nutrients.'strate the, concepts that follow; care-must be ergy and substances required for syntheses Many organisms make from simple com-taken in media preparation. Some common of cellular components. Because . there ispounds all the amino acids, vitamins, andproblems and ways to avoid them follow. such a great diversity of microbiologicalmetabolic intermediates that they need for Mixtures of inorganic compounds may types, there is a corresponding diversity, ingrowth; others have extensive nutritional re- precipitate. Watch pH and chelating agents. the types of nittrkthts required. quirements. Add only materials which have been dis- Some bacteria, such as th,eltgeittii of lep-.solved in water, and add them in the order rosy and of syphilis, have rieverheqktyolti- sdggested in the formula being used. vated on cell-free media. Many organic compounds interact at high Microbial knyrology 9 3i13 a 3" TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION" B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES An example of the problems involved, intemperatures, i.e., Titaclave ...glucose and obtaining the proper growth medium is theKji2PO4 solutions syPara0Y. difficulty experienced in initial isolation of Many organic colnpoimds are destroyed the agent of Legionnaire's disease. by temperatures attained in the autoclave. FrAser, D. W. and J. E.McDade. 1679.These should be sterilized by filtration. Legionellosis. Sci. Am. 241: 82 -99

ty. 2.1 Energy Requirements

2.11 ChemotroPhs The chemotrophs are a; class of, microor- Some examples or-oxidations of inorganic ganisms which obtain energy by the oxida-materials are the following: tion of chemical compounds or elements. H2S o S042- + energy There are' two kinds of chemotrophs. NH4+ --0 NO2-- + energy Some oxidize inorganic elements or com- H2 H2O + energy pounds, and others oxidize organic (carbon- containing) compounds. Bacteria oxidizingOrganisms that carry out these reactions are inorganic molecules are called lithotrophs or important in the cycling of elements in the chemoautotrophs. Most of these organismsbiosphere. obtain all their carbon from CO2 and thus Some examples of oxidatical of organic are true autotrophs (Microbial Physiology,materials are the following: Subtopic 2.211). glucose. (C6111206) 0 6CO2 + energy Microorganisms oxidizing -organic com- stearate (C18113602) 18CO2 + energy pounds are called organotrophs or chemo- Generally, the less oxygen an organic mole- heterotrophs. These organisms generally use the carbon from their energy source as acule contains, the more energy it will release when oxidized to completion. source of cell carbon so are heterotrophic as well as chemotrophic (Microbial Physiology, Subtopic 2.212).

2.12 Phototrophs Microbial phototrophs are a class of pho- Some examples are eucaryotic algae and The Winogradsky column is an effective tosynthetic organisms which obtain energyprocaryotic blue-green algae(cyanobac-way to demonstrate a variety of microbial from light. teria), photosynthetic bacteria. phototrophs. These bacteria grow under an- Eucaryotic algae carry out photosynthesisaerobic conditions in the light, and popula- by using chlorophyll packaged into chloroPtions having different pigments develop in plasts and evolve 02. Cyanobacteria have,different microhabitats in the column. De-, chlorophyll and evolve 02. The green andvelopment of a column 'takes about four purple bacterial phototrophs do not haveweeks (Carolina Tips, September 1978. Vol. chloroplasts but dr..11 specialized mem- XVI, #9). brane structures containin bacteriochloro- phyll; no 02 is formed.

2.2 Required Elements and The elements C, H, N, 0, P, and S make To determine a requirement of a particu- Compounds up 99% of the dry cell mass and must there- lar microorganism, the growth yield is corn- / Microbial Physiology - I I 3 411b TOPICS AND SUBTOPICS A.'ESSENTIAL INFORMATION. B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES fore' be- sunplied in greatest amounts. De- pared on media with and without the corn-. pending on the synthetic capacity of the .pound. particular .microorganism, these' elements may be su plied as organic or inorganic materials and in their oxidized form or reduced , form.

2.2i Carbon I Carbois required for the "skeleton" of All naturally occurring carbon compound's You can dlemonstrate organisms which all of the# organic molecules. can be used as a source of carbon and energy'utilize a partionlat carbon source by setting . by some microorganism. There are man- up an enrichment-. culture. This involves in- made organic compounds (plastica-...MT).oculating 100 ml of a mineral medium, (such which are decomposed slowly, if at all, by.,as the one in Subtopic 2.211 adjusted to pH microorganisms. Some bacteria can use,7.0) with 1 g of soil or several milliliters of more than 90 different compounds, but otiv'pond. water and adding an appropriate ers are liniited to a few. For example; mem- amount ofd the carbon source of your Choic bers/of the, genus Pseudomonas are noted(0.05. to 1.0%, depending on toxicity). Afte for their versatility, irshereas organisms such 1 week , or ,so transfer about 1 ml of this Leptospira are very restricted in theirculture to a new flask made up the same C.,---"Carbon source utilization. way. A-large proportion of the organisms growing 'in the second flask will be able to utilize the added carbon source. J

2.211 Autotrophs The autotrophi can obtain all of their 'Some examples of photoautotrophs are This mediums will support autotrophic carbon requirements from CO2. plants, eucaryotic _algae, blue-green bacteria growth of sulfur: oxidizing kacteria The car- Photoautotroyhs are those which use light(ayanobacteria), and certain photosynthetic,bon comes from the CO2 in the atMosphere, as an energy source and CO2 as a carbonbacteria. Some examples of chemoauto-and the energy comes from added sulfur: or source. trophs are -1Vitrobacter, Thiobacillus, andthiosulfate. Add powdered sulfur to 1%. Chemoautotrophs use chemical sources ofthe hydrogen-oxidizing bacteria. NitrobacA,(NH4) 2504 .0.4 g energy and CO2 as the source of carbon. ter oxidizes nitrogen ,(NO2-' NO3-), and KH2PO4 4.0 g These organisms o inorganic mate- Thiobacillus oxidizes sulfur (H2S SO4 -2). CaC12 0.25 g rials as a source of ergy and can also be 'MgSO4- 7H20 0.5 g called lithoautotrophs. Fe§04 0.01 g titer C 1,000 ml

2.212 Heterotrophs,, The heterotrophs obtain their carbon re- Photoheterotrophs, e.g., some purplac- In addition to an organic compOund, sup- quirement from organic molecules. teria, use light as their energy source andplemental CO2 may be required by, hetero- organic carbon as their carbon source. trophs; e.g., Neisseria: A candle jar provideS Chemoheterotrophs, e.g., fungi, most bac-,adequate amounts of CO2 for these orga- teria, use organic carbon as both carbon nisms. source and-energy source.

- - 2.22 Nitrogen Nitrogen is found predominately in pro- Many microorganisms obtain organic ni- Free-living nitrogen-fixing bacteria may teins and nucleic acids. It is available in ogen excreting proteolytic enzymes to.be obtained by inoculating nitrogen-free me- 1 Microbial gy 13 Op ) 4.3 0

TOPICS AND SUBTOPICS A. ETNTIAL INFORMATION B. ENRIbHMENT IN8ORMAT1ON 'C. PRACTICAL ACTIVITIES many forms.Atmospiieric N2 may*Ae-decompose proteins. They then taliethe re-,dium with a small amount of soil'and incu- . duced andassimilated by nitrogen -fixing or-leased amino acids into their cells. %bating this culture in the dark for 48 h. ganisms such as Azotobacter. Other orga- Only procaryotic organisms seem to beAzotobpcter is commonly isolated by this nisins can 'utilize NO3 or NH4'. 4Organic able to synthesize the enzynies necessary fortechnique. Rhizobium, anitrogen-fixing nitrogen sources such as amine acids may benitrogen fixation. symbiont; can be observed by crushing the required by others. Brill, W. J. 1977. Biological nitrogen fix- nodules on the roots of legumes and staining ation.-Sci. Am. 236:98;82. the cells released.

Phosphorus Cell phosphorous is found predominately Phosphateisactively transported by In growth media phosphorous is usually in nucleic acids, nucleotides, and phospho-membrane-bound carriers. In modt aquaticlaipPlied at 0.01 to 0.1 M by a buffering lipids. Phosphorous isusually added tosystems, phosphate is a limiting nutrient. mixture of KH2PO4-K214PO4. growth media as an inorganic salt. Addition of phosphate, e.g., phosphate con- ,taMed in cleaning compounds, to' streams may result in increased growth of microorganisms. , 2.24 Other Elements Sulfur, found predominantly in proteins, Bulk cations are provided as salts. K4 is Trace elements may usually be supplied may be obtained from organic sources suchrequired for protein synthesis;Mg2+ is usedin adequate amounts by tap water or as as cysteine or inorganic sources' such asas a cofactor for enzymes and functions incontaminants in various media constituents: SO4-2. maintaining the structure of ribosome& Fe2+ There is sufficient Mo' in most Mg2SO4 to Oxygen for nutritional purposes is pro- is required for synthesis of cytochromes and supply trace needs. vided mainly by4oxygen-containing com-other enzymes and coenzymes. Grovth of pounds. some pathown in vivo may be limited by Cations such as K+, Ca', and Fe'the ability- of host; iron-binding, factors to are required by most cells and usually mustrender this element unavailable to the bac- be added to the medium. Cations such asteria. Ca' contributes to heat resistance of Mn", Co", Cu', Mo2', and Zii" are consid-endOspores. The function of the trace ele- ered as trace elements. ments is difficult to establish. MOst are as- (" sociated with specific enzyme activities, but Co' is a component of vitamin B12 and Mo" is required for nitrogep fixation and NO3- reduction.

2.25 Other Compounds Water makes up 75 to 90% of the weight Water in humid air is available to free-, The amount of available water is ex- of most cells and must be provided. living fungi and bacteria. In the tropics, forpressed as water activity (a,,,). You can pre- Growth factors are those precuisors to cell example, care. must be ..taken to inhibitserve food by decreasing available water constituents thatpecific Microorganismsgrowth on materials normally thought to be either by drying or by adding water-binding are unable to synthize from simple mate-too dry to support grOwth, such as leathercompounds such as sugar or salt. Fungi can rials; they must berovidea 'preformed.and glass. grow at lower water activities than most These are organic molecules required in Vitamins, as defined by human require- other microorganisms. small amounts. ments; are also required for microbial metabolism. Many microorganisms can synthesize- vitamins and thus they may not be required in the medium. Vitamins, amino Microbial Physiology-15 ). 3(-)f its 4t I TOPICS AND SUBTOPICS A, INSHNTIAL INFORMATION. B. HNIIICIIMHNT INFORMATION . C, PRACTICAL ACTIV,ITIRS acids, and other growth factors, any be require(' by some species; If growth factors are required, the 'amount of groWth that results \ is directly proporilOQUI to the amount of growth factor added to the Medium. This is 'the basis for many quantitative bioassays. Some bacteria, yeasts, and filamentous I fungi will-synthesize ()teem vitamins. These microorganisms are used for commeklal ploduction of these,substances.

Media Media are cor4osed Of compounds that IA general purpose medium,provides con- Agar, used mdtt frequently as a solidifying are required for growth of microorganisms. ditions that will support growth of a varietyagent at 1.5 to 2.0% for media, is a polysac- No one mediuin will support the require- of Microorganisms, e.g., TrypticauSoy agarcharide obtained from seaweed. It is not ments of all m oorgarusms. (BBL Microbiological Systems). metabolized by most bacterid. Aqueous 2.0% Isolation andowth of any organism are An mulched medium is one to which spe-suspensions melt at 100°C and gel at 45°C. dependent on thBe of an appropriate me-cial nutrients have been ack.ded to enhance Silica gel which has no organic impurities dium. the growth of a specificcroorganfbm, e.g.,may be used as a etaidifying agent when agar' blood agar. is either toxic or metabolized. A differential me ium contains compo- Extracts or enzymic 'digests of plant or nents which permit presumptive identifica-animal tissue provide a rich supply of nutri- tionertain classes of microorganismsents. Examples are tryptone, peptone, and based On their biolOgIcal differences, e.g.,yeast extracts. lactose-fermenting colonies turn pink on Most media sold commercially, are com- McConkey agar. plex; however, there,are defined or synthetic Selective media are media which prefer- media also available. lentially permit the growth of a certain de- siredmicroorganism.Selection may be achieved by addition of inhibitors or by ad- justing nutrient content to optimize groirth of the desired organism, e.g., mannitol-salt agar selectively permits growth of staphy. loocci. The use of media to 'enhance growth of specific physiological types of mi roprga- riisms is comMonly calledrenrichm nt culture Defined or s etic media are those in wlch all the comets are known in kind and aniunt.

, Co a 1,1 4 m is are those which contain pjhnt extracts of variable compo- sitiotty

Microbial Physiology-13; 3(3 C. PRACTICAL ACTIVITI10.1 TOPIc',H AND ti1111TOPICH 'A, lalSIIINTIAL INFORMATION IL IINRICJIMHNTANFORMATIONs 3.0 Growth 'Growth Is the orderly increase it cellular In organisms that reproduce by binary constituents, Normal growth leads to cell 0,11,, meal; Istatiria, growth yields hi. reproduction, Growth of miaroorganismscreased numbers, The organisms tint Are leads to an increase in the population. ' coon cytio increase the size of the individual ratio r than its minima, Montero of metabolism and growth can lead to situations in which Ito normal growth and reproduction cycle is aborted and unbalanced ,groJyth re- 4 sults.

3.1 Measurement of It is possible to'measuro various aspects Growth of microbial growth. One chooses the growth.' measurement technique needed depending on whether the emphasis is on total numbers, viable organisms, or population metabolic rate. Usually several techniques are used, and the results are compared. A sample of washed cells may be dried in 3.11/ Increase in Mass Meaurements of increase in cell mass Nephelometry and specUophotomeCry are emphasize growth rather thanteproduction.the most convenient and generally used the oven, and dry weight can be determined. They may not distinguish living from dead.growth-measuring techniques for bacteria. A sample of washed cells may be analyzed cells. The increase in turbidity of the culture isto determine the total nitrogen or total pro- measured. tein. Spectrophotometers measure the propor- Cellsin suspension scatter light;the 4ion of light which is transmitted through aamount of light scatteredproportional to filled cuvette placed in the light beam.the mass, of cells per milliliter of fluid. A Nephelometers (more sensitive) measurespectrophotometer or nephelometer can be the proportion of light deflected under theused to measure scattered light. same conditions. The direct observation of a gained smear 3.12 Increase in Numbers Cell numbers can be measured by direct 'Direct counts may be done to assist in counting with a microscope. Culture tech-standardizing other methods for routine use. of a known volume of a specimen spread niques which test the ability of each orga-They are also useful for quantitating un-over a known area on slide can be used to nism present to grow to a vidble mass or tousual organisms in natural samples or hard-calculate numbers of organisms per milli- metabolize and produce a detectable prod-to-cultivate species. Automated electronicliter. The Petroff-Hauser counting chamber uct are also used (viable counts). Theservirticle counters may be used. is used for direct counts of bacteria and methods are useful only for unicellular or- Viable counts are much used in studies ofother small cellular organisms, and the ganisms. water samples, urinalysis, and food qualitycount reflects both living and dead micro- determination. A diluted sample is spread organisms. on a solid or mixed into a melted agar me- Use the plate count to determine the num- dium. If this is done with medium in a petriber of CFU in a sample under several nutri- plate, it is called a plate count. Each viableent and physical conditions. organism may grow into a visible mass of Microbial Physiology i9 3 j 39n '1.11P111ti ANI) tillIIT0141(11 A, 11,1114NTIAL 'INFORMATION IIINRICIIMFINT INFORMATION PRACTICAL ACTIVITIblii ceils (a colony). The manlier of colonies 141 er incubation of this medium copresenia the number of viable bacteria in thediluted ti1111100, M11%11110 it in (Wilma to Know if one bacterium or several attached bacteria gave rise to (me colony the number is tumidly given as colony-forming units (CFLI).

3,2 Factors Influencing Microorganisms 111'0 1141tel'OgOilo011ti..in When microorganisms lire not growing be- Growth theirphysical andchemical requkernOnts, cause of inailoquaie conditions, they are not Optimum groWth of curs only when all re- necessarily dead, (mug periods of metabolic quirements are. met. inactivity nmy be tolerated without loss of

3.21 Nutrition Growth pattornAdiffer depending on (Microbial Physiology, Subtopics 3.3 and whether nutrient supply is continuous (open 3.4) system) or discontinuous (closed systent). Most natural situations are open; most laboratory conditions are closed. a

3.211 Open Versus Closed Microorganisms contain regulatory mech- In natural situations such as bodies of Systems of Microbial anisms which alloy/. them to modulate water, organisms receive a continuous if fluc- Growth growth for survival under changing nutrienttuating supply of nutrients. Wastes are also availability. removed. Growth speeds up and slows down, rarely stops. In the test tube, when the 'nutrient is consumed, gtowth stops.

