Manual of ce Practical Microbiology ABLA M. EL-MISHADTO my parents {, Howaida, Afimed and Khali and to Gala ELSaids. Also to my grat d children; Nora, Ali, Hanna, Asmaa, Omar, and Hussien. First Edition June 1978 Second Edition September 1984 Third Edition October 1987 Fourth Edition August 1991 Fifth Edition October 1995 Sixth Edition September 1998 Seventh Edition September 2002 Eighth Edition September 2006 ALL RIGHTS RESERVED TO THE AUTHORFORWORD TO THE FIRST EDITION I had the pleasure of reviewing the present Manual of Practical Microbiology. I was impressed with the systematic approach to different topics, the clarity of illustrations and the wealth of figures and photographs included. I believe that the book will be of extreme value to medical students, general practitioners, postgraduate students in clinical specialties and for every laboratory worker who needs a quick simplified reference to modem microbiological techniques. I congratulate the author, Dr. Abla El-Mishad, for the effort she spent in preparing this book. She has always been capable of producing good work. By Late Yehia El-Batawi, M.D., Ph.D. Professor of Microbiology Faculty of Medicine Cairo University 1978preface the Eighth F dition rela seven editions of the Manual. of Pr, ane. fiat i / a e with the eighth one tical to continu vat is to provide the medical studen crobiologist and those working in labora the different techniques used | th success Of m y encourdy aim of the cian, the 1 escription of postgraduate physi a with cl illustrated ¢ microbiology: The first part of the manual deals with Veto ee Used for eaittic: »f micro-organisms. A chapter on dia, fe ion and Lane oactiiees he second part describe a y rhe third part discusses the essential = aa a nN bnHinention of different species of pa the paieales used for diagnosis of ineaie ee are described, 4 chapter on examination of clinical specimens 18 ineluces. For a complete systematic approach of the pathogenesis, prevention, and treatment of diseases refer to the "Manual of Microbiology ang Immunology" volume I and IL by the same author. The material in this manual is the outcome of my experience in teaching in the Microbiology and immunology Department Cairo University, Photographs were prepared for microscopic slides and culture material used for demonstration. The manual is particularly helpful for medical students in the practical classes and is complementary to lecture courses. This new edition has been completely rewritten and updated. Recent bacteriological methods have been added and illustrated. Some photographs have been changed and new ones were added. _ Towea particular debt to late Prof. Dr. Yehia El-Batawi not only for writing the foreword to the first edition but for my early years of training under his supervision, He inspired a whole generation of medical microbiologists. Many thanks are due to all my colleagues for theif Suggestions and cooperation. 7 aed . oa oan have to pay special tribute for his eras nen encouragement. 1am indebted to my family, to whom I owe Le a ae this manual. Nabarawy ie tlt ane seit Dr. Fakhry Ghobrial and Mr. Yehia Hl ssistance in the production of illustrations. | thank Dr. Gillies, Dr. Dod , Dr, ds and Churchill Livi ion a five figures from "Bacteriology ey sc cultiv molecular biology ¢ ABLA M. EL-MISHAD Cairo 2006Chay 1 10 11 12 CONTENTS Part |— GENERAL METHODS Laboratory Safety Measures... Specimen collection and transport. Microscopy Morphology of Bacteria Preparation and Interpretation ofa ‘Gram Sta ained Smear. Preparation and Examination of a Sputum Sample... Stained by Ziehl-Neelsen for Tubercle Bacilli (TB). Methods of Cultivation of Bacteria Culture Media... Anaerobic Cultivation. Methods of Isolation of Bacteri: Identification of Isolated Bacteri Diagnostic Molecular Biology Methods. Antibiotic Sensitivity Tests. Sterilization and Disinfection..... Part I - IMMUNOLOGY Antigen Antibody Reactions Agglutination. Precipitation. Complement Toxin Antitoxin Neutralization. Virus Neutralization. Immunofluorescence.. Flow Cytometry... Enzyme Linked juin ‘Sorbent Assay ELISA) Radioimmunoassay (RIA ee . Immunochromatographic fechuighieans Assessment of Immune Competence.. Part III — DIAGNOSIS OF MICROBIAL INFECTIONS Staphylococci. Streptococci Strept. pneumococciae Neisseria...... Corynebacterium.. page19 20 sbacter and Serr Klebsiella Salmonella Shigella . Proteus Yersinii 2] Pseudomonas. 22 «Vibrigs....» Campylobacter Helicobacter 23 Brucella... es s and Gardnerelle 25 Bordetella........+++++ 26 Bacillus. 27 Clostridium. Cl. Tetan CL. Perfringens.... Cl Botulinum Cl. Difficile 28 Spirochaetes.. Borrell Leptospira. Treponema...... 29 Mycoplasm: Legionella.. 30 Anearobic organisms, Bacteroides, 31 Actinomyce: 32 Mycology........ Candida albicans. Cryptococcus neoformans. Pneumocystis jirove Dermatophytes, 33 Examination of Clinical Specimens Laboratory Investigations of Microbial Infections.LIST OF FIGURES sund microscope 4. The electron microscope .... §, Anatomy of bacterial cell 6, Distribu [ 8. Bacterial spores asisaee uals 9. A mixture of Gram positive cocci and Gram negative bacil 10. Morphologic classification of bacteria = 11. Tubercle bacilli in sputum. Zeihl-Neelsen stain .- 12. TSL agar slopes .. 13. XLD plate ....... 14, Anaerobic GasPak sy: 15. Robertson cooked meat medium, 16. Plating out techmique ........ 17. Blood agar showing complete and partial haemolysis 18. MacConkey, showing a lactose and anon-lactose fermenter 19, Indole production test . ee 20. Urease test ..... 21. Oxidase test 22. Catalase test. 28. Plasmid profi 29. Antibiotic sensitivity plate . 30, "E" test .....0----+ 31. The simple autoclave 32. The steam jacketed autoclave 33. Vacuum filter ..... 34. Syringe filter .. 35. IgG molecule . 36. IgM molecule 37. IgA molecule . 38, Zone phenomenon 39. Slide agglutination 40. Erythroblastosis foet 41. Antiglobulin Coomb's test 42. Coagglutination .. eae aaairnna 43. Virus haemagglutination and haemagglutination inhibition . 44. Agar gel diffusion (Ouchterlony) Grouping of Streptococe1 Pageal immuno-diffusion. Deter ation test scence technique; direct and inc nemal antibody test 48. Immunoflu 49, Fluorescent t 50. Flow cytomeiry 51, ELISA indirect method $2. ELISA double antibody technique PSA saesueeey = seiseeeees 54. RIA curve — determination of T 55. Immunochromatographic (IC) t 56. E-Rosette 57. Delayed hypers: 58. Staph.aureus on agar 59, Staph. aureus on blood agar .. 60. Coagulase test — tube method 61, Phage typing of staphylococci ations Staphylococci in pus _B. Staphylococei in culture . Strept. pyogenes on blood agar. 64. Viridans streptococci on blood agar 65 A, Streptococci in pus B. Streptococci cultur 66. Antistreptolysin O titration 67. CAMP test. prerettaee 68. Blood culture technique . 69. Pneumococci in animal tissues . 70, Bile solubility test . 71. Optochin sensiti 72, Capsule swelling reaction 73. Gonococci in pus ....... 74, Diphtheria bacilli by Gram stat 75. Diphtheria bacilli by methylene blue stain 76. Diphtheria colonies on blood tellurite medium . 77. Diphtheroids, by Gram stain i 78. TB growth on Lowenstein — Jensen medium . 79. TB by fluorochrome staining................. 80. Methods of diagnosis of tuberculosis ... 81. Myeo. Leprae by modified Zeil Neelsen 82. Esch. coli by Gram stain 83. Esch. coli on MacCnokey 84. Biochemical reaction of Esch, coli 85. Klebsiella pneumoniae in animal t sues fee i aed lactose fermenting colonies of KL pneumoniae. » Pale colonies of salmonella on MacConkey .. 88. Biochmeical reactions of 5 Typhi Vili 61 63 65 66 67 69 69 70 70 7 2 3B % 16 7 8 79 80 81 82 83 84 85 86 87 87 88 91 92 93 94 95 96 97 98 101 102 103 103 105 106 106 10789 90. 91 92. Vibrio c 93. V. chole colonies on TCBS 94. String tes 95. Clue cells coated with G. vaginaiis 96. Anthrax in animal tis 97. Anthrax spores 98. Anthrax culture by G 99. Cl. tetani 100. Commensal spirochaetes, by Fontan: 101. Borr. vineentii and fusiform bacilli, Vincent : angina 102. Borr. recurrentis in blood film. Leishman stain .. 103. Tr. Pallidum haemagglutination last 104. Mycoplasma colonies by Dienes stain 105. P. melaninogenicus on blood agar.. 106. Sulphur granules .. eres 107. Actino. israelii Gram stain. oa 108. Yeast cells and pseudohyphae . 109. Candida by Gram 110, Germ tube . 111. C. neoformans by India ink 112. Pneumocystis jirovici .. 113. Branching hyphae among epithelial cells . 114. Ectothrix in hair ....... 115. Endothrix in hair .... 