3.22 Physical and Chemical ,Influences on Growth

3.221 Gaseous Atmosphere Microbial growth is affected by the con- Obligate anaerobes may be recovered Aerobes and facultattve anaerobes must ,') centrations of 02, CO2, and other gases infrom conditions in nature which appear tohave an 02 solace for best growth. In liquid the surrounding medium. Most higheNforrns be. aerobic. These organisms grow in mi-culture best yields are obtained with 'small ,of life require 02; many microorganisms docroenvironments that are anaerobic. Bacte- volumes of fluid in large vessels on shakers. not. Obligate aerobes require 02 for growth. roicte8, an obligate anaerobe, can be isolatedAnaerobic growth conditions can be ;pros Their metabolism is respiratory. Facultativefrom the mouth. It inhabits anaerobic crev-vided in sealed jars with catalytic oxygen anaerobes grow either with 02 or without it.ices around the teeth. Many anaerobes areremoval or inanaerobic incubators. Thiogly- Growth is usually better in the presence ofkilled by oxygen. Isolation from natural hob-collate, chopped meat medium, and media 02. These orga 'sms are usually ferments-/ itats (anaerobic mud and soil, the guts ofcovered with mineral oil may be used to tive in the abs/7 nce of 02. Obligate anaerobesanimals, wounds, and absesses) requires spe-provide anaerobic conditions. Fligh-0O2, do not grow in the presence of oxygen. Mi-cial sampling techniques, transport condi-low-02 atmosphere can be provided in a croaerophiles require reduced 02 concentra-tions, and prereduced culture media. candle jar by combustion pr in specially require, tion and may increased CO2 concen-. designed incubators. 3 Microbial Physiology 21 3;)? TOPICS 'AND SUBTOPICS A, ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION, C. PRACTICAL ACTIVITIES tration. Kerotolerant anaerobes are orga- The catalase and oxidaie tests may be nisms livinE totally by fermentation in the useful in identification of bacteria. presence orair. They are not sensitive to 02. E. H. Lennette, A. Balovis!t_W. J. Haus- ler, Jr., and J._P. Truant (ed:):: 1980. Man- ual of clinical tnicrobiology,, 3rd ed. Amen- can Society for Microbiology, Washington, D.C. - For most organisms the optimum pH for Extreme pH occurs in some natural envi- The complex peptones, cell extracts, and growth lies between 6.0 and 7.5. Some highly ronments'. Drainage from mining operations, other protein derivatives in most complex specialized organisms can tolerate a pH as high mineral areas, and certain fermentationmedia exert a strong buffering effect. Note low as 1.0 or as high as 10.4. The internal conditions yield high-acidic environments.pH information on media labels. A variety pH of most microorganisms is close to neu- The inland salt or alk,ali lakes may have a of inorganic buffers may be added to defined trality regardless of the pH of the medium. pH as high as 11. media. Microbial metabolism produces acidic or Thiobacillus is an, autotroph which pro- Detection of pH change in media is usu- basic waste products. These may alter theduces H2S0, and lowers pH of medium toally done with NO indicators, e.g., phenol environmental pH to a point where growth 1.0 or below before growth stops. Fermen- red is yellow below pH,,,6.8, and reilbove,,,, stops.Laboratorymediaareusually tative organisms such as those used to make6.8; methyl red is tied below pH 4.5 and buffered to reduce this effect. yogurt or sauerkraut lower pH by produc-yellow above 4.5. fitaroni6 pH meters can tion of organic acids. These acids eventuallybe used for exact readinginin liquid media, arrest growth. They preserve food by pre- Selective media for the isolation of fungi venting growth of less acid-tolerant orga-usually have a pH beloiG.0, whereas most nisms that would spoil foods. general-purpose bacteriological media have Many important biochemical tests used ina pH of 6.8 to 7.4. identification of bacteria depend on pH indicators to detect production of acidic or basic end products.

3.223 Temperature A given strain of microorganism will have Microorganisms grow over their entire Soirtir water samples may be plated and anoptimumtemperatureforgrowth. growth temperature range. At suboptimal incubated at different temperatures to dem- Growth will occur between its minimum temperatures their generation time is ex- onstrate presence of all three types of orga- temperature and its maximum temperature. tended from minutes to hours. Above the nisms. Many psychrophiles are killed by ex- These points are genetically determined. optimum, growth slows as the microorgan- posure to room temperature, so precautions The effect of temperature on enzyme ac- ism diverts'ever-increasing amounts of cel-must be taken to keep samples and media tivity (Microbial Physiology, Subtopic 4.53) lular energy to repair heat-induced damage. cold. is important in determining the range over The upper cut-off point is usually quite which an organism will grow. There are sharp. Therrnophilic procaryotes can with- three groups of microorganisms with differ.- stand higher temperatures than eucaryotic ent ranges of growth temperatures: psychro- thermophiles. The hottest inhabited ther- philes, mesophiles, and thermophiles. mal springs contain only procaryotes. Ther- One commonly used definition of psychro- mus aquaticus, a therrnophilic bacterium, is philes is that they have a growth range found growing in hot water heaters as well somewhere between 5 and 20°C. Meso- as in hot springs.

Microbial Physiology-23 3.'4a 3 4J 3 S AND SUBTOPICS A. ESSENTIAL INFORMATION B.. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES philes grow between 20 and 45°C. Thermo- philes grow between 45 and 90°C. 4 Osmotic Pressure 'Osmotic pressure is determined by the All types of dissolved and colloidal parti- Mannitol salt agar may be used for the concentration of dissolved particles in water.cles contribute to the osmotic pressure of aselective isolation of Staphylococcus from Microorganisms are round in environmentssolution. Cytoplasm haVa relatively highhuman clinical samples because its high of varying osmotic pressures. Most micro-osmotic pressure. The, rcell can vary this(7.5%) NaCl concentration inhibiti almost organisms with the exception of pintozoanswithirtlimits by concentrating or excretingall other nasopharyngeal organisms. The os- are protected from lysis in dilute environ-K+. When the external environment con-motic pressure of media for isolation of cell ments by a rigid cell wall. tains more dissolved material than the cy-wall-deficient organisms, e ,,Mycoplasm, Exposure to high osmotic pressure causestoplasm, it is hypertonic,and the cell will must be carefully adjusted. loss of water from cells. This arrests growth. tend to lose water to the environment. This The halophilic organisms live in highly sa-drying effect arrests metabolism in most or- line environments (15 to 30% dissolvedganIsms; the effect explains the preservative salts). They have a specialized membranevalue of salting or sugaring food. Dilute en- and salt-tolerant enzymes. vironinents pose a challenge to bacterial survival. When the exterior environment is hypotonic, water tends to enter the 'cell. Ex- pansion and death are prevented by the rigid cell wall. Cell wall-deficient forms of bacteria ents. Ex- -c. survive only in isotonic environ amples are the Llorms and inyvo lasms.

Closed System Growth Within a closed system the amounts of All laboratory cultivat115n microorga- nutrients and their energy content are fixed. 'nisms .except continuous culture follows this Growth produces waste products which ac- model/. The phases of growth may be dem- cumulate and may be toxic. Space for by inoculating fresh, prewarmedonstrated growth or attachment to substrate becomes Log no medium with an inoculum of log-phase cells limiting. of bacteria and following the increase in medium turbidity. Any other accurate measurement of cell components may be used. By taking simultaneous samples for plate count and direct count, a good approximation of actual Time numbers may be obtained for each reading.

FIG I Phases of microbial growth tn closed vulture

Lag Phase Introduction of a microorganism that di- Each cell in the innoculum needs to carry The duration of lag phase' is extended if vides by binary fission into a sterile closedout internal regulatory shifts to start growth.the inoculum is from an old culture, if the system such as a flask of nutrient brothAccumulation of cytoplasmic componentsnew medium is quite different in chemical starts growth. For a while there is no appre- and increase in cell size betin. During lagcomposition from the old, or if it is at refrig- ciable increase in numbers. phase, new proteins may need to be made. erator temperature. During lag phase, mea- A complete round of DNA replication willsurement of cell numbers shows no change. precede the first and every subsequent cell Some cell constituents may increase. diVision. Microbial Physiology 25 3 ) 3(1 I' \ )441 C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS 6. FSENTIAL INFORMATION B. ENRICHMENT INFORMATION During log-phase'growth, readirigii should 3.32 Log Phase (Exponential Once growth begins, increase in numbers Growth) is exponential for a period of time. Thebe made frequently because turbidity cap culture will commence 'division at the max- double in as little 4--20 to 30 min for some imum rate possible given the genetic poten- Growth'cluive data may be plotted tial of the culture and the suitability of theon linear graph paper with time as the ab- medium. scissa and the log of the turbidity or the log of the number of organisms as the ordinate.

It is more convenient to use semilog pi 1 the cell number or turbidity is plotted on II the log axis, and the time is plotted on the ,j linear axis. Both of these plotting methods will produce a straight line if the culture is in exponential growth (Fig. 2). During exponential growth there i* a Explanation of Expo- For most microorganisms one cell divides Allpulations grow or decrease exponen- 3.321 straight-line relationship_between the log of nential Growth into two, Thus, during each generation time tiallyeach change reflects addition or sub- the population doubles. Starting with onetractioof some 'constant percentage of thecell number versus time. , bacterium, you can illustrate ekponentialtotal individuals present at that time. When data are plpttecl, the generatioh growth with the geometric progression: If exponential growth proceeded for 48 h,time can be estimated bj determining the time needed for a do'iibiing of numbers or 1,2, 4,8,ffi32 ... or 2°,2',22,23, 24, one bacterium which divided every 20 niln would yield a quantity of bacterteighingcell mass. Specific data points may be taken 4,000 times the weight of the earth. Com .the straight-line portion of the' plot to use in a more precise calculation of genera- , tion time. --

Calculate generation time as follows: 0.322 'Generation Time The generation time observed is the av- Generation times for some bacteria may erage time needed for one cell to complete.rbe as short as 12 min. Thus, bacteria can t, to G a round of division into two cells. In thismultiply extremely ..rapidly under optimum n time the population in the closed system will°conditions. geration. times for other bac- double. The total amount of each cell con-ieria and ingny eucaryotic microorganisms \or stituent will double. may be as long as several days. ti,ta (3.3)loglobi log,obo = doubling time or generation time = time at first measurement t, = time at second measurement bo = number of cells at to b, = number of cells at t, n = number of generations

Nutrient limitation is they - .major restric- The stationary phase may persist for a 3.33 Stationary Phase Environmental limitations put an end to exponential growth. The growth curve ap- tion for most aerobic organisms. Accumula- long period. Readings should be less fre- proaches a horizontal line. During the sta-tion of toxic wastes is the most frequent quent. tionary phase, some cells grow and divide. cause of growth arrest in anaerobic culture. Microbial Physiology 27 ,

3LI3 a

8.0 10 9 7.9 8 0 log 8 x 10' r - 7 "27.8 7.903 6 7.7 - 5 Data 7.6 log 4 X 10' 4 V Log,o no, Time No. of organisms ==, 7.602 of bacteria/ml 7.5 - 3 0 1 x 101/m1 7.4 - No. of bacteria 20 2 x 107/m1 olog 2 x 10' =7.301 2 7:3 - PLOT B 40 4 x 107/m1 7.2 - gen. time 60. 8 x 107/m1 PLOT A 7.1 - (all x107)/m1 1 7.0 I Time 10 20 30 40 50 60 Time 10 20 30.E 40 50 60 (minutes) (minutes) Via. 2. Plots of exponential growth.

3 3 Microbial Physiology-29 a

'OPICS AND SUBTOPICS 'A. ESSINTIAL INFORMATION ;ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES Somfkare active, others dig, and the popu ! , !edgy number is unchanged., The decline is usually exponential. Small 3.34 Death Phase A decline in number of viable cells Death phase- resultsjn a reduction in. via- bites death phase. ble cell numbers. It may or may not have anumbers of viable cells may persist indefi- . corresponding decline in total cell numbers. nitely at the end of the death phase. ' ' , :

3.341 Mechatiisni Acute nutrient limitation or high concen- When cent lose viability without lysing, trations of toxic waste Products-may triggerthis loss can usually be attributed to inabil- activity of autolydc Ifizymes causing cellity to continue supplying the energy to re- lysis. painkey genetic structures. When autolysis occurs, it is usually related to low cellular adenosine triphosphate (ATP) levels, signaling activation of intracellular lysozyme. Clearing of the culture may be quite rapid if autolytic mechanisms are set in motion. There may be no viable survivors. a 3.4 Open System Growth Open system growth occurs when constant environmental conditions are maintained. Nutrients are continuously provided, and wastes are removed. The number of cells per alit volume is kept constant. 0 Grgwth occurs in a growth chamberyro- 3.41 Conditions Laboratory devices for producing open The grqwth rate of the bacteria in the system growth provide a source of cells invessel adjusts to the rate at which thenutri-vided with a resetvoir of sterile medium that the log phase of growth. They allow studyents are provided.'After a period of adjust- is added at,a steady rate. Culture fluid leaves of a culture under optimum physiologicalment, the rate of increase of the cells`the chamber at the Bathe time. Aeration and conditions. I through growth will just equal the rate ofmixing are-also provided. cell loss. The cells in a contintibus culture apparatus are in lug phfise. The doubling time is determined by the grbwth conditions. If all else is constant, growth rate depends directly On the concentration of a limiting -nutritional factor. In a chemostat, the flow rate is set at a 3.42 Contijous Culture These are two types °lain- tinuous culture Continuous culturyfftems provide a con- Devices devices, chemostats and turbidostats. Theyvenient con source of log-phase cells for.certain value. The rate of growth of the culture adjusts to the rate at which nutrients '4. are used for study of all activities of log-study. T y are used for research on mech- phase cells. anisms f regulation, concentration of nutri-are added. In a turbidostat, an electronic ents,lection of growth rate mutants, anddevice monitors turbidity, i.e., cell mass, of inter ctions among species in mixed cultures the fluid and electronically signals the ad- andconditions which stimulate naturaldition of fresh medium to maintain the de- envu- nments. sired populatiOn density. Microbial Physiology 31

401 4 k. A. ESSENTIAL INFORMAION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES B TOPICS AND SUBTOPICS 4.0 Enzymes a 4.1 Definitions Starch + Water Glucose 4.11 Catalyst A catalyst is a substance whic affetts the Catalysts are, used in a number ot reac- rate of a chemical* reaction with ut beingtions of everyday practical interest, and in- The above reaction will proceed at a very permanently altered itself. dustrial importance. For example, platinumlow rate in warm water. Upon addition of a catalytically enhances the oxidation of un-small quantity of amylase, accelerated glu- burned hydrocarbons in automotive exhaustcoseproduction (and starch hydrolysis) is (catalytic converters). Also, the industrialnoted. After a time, the enzyme can be iso- extraction of apple juice from apple pulp is lated from the system with its original activ- aided by treatment with pectin-hydrolyzing ity essentially undiminished. enzymes (pectinases). The fact'that enzymes are protein can be 4.12 Enzymes An enzyme is a highly specific catalyst The sOcificity of enzymes extends even produced by 2 living cell. Enzymes have a to the ability to "recognize" specific isomeric demonstrated by their reaction with protein- protein component. They may or may notforms of a potential substrate. For example,detecting reagents such as Millon reagent or have other components such as metal ions,a particular lactate dehydrogenase may dis-the Folin phenol reagent. Enzyme solutions vitamin, or carbohydrate molecules. criminate between ill-lactic acid and L-lacticwill also exhibit maximum' ultraviolet ab- acid. The only structural difference is thesorbarice at 280 nm, which is characteristic relative position of "H" and "OH" groups onof protein solutions. the second carbon of this three-carbon compound.

4.13 Substrate. A substrate is a substance which isaltered in an enzyme-catalyzed reaction.

4.2 Structure Some enzymes consist only of protein.

4.21 Holoenzyme A holoenzyme is the functional form of an enzyme. Some holoenzymesconsistof apoenzymes, coenzyme, or an inorganic factor.

4.22 Apoenzymes The apoenzyme isOhe specific protein portion.

4.23 Coenzymes Coenzymes are vitamin derivatives which Coenzymes will often act as donors or serve as carriers in enzyme reactions. acceptors of electron pairs or functional groups. In the oxidation of malic acid to oxaloacetic acid in the tricarboxylic acid cycle,nicotinamideadeninedinucleotide (NAD) which is derived from the vitamin niacin, acts as an electron and proton acceptor. Pyridoxamine phosphate (derived from Microbial Physiology-33 4 3 4 TOPICS AND SUBTOPICS --A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION , C. PRACTICAL ACTIVITIES the vitamin pyridoxal phosphate) acts as an amino group donor in tiansamination reac- - tions. Other commonly encountered coenzymes include; flavine adenine dinucleotide (FAD, derived from riboflavin); nicotin- amidecdeninedinueleotidephrAphate (NADP,derived from niacin); coenzyme A (CoA, derived from iiantothenic acid); and thiamine pyrophosphate (TPP, derived from thiamine). Many microorganisms can synthesize all of these cofactors.

4.24 Inorganic Cofactors An inorganic cofactor.is a metal ion, such In some enzymes the metal ion may serve as Mg' or Fe', which is required by aas an important part of the catalytic,site. In specific enzyme for catalytic activity. , other reactions the metal ion is involved in Potential Substrates substrate binding or in maintaining the ac: tive conformational form of the enzyme.

4.25 Active Site An active site is a small area on the en- Tilt active site can be pictured as a structure which matches up with a specific com- zyme's surface which binds in a highly spe- A Will not bind cific manner to the substrate. It is also calledplementary conformation in the substrate in Will Bind the catalytic site. a ;'lock and key" sort of arrangement (Fig. 3). Fin. 3 Complementary structure of active site and substrate

- 4.26 Allosteric Site An allosteric site is a region on the enzyme The allosteric site can be pictured as Allosteric Site surface, apart from the active site, where ashown in Fig. 4. Note that the active site will regulatory substance may bind and affect"fit" the substrate but not the allosteric the affinity of the enzyme for the substrate.effector. The effector, if bound, results in a modification of the three-dimensional structure of the apoenzyme. This, in turn, modi- fies the structure of the active site. Active Site (3-- Alternate Enzyme Conformations

Substrate Effector A Effector B I I If effector A is present, enzyme will be in a favorable conformation for interaction with the substrate. If effector B is present, substrate bind. ing will be reduced because this effector stabilizes conformation B which cannot bind substrate. FIG. 4. Allosteric site and binding of positive and negative effectors. Microbial Physiology-35 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 4.3 Mechanisms

4.31. ActiVationEneru The minimum energy required by a chemical system in order to react is referred to as the activation energy. Enzymes work by lowering the reqUired activation energy, thus facilitating the reaction (Fig. 5).

4.32 Enzyme Substrate A substrate (S) binds temporarily to the

Complex _ active site of an enzyme (E) to form an enzyme - substrate' complex (ES). The ES dissociates to yield product (P) and enzyme (E), which is free to catalyze another reaction.

4.33 Equilibrium Under optimal conditions the concentra-/ Once equilibrium js established, constant Equilibrium can be demonstrated with a tions of enzyme, substrate, and product in aconcentrations of su I'Vfrate and enzyme are hypothetical system consisting of radioac- system reach equilibrium. This involves thepresent in the system unless the equilibrium tively labeled substrate and unlabeled prod- simultaneous conversion of some molecules is disrupted in some manner. In fact, someuct. of substrate into product and product intosubstrate is continuously being converted to A' B substrate. product and some product is converted to substrate. Thus, although the concentra-Initially, all the label is in the substrate. tions remain constant, the individual mole- Eventually however, the following will be cules are in a continuous state of interaction.observed: In biological systems, utilization or re- A' B* moval of products can keep reactions fro In this second instance, the label is distrib- coming to equilibrium. uted between the substrate "A" and the product "B". Yet,.the actual concentrations of A and B could be the same as in the initial example.

4.4 Nomenclature Enzymes are usually named according to Enzymes. have common names estab- the type of reaction catalyzed; they oftenlished by long usage as well as official names have the common suffix -ase. and numberp designated by the Enzyme Commission. For example, invertase, the enzyme that hydrolyses sucrose to fructose and glucose is officially called /3-D-fructofurano- side fructohydrolase (EC 3.2.1.26).