116. M. audoinii colonies 1 113 114 11s 116 116 122 124 125 126 127 132 133 134 138 139 142 143 143 145 145 146 146 146 148 148 148 148CHAPTER 1 LABORATORY SAFETY MEASURES In a microbiology laboratory many experiments and procedures involve handling of living pathogenic micr These may cause infection of attending personnel by contact, aerosol inhalation, ingestion, inoculation into eyes (by splashing or contaminated fingers), or injection through the skin (by needle stick injury ot broken glass). Therefore, it is of paramount importance to apply certain safety measures to protect yourself and the surrounding environment:- 1- Avoid unnecessary talking and moving around. Eating, drinking, smoking, mouth pipetting and handling contact lenses are prohibited. 2- Wear a laboratory coat and any other protective clothes if necessary (such as gloves, masks, eye protection and face shields). 3- Disinfect the laboratory areas at the beginning and end of daily work by wiping with 70% alcohol or 1% hypochlorite solution 4- Sterilize the inoculating needle in the Bunsen flame or in the micro-incinerator immediately before and after use. 5- All specimens and cultures should be regarded as potentially infectious and should be placed in containers that prevent leakage during collection, handling, processing, storage and transport. 6- Work should be conducted in biological safety cabinets when working with highly potentially infectious material ¢.g- those likely to contain TB, HIV, hepatitis and haemorrhagic fever viruses. 7- Wash your hands with soap and water frequently specially before leaving the laboratory. 8- Disposal of used material: All used syringes, needles, other sharps and broken glass should be placed ina puncture-proof container until incinerated. Used. slides and covers are placed in jars containing disinfectant (e.g. 2.5% Na hypochlorite). Used glassware e.g. culture tubes and plates are placed in stainless steel containers to be autoclaved before cleaning. 9- In case of a laboratory accident, call the instructor. If any culture material is broken, spilled or scattered on the floor, bench or elsewherc‘usually it is best to flood the area immediately with a disinfectant (e.g. 5-10% Na hypochlorite solution). Leave the material undisturbed for 20-30 minutes; wipe it up and discard the waste. 10- Laboratory personnel should be vaccinated against infectious agents e.g. TB, HBV and rubella for females of child bearing age. Other vaccines may be considered according to the field of work. 11-A biosafety manual should be available for all laboratory personnel.CHAPTER 2 SPECIMEN COLLECTION AND TRANSPORT the - Proper collection of a specimen and its proper transport to the laborator most important steps for proper laboratory diagnosis of any infection ~ All specimens should be properly labeled and should be associated with a request hospital number, type, souree and time antimicrobial treatment taken, the form that includes patient's name, age, s of collection of sample, suspected diagnos! test requested and the referring physician, ~ Proper technique during collection. Avoid contaminating specimens from different sites with nearby flora. - Proper skin disinfection before blood and CSF - A sufficient specimen should be collected from the actual infection site (e.g. sputum not saliva for respiratory infections, cervical and not vaginal swab for gonorrhoea) using sterile collection device and containers. collection. - Specimens should be collected before starting antibiotics, whenever possible. -Numbers and time of specimens collection depends on the type of infection e.g. 2- 3 samples, at intervals, are required for blood cultures during the high fever as in endocarditis; three morning sputum samples for diagnosis of tuberculosis; paired sera separated by 7-10 days for serologic diagnosis - Types of specimens depend on the site of infection. Examples are; deep aspirate of pus or swab from wound infections; sputum or bronchial aspirate for lower respiratory tract infections; throat or nasopharyngeal swabs for upper respiratory tract infections; stool or rectal swab for GIT infections and midstream urine for UTL - All specimens should be rapidly transported to the laboratory preferably