Microbial Physiology-37 4.3 Non-Enzyme Catalyzed

Sub rate molecules are visualized as balls near t,he top of a hill. If they could roll down the hill, they would have tremendous kinetic energy. However, they are trapped in an "energy depression." To escape this requires a "push" of activation energy.

Energy Product

Progress of Reaction

Fm. 5. Enzymes and activation energy of reactions.

Microbial Physiology-39 v TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 4.41 Oxidoreductases Enzymes of this 'type catalyze the inter- Generally electrons are transferred with molecular transfer of electrons. protons. NAD+ and FAD are common proton acceptors in oxidoreductase reactions and,intheirreducedform (NADH, FADH2), are proton donors. An example of an oxidoreductase reaction is the interconversion of lactate and pyruvate by lactate dehydrogenase.

4.42 Transferases Enzymes of this type catalyze the inter- The conversion of a-ketoglutarate. to glu- molecular transfer of functional groups. tamic acid is catalyzed by a transaminase which moves an amino group from aspartate to a-ketoglutarate.

4.43 Hydro lases Enzymes of this type catalyze hydrolytic A hydrolytic reaction is the breaking of a reactions. covalent bond by the addition of one molecule of water. Preliminary digestion of food molecules is accomplished by exoenzymes of this type. 4.44 Lyases Some enzymes of this type catalyze the An example of the first type of lyase re- 1 addition of groups of atoms across a doubleaction is the addition of water across the bond. Other enzymes of this group catalyzedouble bond of fumarate to form malate. cleavage of CC, C-0, or CN bonds byThis is catalyzed by fumarase one of the elimination reactions leaving double bonds. tricarboxyclic acid cycle enzymes. Aldolase is an example of lyase able to cleave CC bonds.

4.45 Isomerases Enzymes of this type catalyze the- inter- This is demonstrable with two cultures, conversion of isomers. one of which is genetically able to produce galactose isomerase, whereas the other is not. In a medium containing galactose as the sole carbohydrate, the organism which produces the enzyme will exhibit evidence of galactose utilization (such as gas production in a fermentation tube), whereas the other organism will not. Such reactions are of significance in the identification of unknown microorganisms.

4.46 Ligases Enzymes of this type catalyze energy-re- Assembly of macromolecules from mono- quiring reactions involved in biosynthesis. mers typifies ligase action. Nonphotosyn- thetic CO2 fixation catalyzed by pyruvate carboxylase is another example. CO2 ± Pyruvate + Energy ---o Oxaloacetate 412 4 Microbial Physiology 41 or

TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C, PRACTICAL ACTIVITIES 4.5 Methods of Measure- Rates of reactions may be measured in Enzyme activity is commonly expressed A variety of techniques are possible: .. merit terms of disappearance of substrate or ap-either as a "turnover number," the number Spectrophotometry: Frequently, it is pos- pearanco of product with time. of molecules of substrate converted to prod= sible tb observe enzyme activity in terms of uct per unit time, or as specific activity,the production, with time, of materials with which is the number of molecules of sub.:characteristic absorption spectra. For ex- strate converted to product per unit timeample, polygalacturonic acid lyase, an en- IT? per milligram of protein. zyme involved in the degradation of pectin,

tv produces a product which absorbs maxi- mally at 235 nm. Photometry; Firefly extract (luciferin-lu- ciferase) emits light in the presence of ATP. The rate of ATP utilization in a ligase reaction can be quantitated by measuring the light emitted by an enzyme-substrate system Reaction at various intervals. Product generation or Rate substrate utilization can also be detected by a variety of chromatographic and radioiso- tope techniques.

4.6 Factors Affecting Rate The most important factors affecting the of Enzyme ReacOna rate of enzyme reactions are: enzyme con- I I I I I I centration, substrate concentration, temper- Enzyme Concn ature, and pH. Within limits, a linear relationship exists FIG. 6. Enzyme concentration and reaction 4.61 Enzyme Concentration rate. between enzyme concentration and rate of reaction (Fig. 6). Even sally, however, all available substrate molecules are bound to 'enzymes; thus, an additional increase in en- Reaction zyme concentration will not cause an addi- Rate ;tional increase in the reaction rate.

4.62 Substrate Concentrationw. As with enzyme concentration, a linear relationship exists between substrate concentration and the rate of reaction (Fig. 7). Siinilarty, a point is reached where all active Substrate Concn sites are bound to substrate molecules. Fur- ther additionaof substrate beyond this point do not further increase the rate of reaction. FIG. 7. Substrate concentration and reaction rate. 4.63 Temperature Each enzyme has a specific 'optimum tem- Generally, for each 10°C rise (up to the perature. Starting below the optimum, rais-optimum temperature), a doubling of en- ing the temperatUre increases the reactionzyme activity is observed. At temperatures rate until the optimum is reached. Further-above the optimum, irreversible modifica- Microbial Physiology-43 4 1 3 4' C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS . -A, ES91 1NTIAL INFORMATION B."ENRICIIMENT INFORMATION more, heating'`Will load to thermal inactiva- Mons of the three-dimensional structure of tion of the enzymes, the enzymes (denaturation) will occur, lead-, sl'Tsro' r',1.' ing to a decrease in enzyme activity.

4.64 pH Ed'ph enzyme had' a specific optimum pH. The optimal pH for most microbial en- De./pions (above or below) from this in thezymes is between 6 and 8. arit9n result in reduced rates of reaction as ot: (Fig. I),

4,7 Enzyme 'I,hibition (Mic 1 Genetics Subtopic 6.1),

4.71 . Comp eive Inhibition InhibiIs o s which are structurally similar Consider the following structures: to the trate compete with the substrate I molecules for enzyme-active sites. As the ratio of inhibitor to substrate is increased, a concomitant decrease in product formation occurs. Competitive inhibition can be re- versed by increasing the ratio of substrate to inhibitor. NR2 NH2

(PABA) Sulfanilamide para-aminobenzoic acid Some, microorganisms utilize para-aminobenzoic acid (PABA) as a substrate in the synthesis of folic acid, a material required for metabolism. The enzyme which normally bindS PABA at its active 'site will also bind Reaction sulfanilamide. Therefore, if the: ratio of sul- Rate fanilamide to PABA is high, the probability of the enzyme reacting with sulfanilamide rather than with its normal substrate is also high. The result is a decrease of folic acid 6 , pH synthesis and suppression of metabolism. Such inhibition is based upon the similarity Fla. 8. pH and reaction rate. in structure of normal:substrate and inhibitor. Sulfanilamide does not effect the supply of folic acid- to mammalian cells because they cannot convert PABA to folic acid and thus require folic acid preformed. s NoncompetitiveInhibi- Noncompetitive inhibitors bind at a site An example of noncompetitive inhibition tion, on the enzyme oiher than the active site. is the action of heavy metals (He'; Ag*, and V Microbial Physiology-45 4.16

I '1'01'108 ANI) HUBTOPICH A. 1048ENTIAL. IN B, ENRICHMENT INFORMATION 0, PRACTICAL ACTIVITIEN Since the active site Is not directly affected, othura) on the sulthydyi groups of enzymes, increases in the ratio of enbotrtite to inhibitorThis may, In part, explain the inhibition of cannot reverse the inhibition, growth Of some microorganisms by heavy metals.

4 4,721. Feedback Inhibition Feedback inhibiti9n is a special case of An excess of "F" interacts with the alio= noMsmpetitive inhibition. When the endsteno situ of the enzyme catalyzing the con- riiotItiet of a metabolic pathway is present inverak n of A to H. Since production of B is excess, it may inhibit the action of one of theblocked IciP will not be produced. early enzymes in the pathway; This inhibition involves the allosteric site (Microbial A - C E 4F ) Genetics, Subtopic 6.2). _ Inhibitor Mo.9. Feedback inhibition.

.4.8 Enzyme Synthesis (Microbial Genetics, Subtopic 6.1)

5.0 Metabolism Metabolism is the sum of all the chemical In general, microbial metabolism is quite reactions in the cell. similar to the chemical reactions that occur in higher animals and plants. .. However, upon examination of individual microorganisms there is a marked diversity of metabolic pathways and end products. This diversity is useful in identifying bacteria and fungi. Many metabolic pathways known to be similar in all organisms were first worked out by investigators with microorganisms. Escherichia coli and Saccharo- myces have been particularly useful in metabolic research.

5.1 Energy Energy is the ability to do work. Microbial movement, growth, and reproduction occur only if there is an energy source. The ultimate source of most biological energy is the sun. A possible exception to this is the deep-sea volcanic oases system

4 where the driving energy is the oxidation of H2S by bacteria. Ballard, R. D., and J. F. Grassle. 1979. Return to oases of the deep. Natl. Geogr. 156:689-705.

Forms of Energy Energy can exist as light or other kinds of The form of energy required directly for 5.11 4_13 1 h siology-47 S.

TOt'I('H A NI) MlIIIT0101IN A, ESSENTIAL INFORMATION 1111 N11101IMENT INFORMATION 0, PRACTICAL ACTIVITIES radiation etiergy, chemical energy, meeltan- nthaloiSilluevellaillt, growth, and reprotinc- lea! energy,fa' 1100, I ion OnfrtlY,

5,12 Tril 1111611.1111111011 OrEn, Energy can be transformed ftoni ono101111 Not only are forma of energy ittlercon- orgy 10 another, yertible, but differentforma of mat are ConVertilb to energy, Oxidation of ergot& molecules is often said to yield energy, 'Flits does not mean that thew reactions make energy, but simply result in energy transformation,

5.121 First Law of Thert»ody- The total energy and matter in the lint- nmrdcs verse are constant. !teat produced during the chemical reac- .5.122 Second Law of Thermo- During energy transformations, a loss In All biological processes are inherently in- dynamics usable energy occurs as heat is produced, or effidient and thus heat pro.ducing. tions of microbial metabolism can be mea- the system becomes less organized, or both, sured. When systems become less organized they A demonstration of this is the increase in can do less work:This tendency towarddio- internal temperature in compost heaps. order is called. an increase in ontrophy. If you burn a carbon-containing substance 5.13 Chemical Bond Energy The energy of a molecule can roughly he represented as the sum of the forces holding (tottilly oxidize it to CO2), the heat produced the atoms together. is a measure of the potentially useful chemical energy of that substance.

5.14 Endergonic and Exer- Making certain bonds between atoms re- A typical endergonic reaction may be rep- gonic Reactions quires energy input (endergonic reaction), resented by the following. but this energy cap later be released by A + B + energy AB breaking the bond (exergonic reaction). A typical exergonic reaction may be represented as follows. BC -- B + C + energy

5.15 Coupled Reactions Biological transformations of chemical en- A coupled reaction is the sum of the fol- ergy can occur because exergonic (energy- lowing. yielding) reactions are linked to endergonic endergonic: A + B + energy AB (energy-requiring) reactions by common in- exergonic: BC B + C ± energy termediates., SUM A + BC AB + C Coupled reactions may also be written as follows. A endergonic AB

exergonic BC Microbial Physiology-49 4 "1 eL2, ) A C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION The reactions will proceed in the direction of the arrows as long as the energy releasefi 1 by the exergonic reaction exceeds that required by the endergonic reaction.

5.16 Nucleoside Diphos- These compounds are the most used in- Although removal of the terminal phosphates and Triphos- termediates between exergonic and ender-phate group from ADP releases about as phates ADP, GDP, gonic reactions. The conversion of adenosine much energy as the hydrolysis of ATP to UDP, CDP, ATP, GTP, diphosphate (ADP) to adenosine triphos-ADP (7.3 kcal.mor 1, 30.5 kJ mo1-1), ADP is UTP, CTP phate (ATP) for example, provides an effi-not commonly used as an energy interme- cient short-term energy storage and transferdiate. mechanism in biological systems. The addi- In some specific reactions, hydrolysis of tion of a third phosphate to a nucleosideGTP, UTP, or CTP provides the energy. diphosphate is an endergonic reaction and A conventionalsymbolismforthe thus can only occur when this reaction is"high-energy" bond is so that ATP = linked to an exergonic reaction that releasesAMP P P. sufficientenergy.Enzyme-catalyzedre- moval of the third phosphate is exergonic; thus, it can be used to drive cellular energy- requiring reactions. I.

5.2 Catabolism Catabolism is the sum of the cellular re- According to Mitchell's hypothesis (Mi- actions resulting in conversion of the energycrobial Physiology, Subtopics 6.1) the gra- from various organic molecules to energydient of protons across a cell membrane or usable for cellular work. ATP is the mostorganelle membrane can be used directly to common "useful" cellular energy form. do cell work. Catabolism can result in production of proton gradients across membranes. Some microorganisms obtain all their en- Fermentation and Res- Fermentation and respiration are the two It is often important in characterizing bac- 5.21 ergy by fermentative pathways, some only piration basic catabolic schemes. teria to determine the type of catabolism In fermentative pathways, the organicthey carry out. Strict aerobes carry out res-use respiratory pathways, and some can use molecules being utilized as a source of en-piratory catabolism only. Facultative anaer-either, depending on the presence or absence ergy are incompletely oxidized. No externalobes can utilize either fermentation or res-of oxygen. inorganic electron acceptor is necessary (nopiration. Aerotolerant anaerobes use fer- Simple laboratory tests can be used to 02 is required). ATP is produced only bymentative pathways but are not harmed bydetermine whether a particular organism is substrate-level phosphorylation (Microbial02. Strict anaerobes use fermentative path-obtaining energy from ferme tation or res- Physiology, Subtopic 5.25). The number ofways and are harmed by 02. A few are ablepiration. One such test depen s on the abil- ATP molecules produced per molecule ofto respire by using alternate electron accep-ity or inability of an isolate to utilize a par- substrate catabolized is small. tors(Microbial Physiology,Subtopicticular organic energy source in a sealed tube. Information on oxidation-fermentation .Inrespiratorycatabolism(sometimes 5.2432). called oxidative catabolism), the organic Two industrial processes using yeast can (OF) test media can be found below. molecules being utilized are usually oxidizedbe used to demonstrate differences between Lennette, E. H., A. Balows, W. J. completely to CO2. The electron transportfermentation and respiration. When yeast isHausler, Jr., and J. P. Truant (ed.) 1980. Microbial Physiology-51 421 4' TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT I FORMATION C. PRACTICAL ACTIVITIES system(Microbial Physiology, Subtopicused for alcohol product n the vats are keptManual of Clinical Microbiology, 3rd ed. 5.24) serves to transfer electrons to an inor-strictly anerobic. Thehanol is a product American Society for Microbiology, Wash- ganic electron acceptor. Most of the ATP isof fermentation. Whenast is produced forington, D.C. produced by oxidative phosphorylation cou-sale as bakers' yeast it wn under highly pled to the electron transport system. Theaerobic conditions b use the best yield of yield ,of ATP per molecule of growth sub-cells for the substo added can only be strate metabolized is much greater than theobtained if the yeast is living by respiration. yield of ATP in fermentation.

5.22 Oxidation-Reduction Oxidation is removal of electrons and of- In biological systems, oxygen is one of the An oxidation-reduction reaction can be Reactions ten protons (H+) from a molecule. The elec-most avid available acceptors of a electronsrepresented as follows. trons removed must be donated to another (most electronegative). Thus, an oxidationA H A molecule, and this recipient is said to bein which oxygen ends up with the trans- reduced. Thus, an oxidation reaction must ferred electrons releases the most energy. B B H always be, accompanied by a reduction reaction. Oxidation-reduction reactions result = electron in a transfer of energy. Much of the energy H = proton involved in the transfer may be lost to the A = electron donor system as heat, or if the reaction proceeds B = electron acceptor. in a stepwise fashion, some of the energy may be conserved by coupled reactions.

5.23 Nicotinamide Adenine Nicotinamide adenine dinucleotide The use of NAD+ and NADP as electron For directions for performing a lactic de- Dinucleotides as Elec- (NAD+) and nicotinamide adenine dinucleo- acceptors in catabolic processes is very im-hydrogenase enzyme assay which will dem- tron Carriers tide phosphate (NADP+) are the most com-portant to the conservation of energy re- onstrate use of NAD as an electron acceptor mon immediate electron acceptors of or-leased by oxidation. NADH and NADPH do consult: ganic molecules. Reduced forms of thesenot donate electrons to oxygen directly and Bergmeyer, H. U. 1974. Methods of en- molecules (NADH and NADPH) act as elec- are relatively stable in the absence of en-zymatic analysis, vol.1. Academic Press, tron donors in other reactions. zymes specific for transfer of the electrons Inc., New York. to a given substrate. This enables the organism to maintain control of its oxidation- reduction reactions. NAD' is made from niacin and is a good example of the place of vitamins in metabolism.

5.24 Electron Transport Electron transport is a cellular mechanism for conserving the energy released during oxidation of organic molecules. Catabolic processes involving participation of the electron transport chain are called respiration.

5.241 Components The electron transport system consists of Components of the electron trnsport sys- Procaryoticmicroorganismshave the ; Microbial Physiology -53 4"3 4L, Fi AND SUBTOPICS A, ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES a series of molecules (flavoproteins, qui-tern are muoh more variable among procar-molecules of the electron transport system , nones, cytochromes) that are embedded inyotic organisms. The position of the flavinassociated with the plasma membrane. Eu- a cellular membrane. This series of mole-component (FAD) is in question and cyto-caryotic microorganisms have these compo- cules is referred to as the respiratory chain.chrome c is not present in all bacteria. Those nents in the inner mitochondrial membrane. The Se molecules have the ability to be bothwith certain cytochrome components give aIf the components are extracted from the good electron acceptors and good electronpositive oxidase test and this is'an importantmembrane, they will no longer function to donors. They are ordered in a sequence de-diagnostic characteristic for certain bacterial transport electrons in a coordinated way. termined by their avidity in accepting elec-groups. trons.

Mechanism The prime cellular donor of electrons to Branched pathways of electron transport A 'typical electron transport chain is illus- the electron transport system is NADH.are not uncommon in bacteria. Flavin en-trated in Fig. 10. When NADH provides an electron pair, thezymes can donate electrons directly to 02, electron transport molecules are alternatelybut H202 is formed instead of water. The reduced and reoxidized in a particular orderperoxide is decomposed by catalase. The as the electrons pass from one to the next.presence or absence of this enzyme is impor-

As these stepwise oxidation-reduction reac-tant in characterizing bacteria. 1 tions occur, energy is released, and this energy can be conserved by oxidative phosphorylation (Microbial Physiology, Subtopic 5.25).