within 2 hours. They should not be frozen before culture. They may be refrigerated at 4- 10°C to preserve cells and reduce multiplication of flora (not more than 24 hrs). Specimens for isolation of Sir. pneumoniae, haemophilus and neisseria species must never be refrigerated because cold kills these pathogens (CSF, genital, eye, or internal ear specimens should never be refrigerated). - Specimens should not be allowed to dry. Transport media may be needed in different situations e.g. thioglycollate media for anaerobes, Amie's transport medium for delicate organisms on swabs e.g. N. gonorrhoea, Cary-Blair medium may be used to transport faeces that may contain salmonella, shigella campylobaeter or vibrios. : - All clinical specimens should be considered as potential biohazards and should be handled with care using universal precautions. - Species can be rejected if improperly labeled or received in improper conditions e.g. dry specimens, leaking container, samples in formalin or urine at room temperature for more than 2 hrs etc...CHAPTER 3 MICROSCOPY Bacteria are very small in size, measured in terms of microns (}) Hence they can not be seen by the naked eye. The microscope is used to produce magnified images of these organisms, so that they can be visualized and their motility, morphology and staining properties studied. THE STUDENT LIGHT MICROSCOPE It consists of the stand, the body, the system of optical lenses, the stage and the sub-stage (Fig.1). The stand comprises a heavy foot often horse-shoe shaped, and the limb which is attached to the foot by a hinged joint, so as to set the microscope at a comfortable angle for the observer. The body, which is attached to the limb, carries the optical tube. At the upper end of the tube is the eye piece (it may be monocular or binocular), at the lower end is attached a revolving nose piece which carries the objective lenses. The latter can be moved up and down to focus on the object by using fine and coarse adjustment serews which are attached to the sides of the body. The stage is a platform which accommodates the microscopic slide, on which the object is mounted. It is attached to the limb below the level of the objective lens, and has an aperture in its center to allow light to reach the object. Beneath the stage is the sub-stage, which carries a condenser whose lenses focus light from the illuminating source on the plane of the object. The height of the condenser can be varied by a screw attached to the lower part of the limb. The condenser is screwed high up when using the oil-immersion lens, whereas it is screwed down when using the low or high power objective lenses. Below the condenser is the iris diaphragm which controls the amount of light reaching the objective. Fitted to the tail piece below the condenser is a hinged mirror, which is plane on one side and concave on the other; it reflects light on the object. The plane side is used with the oil-immersion lens and the concave side with the other objectives. The source of illumination may be a built-in lamp in the foot of the microscope which passes a beam of light upwards through the iris diaphragm. A * y= 1/1000 of ammedjustment __ Revolving pose-piece Fine » jective ' oe Iris diaphragm (Fig. 1): The student light microseape Magnification: The magnification of a microscope is the product of the separate magnifications of the objective and the eye piece lenses. The magnification of the eye piece may be 5, 10 or 15 times. The one commonly used in the student microscope is X 10. There are three objectives; the low power which has a focal length of 16 mm (2/3 in.) and a magnifying power about 10 times, the high power which has a focal length of 4 mm (1/6 in.) and a magnifying power of 40 times and the oil-immersion lens which has a focal length of 2 mm (1/2 in.) and 90-100 times magnifying power. -- Magnification of the microscope when using the low power lens: 10x 10 = 100 times. ~- Magnification of the microscope when using the high power lens: 10 x 40 = 400 times. ~- Magnification of the microscope when using the oil-immersion lens: 10 x 90 = 900 - 1000 times. Numerical Aperture: Objective lenses are rated not only by their focal length but also by their light gathering