TerminalElectron Ac- For the continuation of electron flow If there is no terminal acceptor or if the ceptors through this pathway, there must be sometransport is blocked at the last step, all of exogenous compound that will accept elec-the electron transport components will be- trons from, and thus reoxidize, the last cy-come reduced and NADH cannot be reoxi- tochrome in the chain. dized. If there are not alternate methods of reoxidizing NADH, metabolism will not continue.

Aerobic Respiration Most microorganisms utilizing electron Obligate areobes are microorganisms that transport require oxygen to accept electronscannot grow a roduce in the absence from the final reduced cytochrome. of molecularorcTgreW.They require oxygen as a terminal electron acceptor. Anaerobic Respiration A few types of bacteria can use oxidized Bacteria that use NO3- as a terminal elec- Nitrate reduction can be observed in the inorganic ions as electron acceptors in thetron acceptor (denitrifiers) will use oxygenlaboratory by culturing dentrifying bacteria absence of oxygen. NO3" (nitrate) and 5042 preferentially The conversion of soil nitrate under anaerobic conditions with NO3- added (sulfate) are the most common alternate(NO3-) to gaseous nitrogen products by den-to the medium. NO2- may be produced, e.g., electron acceptors. itrifying bacteria is one way that soils loseE. coli. Dentrifiers reduce NO3- to N2 or nitrogen. The use of S042- as a terminal other gaseous products, e.g., Pseudomonas electron acceptor is restricted to a special-aeruginosa. ized group of anaerobic organisms. H2S is Winogradsky columns (Microbial Physi- produced. Water-logged sediments with an ology, Subtopic 2.12) usually develop a good Microbial Physiology-55

4 "G (Oxidized) Reduced Oxidized Reduced Oxidized Quinone Cytochrome 6 Cytochrome c Cytochrome a Water FAD .

Reduced Oiddized Reduced .A...... Oxidized Reduced 02 FADkj2 Quinone Cytochromeb Cytochrome c Cytochrome a

41 , 4 .Flo. 10.Typical electron transport system. ,.

Microbial Physiology-57

I 4.,"3 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES abundance of organic material and S042-population of sulfate-reducing bacteria. It is (tideflats, for example) are noted for theirpossible to smell the H,S produced in the H2S-producing Deaulfovibrio populations. cylinders or test for it with filter pap r soaked in 5% lead acetate.

5.25 Substrate Level and Ox- The principal method of conserving, en- Although no one mechanism for oxidative idative Phosphorylationergy released during cellular oxidations iqphosphorylation has been proven, the chem- coupling the energy-yielding reaction to aiosmotic hypothesis of Mitchell seems best reaction adding phosphate to a nucleosidesupported by available evidence. This pre- diphosphate (Microbial Physiology, Sub-sumes that energy released causes a flow of topic 536). protons across the membrane and the pro- In substrate-level phosphorylation, theton gradient drives the phosphorylation. phosphate is transferred directly from aEach 2W equivalents transported results in high-energy fihosphorylated intermediate ofphosphorylation of 1ADP 1 ATP.It has metabolism,'fro the nucleoside diphosphate. been difficult to demonstrate that 3ATPare In oxidatiVe phosphorylation, the energyproduced for each NADH oxidized in bac- released by4h*N oxidative steps of the elec-terial electron transport systems. Bacterial tron transpcirt chain is conserved by someelectron transport systems may be leas effi- intermediarY process and then is used tocient than mitochondrial electron transport phosphorylrite ADP toATP.The energysystems (Microbial Physiology, Topic 6.0). released by44nOving one pair of electrons from NADIR t14ough the electron transport chain to oxigeri is sufficient to phosphoryl- ate three ADP molecules to three ATP molecules.

5.26 Degrativeor Cata- Cells break down organic molecules by bolic Pathways series of enzyme-catalyzed reactions. A particular series of reactions is called a metabolic pa sway.

6.261 Glycolysis (Embden- Glycolysis is a pathway for degradation of Glycolysis is essentially the same in mi- Meyerhof Pathway) glucose that is found in most microorga-croorganisms, plants, and animals. It is a nisms. ,,There are nine reactions needed tocytoplasmic cellular process, and the en- convert glucose to two pyruvate molecules. zymes are not membrane-bound. The intermediate substrates are all phosphorylated. Glucose 2 Pyruvate pathway can be divided into three + ,,FA Ei es. The preparative phase converts glu- 2NAD* 2 NADH cose to two three-carbon phosphorylated + + molecules. This requires two moleculesof ADP 2 ATP + ATP. The oxidative phase converts the '2 Pi three-carbon molecules to the highly reac- tive1,3-diphosphoglycericacid(3-phos- There is one oxidative step in whichphoglyceroyl phosphate). This requires two

Microbial Physiology69 '' 43 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION . C. PRACTICAL ACTIVITIES NAD* accepts a pair of electrons from amolecules of NAD* and generates 2 NADH. three-carbon intermediate. The energy-transfer phase results in sub- The ATP molecules are produced by substrate-level phosphorylation of 4 ADP to 4 strate-level phosphorylation. The pyru-ATP. Two reactions occur whereby energy il.gte and NADH generated have a differentand phosphate are transferred directly from fate in fermentative organisms than in res-the 3-carbon phosphorylated high-energy piratory organisms. intermediates to ADP (Fig. 11). The pathway by which glucose is catabo- 5.262 Other Pathways There are other routes bywhiclieugars A unique characteristic of procaryotic can be catabolized. For example, glucose cancells is the diversity of catabolic pathwayslized may be an important taxonomic char- be catabolized to pyruvate by the pentosethat may be found in different species. This acteristic of some Microorganisms. Most phosphate pathway and the Entner-Dou-may be a reflection of the intensive investi- members of the genus Pseudomonas char- doroff pathway. Catabolic use of these path-gation of this aspect of bacterial metabolism. acteristicallyusetheEntner-Duodoroff ways is restricted to certain ba terial groups. pathway.

5.263 Fates of Pyruvate and NADH

5.2631 Fate in Absence of Res- Fermenting microorganisms are unable to Because the energy yield per molecule of It is possible to identify certain taxonomic piratory Pathways-Fer-carry out the reactions of electron transportsubstrate utilized is low, a fermenting micro- groups of bacteria by their fermentation mentation either because they lack a terminal acceptororganism must catabolize more molecules ofproducts. The identification of anaerobic or because they lack the necessary enzymessubstrate than a respiring microorganism for pathogenic bacteria by gas chromatographic or cofactors. They can grow and reprdducethe same yield of growth and reproduction. analysis of their volatile fermentation prod- on the ATP produced by glYcolysis of other In fermentations there is no exogenousucts may become an important clinical tech- anaerobic pathways, but; because N4Wf iselectron acceptor, so the average tnidation nique, in very limited supply, they must be'able tolevel of the fermentation products must The Voges-Proskauer test for the pres- reoxidize the NADHproduced,to permit theequal that of the substrates utilized. Thisence of acetoin is frequently used in identi- anaerobic energy-yielding pathway to con-,requirement, and the fact that no oxygen isfication of enteric bacteria. tinue. Fermentative microorganisms utilizeavailable to carry out oxidative bond cleav- Two fermentative pathways are illus- pyruvate or other products directly or indi-age, limits tip range of substrates that cantrated in Fig. 12. For additional examples rectly as recipientsfor electrons frombe fermented. In general, compounds more consult microbiology and biochemistry to41:k 4.7.- NADH. Thus, mucliof the pyruvate carbonoxidized than pyruvate or more reduced books. ok. can be found in the reduced organic mole-than the aliphatic amino acids do not serve cules (fermentation products) excreted byas substrate for fermentations. fermenting organisms. ., .

5.2632 Fate of Pyruvate and In microorganisms with a functional elec- NADH in Organisms tron transport system, NADH can be readily Utilizing Respiratory reoxidized by this system. Pyruvate can be Pathwhys completely oxidized to CO2 by the tricarboxylic acid cycle.

5.26321 Oxidative Decarboxyla- Pyruvate is converted to acetyl CoA This reaction is carriedo\-by. a multien- Figure 13. don of Pyruvate which then enters tricarboxylic acid cycle.zyme complex containing three different en- Microbial Physiology-61 A ct 1 1-40 4 " COH Dihydroxyacetone phosphate

Preparative Phase: 2ATP 2ADP POC COP CON

1 Reaction 3 Reactions I I COP

Glucose OH OH OH Fructose - 1.6-di p hosphate MMI. IMO INNEN. 1111 111 =IMO 2

0 COH Oxidative Phase: // COP C OltrP 2P, 2 COH Glyceraldehyde-3-phosphate 4 COP 2NADH.I-1 3-Phosphoglyceroyl Phosphate

iIMMID MEM& MI01. I1=11 4MMI OM. 2ADP 2H20 CH2 2ADP 2ATP O Energy Transfer Phase: I // 2 2 C-0 2 Reactions COP 1 Reaction COH N0 N0 C-013 Phosphoenolpyruvate Pyruvate 3 Phosphoglyceric acid FIG. II. Glycolysis.

Microbial Physiology-63 4 " The reduced organic molecules produced by fermenting microorganisms are of great commercial and diagnostic importance. Ethanol is produced mainly as a product of yeast fermentation.

Substrate: Glucose (6 Carbons) 2 Ethanol: Fermentation Product: (2 x 2 Carbons)

2ADP 4,,,.---7----411C 2NAD Reoxidation of NADH,H Release of 2P, Energy 2ATP 2NADH,H

2 Pyruvate 2 Acetaldehyde

2CO, (2 carbons)

Lactic acid, a product of the fermentation of milk sugar (lactose) by lactobacilli and streptococci, is important in production of cheese and yogurt.

Fermentation Product Substrate: Lactose (2 x 6 e 12 Carbons) 4 Lactic Acid (4 x 3 =. 12 Carbons)

4 ADP GIC 4 NAD Release of 4 P, Energy Reoxidation of NADH,H*

4 NADH,F1' 4 ATP

4 Pyruvate In the clinical laboratory, bacterial isolates may be identified on the basis of fermentation substrates and products.

FIG. 12. Examples of fermentative pathways.

Microbial Physiology-65

4 "5_ CH., CH.,

+ NAD + Coenzyme CO; C=0 + NADH.H* A SCoA C=0 ICoA) Acetyl-CoA

Pyruvate

Pm. 13: Oxidative decarboxylation of pyruvate.

Microbial Physiology-67 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES Coenzyme A (CoA) is essential to this reac-zymes and five cofactors. ATP concentration tion and serves to carry an activated acetylregulates activity of the enzyme complex. group into the cycle.

5.26322Tricarboxylic Acid Cy- The further oxidation of acetyl CoA is The tricarboxylic acid cycle has more than Figures 14 and 15. cle (Krebs' Cycle) (Citric dependent on enzymes of the tricarboxylicjust a catabolic function. Many of the inter- Acid Cycle) acid cycle. These enzymes are associatedmediates ere starting material for synthesis with the cytoplasmic membrane of procar-of important cell components. Anaerobes yotic ',Cells and are in the mitochondria ofthat do not use the cycle for respiratory eucaryotic cells. metabolism may still have many of the cycle enzymes. These have biosynthetic function. When tricarboxylic acid intermediates are removed for biosynthesis, the cycle can be kept in operation by synthesizing new oxaloacetate. The reactions responsible for synthesizing oxaloacetate from pyruvate, phos- phoenol pyruvate or acetyl CoA are called anaplerotic or replenishing reactions. Be- cause of the central position of many of the tricarboxylic acid intermediates in metabolism, an expanded version of the tricarboxylic acid cycle is presented in Fig. 15.

5.264 Pathways for Degrada- Microorganisms can use a wide range of Some synthetic molecules are very resist- Microorganisms vary greatly in their abil- tion of Other Substratescarboncompounds as sources of carbon andant to microbial degradation. DDT is one ofity to degrade carbon compounds. These energy. Series of degradation reactions, of-these recalcitrant molecules. need not be water soluble, e.g., crude oil. ten catalyzed by inducible enzymes (Micro- Microorganisms able to degrade large mol- bial Genetics, Stibtopic 6.1), result in prod- ecules often excrete the degradative en- Pesticide Approximate ucts that can enter the glycolytic or tricar- chemical half-life (yr) zymes into the medium containing the sub- boxylic acid cycle pathways. strate. These excreted enzymes are called Ctlyclane, 2-4 3-10 exoenzymes and can be detected by growing Dieldrin 1-7 the microorganisms on solid medium con- Heptachlor 7-12 taining the substrate and observing substrate disappearance around the areas of microbial growth.

5.2641 Carbohydrates Enzymes catalyze removal of monosac- Certain polysacchaildes such as starch A solid nutrient agar medium containing charide or disaccharide units from polysac- and glycogen are readily degraded by exoen-starch can be inoculated with an amylase- charides. Disaccharides are broken into twozymes secreted by many microorganisms.producing microorganism; after incubation monosaccharidis. Glucose and fructose en-The breakdown of cellulose is limited to aand growth of the organism, flood the plate ter the glycolytic pathway directly. Most few groups of bacteria, some fungi, and a few with a 3% iodine solution and note the other monosaccharides can be converted to protozoan types. starch-freeareaaroundthemicrobial either glucose or fructose. growth. An example of an amylase-producing bacterium is Bacillus. subtilis. 4 kJ" 1" 'Microbial Physiology-69 4 (2 Carbons) Acetyl-CoA. CoA

Citric Acid (6 Carbons) (4 Carbons) Oxaloacetic Acid

111\

GTP CO2

GDP 3 NAD CO2 O 1 FAD 3 NADH,H*

1 FADH2 A summary of the cycle. (i) AcetylCoA reacts with a 4-carbon dicarboxylic acid (oxaloacetic acid) to produce a 6-carbon tricarboxylic acid (citric acid). CoA is released. (ii) Citric acid undergoes a series of reactions which convert it back to oxaloacetate, thus completing one cycle. In each cycle four oxidative reactions occur (three NADH,Ir and one FADH2 produced), 2CO2 are released, oxaloacetate is regenerated, and one substrate-level phosphqlotion occurs (GTP). (iii) The 3 NADH,II" anc1,1FADH2 donate electrons to the electron transport chain and ATP is produced by oxidative phosphrlation -(see 5.24). Fm. 14. Tricarboxylic Acid cycle summary.

4 t"a Microbial Physiology-7I Acetyl-CoA CH ,COSCoA CoA

Fl---C/343- Citric 1 HOCCOO H:C000 H2C COO Isocitric Oxaloacetic HOC000 HC000 Ma lic C000" HOCCOO NAD H:0 CO: C000 H2C000 Fumaric II CO: C000 H:C

I a-Ketoglutaric H2C000 HNC COO 1:.C000**

, I HX000 H2C NAD

Succinic C.CSCoA, Succinyl-CoA FAD Cp

u .GDP

c..%. " 15. Triectr box.911e add c11E.1411 .wls.o

ial Physisol 71 el0 PICS ANIY SUB;POVICS A. ESSENTIAL INFORMATION 13. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES Lipids Lipids are cleaved into their component Many of the normal mammalian skin bac- The breakdown of fatty acids and hydro- n. -.1 parts. Fatty acids and other hydrocarbonsteria, e.g., diptheroids, are good at degrading carbons usually requires molecular oxygen. S. 4', are degraded by oxidative reactions to acetylfatty acids, 'rho odor produced by variousThis explains the environmental persistence CoA which enters the tricarbdxylic acid cy-mammals, including humans, is in part gen- of these compounds under anaerobic condi- cle. Glycerol can enter the glycolytic path-erated by the action of skin bacteria on skintions. Several media can be used to detect way. fatty acids. bacterial ability to degrade lipids, e.g., media containing egg yolk or Tween 80 (sorbitan mono-oleate polyoxyethylene). A precipitate around the colony indicates lipolytic ability. Various members of the gentiS Clostrid- I ium may be identified by their phospholi- pose activity.

5.2643 Proteins Proteases degrade proteins into amino Some oftheproteolytic exoenzymes Many microorganisms are able to degrade acid, units. Amino acids are converted toformed by the members of the genus Bacil- proteins. A common laboratory test for abil- pyruvate, acetyl groups, or tricarboxylic acidlus have found commercial use as additives ity to hydrolyze protein involves inoculating cycle intermediates. to washing products. They degrade protein-an organism onto solid gelatin medium and containing stains like blood and gravy. noting liquefaction after growth.

5.3 Inorganic Compounds as A small number of procaryotic species can Usually lithotrophs are quite specific for Media made with a particular reduced Energy Sources oxidize reduced inorganic compounds anda given inorganic material. For example, sul-inorganic compound as an energy source and use the energy released by these oxidationsfur oxidizers do not oxidize reduced nitrogenCO2 as a carbon source will develop a pop- to form ATP. The electrons from the oxi-compounds. For most lithotrophs, this modeulation of lithotrophs when inoculated with dative reactions enter the electron transportof existence is an obligate one. They do notsoil or water and incubated in the dark.4, system (Microbial Physiology, Subtopicoxidize organic molecules, and, in fact, some Some of the reduced inorganic molecules 5.24), and ATP is produced by oxidativewill not grow in the presence of moderatethat can serve as energy sources are: H2 phosphorylation(MicrobialPhysiology,concentrations of organic materials. The hy- (hydrogen oxidizers); NO2(nitrite oxidiz- Subtopic 5.25). These organisms, called lith-drogen bacteria, however, can also obtainers); H2S, S, S203" (sulfur oxidizers); NH3 otrophs, usually obtain their carbon fromenergy by oxidizing organic molecules. (ammonia oxidizers); Fe' (iron oxidizers). CO2 and thus are autotrophs as well.

5.4 Light as an Energy Organisms able to use light as an energy There are procaryotic phototrophs, e.g., Source source are called phototrophs. Phototrophsphotosynthetic bacteria and cyanobacteria convert light energy to chemical bond en- (blue-green algae) and eucaryotic microbial ergy by some light-absorbing pigmentusu-phototrophs (algae). Nearly all the organic ally a form of chlorophyll. Chlorophyll ischemical energy on the earth is derived from oxidized by light of certain wavelengths. Thelight energy trapped by phototrophs. Micro-. electrons removed from chlorophyll are then bial phototrophs (predominantly marine) high-energy electrons. They enter an elec-are responsible for about 50% of the global tron transport system and ATP is producedconversion of light energy to chemical bond (Microbial Physiology, Subtopic 5.24). energy. Microbial Physiology-75 7 4Y1 4 A' TOPICS AND SUBTOPICS A. INHINTIAL INFORMATION 13, ENRICHMENT INFORMATION C. PRACTICAL ACTIVITI E8 5.41 Anoxygenic PhotoSyn- Photosynthetic bacteria (other than cy- Some bacterial photosynthesizers (purple thesis imobacteritt)1,0ary out photosynthesis only and green sulfur bacteria) require reduced in the absence Hof oxygen. Their photosyn- sulfur compounds as a source of electrons to thesis does nati produce 02. In these orga- reduce NADI'', This limits their distribu- nisms, etechime released from chlorophyll tion to illuminated anaerobic areas which provideOeray for synthesis of ATP (Micro, have a source of reduced sulfur compounds. bial PhySiolagy, Subtopic 6.1232). Ele.ctrons Electron flow in a photosynthetic bacterial reducing, NOP' to the NADPH needed to system is often described as cyclic and can fix CO2iro kie"yed from other sources. he diagrammed 'as in Fig. 15,

5.42 Oxygenic Photo- In OanoliS:teria (blue-green algae) and Cyanobacteria (blue-green algae) do .not syntheses eucaryotic algae(as well asallhigher have chloroplasts. Although the reactions of plants), electrons from chlorophyll provide their oxygenic light reaction are quite similar energy for syntheses of ATP and serve as a to those occurring in eucaryotic photosyn- reductant NADP+. Electrons from water thesizers, all of the components of the pho- reduce chlorqphyll, and the oxygen from the tosystem reside in membranous sheets or water is releksettas 02. sacs in the cytoplasm. Some investigators think that chloroplasts may have evolved from cyanobacterial symbionts residing in a larger host cell. The oxygen-producing noncyclic photophosphorylation and photored- uction system can be diagrammed as in Fig. 17. 6:r

5.5 Anabolism AnabolismiS,the sum of the cellular chemical reactions that produce the organic molecules necessary for maintenance, growth, and reproduction of the organism.

5.51 CO2 Fixation in Auto- The conversion of CO2 into the carbon The primary reaction in autotrophic CO2 CO2 fixation (the Calvin cycle) was first trophs compounds of cell material requires energy fixation is the binding of CO2 to the five- investigated in green algae. The path of in- and reducing power. carbon sugar ribulose 1,5- diphosphate. This corporated 14C- labeled CO2 was observed in results in production of two phosphoglyceric photosynthesizing organisms. It was later acid molecules (each three carbons). These discovered that this pathway is used by al- can be used as starting material to synthe- most all autotrophs. size the carbon compounds needed by the autotroph.

5.52 Relationship Between Catabolic metabolism provides sources of Anabolism and Catabo- energy (ATP), reducing power (NADH and lism NADPH), and carbon skeletons for bio- syntheses.Withdrawalof intermediates from catabolic pathways diminishes the net ATP yield. Control mechanisms function so Microbial Physiology-77 4 Al 3 44' Primary electron acceptor ADP,Pi, Electron Photophotiphorylation ATP Chlorophyll Light

Chlorophyll acts aN the donor and the acceptor of the electrons.

Fin. 16.cyclic electron flow in phototrophic bacteria.

Microbial Physiology 79 NADI' Electron u Electron acceptor acceptor ADP

ATP NAIW11,11*electron 00/ photo 1140 Chlorophyll 'phosphorylation Chlorophyll Reaction Reaction Center I Center 11 1402 Light

(center)

Flo. 17..Noncyclic electron flow in cyanobacteria and eucaryotic photosynthesizers.

Microbial Physiology- 81

4k, UP

IL PRACTICAL ACTIVITIES TOPI1-1 A NI HIIIITOIIICS A, ESSENTIAL INFORMATION H, INIHIMATIN that stuholie proviso's's (itiosynt het le Intl h- ways) are maximized when the A'l'l' supply ishigh and c11181)0110 prtieessist predominate when ATP slimily is low.

5,53 Synthesis of MoluMII M ier00411111401111 CIO) syttl !WOO, Or the email molecule', (monomers) needed for growth. The starting carbon compounds for therm pathways come from intermediates in cstabolle pathways, Motentors not sty tithe- 1111114110 prov hied In the medium (Mi- crobial Physiology, Subtopic 2.25).

5.531 itemise Phimphates Oxidoncetate, a trIcarboxylic acid cycle MIcroorganinins able to synthesize !mi- intermediate, ClUI be used met Marling mate-mes front acetyl CoA utilize a special pat- rial for hexose synthesis. Since oxaloacetate way, the glyoxylate bypass, in conjunctiim can be synthesized front pyrtivate and CO2 with the tricarboxylic acid cycle. Acetyl CoA and from other tricarboxylic acid cycle in- can be condensed with glyoxylate to forma termediates, and in certain organisms from imitate. This permits accumulation of tricar- acetyl CoA, arty compound that. can be con-boxylic acid cycle intermediates for biosyn- verted to these will also serve as a precursor thesis. of hexoses (Fig. 18).

5.532 Pentose Phosphates Deoxyribose and ribose are needed for The pentose phosphate pathway has two synthesis of nucleotides. Ribose is synthe-stages. The oxidative portion converts glu- sized by the pontose phosphate pathway.cose 6-phosphate to ribose 5-phosphate. The This pathway, often called the hexose nkono-rearrangement portion converts six pentose phosphate shunt, involves an oxidativb de-phosphates to five hexose phosphates. De- carboxylation of hexose phosphate to a pen-pending on the biosynthetic needs of the tose phosphate. The NADPH produced dur- organisms one or both portions may hte- ing the oxidative steps of this pathway is important in providing electrons for reduc- tive steps in biosynthetic pathways. Ribose can only be converted to deoxyribose when it is incorporated in a nucleotide. 6 The types of fatty acids synthesized by a 5.533 Lipids There is considerable diversity in the lip- For fatty acid synthesis the general path- ids synthesized by microorganisms. Fatty way involves successive addition and reduc- particular microbial group are often char- acids, phospholipids, carotenoid pigments, tion of two carbon units (derived from acetyl acteristic of that group and me identified quinones, poly-fl-hydroxybutyrate, and CoA) to a growing fatty acid chain held onby gas or thin-layer chromatography. f.hlorophyll can be synthesized by variousa carrier protein. Double bonds are either It may be possible to detevmine the rela- prociiryotes.Eucaryoticmicroorganismsleft in during synthesis or added when the tive amounts of eucaryotic and procaryotic synthesize a different range of fatty acidschain is finished. microbial biomass in environmental samples 4 Microbial Physiology-83 43 4 Acetyl-CoA ----:,... 2 Oxiiloacetide 2 Phosphoenolpyruvate 2 Glyceraldehyde --0.- Hexose --k :i phosphate 2 Mutate 2 ATP 2 ADP 2 NADH,H 2 NAI) 2 Pvrtivate Energy Reducing required power required 2('O,

Hexose synthesis Once glucose P or fructose P is .synthesized these sugars can he meric(' to a wide range id amino sugars, sugaracids, .. anddeoxysugars Often sugar conversions are carried out while the sugar is in an admitted form Example11)Pvlacose - l'UP galactose. Glyoxylate bypass is nut found in all organisms

Microbial Physiology85 C. PRACTICAL ACtIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION than procaryOtic microorganisms. Eucary- Bacteria, with the exception of cyanobac- by analyzing the characteristics of extracted otes usuallysynthesize considerableferia, do not synthesize fatty acids with more lipids. amounts.,Ofsterol as well. than one double bond. alycerol pphosphate needed for phospho- lipid synthesis is synthesized from dihydroxyacetone phosphate, an intermediate of the glycolytic pathway. Fatty adds are synthesized from acetyl CoA.

O Glycerol phosphate, fatty acids, and additional components are combined to produce phospholipids. Other types of lipids are synthesized from CO2 and two carbon units derived from acetyl CoA. Some microorganisms can synthesize all 5.534 Amino Acids The addition of an amino group to pyruvate, oxaloacetate, or a-keto-glutarate is the 20 amino acids required for protein synthesis starting point for the biosynthesis of most and some cannot. The amino acids synthe- amino acids. sized by short pathways are the most uni- versally synthesized. An example would be glutamate. The pathways for synthesis of some other amino acids are long and expen- sive in terms of ATP and reducing power (NADH). These amino acids are required as growth factors by the organisms unable to synthesize them. Some of these longer pathways are branched, giving rise to several amino acids. An example might be the iso- leucine-valine pathway

5.535 Nucleoside Monophos- Small chemical groups containing carbon phates and nitrogen derived primarily from amino acids are assembled into the purine and pyrimidine rings. The purine ring is assembled starting with an activated form of ribose phosphate. In the case of pyrimidine ribon- ucleolide, the pyrimidine ring is synthesized 'and then attached to the activated ribose phosphate (Fig.19).

5.54 Synthesis of Polymers Polymers are synthesized by condensation of monomers. Energy is required for this 4- condensation. Monomers are activated by a 4 Microbial Physiology87 FIG. 19. Ongtn of carbons and nitrogens in purines and pyronidines The eurine ring issynthesized from seven units derived from six different sources. It takes seven ATP molecules to build a purtne ring The pyrimidine ring issynthesized from two units. One of these units is carhamyl phosphate. an activated compound

Microbial Physiology R9 B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES TOPICS AND SUBTOPICS A. ESSINTIAI, INFORMATION , reaction with a high-energy compound (usu- allY a nucleoside triphosphate like ATP).

Monosaccharides are added one at a time 5.541 Polysaccharides to a growing polysaccharide. Each monomer is first activated by reacting with a nucleoside triphosphate. The monosaccharide can be transferred from this active unit to the end of an existing polysaccharide chain with- outfurtherexpenditure of energy (Fig. 20). Bacteria have been extraordinarily useful 5.542 Informational Polymers: These template-dependent polymers are as experimental systems ininvestigations of DNA, RNA, and Protein synthesized from their respective mono- merit units by mechanisms described in Mi- the synthesis of DNA, RNA, and protein. crobial Genetics, Subtopics 2.13, 2.23, and 2.33.

6.0 MembranePhenom- ena 6 I Membranes and Energy Membranes are energy transducers. That is, within the membrane structure, exergonic reactions occur and are coupled to endergonic activities. The membrane itself is essential for coupling.

6.11 Membrane Semiperme- Membranes are semipermeable. They ability permit the free passage of water in either direction, but restrict the movement of ions and small organic molecules. These may enter and leave only at certain points.Trans- port of such substances is under tightcellu- lar control.

6.111 Gradients Often the concentration of a substance may he much greater on one sidethan on the other. When this occurs, a gradient is said to exist.

6 112 Movement in the Gra- When molecules of a substance move from dient an area where they are moreconcentrated to one where they are less concentrated, they move down the gradient is can occur spontaneously without the exnditure 4:,1 4.3 of energy. In fact, such movement is exer- Microbial Physiology 91 A

PolysaccharideIn I

NTP + Monosaccharidea` NDPMonosaccharide 4 phosphate (activated monomer/ NI)P + Pyrophosphate N weleoside k triphosphate PolysaccharideIn +

Aft' ATP

Fir;21)Syntheses of polysaccharides.

Microbial Physiology93 4L5 TOPICS AND SUBTOPICS A. ESSENTIAL. INFORMATION B. ENRICHMENT INFORMATION gonicit releases energy. The'lnembrane prevents this from happening in a randoin fashion. When molecules move from a less to a more concentrated area, they are moving against the gradient. This requires the ex- penditure of energy, which is endergonic.

6.113 Gradients as Energy Since movement of substances down gra- Sources dients releases energy, a gradient, like stored water water behind a dam, is a source of energy the cell can tap.

6.114 Proton Gradients Several cell processes generate proton gradients where the concentration of protons (H ') exterior to the cell is greater than that within. Since the exterior protons, if allowed to reenter the cell, act as an energy source, the gradient and resulting membrane poten- tialis referred to as proton motive force (PMF).

6.115 Proton Reentry The membrane is not permeable to pro- Part of the ATPase complex is a proton tons except at certain points (proton chan-channel. Artificial membranes containing nels). Reentry at those points is coupled tothis section are proton permeable. ATP synthesis. Membrane-bound Mgt' -dependent adenosine triphosphatase (ATP- ase) is responsle for synthesis of ATP as protons move across the membrane. Proton reentry can also be coupled to transport of small molecules.

6.12 Means of Producing Proton gradients are produced in several Production of proton gradients requires Proton Gradients different ways in various organisms underclosed membrane systems. Broken cells or specific conditions. Three discussed beloworganelles no longer maintain proton gra- are electron transport, ATP hydrolysis, and dients or synthesize ATP. light-driven transport. Hinkle, D. C., and R. E. McCarty. 1978. How cells make ATP. Sci. Am. 238:104-123.

6.121 Electron Transport (See Microbial Physiology, Subtopic 5.25 Figure 21 represents a possible interpre- for information on electron transport.) Thetation of an E. coli proton translocation chemiosmotichypothesisexplainsthe system. ATP synthesis occurs during reen- means by which electron transport (exer-try of the protons. gonic) is coupled to ATP synthesis (ender- Microbial Physiology-95 4:.J ATP

thOl Flavoprotem Fe ti, non heme iron protein Coq quinone component of the electron transport chain. 2W

FIG. 21Proton translocation and ATP synthesis.

Microbial Physiology 9'7 4 3 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. 1.:NRICHMENT INFORMATION C. PRACTICAL ACTIVITIES gonic) within the framework of a membrane. It states that when certain electron carriers are reoxidized, protons are extruded through

the membrane. Thus electron transport re- iw sults in a proton gradient being' established across the membrane. The reducing power of the electrons is converted to PMF. This is the primary effect of electron transport.

6.1211 Oxidative Phosphoryla- The protons are allowedtoreenter tion through a channel where the PMF may be harnessed to ATP synthesis. This is the secondary effect of electron transport. Mem- brane-bound ATPase is essential to ATP synthesis.

6.122 ATP Hydrolysis The hydrolysis of ATP to P and inorganic phosphate (P,)releases energy to pump protons out of the cell. Membrane- bound ATPases have two functions. They may break down-ATP to establish proton gradients and they may synthesize ATP as protons reenter (Microbial Physiology, Sub- topic 6.121). Cytoplasmicreactions(substrate-level phosphorylation) may provide the ATP necessary to produce proton gradients. This permits bacteria lacking electron transport systems to generate the PMF needed for active transport and motility.

6.123 Light-Driven Proton Two systems of light-driven proton tns- Transport port exist. One is the system found in ha bacteria; the other is the light-dependent electron transport found in photosynthetic procaryotes and eucaryotes.

6.1231 Holobactenum (purple The halobacteria synthesizeapurple Stoeckenius, W. 1976. The purple mem- membrane I membrane protein when they are grown inbrane of salt-loving bacteria. Sci. Am. 234: light without adequate oxygen. 38-57. When light is absorbed by the pigment, the pigment is bleached and protons are extruded from the cell. A proton gradient is established and per - Microbial Physiology-99 4'" El' TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMkNTINFORMATION C. PRACTICAL' ACTIVITIES sists as long as the cells are illuminated. This proton gradient allows the cells to synthesize ATP.

6.1232 Light-Dependent EleC- The ATP generated by the light reaction Miller, K. R. 1979. The photosynthetic tron Transport of photosynthesis is thought to be a resultmembrane. Sci. Am. 241:102-113. of the production of proton gradients across chlorophyll-containing membranes. Light striking chlorophyll results in vectorial electron transport reactions which in turn cause proton movement across the membrane. This generation of PMF and subsequent movement of protons back across the membrane through proton channels controlled by an ATPase leads to synthesis of ATP.

6.2 Biological Activities of Two important functions of membranes Membranes are transport of small molecules and provision of energy for flagellar rotation in pro- varyotes.

6.21 Transport of Small Mot: Except for water, ammonia, gases, and E. coli may produce over 100 distinct ecules some lipid-soluble molecules, metabolitestransport systems. These are all genetically move across membranes by specific energy-coded and many are inducible (synthesized requiring transport systems. There are three only in the presence of the substance to be basic types of transport in bacteria: trans- transported). port driven by proton circulation, transport driven by ATP, and group translocation.

6.211 Proton-DrivenTrans- These systems involve specific membrane port proteins that act as carriers. The proton gradient (PMF) provides the energy and the direction of transport.

6.2111 Symport: Transport Certain molecules or ions are cotrans- The lactose transport protein M of E. coli with the Down-Gradient ported with one or more protons. The spe-is an example of a symport carrier. One or Movement of Protons cific carrier binds both proton(s) and itsperhaps two protons move to the inside of triinsportee molecule and by some confor- the cell with each lactose molecule. mational change moves them to the other side of the membrane. The coupling of the down-gradient(exergonic) movement of protons provides the energy for the up-gradient ( endergonic) movement of the transported molecule. Microbial Physiology -101 4. 4° .,

TOPICSltDSUBTOPICS A. ESSENTIAL INFORMATION 'IPENAHMENTINFORMATION C. PRACTICALACTIVITIP 13.2112 _Antiport: Transport When protons move from a higher to a Antiport-produced Na l. gradients maybe Airipst the Down-Gra- lower concentrlition they provide energy fpr used by the fell to drive influx of other dient Movement of Pro- movement of some ions and molecules in the moleculps. Melibose and glutamate may be tons o 'opposite direction-. For exampleNa' andcotransrorted with Nn' by E. coli. Ca' are moved out by the carriers that move protons,in.

6.212 ATP-DriPen Systems These transport systems are commonly Soine sugars, amino acids, phosphate, and found in gram-negative bacteria. They haveK.' may be transported by such systems. a periplasmic binding protein plus a specific membrane carrier. In the presence of ATP these systems can transport molecules so that the internal concentration far exceeds the external concentration. Systems such as this also exist in gram-positive bacteria, but the mechanism is not quite as clear. fy .61 6.213 Group Translo-clItion These systems catalyze a specific enzy- The phosphotransferase system responsi- matic reaction as the molecules ar4 trans- ble for transporting glucoSe is found in many ported. The best known system depends onanaerobic and facultatively anaerobic bac- phosphorylation of sugars with phospho- teria, e.g., E. coli, but does not seem to occur enolpyruvate as a phosphate donor (phos- in strict aerobes like Pseudomonas and My- p hotransferase system). try.,s° cobacterium. Provision of Energy for Flagellar Fotation

Structure of Flagella Bacterial flagella are rigid helical struc- Flagella are good antigens and are respon- Flagella can be disaggregated into indivik tures composed of Protein subunits. Eachsible for the, H. antigenic ;determinants, ofual protein Subunits. Given proper con di- , flagellum has a hook-shaped structure at itsgram-negative tions these units will self-assemble into flag- "hase..Two di:sks on the end of this hook are eller structures. "associated with the plasma membrane.