power, which is determined by their angle of aperture or numerical aperture (NA), The NA may be defined simply as; the ratio of the diameter of the lens to its focal length. It is expressed mathematically as NA = a Sin U where n is the refractive index of the medium between the object and 4ns (air.]; immersion oil, 1.5) and 2 U the angle of aperture i.e. ed by the two extreme rays of li it, which starting from the (of the object, reach the eye of the observer (angle GAD, Fig.2). i Sin U=CE /CA So, if other things are equal, the numerical aperture will depend on CE therefore, may have equal A | apertures depending on the diameter of the front lens. The NA of an oil-immersion objective varies between 1,3- 1.4. focal lengths but different numeri (Fig. 2): Diagram to illustrate the numerical aperture Resolution: The resolving power of the microscope is its capacity to distinguish two neighbouring points as separate entities. It is this power which determines the amount of structural details that can be observed microscopically. It depends on the wavelength of the used source of light and on the numerical aperture of the objective lens. The ordinary microscope can visualize objects as small as 0.25 p. In the electron microscope the illumination is provided by a beam of electrons that has a wavelength 1/100,000 times shorter than that of ordinary light. Its resolving power is 250 times better than that of the light microscope; i.e. the shorter the wavelength of the light source, the greater is the power of resolution. THE DARK-GROUND MICROSCOPE This microscope is used to visualize delicate organisms such as spirochaetes of syphilis, which can not be seen in unstained preparations by an ordinary microscope. In this microscope a special condenser is used, it allows only oblique rays to pass to the object (Fig. 3). The rays do not enter the tube of the microscope, and so they do not reach the eye of the observer unless they are scattered by objects (e.g. bacteria). Asa result, the organism appears brightly illuminated in a dark background.oblique roys am showing Condenser ays in the dark- ground microscope. \| Porallel roys PHASE CONTRAST MICROSCOPY It provides a second method for observing unstained living organisms with good contrast and high resolution. It does not show up very small or slender objects, such as spirochaetes, as vividly as dark-ground microscopy, but is more useful than the latter for studying the structure and structural changes in larger microorganisms and tissue cells, The microscope includes special condensers and objectives, by which objects are made to appear in dark-gray contrast against a bright background by causing direct and diffracted rays from them, which differ in their wave phase, to recombine in such a way that they interfere with each other and so reduce the light intensity in the area of the image corresponding to the abject. THE FLUORESCENT MICROSCOPE In the fluorescent microscope, the source of illumination is ultra-violet (UV) radiation and not ordinary light. When certain dyes are exposed to UV, they convert this invisible, short wave radiation into the longer wave-length radiation of visible light, \ (yellow fluorescence), inge R stains nucleic acids and can be used to differentiate RNA (fluorescing orange-red) from DNA. (fluorescing yellow-green) and may be used fo stain viral nucleic acid in infected cells and tissues, i Fluorescein and rhodamine B dyes can be conjugated to immunoglobulins. Such labelled antibodies are used in immunofluorescence techniques for detection of antigens or antibodies (Chapter 12). Under the fluorescent microscope, such stained materials can be seen ae flnarecent Structures in a dark background (Fig. 48),THE ELECTRON MICROSCOPE The electron mic scope consists of a column, at the top of which is mounted the source of illumination, i.e. the "electron gun", which emits electrons from a hot tungsten-wire filament ( 4). The electron beam passes through the evacuated column and is focused on the object (prepared in special ways) by an electromagnetic condenser lens system, instead of the glass lenses used in the ordinary microscope. A final image of the object is formed by electromagnetic lenses on a fluorescent screen at the lower end of the column, and can be viewed through a glass window. Photographs are taken by a camera placed below the screen. The electron microscope has a resolving power of 1 nanometer (nm = 1/1000 of a p). Its high resolving, power is due to the short wavelength of the electron beam. It has, also, a high magnifying power 100,000 times or more The electron microscope is the only means by which most viruses can be seen. It is, also, used to study the fine structure of viruses, bacteria or tissue cells —_ ‘Electron gun Electron beam Electromagnetic condenser Object Electromagnetic objectives Pinal image on fluorescent screen (Fig. 4); Diagram showing the optical system of the electron microscope.CHAPTER 4 MORPHOLOGY OF BAC TERIA The anatomical and morphological features of bacteria will be beetle Jescribed since these features are essential for their laboratory identification, described s' atures ize: — Bacteria are measured in terms of microns (1 = 1/1000 of a millimeter), bacilli vary e.g. anthrax bacillus 410 8 by | Most cocci are | p1 in diameter, 0.3-0.5 p. to 1.5 wand the whooping cough bacillus 1.5 tol.8 by Shape: F ae : Bacteria can be broadly classified according to their shape into: Cocei: spherical or spheroidal. Bacilli: relatively straight rods. Vibrios; definitely curved rods. Spirilla: spiral non-flexuous rods. Spirochaeies; thin spiral flexuous filaments. Actinomycetes: long filamentous branching rods, Arrangement: Cocci may occur in clusters or groups (staphylococci), in pairs (pneumococci), in chains (streptococci), in groups of 4 (micrococcus), or in groups of 8 (sarcina). Bacilli may be separately arranged (salmonella), in pairs (Klebsiella), in chains (anthrax), Chinese letters arrangement and club shaped ends (diphtheria), or in short oval parallel rods (diphtheroids). These arrangements depend on splitting or adhesion of daughter cells, and on the plane of cleavage during cell division, Staining properties: _ Staining reactions are of primary importance for the identification and differentiation of bacteria. The most important differential stains commonly used are:- Pepe een in ie a can be divided into two dornplex wif aypear purple and Gres a fain the methyl violet-iodine dye ; negative bacteria that destain with 95% alcohol; and appear pink due t ini 5 I © counter-staining wi i i mechanism of Gram's staining reaction is not tichtah rae to difference in the 2-Ziehl-Neelsen's acid-fas groups of bacteria which not teadily stainable wit Stain e.g. strong carbol they will resist decolou it stain: This 1 are described as h ordinary Stains, bu -fuchsin with the ay rization with miner: s stain is used for the detection of acid-fast", These organisms are t they. heed exposure to a strong pplication of heat, Once stained, al acids ©.g. H2S0, or HCL 8 aThis property of acid-fastness mz and fatty acids particularly be due to the large amount of lipids colic acid wax which these organisms contain. There are three groups of acid-fast bacteria or bacterial structures: (1) The Mycobacterium group M. tuberculosis, M. leprae and saprophytic acid-fast bacilli. (2) Bacterial spores. (3) Actinomyces and Nocardia 2 Their degree of acid-fastness v: carbol-fuchsin stain when decolov alcohol whereas, lepros tubercle bacilli will retain the red zed with 20% HzSOx or 3% HCI in elect nd saprophytic acid fast bacilli will only withstand decolourization by $% H2SO. or 1% HCl in alcohol, Spores tolerate only 0.25 to 0.5% H2SOx., while in decolourization of actinomyces clubs and some Nocardia species, the strength of acid used is 0.5 to 1% Anatomy of the bacterial cell (Fig. 5): Bacterial cells are surrounded by a rigid cell wall which maintains their shape. Few species do not possess cell wall and are pleomorphic e.g. Mycoplasma and L-forms of bacteria. Under the cell wall is the cytoplasmic membrane which is osmotically sensitive. It selectively allows absorption of nutrients and excretion of waste products. This membrane together with the cell wall are probably involved in determining the response of the cell in Gram stain. The cytoplasm contains the nuclear apparatus which carries the genetic information of the bacterial cell. It contains also, ribosomes and various aggregations