6.222 Function of Flagella Fltigella are rotated by a force applied Certain modified, bacteria can be "teth- through the membrane. Energy for this ered'; to glass slides by' antibody .to their turning is generated by the prottin circula- flagella. When the 'flagellum is 'unable to r don of the plasma membrane (PMF). Fla- turn, the rotation force results in the cell gella rotating in'One direction propel the cell body turning. This can be 'observed under litre straight line (a run). Reversal of rotation the microscope and the rotation directiOn causes a disruption of this run (a tumble or .can be noted. twiddle). One tEst for chemotaxisrmvolvesusing a 6.223 Chemotaxis e Bacteriaareattracted to some chemical The. rotation direction and frequency of, capillary tvith an attractive or repel'tsub- "substances and are repelled byt others. Pen- change of direction of "tethered" bacteria' 1 Microbial Physiology ]03 4' d

TOPICS AND SUBTOPICS , A. ESSENTIAL INFORMATION B. ENRICHMENT INFp.IATION C. PRACTICAL ACTIVITIES plasmic proteins, the same ones involved in (Microbial 13:1; Biology, Subtis 6.222) canstance in it. The capillary is put into a sus- transport (Microbial Physiology, Subtopiebe observe' hen potential.attractant orpension of bacteria. After a designated time 6.212), are involved in sensing concentrationrepellent chemicals and added to the slidethe number of bacteria in the capillary is AO: gradients of attractants or repellents. 'Thepreparation. 0 compared with the number in a control cap- plasma membrane is affected by this sensory illary without attractant or repellent. binding of Molecules. If the bacterium is swimming into a higher concentration of an attractant, the run will be prolonged. If it is swimming into a lower concentration, the flagellar rotation direction will reverse more frequently and swimming directionwill change until an up-gradient swimming direction is resumed.

d. a.

0 4 t,

4 Microbial Physiology-105 Section IV..MicrobialGenetits'

TABLE OF CONTENTS TABLE OF ILLUSTRATIONS

1.0 GENERAL INTRODUCTION FIG.I. ChromOsomeReplicationin Epcteria 1.1 1.1 Definition of Genetics F,16.' 2. Overview of ProteinSyntheses , 19 1.2 Novel Features Making Microbes Useful Tools fur Gent lir Studies 3 Fiji.3: Protein Synthesis 1 21 Frame Shift Mutation 33 2.0 GENETICALLY IMPORTANT MACROMOLECULES 5 DNA Fre. .5, Replication o.1 Lytic-Rai tr6:11Ye,riet,. 41 2.2 RNA 13' 6. Life Cycle ofd Tentree"rateRai-ten'ophige 43 Protein 15 Fin.7. Specialized Transduction = 49 3.0 ORGANIZATION OF GENETIC MATERIAL FIG.S. C.:en:Wired Transductum 51 /' 3.1 Procaryobc Organization. Fit..9. CloningForeignDNA 55 10 3.2 Euraryotic Organization' 25 GeneralizedEuraryotic Life Cycle 59 ;." '3,3 Viral Chromosomes 27 En,. 11 Repression of Enzyme Synthesp: 63 4.0 MUTATION 27 Tu. 12 Induction of .1Degrddativr Enzyme.System 65 471 Types of Mutations 29 FR..13 Feedback Inhibition 42 Mechanisms of Mutation 31 '4.3 Rates of Mutation 31 4_4 Mutagenic Agents 31 4.5 Selection of Mutants 35 4.b DNA Repair 37 5A3 RECOMBINATION 37 5.1, 'Viral Genetics 39 u 5.2 Procaryotie DNA Transfer '45 ). Recinnbinant DNA: 53 w.4,)E ucaryoty Recotnt?inati(in, 53 0 REGULATORY 11.4ECf;1 1ISM5 57

Regullyri of cent/ ActfY,Ity . 57 rdi of'Enzyme- Activity 61

400

a Pao

0 (I4 it?

TOPIOS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 1,0. General Introduction 1.1 Definition of Genetics Genetics is the study of the means by which hereditafy information of cellsis stored, expressed,sed, and modified and the mechanisms by which this ;information is br transmitted to othercellapi the population and to future generatiotis)of organisms.

1.2 Novel Features. Making Mierriorganisnis have short generation Generation times determined for some For microorganisms reproducing by bi- Microbes Useful Tools times. Very large ovulations of almost idea- typical bacteria growing in liquid media un- nary fission, generation time can be deter- for Genetic Studies ticsl cells can be produced asextailly from a der optimum conditions are as follows: mined by inoculating a culture with a known single cell in a short time. Benechea natriegens 9 min number of cells, determining the population EsCherihui colt 17 min size at a later time, and applying the for- Staphylococcus ailreus 30 min mula:

Mycobacterium tuberculosis -800 min II t. The time required for one cell to give rise 3.3 log,,,h, to 10" cells under optimum conditions: where: B. noiriegeiis 4.5 h G = generation time E. coli 8.5 h t..= time at first measurement S. aarcus 15 h 1 = time at second measurement M. tuberculosis -400 h h = number of cells at beginning of time period = number of cells at end of time period. any microorganisms are haploid, so mu- Where microorganisms divide by binary fis- tation can be expressed immediately. sion, a single cell will give rise to over 10" cells in 20 generations, and to over 10" cells in 30 generations. Some mutant white colonies of Serratia marcescenswill develop if cells which are streaked onto a solid medium are exposed to sublethal doses of ultriviolet light. The nonmutant colonies are red.

.5, re transferred between..microor- Gene transfers t4urring in Inatureare, in Gene transfers can be studied in the lab- part, respoipsible for the dramatic increase oratory (Microbial Genetics, Subtopics 5.2- in antibiotic-resistant organisms that can be 5:5). found today. Organisms resistant to several antibiotics can .even transfer thiS t-au' ltiple resistance to other organisms. Transfers cur withfri the same species and, in some -4" instances, between species (Microbial Ge- netics, Topic 5.0).

Microbial Genetics-3 . 4"'" TOPICS AND SUBTOPICS A. 1.:SSENTIAI, INFORMATION It. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES A wide variety of mutants can be isolated. Mutant strains of microorganisms are de- Pigmentation mutants can readily be ob- veloped and used in industry. Mutants withserved in' colonies on solid media. enzymatic blocks often accumulate large Phage-resistant mutants develop as colo- quantities of useful end products. Mutantsnies on agar plates inoculated with sensitive which are super-producers havvibeen devel- bacteria and sprayed with phage. oped to increase' the production of antibiot- Antibiotic-resistant mutants can be, iso- ic's. Custom-designed organisins are beinglated on media containing the antibiotic. engineered to perform specific tasks, such as Nutritionally deficient mutants, called degrading petroleum and syntheOzing insu- anxotrophs, can be identified by their failure lin and interferon. to grow except when a specific growth factor, which they can no longer synthesize, is present in the medium.

Large populations can be grown, in a small Cell populations of bacteria can exceed volume. re 109 cells per ml in liquid cultures. Denser 1 ryc populations can be groWn on the surface of NOW media. c 2.0 Genetically Impor- tant Macromolecules The major macromolecules important in Exceptions exist, such as in some RNA ,...411e study of genetics are deoxyribonucleicviruses in which an enzyme, reverse tran- acitVIDNA), ribonucleic acid (RNA), and scriptase, produces a DNA cOpy of the RNA protein. The genetic information coded in genome of the virus. -,_DNA is transcribed into messenger RNA Temin, H. 1972. RNA-directed DNA syn- (mRNA), which is then translated into pro- thesis. Sci. Am. 226:24-33. tein.

2.1 DNA

2.I1" DNA Structure The DNA molecule is composed of two Watson and Crick, in 1953, deduced the The two strands of a DNA molecule are complementary chains of nucleotides woundstructure of DNA from X-ray .crystallo-aligned in an antiparallel manner, i.e., one around each other to form a double helix.graphic data. Arend runs in a trOV direction and the other strand rtinian Each nucleotfile consists of a phosphate Chargafrs work Of the late ,1940's and. +, group, the sugar deoxyribose and one of fourearly 1950's showed that the faur,nucleogds-numbers refer to the carlio&atonisin deox- bases; 'adenine (A),_guanine (G) ,-thymineof DNA are not present in equattriiints yribose. (T), and cytosine (C). The sequence of nu DNA and that the amounts vary frion one The base composition of DNA -can he determined by its isolation, acid hydrolysis, Cleo 'des on one strand determines the se-Species to another. He also showed that in DNA, the number of moles of adenine equals and then chromatography to separate the gue ce on the second strand. Adenine onthat of thymine; the number, of moles ofbases. Detection and quantitation of these onstrand pairs by hydrogen bond iyithcytosinc,,equals that,of guanine. bases yield the concentration of each. The thymirie; on the other strand guanine pairs The hase sequence of I)NA can be xleier-percent guanine plus cytosine IC G+C) con- with cytosine. The two strands are thus Com- mined. The`entire genoine of several VP'estent is a, convenient description of base complements of each other: has been sequenced. 1 1,- position of I)NA. 4"4. Microbial Genetics-5 , a', 04- c, IlltACTICA I. ACTI V ITIEH To1)101 ANO MIIIITONcs A, PIN810,NTIAI, INFOltMATItiN IL ENRICHMENT INFORMATION Crick, L. H. C. 1954. The structure of the Hitch's, .1. C. 1977. Thu nucleotide se- The 0+C content of any DNA an be hereditary material. SCI. Am. 191:54-01. quence of a viral DNA. Sill Am.237:54-67.calculated by the- following formula:

101-1 -[C1 x 100 S'iO +C [A] + [T1 + [C] + [01

(where each base is measured in motes). The base composition of the DNA determines the temperature at which the two strands separate, this temperature being the melting temperature, or T,. The higher the % G+C value, the higher the To determine the buoyant density of a DNA molecule, the CaCI density gradient centrifuga NV. don. technique is used. The buoyant density N of a DNA molecule is a linear function of the ratio of G-C to A-T complementary base pairs. ,

Early evidence that DNA is the carrier of 2.12 DNA Function DNA has two major functions: to ntlicate Khorana and colleagues (1970) succeeded itself and to code for the synthesis of protein. in chemically synthesizing the gene codinggenetic information came from several dif- A gene is the sequence of nucleotides which for the production of yeast alanine tRNA. ferent researchers. codes for a single protein (or polypeptide). Walley, R. W. 1966. The nucleotide se- The entire array of genes in an organismquence of a nucleic acid. Sci. Am. 214:30- comprises its genome. 39. The transformation experiments of Grif- fiths (1928) demonstrated that a transform.. ing principle existed in bacterial cells. Avery, MacLeod and McCarthy (1943- 1944), using the same species as Griffiths; et) demonstrated that DNA was capable of transferring inheritable properties to. a cell torn receiving the DNA. Hershey and Chase (1952) demonstrated that in viral infections of bacteria ord,LDNA, an-enk protein, entered the bacteiW4ell to' initiate the production of more virus iarti-

Beadle and Tatum (1941) formulated the "one' gene, one enzyme" hypothesis from *their studies of the mold Neurospora. Their ' hypothesis states a particular gene is responsible for the, production of a particular en- Microbial Genetics-7

4'4-1 t4..! i1,iglitiolmor1' IN100164A190N- 11. idtACTIPAI. ACTIVITM '11111101 AND fit 111T0111(14 A. 104-1t-IF '1A1, INV( )I1M AT1ON zyme, emit enzyme In ended for by a single gene. lladle, (1. .Ill IM, The gotten of men_ and molds, 710(,4AM;1170:30-30,

Iteplicativa is a colnilex process 111114/MI1 Mt4Ition 11111i Stahl (1958) used DNA Replication The two DNA strands Hutatmte id 71 spy- 2.13 by a series Of 1111mm-105 which require a ric'hhi cell DNA and ',I U1-0 (lewdly cent rg'. title point called the origin, An the strands stuni- separate, enzymes catalyze the synthesis ofsing-stranded template, The getters! stopsligation technique to demonstrate conservative mode of DNA replication. The two mat strands complementary to eachofin the process have been identified; mil cells were grown fill .14 generations. 11w original two strands, The two now DNA -DNA. unwinds at the origin to form a molectOes formed consist of one originalreplication fork and expose single-stranded,"N medium, is a nintradioactiVo iso- tope of nitrogen. used to synthesize I)NA strand and its newly synthesized com-sections. plementary strand. This p ()cuss of DNA An enzyme synthesizes a short segment ofDNA Instead of the usual l'N,1 he ''N DNA replication is termed "senticon. rvative. complementary RNA, about 50 nucleotides will be denser, f3,y switching tit -I'N- grown ,long, to part of the exposed single strands ofcells to "N medium for one genefation and DNA, thus forming DNA-ItNA hybrids. then isolating the DNA, followed by CsCI Using ispecific end (called 3') of the RNA density gradient centrifugation, they found segment as a primer, another enzyme initi- the DNA from these cells to be intermediate ates DNA synthesis in a specific direction in density to that of cells grown only in'N. along the single-stranded DNA. This proc- After a second generation, they found that ess results in ti DNA-DNA sectionattachedone-half of the DNA was of intermediate to the RNA- NA hybrid section.. density and one-half of the DNA was'the The printer iNA segment is removed by,density of that of cell grown only on "N. another enzyme leaving the single-strandedThese results are consistent with semi- ru DNA section. conservative replication rather than a model Another enzymsynthesizes DNA com- in which the parent DNA is conserved in- plementary to th sections from which the tact, or a model in which the parent DNA is RNA was rem( ed. This produces tworandomly dispersed between the' progeny DNA-DNA doe le-stranded sections, each molecules. identiCal to.theriginal DNA'section. An enzyme s .als any gaps in tfie DNA strands. The process in steps 1. through 6 is repeated around the circular DNA of bact ria until replication is corilrilitic, resulting in to DNA molecules exactly like the original DNA molecule (Fig. 1). Cairns (1963) used radioactive thymidine to confirm semiconservative replication in bacterialcells.Photographic emulsions placed over slides spread with E. coli cells 016 grown oii'radioactive thymidine showed blackened grafts-f7h*Ore radioactive particles Microbial Genet ics 9 it" 4 PAGE 11 REMOVED DUE TO COPYRIGHT RESTRICTIONS.

4., 7.;

TOPICS AND SUBTOPICS A. FSSENTIAL INFORMATION B.. EI1111CHMENT INI ORMATION O. PRACTICAL ACTIVITIES '' II had exposed the photoemulsion. The repli- . cating DNA was photographed in a configuration that is consistent with semiconservative replication. Cairns J. 1966. The bacterial chromosome. Sci.m. 214:36-44. 1

RNA

2.21 RNA Structure RNA is a single-stranded chain of nucleo- mRNA is a single, nonhelical strand. In Thethree kinds of RNA vary in size. One tides. The nucleotide subunit of RNA differsbacteria, an average mRNA is 900 to 1,500method of measuring RNA size is by an from that of DNA in that ribose replacesnucleotides ong. ultracentrifugetechniquedevelopedby deoxyribose and uracil replaces thymidine. 'Hurwitz J., tio J. J. Furth. 1962. Mes.-1Svedberg. The-sedimentation-velocity There are three major types of RNA:senger RNA, Sci.t.Ain.206:41-49. rnethcid yields a sedimentationoefficient,-- 4 i ' mRNA, transfer RNA I tRNA), and ribo- 1 value for the material being ceidtVugerit somal RNA (rRNA)4 high speeds in a solvent. The sedimentation -1) coefficient is called a Svedberg unit and is denoted by the letter S.

rRNA constitutes up to 65e; of'ribosomal mass.- The bacterial ribosome has two basic subunits: a 505 unit and a 30S unit. These subunits are composed of rRNA and poly- peptides. The intact ribosome has a 70S value. Bacterial ribosomegenerally have 0/\ three different linear, single - stranded mole- culgs of RNA with the following S values: 16$, 5S, 23S. tRNA is of relatively small molei.:.idar weight, 23,000 to 28,000, with 75 to 90 nucleotide subunits. The bacterial.cells contain about 60 different tRNA, species. Eucaryoti cells may .have 100 to 200 differ. ent tHNA's. 1 -'c' tRNA is folded in alspecific.. thaalner, re-! a :,,embling a cloverleaf. At otie4p the "leafs'; of the cloverleaf is a region c4 ed the-adii- codon Jite. Thif; site is thii,ei nuclyidet.: long; at this region the anticodon by` ,ii.wich ---,,,....2). the codon of the mRNA. At arki ei-Aend of the t RNA molecule is an amine d banding' 4 "9 site (Microbial Genetics, Subtopit243. Rich, A., and S. H. Kim. l978. three-dimensionalstructtireoftrdivifer 4 ',13 RNA. Sci. Am. 238:52..12.. a i. . Microbial Genetics-13 /1 tee TOPICS'AND SUBTOPICS A. VSSENTIAL INFORMATION B. ENRICIIMEN'l INFORMATION C. PRACTICAL ACTIVITIES Ettcaryotic ribosomes of the cytoplasm a have four diffeient RNA molecules: 5S, 28S, 7S, 18S. Ribosomes in the mitochondria of eucaryotic cella resemble those of bacteria in S values. Ribosomes from the cytoplasm of eucaryotic cells are larger than those of bacteria. The two subunits are 60S and 40S, and the intact ribosome is 73 to '80S.

9.22 RNA Function The three types of RNA have specific mRNA molecules of etteakvotic cells have Nirenberg and Matt hei (1960) developed . functions in proteinsynthesis. mHNAis a ,rather long half-life when compared witha cell-free system for protein synthesis. In- translated into protein; it is a transitory mol- thoseof procaryoti mRNA moleculescluded in the test system were bacterial ri- ecule coding'for the synthesis of one specificwhich have half-lives of a few minutes. bosiimes, amino acids, ATI', RNA, enzymes . .protein. tRNAmolecules carry amino acids and cofactors (Microbial Genetics, Subtopic to the ribosome. rRNA interacts with spe- 2.33). cific proteins to forma structure called the ribosome. The ribosome functions'as the site or protein synthesis (Microbial Genetics, Subtopic 2.33). ... 2.23 RNA Synthesis RNA molecules are synthesized enzymat- Hybridization techniques have been used Under appropriate laboratory conditions, ically, with one strand of DNA as the tem- to isolate specific genes. In fact, tNs tech-RNA molecules can form double-stranded plate (transcription). Specific genes code for nique conveniently selects a gene on thestructures (hybrids with the DNA sequences' each kind of mRNA, tRNA, and rRNA.DNA for isolation and study. from which they were transcribed). The hy- mRNA synthesis represents the first step in bridization process involves base pairitig be- protein synthesis (Microbial Genetics, Sub- tween the RNA nucleotides and the DNA topic 2.33). nucleotides, adenine on the DNA strand pairing with uracil on the RNA strand. Certain antibiotics inhibit RNA synthesis. Actiponlycin D binds to DNA so RNA polymerase does not complete synthesis of RNA. Rifamycins inhibit bacterial RNA po- L lymerase activitS/. Hfilt. 1974. How actinomycin binds to DNA. Sci. Am. 231:82-91.