of material or granules which vary in composition. The volutin. granules occur in the Corynebacterium group and help in their identification and differentiation. These granules can be stained by special stains. Capsule and extracellular slime: Some bacteria can form a thick gelatinous circumscribed layer outside the cell wall. This is called the capsule. Capsules develop readily when such bacteria are growing in host tissues. They are highly specific chemically and immunologically and thus allow for type-differentiation as in the case of "quellung reaction” for typing pneumococci. Capsules are not usually stained by ordinary staining methods and so they appear as unstained halos around organisms growing in tissues. ; ; Some bacterial species produce an extracellular loose slime. This colloidal material gives surface colonies a mucoid appearance, as in Klebsiella. Flagella and Mot : ; ; With the exception of spirochaetes, motile strains of bacteria possess one or more thin filamentous appendages known as flagella. The distribution of flagella is constant in any species (Fig. 6). Peritrichous and monotrichousFimorive Flagellum CP \ Capsule Cem watt cytoplasmie membrane Nuclear material. (Fig. 5); Anatomy of the bacterial Inclusion cell. granules. aa Mesosome arrangements are the most frequent in pathogenic species. The flagella can be seen by the electron microscope or by the ordinary microscope, provided they are thickened by deposition of special stains on their surface. Flagella are of great practical importance to the bacteriologist since specific anti-sera can be prepared in animals and are used for serological differentiation of various flagellar types. This is the basis of type-identification of the various members of the genus Salmonelia. aw 1. Monetrichate 2, Amphitrichare a 3. Lephoreichare Morile i Non-motile orgonism organism 4, Peritrichare (Fig. 6): Various distributions of flagella. (Fig 7) Bacterial motility _ For diagnostic purposes, the motility in flagellated bacteria is observed either microscopically in a wet mount, or by observing the spreading growth in culture. The latter is done by using a U-shaped tube containing semi-solid agar; the organism is inoculated in one limb then incubated at 37°C for 24 hr. Motile bacteria will grow in both limbs of the U tube giving a diffuse opacity, non-motile bacteria will only grow along the inoculum track (Fig 7). : 10Preparation and examination of a wet mou Th living free for od is used for examining u stained bacterial suspensions in the lis “dt mainly used to study motility of the organism. Procedure: i i it se a clean slide and a clean cover slip. opread a i i 2) Spread a thin film of vaseline around the edge of the cover slip. 3) Plac . 3) Place a drop or loopful of material to be examined in the center of the cover slip. 4) Invert the s f lide directly over the liquid on the cover slip. 5) Press the slide gently onto the cover slip so that the surfaces will adhere. 6) Tum the slide right side up so that the cover slip is upper most. Examine under the microscope with the low and high power. The iris diaphragm should be sufficiently closed and the condenser racked down tell you get good contrast. N.B: True motility must be distinguished from Brownian movement; which is a local oscillatory movement of the bacterium due to bombardment by the water molecules. Motile bacteria change their site and direction. Darting motility is characteristic of V. cholerae. Spores: These are highly resistant forms of bacteria. The increased resistance of spores is due to the hard spore case, their low content of unbound water and their very low metabolic activity. The position of the spore in relation to the body of the bacillus varies, and is characteristic of the species. With Gram stain they appear as unstained (oval or rounded) areas. They can be stained with modified Ziehl-Neelsen in which we decolourize with 0.5% H2SQx. The spores appear pink, and the body of the bacillus blue (Fig. 8). Modified Grams sfoin Zieh! Neelsen | | spherical, ferminol, projecting ovoid, centrale non- projecting (Fig. 8); Shape and position of bacterial spores. | subrerminal non-prosecring ovoid, oO - Freee speresCHAPTER 5 PREPARATION AND INTERPRETATION OF A GRAM STAINED SMEAR Direct examination of properly stained smears is of great v alu ns seen in smears of 1) The type and concentration of different orgar ; rexample, detection fresh clinical material may be sufficient for diagnosis, é " of Gram negative diplococei intracellularly in urethral discharge smears is diagnostic of gonorrhoea. Pneumococci in large numbers in sputum smears are suggestive of pneumococeal pneumonia. Tubercle bacilli in sputum smears stained by Ziehl-Neelsen is a fairly strong indication of tuberculosis, Motile coma-shaped Gram negative bacilli in stools of a secondary case of cholera is diagnostic. 