2.3 Protein

2.31 Protein Function A protein or polypeptide is a 'near chain The prim'ary structure of a protein mole- Amino acid sequences of proteins can be of amino acids joined together by peptide.cule is the sequence of amino() acidi determined in laboratories equipped Aiith bonds. Proteins have unique, three-dimen- Kendrew, J. C. 1961. 'the three-dimen-sequenators and amino acid analyzers. The sional structures determined by their aminosional structure of a protein. Sci. Am. 205: three-dimensional structure results from in- acid sequences. 96-410. tramolecular bonding, both covalent and 430 Microbial Genetics-15 401 C. PRACTICAL ACTIVITIOS A. tiSENTIAl. INFORMATION 11. ENRICHMENT INFORMATION TOPICS AND SUBTOPICS noneovalent. In addition, in Home proteins two or more plypoptideichains.are associated to form it IMIC1101101 protein.

2,32 Protein Function Proteins function'as enzymes and as structural elements. All the properties of a cell are determined by its proteins: The two processes,are separated by a ni- EleCtron micrographs of procaryotes havo 2.33 Protein Synthesis ' Genetic information flows from the nucleotide sequence of the DNA to the nucleo-clear men1brane in eucaryotes. Transcrip- shown the two processes of transcription and tide sequence of mRNA to the primaiytion is a nuclear process; translation occurstranslation of the same gene occur almost structure of the protein. The first part of the on the ribosome. simultaneously. process is transcription; the second partis Nirenberg and Mntthei (1961) developed a cell-free system to synthesizepolypeptides. translation (Fig. 2). This system included ribosomes, amino. acids, and a synthetic RNA polymer of known composition used as mRNA. A synthetic RNA With only uracil as the base . coded for a polypeptide of only phenylala; nine. DNA is transcribed by RNA polymerase 2.331 Transcription The mRNA molecule is synthesized complementary to a gene on one strand of DNA. vizyme which has several subunits.Oneof 4,1 Transcrigtion begins and ends at specificthe subunits, the sigma factor, is responsible sites on the DNA. A sequence of three basesfor recognizing the specific DNA site where of the mRNA is called a codon and specifies transcription is initiated. For some genes, one amino acid.. this site is on. one strand' of DNA, and for other genes, this site is on the other strand. In eucaryotes, there are several RNA po- )' lymerases which transcribe different kinds of RNA. The complete genetic code can be found in most biochemistry texts.

Translation of mRNA involves four major 2.332 Translation' The mRNA binds to the ribosome, where it is decoded into a sequence of amino acids.steps. Each tRNA molecule functions as an adap- (g Activation of tRNA. Each amino, acid tor by binding to a specific amino acid athas a specific enzyme, aminoacyl-tRNA syn- one end and recognizing and binding to a .thetase, Which covalently liuks a.si::ecific specific codon in the mRNA on the ribo-amino acid to its tRNA. Tfth amino acid- some. As.the mRNA moves alongthe ribo-tRNA combination is termed aminoacyl- some successive codons aretranslated and tRNA. amino acids polymerize in the proper se- (ii) Initiation of translation. A complex is .4 quence (Fig. 3). formed among the first aminoacyl tRNA, the mRNA and the ribosome. 4' r) Microbial GenetilCs--17 ° A '

PAGE 19 REMOVED DUE TO COPYRIGHT RESTRICTIONS.

41,

4) J PAGE 21 REMOVED DUE TO COPYRIGHT RESTRICTIONS, It ACTICAL ACTIVITI 'Ft Pt AND:311113'01'1CH A. Islti;iltINTIAL INIMUMATION 11, 11N11411 INAHNT 11\1 F(IRMATiON 1111) P10111411'1'411( 1011 111 11111111111teida, A aec- ond hliali, to 11w cotlial adjacent to the InitiolIng IItNA. Ao a peptide bond forma enzymatically belveen the Iwo lo1.111. cent amino arida, lilt DINA dis- charges 11 1-111111111U111'111. Additional IliNA molecules bind in riequence to 1111111' 11111111- 111illit/ (41(1011(1 1111(1 IIICk 1111 11111 growing peptide chain in turn. (iv) Termination. The protein chain 10 completed w11on 1 he rihosonte 1141010S II ('0- (1011 W111(11 101110 111I' 110 1111111111 11C1(1(11 non senso(.0(10111. T114.! 111:11V1111011 Of 1 UNA 1111(1 pel111(111 chain elongation. requires energy. Crick, F. II. C. 1962. The genetic code. Sri. Am. 207:66-74. Nirenberg, M. 1V. 1963. 'I'he genet it 1.).(ale II. tici. Ain. 208:50-94 Crick, I'.11. C. 1911(1. The genetic toile III. tici. Am. 215:55-112.

3.0 Organization of Ge- netic Material 3.1 Procaryoti Organiza- tion

3.11 Chromosome The procaryotic chromosome generally A typical bacterial chromosome(Esche- The nuclear region can be seen within the exists as a closed circular DNA molecule. richia soli) has a molecular weight of 3 x cell, both by staining and by phase micros- The chromosome is not surrounded by a a and contains 4.5 X RP nucleotide pairs. copy. nuclear membrane. No structural proteins have been identified. It exists as a very long, looped *(super- The circular eproniosome may 1w visual- coiled) molecule. ized by electron microscopy. The % G+C of. the DNA differs between The chromosome may he isolatedat- species. It may vary from 25 to 70% G+C. tached to the bacterial membrane by deter- The sequence of bases in the DNA may gent lysis of the cell, followed by centrifu- be used as a measure of the relationship of gation. different species. The more identical the sequence, the closer the relationship. DNA replication and cell divisionmay proceed at different rates in babteria. The 4 'L)) 4 `)uJ aveyage number of chromosomes, therefore, Microbial Genetics 23 C. PRACTICAL AC'fIVITIES TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION 43. ENRICHMENT INFORMATION varies with the growth rate of the cell. The faster the cell grows, the greater the number. Plasmid DNkrnay be isolated intact by 3.12 Plasmid A plasmid is arl extrachromosomal, closed The molecular weight of plasmids varies circular DNA molecule. It may be 0.1 to 10% from 10" to >10". equilibrium density centrifugation in a salt- the size of the cell's genonle. Phismidi are dye solution. The closed circular plasmid not essential for cell growth, but provide the DNA separates from the linear fragments of cell with properties that may be useful under the chromosomal DNA. certain conditiong. z. Multiple copies of a plasmid may he pres- Plasmids may be compared in a number ent in a cell of ways. Their molecular weight rhay be determined or their sdquence homology may he compared. They may be cleaved into fragments by enzymes (restriction enzymes) which cut of specific. sites. Each plasmid yields a distinct and reproducible fragment pat tern: Plasmid replication may he under its own control or that of the chromosome Plasmids may contain genes that control their replication and transfer to other cells. They may also contain structural genes for a variety of proteins. Plasmids may be grouped into classes depending on the gene products for which they code: drug resistance, bacteriocin, toxin, degradation of hydrocarbons, and proteins concerned with gene transfer. Clowes, R. C. 1973. The molecule of infectious drug resistance. Sci. Am. 228:18-27 Some plasmids may exist either independently in the cytoplasm or integrated into the bacterial. chromosome Transposons carrying a variety of genes 1.1 "I'ransposon A transposon is a genetic element t DNA / which cannot exist autonomously but may have been characterized. These include drug insert itself at numerous sites eitheranto the resistance(ampiillin, streptomycin, and chromosome or into plasmids in the cell.It kanamycin), toxin synthesis, and /I-galato- may move (jump) from one DNA moleculesidase synthesis. to another within the cell Cohen, N., and J. A. Shapiro. 198t) Transposable genetic elementsSul Am 242:4(1-5(1

3.2 Eu 'ryotic ()rganizat 4 4s_ t,6 Microbial (;erielu.s 25 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES 3.21 Nuclear DNA The majority of genetmaterial (DNA) Eucaryotes contain several basic proteins The chromosome number can be deter- of the eucaryote is located in the nucleus. It(histones) bound to their DNA. There aremined in some cases by staining chromo- is found as multiple structures (chromo- five classes of histones which are very closely somes and counting and can be estimated by somes), each containing a large DNA mole-related in all higher organisms. These help linkage analysis. cule and bound structural and regulatorydetermine the chromosome structure. Acidic proteins. proteins bound to the DNA are involved in the regulation of gene expression. The nucreus may contain one (haploid) or two (diploid) copies of each chromosome. Eucaryotic microorganisms contain about The size of the haploid genome can be 10 times more DNA per ,haploid genomedetermined by measurement of the rate at than/procaryotic microorganisms. which single strands of DNA reassociate. , The kinetics of reassoiation can also demonstrate the presence of multiple copies of some sequences in the genome.

:3.92 Eucarvot lc Organelles Both mitochondria and chlooplasts con- DNA of eucaryota organelles ranges in tain small yNA molecules similar to those molecular weight from -2.5 x 10' to Hr. of prta-arvots Organelle DNA contains genes for rliNA,61 ribosomal protein, and some, but notall. t RNA's. It also contains geiles/for some en- zvnies such as cytochrome oxidase in mitochondria" and rihulose d iphospha te cabox- ylase irk chloroplasts. 4 The circular organization of the DNA, the lack of hound protein, 'and the presence of these genes support tilt. theory that euar- yotic organelles evolved from prat aryotic organisms. Goodenough, U. W,,j, and R. P. Levine. 1970. The genetic activity of mitochondria and chloroplasts. Sri. Am. 223:22-29.

3.3 Viral Chromosomes The genetic information of viruses may be Some viral DNAs contain modified base single- or double-stranded DNA, or single- residues, such as glucosylated cytosine found or double stranded RNA, depending on the in bacteriophage T. viral species. No structural proteins are as- Viruses contain many fewer genes than sociated with the nucleic acid. The nucleic bacteria; their total number ranges from 3 to acid may be either a linear or circular mol- 250. ecule, depending on the virus. Sinsheimer, R. L. 1962. Single-stranded DNA. Sci. Am. 207:109-116.

4.0 Mutation A mutation is an inheritable change in the Mutation events in a population can he Microbial Genetics 27 4JJ 4

TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES nucleotide sequence of DNA (or RNA, ifdistinguished froin reversible phenotypic that is the genetic material)._ changesresultingfromenvironmental changes., Only a few cells in the population in the former .instance and all cells in the -.e latter instance show the new phenotype.

Slane mutants MasKbe detected by,direct .1 Types of Mutations A mutation may change any characteristic Examples of types of mutations include of a cell or virus. Mutations are detected bystructuralloss or gain ofability to produce obskvation, as, for example, a change in their altered phenotypes resulting from a capsule or flagella; change incolonial mor; pigmentation or a change.in growth pattern changes in an enzyme or other protein in the pholoy; drug or virus resistancegrowth in on a differential medium. mutant cell. the ReSence of a drug or virus which kills or The replica-plating, technique developed inhibits the growth of the parent; loss of by the Ledeirtergs particularly useful for catabolic enzymes; nutritionalloss of abil- detecting and isolating mutants which re- ity to synthesize a biosynthetic enzyme quire a specific grcykth factor. A velvete4n which produces a requirement for a partic- pad is used as an inoculating device to trans- ular nutrient or growth factor not required fer colonies in their exact position from-70a by the parenttell (parent is the prototroph plate containing an enriched medium to a and the- nutritional mutant derived from it variety of media lacking particular growth is an auxotr,oph); and conditional lethal factors. Mutants are identified by their fail- defect in enzymes required for macromolec- ure to grow on media lacking a particular ular synthesis (essential end product cannot growth factor. be supplied as in nutritional mutants, and An enrichment technique may be used as the mutant will grow only if certain environ- a first step in the isolation procedure. The mental conditions are present). These mu- cell population is incubated under condi- tants are often temperature sensitive, unable 'tions in which the parent cell type grows ttnd to grow at the higher temperatures at which is thereby killed or eliminated. Under the the parent cells can grow. same conditions, the mutant cell neither grows do! is killed. The remaining cells are 4 then trarisferred to media in which the mu- k tant can grow. Examples of this technique include the penicillin selection method fof bacteria and filtration techniques for mold, Mutations may or may not cause an obi servable change in phenotype, depending on the extent or site of change. Since more tfian one codon may represent a particular amino acid, it is possible to have a 'change in a single base of the DNA molecule resulting in an alternate codon for the same amino acid. It is also possible that slight changes in amino acid sequence in crimmes may have little or no effect on entylne activity and may go undetected.

Microbial Genet ics 29 4 4 ,'3 TOPICS AND SUBTOPICS A. I1SSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACT(SAL; ACTIVITIIS The Ames test 'is based on the reversion 4. 11 Rey ersio n of Mutations Mutants may revert` to their original phe- RestorpOon of the original phenotype may nOtyPe. occur in th.6. following ways: true reversionof mutants '(Microbial Genetics, Subtopics the tield?red nucleotide sequence is restored 4.4). the original, or suppression, a' ttinitation 1 ocrs elsewhere in the gen e that restores *. the original phenotype. T s niay be a mu- -'1 " tation in the same genentrageni's) or in another gene (extragenic). A supprasor rOu- tation usually refers to extragenic suppression.

4.2 Mechanisms of 'Muta- The nucleotide sequence of DNA may be Mutational events involving a change in tion changed by substitution/of one nucleotidea single nucleotide or nucleotide pair are for another or by addition or deletion of onecalled point mutations. or more nucleotides. , Figure 4 illustrates the effect of adding a nucleotide pair, resulting in a'type of mutation referred to as a frameshift mutation. Large deletions are not revertible; small deletions may be Rafertible by suppression.

4.3 Rates of Mutation The frequency with which mutations oc- A "spontaneous mutation" arises with no cur''specific sites on the DNA moleculeintervention on the part of the investigator. differs. Mutation rate expresses the proba-The frequency range for spontaneous mu- bility that a Cell will undergo mutation at atations is 10-,4 to. 10'. The probability of particular gene. An average frequency oftwo mutational events in one cell is the spontaneous mutation at any given gene isproduct of the probability of the two sepa- 10', or one change in ainillion. rate mutations. Because of mutations, not all cells hrising from a single cell are genetically identical. of ultraviolet 4.4 Mutagenic Agents Chemical and physical agents which in- Mutagenic agents are used experimentally The mutagenic activity crease the rate of mutation are called mu- to increase the frequency of mutation; there(UV) light can be demonstrated by irradiat- tagenic agents. is a dose-response effect with any mutagenic ing a bacterial culture (Serratia marces- agent. cens) and then comparing the frequency of mutations iiithw irradiated cultures and r unirradiated cultures with regard to pigmentation and colony morphology. No mutagenic agents ace known which The Ames test, a screetaing,test for poten Cause mutations only in particular genes. tial carcinogens, is based on the fact that most known caminogens are mutagenic agents. Since mutagenic agents increase the rate of mutation, they also increase reversion rates. In the Ames test, histidine-requiring Salmonella mutants are used; the Microbial Genet ics 31

CY5 a.

Parent.Ce11-7-Nonmutant

DNA segment T CA C GA A TK.TGACTIA. mRNA segment AGDGCUUAUACUGAU (transcription) ,.^-.-1 "--." AAsequence ser ala tyr thr asp (translation)

eutantCellInse'rtion of Base Pair DNA segment A T A mRNA segment A G U G A C U U A U A C U D A . (transcription) sC s...... , -...... r,...... " -...... -...... -,s....:-..,-.....,----...... , ser , asp leu t'r. "Nonsense" (translation) AAsequence chain termination Ite-WZ .6 Fm. 4. Frame Aft matalion. 1

Microbial Genetics-33 4J3 TOPICS AND. SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES rate of reversion to prototrophy is measured in the presence and absencaof the chemical being tested. If the chemical is mutagenic, the reversion rate increases. Devoret,.R. 1979. Bacterial tests for potential carcinogens. Sci. Am. 241:4Q-49. Categories of mutagenic agents include the following. (i) DNA base analogs substitute for normal bases and result in an in- \ creased incidenceofincorporationof "wrong" bases. Examples are 5-bromouracil (HI.) and 2-aminopurine (AP). (ii) Certain , chemicals react with I)NA bases in such a way as to change their chemical structure, resulting in a change in hydrogen bonding properties and conseq.u(nt changes in bale pairing, addition or deletion of bases. Ex- amples include nitrous acid, hydroxylamine, monofunctional alkylating agents, such as ethyl methane sulfonate (EMS), bifunc- tional alkylating agentA, nitrogen mustards, mitomycin, and nitrusoguanidine. This last group causes cross-linking betwebn the two I)NA strands, prevents unwinding, and may result in deletion of a segment of I)NA. Acridines insert between bases in DNA en. and c.ausethe addition or deletion of a single base, producing frame-shift mutations. UV radiation causes formation of covalent bonds between two adjacent thymines on the same strand of DNA (thymine dirtier), producing changes in base pairing. In some instances, the actual expression of mutations may depend on the excision and faulty repair of the damaged segment. Deering, R. A. 1962. Ultraviolet radiation and nucleic acid. Sci. Am. 207:135-144: Ionizing radiation (X rays and gamma rays) produces multiple effects on the DNA molecule. Transposons insert into genes, thereby inactivating them (Microbial Ge- netics, Subtopic 3.13). 4.5 Selection of Mutants Changes in the environment may give a An example of natural selection is the Antibiotic-containing media can be used mutant an advantage so that it will grow increased incidence of penicillin-resistant to select antibiotic-resistant mutants in a Microbial Genet ics 35 4.J3 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES. faster than the parent and replace it in theStaphylococcus strains after the initiation ofpopulation. The replica-plating technique population. The change in environment doespenicillin therapy in the 1940s. This muta- can be used to demonstrate that the'mutants not cause or induce the mutation but selectstion was not induced by penicillin, but pen- were preexistent and not induced by the for preexisting mutants. icillin acted as a selective agent for preexist-antibiotic. ing mutants. A population tends to remain genetically stable in the absence of an environmental change selective for mutants; each mutant is in equilibrium at a frequency in the total population proportional to its mutation rate. Since no environment is completely nonse- lective, cultures maintained over long periods in a laboratory tend to change. Bacteria adapt more readily to new environments than eucaryotic cells because their haploid nature makes possible the immediate expression of mutations, their growth rate is faster, and the large umber\ of cells in a population increases the probal. ity of a wide variety of Mutants.

4.6 DNA Repair Microorganisms have enzymatic mecha- Photo-repair or light repair mechanisms To demonstrate repair mechanisms which nisms to repair certain types of DNA. Some require visible light. This mechanism repairs require light, UV-irradiated cells may be mechanisms require visible light. UV-induced damage and breaks the cova- exposed to light and the frequency of mu- lent bonds joining thymine dimers; it re- tations in cells exposed to light may be com- quires a3 specific enzyme system. pared with that of UV-irradiated cells kept Dark repair mechanisms do not require in the dark. visible light. One type involves specific cellular enzymes which excise the damaged portion of a single DNA strand and other .enzymes which then synthesize a segment of DNA complementary to the normal DNA strand. Rare errors may be made in this repair process.

5.0 Recombination Recombination is the joining together of The frequency of recombination between DNA molecules from two parents, whichtwo genes is proportional- to the distance results in traits in the offspring not found between them. together in either of the parents. The process The location of a large number of genes requires the DNA molecules to he homolo- has been mapped on chromosomes of viruses gous to each other. and bacterial and eucaryotic organisms. In E. coli,over 600 genes have been mapped. Mapping studies of several viral and bac- 5 CI .r) 4j3 M icrobial Genetics -37 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES terial chromosomes have shown that many are circular.

5.1- Viral Genetics Viruses can undergo recombination. infection of the same cell with two viruses, each having a different mutation, results in the release of some virus particles having properties of both infecting particles.

5.11 Virus Replication Lytic Replication of bacterial, animal, and plant Animal and bacterial viruses invade cells viruses is similar. The nucleic acid of theafter binding to specific receptor sites on the virus separates from the protein coat andwalls of the host. This leads to specificity of serves as the template for the synthysis ofthe virus and the cell it infects. Plant viruses viral components. Host cell enzymes func-do not attach to specific receptors on the tion to supply energy and the building blockscell walls of the plant and, therefore, have a of the virus particles. Multiple viral particleswide host range. are synthesized and released from the cell. The replication of virulent viruses can be Some viruses are released ,from the cells Cell lysis can be observed as plaques, or illustrated by the replication of the T-eventhey infect by lysing the cells. Other' virus zones of clearing. bacterial virus (bacteriophage or phage) inparticles are extruded from cells witholit E. coli (Fig. 5). lysing the cell. Stent, G. S. 1953. The multiplicationf bacterial viruses. Sci. Am. 188:36-39.

5.12 Virus Replication Ten's- Some bacterial viruses have the ability to The integrated viral DNA may alter the perate multiply either as a lytic phage or as a partproperties of the host cell. For example, of the bacterial DNA following the integra-Corynebacterium diphtheriae produces a tion of viral DNA into the DNA of the cells toxin only when lysogenized by a specific they invade. Viruses that have the ability tophage. Scarlet fever is caused by a lysogenic replicate in this fashion are termed "tem-strain of Streptococcus pyogenes. Animal perate." The viral nucleic acid (prophage) tumor viruses are also capable of integrating replicates whenever the bacterial RNA rep-their DNA into the host cell DNA. The host licittes. Bacteria carrying a prophage arecells are transformed by the integrated DNA termed "lysogenic." The protein of the virus into cells having many properties that are is not synthesized as long as the viral DNA different from those of the uninfected cells. remains integrated in the host DNA. UnderRNA tumor viruses contain an enzyme that certain conditions, the viral DNA becomescopies the RNA into DNA, which is then excised from the bacterial DNA (induction)integrated into the DNA of the host cell. and virus particles are formed by the same Herpesvirus DNA may become integrate sequence of reactions involved in lytic rep-into the DNA of the host cell after herpes lication. (Fig. 6). infection. Campbell, A. 1976hlow viruses insert Some temperate phages exist as plasmids their DNA into the DNA of the host cell.as part of their life cycle. Sci. Am. 235:102-113. 5 Microbial Genetics-39 501 PAGES 41 & 43 REMOVED DUE TO COPYRIGHT RESTRICTIONS.

53 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES Trinsfer can be demonstrated if the donor 5.2 Procaryotic DNA A part of the bacterial chromosome may Tranafer has been demonstrated in only a Transfer be transferred from dbnor to recipient cellssmall minority of bacterial species. The lackDNA differs from the recipient DNA and by three different mechanisms. The threeof transfer may merely reflect not havingthe mixture of the cells is plated on a me- mechanisms are: DNA-mediated transfor-the conditions necessary to elicit gene trans- dium on which only recombinants will be mation, conjugation, and transduction. Once fer. able to form colonies. inside the recipient cell, the donor DNA may Procaryotic DNA transfer results in the recombine with the recipient chromosomerecombinant remaining haploid, since inte- (Microbial Genetics, Subtopics 5.21-5.23). gration usually occurs quickly after DNA transfer. Occasionally, partial diploids may result if the entering DNA is not integrated.

5.21 DNA-MediatedTrans- Short segments of DNA are released from To take up DNA, the recipient cells must Addition of deoxyribonuclease to the formation donor cells when they lyse. The DNA canbe grown in a certain way that varies fromlysed donor cells prevents transfer by de- often be taken up and expressed by recipientspecies .to species. Such recipient cells aregrading the donor DNA. This experiment cells. termed competent. demonstrates that the donor material is na- Hotchkiss, R. D., and E. Weiss. 1956. ked (unprotected) DNA. Transformed bacteria. Sci. Am. 195:48-53.

DNA transfer occurs when donor and re- 5.22 Conjugation DNA is transferred while cells are in phys- Cells commonly attach to one another by ical contact with one another. means of pill DNA is transferred as a single-cipient cells are mixed together and the cul- Wollmann, E. L., and F. Jacob. 1956.stranded molecule only as long as cells re-ture is not disturbed. If the culture is shaken, Sexuality in bacteria. Sci. Am. 195:109-118.main attached. Transfer of the entire chrom-the cells separate from one another and some in E. coli requires 90 min, and thistransfer stops immediately. occurs very rarely. Conjugation has been observed primarily in gram-negative organisms, although it does occur in the streptococci.

5.221 Conjugation F Transfer Many strains of E. coli contain plasmids The donor cell is commonly termed the If r and r cells are mixed together, the which transfer from donor to recipient cells.male, or Fl" cell; the recipient is the female, F- cells are converted to F:1- cells, which can The most thoroughly studied representativeor F- cell. Transfer of the entire particlethen serve as donors of F plasmids. plasmid is the F plasmid, which codes foroccurs within minutes after mixing r and the synthesis of the. pili. F- cells. F plasmids can be transferred to The chromosome is not transferred whenspecies closely related to E. coli. the F plasmid is transferred.

5.222 Conjugation Chromo- The F particle can exist as an independ- The chromosome is transferred as a linear When a population of Hfr cells is mixed some Transfer ent, self-replicating entity or it can integratemolecule of single-stranded DNA. Differentwith F- cells, the ordered transfer of the into the chromosome. When it integrates,F particles integrate at different sites, anddonor chromosome can be shown by agitat- the chromosome is capable of being trans-this determines the order in which the genesing the cells at various times and plating ferred if the donor cell (termed "Hfr") con-are transferred. The integrated F particle isthem out on appropriate media for demon- tacts an F- cell. A genetic element that canalways transferred last. Only a small fractionstrating transfer of specific genes. Some do- Microbial Genetics-45 504 5'35 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES replicate independently in the cytoplasm orof the recipient cells undergoes recombina-nor genes will be transferred early, others as part of the DNA of the bacterial chro-tionVith chromosomal DNA. later. mosome is termed an "episome." The process of integration and excision isreversible) Cells carrying the F particles can lie de- 5.223 Conjugation F' When the F particle is excisedfrOm the Since only chromosomal DNA adjacent Transfer chromosome, a fragment of the chromosometo the site of integration of the F particletected by mixing them with F- cells. The F may remain attached to the F particle. Thiscan become a part of the F' particle, differentparticle is rapidly transferred throughota: particle with its attached chromosomalF' particles carry different chromosomalthe F population. This can be determined DNA is termed an F' particle. It is trans-genes. by plating on a medium which will detect ferred to F' cells very rapidly and at a high the transfer of the chromosomal genes in the frequency. F' particle. Recipient cells are, transducea-when a ly- 5.23 Transduction The donor DNA is transferred from donor The bacterial DNA takes the place of to recipient cells within the coat of a bacte- some or all phage .DNA inside the proteinsate from, a phage-infected culture of donor riophage. Once inside the recipient cell, thecoat of the pha Thus, the phage is "defec-cells is added to recipient cells. The recipient bacterial cell, the bacterial genes carried in- tive" since it es not have all of the genescells are grown under conditions which min-s side the phage coat undergo recombinationrequired for own replication. imize lysis and are then plated on a medium with the recipient chromosomes. There are which will detect the transfer of genes from two types of transduction, specialized 'and donor to recipient cells. generalized, depending on the bacterial genes that are transferred. I Zinder, N. D. 1958. "Transduction" in bacteria. Sci. Am. 196:109-118.

5.231 Specialized Transduc- Specigized transduction results from the The same phage may transduce different tion transfer of 'a certain group of genes located bacterial genes because the phage DNA may next to the site at which temperate phagebecome integrated at different sites. There DNA has become integrated into the donorare preferred and less-preferied sites for in- chromosomeWpon induction, a piece of the tegration. donor DNA remains attached to the phage The bacteriophage Ilfmbda and P22 are DNA (Fig. 7). specialized transducing phages in E. coli. In many cases, the coat of the phage is 5.232 Generalized Transduc- In generalized transduction, any segment tion of the bacterial chromosome can be trans-,filled completely with bacterial DNA dctl:ep ferred. The usual case involves:a temperatedonor cell. phage which packages a piece of donor DNA inside its protein coat as it goes through its .virulent life cycle. This DNA is transferred to a recipient cell when the phage infects (Fig. 8).

5.24 Plasmid Transfer An entire plasmid, or parts of it, may be Plasmids may be transferred between transferred by the same three mechanismswidely different genera. These include Esch- Microbial Genetics-47 5,,' 5U6 PAGES 49 & 51 REMOVED DUE TO COPYRIGHT RESTRICTIONS. te

PRACTICAL ACTIVITIES 'TOPICS AND tiun'tppico, A. lo,SSENTIAI4 INFORMATION B. ENINIIMENT INFORMATION 1 that operate for the transfer of chromosomalerichia,Shigella,Salmonella,Protean, genes. The plasmic! DNA,however, does notPneadomonan, and Vibrio. have to be homologous to the recipient The increase in the incidence of antibiotic- DNA.- resistant organisms in the hospital environment is due largely to the transfer of plea- 'nide that code for antibiotic resistance into previously sensitive cells. A single plasmid may carry the genes coding forresistance to seven different antibiotics.

Restriction enzymes, which recognize spe- 5.3 Recombinant DNA Recombinant DNA currently refers to two different_DNA molecules joined together in cific nucleotide sequences in DNA, are often vitro. Commonly, a plasmid which can rep-used to cleave the DNA molecules which 5 licate in bacteria is one component of theare to be joined together. It has been possi- molecule. The second component is DNAble to cloneeucaryotic genes in E. cbli and A from any source. The hybrid plasmid is thento produceells of E. colt which synthesize introduced by DNA-mediated transforma- insulin andgrowth hormone. tion into E. colt, where it replicates. Thus, Cohen,. N. 1975.The manipulation, of the foreign DNA introduced into the plas-genes. Sci.m. 233:24-33. mid becomes cloned, resulting in the pro- Foreign DNA can also be clone in duction of many identical copies (Fig. 9). lambda phage. Eucaryotes exhibit' several mating 'pat- Fungi are the most widely used system for EucaryoticRecombine- There are two means of achieving genetic 5.4 terns: they may or may not produce special- genetic analysis of eucaryotes. In the Asco- tion recombination: two haploid gametes, usually mycetes, sexual union occurs within a sac. from different parents, fuse to produce a newized mating structures; and in some, but not Fusion of nuclei is followed by meiosis and individual with a combination of traits fromall cases, they exhibit mating types. A and the products are called ascospores. each parent; or in meiosis. (reduction divi-a in yeast is one of the bestknown. Special sion), a diploid cell divides so that eachfeatures: fungi can fuse so that nuclei with nucleus receives only one member of eachdifferent genetic complements can exist in ,chromosome pair. During this process, whenthe same cytoplasm (heterocaryons). In this the chromosome pair aligns, they may ex-case, they may exhibit recombinationand change pieces. In this way, genes are ar-segregation during mitotic division by a ranged in new combination or recombineprocess Called the parasexual cycle. In some during meiosis. protozoans, differentiated nuclei exist; one type is concerned with expression and cell function and the other is involved with sexual reproduction% Some eucaryotic microorganisms have Because of the way in which the chromo- both haploid and diploid phases in their lifesomes segregate during nucleardivision, cycles. In the haploid phase, the nuclei con-chromosome mechanics have been studied tain one copy of each chromosome; diploidby analysis of the ordered ascospores in Neu- nuclei contain two copies of each chromo-rospora. some and, therefore, of each gene. An orga- 5'39 4- Microbial Genetics-53 5i PAGE 55 REMOVED DUE T6 COPYRIGHT RESTRICTIONS. 4' O. l'ItAOTIOAAOTI MIMS TOPICS ANI) H1111T01108 A, EHNHNTIAIN MATION I 1011 M NM` IMATION Mom can maintain either plume by :eana of mitotic division; each nucleus, whether haploid or dipkild, reproduces exact Opium of itself, Mitotic division occurs with no concomitant recoinhination of genetic material (Fig, 10), By crossing two strains of &Norio which produce difl'erent-colored am:mamma, the arrangement of spores in each ONUS provides immediate linear evidence of the chromosomal events which occurred during meiosis.

An example of an environmental influence Mecha- Independent mechanisms exist for regu- Jacob and Monod developed the operon 6.0 Regulatory theory which provides an explanation foris the production of pigment in Serra tic nisms lating gene expression and the activity of productionoccurs preexisting enzymes. Alteration of the envi-regulation of gene activity (repression, andmarcescens. Pigment only when cultures are incubated at temper- ronment may reversibly alter phenotypeinduction). They also developed the model atures below 37°C. without altering genotype, by which preexisting enzymds in a metabolic pathway are rendered inactive in the presence of the end product of thepathway (feedback inhibition). Regulation of Gene Ac- Repression and induction regulate theini- Regulation of gene activity prevents syn- , 6.1 thesis of needless enzymes. gnergy and ma- tivity tiation of gene transcription. terials are conserved. The functional state of the repressor is determined by the presence or absence of the specific metabolite. An operon is a cluster of genes which' is transcribed as a unit. The operon contains structural genes which code for the produe- tion of several etikymeshi ,5 metabolic path.: way, and an operator i4gion.' Aregulator gene, often located at a distanceffom the operon, produces a repressor 174einwhich, " when activated, can bind 'to, thte operator and block transcripilop of the structural genes by preventing the binding ofRNA polymerase to DNA. The initiation of transcription of all the genes in the operon is thus prevented. Repressor proteins are al- losterib; one site 'attaches to the operator and the ()tiler attaches to a specific metabolite. Operons have been found in many 51 bacteria but apparently are ke in eucary- 513. IOW otes. Microbial Genetics-57 Mit.4!rilt! Mitosis Mitosis Diploid parent spore 21's, zygote Meiosis 'Recombination)\ Fusion Haploid spore Haploidgamete Mitosis

Haploid parent

Flo. W.Generalized eucaryotic life cycle.

Microbial Genetics-59 5' 4 TOPICS AND SUBTOPICS A. ESSENTIAL INFORMATION B. ENRICHMENT INFORMATION C. PRACTICAL ACTIVITIES Ptashne, M., and W. Gilbert. 1970. Ge- netic repressors. Sci. Am. 222:36-44. In the absence of the end product, the When-E. coli is grown in a medium con- 6.11 Repression In end-product repression, the end product of a biosynthetic pathway blocks therepressor protein is inactive and cannot at-taining tryptophan, the organism does not transcription of genes coding for the entiretach to the operator, and the operon ismake the enzymes for the biosynthesis of group of enzymes involved in that pathway.turned on. When the end product of thetryptophan. If tryptophan is not present in pathway is present, it binds to the repressorthe medium, the enzymes for tryptophan protein, changing its conformation, therebysynthesis are produced until the concentra- activating it, so that it can bind to the op-tion of tryptophan synthesized by the cell erator and "turn off" transcription. Theshuts down further enzyme synthesis. functional state of the repressor is determined by the presence or absence of the specific metabolite (Fig. 11).

In inducible systems, the operon is nor- E. colt produces the enzyme fl-galactosid- 6.12 Induction In enzyme induction, the substrate in a degradative pathway triggers the transcrip-mally turned off because repressor protein isase only when lactose is present in the me- tion of genes coding for enzymes in thatbound to the operator. When the substrate dium. pathway. of an inducible pathway is present, it binds to the repressor protein, changing its conformation, thereby inactivating it so that it can no longer bind to the operator. Transcription can then occur. In addition to substrate, cyclic adenosine monophosphate (AMP) and a protein (CAP) are required for transcription (Fig. 12). Repressible systems, such as the synthesis Modification of Enzyme In feedback inhibition, the end product of Feedback inhibition conserves energy and 6.2 of tryptophan in E. colt, also show feedback Activity a biosynthetic pathway inhibits the activitymaterials. of the first enyzme in that pathway. Since Feedback inhibition operates when an eri=inhibition. In the presence of tryptophan, no product of the first reaction isformed zyme is an allosteric protein. When the end the first enzyme in the biosynthetic pathway subsequent enzymes in that pathway willproduct combines with the allosteric site onfor tryptophan synthesis is inhibited by have no substrate and the entire pathwaythat enzyme, its conformation is altered, tryptophan. will be shut down. thus altering its activity (Fig. 13).

5 1'

5 5_ Microbial Genetics 61 PAGES 63 67 REMOVED DUE TO COPYRIGHT RESTRICTIONS.