2) The smear may be the only source of information regarding the aetiologic agent if the organism can not be grown in culture; for example “vincent angina” is diagnosed on detection.of Borr. vincenti and fusiform bacilli in smears from mouth and throat ulcers. Again, the detection of lepra bacilli in smears prepared from nasal scrapings or pathologic lesions stained with modified Zichl-Neelsen is the diagnostic method of leprosy. Detection of Tr. pallidum in chancre fluid smears, by dark ground microscopy or by direct immunofluorescence, is diagnostic of primary syphilis. 3) The quick information given by smears may be urgently needed to start treatment of certain conditions before the results of culture appear; for example, smears from cerebrospinal fluid (CSF) deposit may determine the actiologic agent of meningiti = Preparing a smear for staining: Smears may be directly prepared from pathologic material ¢.g. sputum, pus, urine deposit, CSF deposit, or from bacterial Suspensions in the following way: 1- Take anew glass slide, clean it by passing in the flame, then wipe it while warm with tissue paper. If the slide is not properly cleaned, the smear will not spread evenly, 2- Sterilize the inoculating needle in ene the Bunsen flame or micro-incinerator till it turns red hot, 3- When the needle has cooled, dip it bacterial suspension, Rub glass slide, in a circular mi 4- Resterilize the inocul: in the pathologic specimen or the tip of the needle, on the middle of the tion to provide a uniform thin smear. : ating needle before placing it on the bench, 5- Allow the smear to dry in air, or by holding it close to the flame. 12 i iaali ; with the smear-side upper most, over the flame for 2-3 \-fix the preparation to the slide. Do not overheat the smear. he slide should be ‘i ‘ ; ae hould be only warm when you touch it with the back of your rand, To prepare a smear from bacterial growth on solid media e.g, agar slope or var plate, put a drop of water on the middle of the slide using the inoculating loop. With the sterile loop collect bacteria from the surface of the agar by touching (lightly) the bacterial growth. Rub the tip of the needle on the glass slide in the drop of water in a circular movement till you get a homogeneous smear. If the specimen is a swab, roll the swab on the middle of the slide, then proceed as above described. Gram Stain: This is the most important “differential stain” used for diagnostic identification of various organisms. To stain a smear: 1- Cover the smear with crystal violet or methyl violet” for 30-60 seconds. 2- Pour it off and wash with water. 3- Add iodine solution* and leave it to act for | minute, then pour it off and wash with water. 4- Decolourize by adding 95% alcohol or acetone-alcohol and rock the slide from side to side. Pour it off, and reapply till no violet colour comes off the smear. §- Wash rapidly with water. 6- Counter-stain with safranins or dilute basic fuchsin* for 1 minute. 7- Wash with water, then place the slide at an angle to air dry or blot dry. Examination of a stained smear: 1) Set up the microscope facing a good source of light. 2) Rack the condenser up and open the iris diaphragm. 3) Place a small drop of immersion oil on the smear. 4) Put the slide with the smear-side up on the stage of the microscope. ee Gs i ; Basic fuchsin Crystal violet or Methyl violet stains Crystal violet or Methy! violet OB 10 gm Ba: cot 05-1 gm Dissolve in absolute alcotvol 100 ml Distilled water 1 liter 1000 mi Distilled water lodine Soluti Safranin 0,5% in distilled water Iodine 1 gm Potassium iodide 2 gm Distilled water 100 mi