Part 2 book “Practical manual of medical microbiology” has contents: Bile solubility test, antimicrobial susceptibility testing, brucella agglutination test, treponema pallidum haemagglutination assay, enzyme linked immunosorbent assay, experimental animals, fungal slide culture, medical entomology,… and other contents.
28 Nitrate Reduction Test ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ PRINCIPLE This test detects the presence of enzyme nitrate reductase which causes the reduction of nitrate to nitrite in the presence of a suitable electron donor Almost all Enterobacteriaceae members reduce nitrate Nitrate reduction can be detected by an appropriate colorimetric reagent Requirement Medium Potassium nitrate - 0.2 gm Peptone - gm Distilled water - 1litre Tube in ml amounts and autoclave at 121oC for 15 Test reagent Solution A - Dissolve gm sulphanilic acid in litre of acetic acid mol/lit Solution B - Dissolve 5.0 gm of α-naphthylamine in litre of acetic acid mol /litre immediately before use, mix equal volume of solution A and B to give the test reagent Nitrate Reduction Test 119 Method • Inoculate the medium with test organism • Incubate at 37 oC for 96 hours • Add 0.1 ml of the test reagent to the test culture Result A red color within a few minutes - Nitrate reduction positive e.g.Esch.coli No.red color - Nitrate reduction negative e.g Acinetobacter 29 Hydrogen Sulphide Production Test ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ PRINCIPLE Some gram negative bacillii produce H2S by breaking down sulphur containing amino acids like cysteine or cystine Hydrogen sulphide is demonstrated by its ability to form black insoluble sulphides Method There are different methods and media are used for detection H2S production List of media used for H2S detection Bismuth sulfite agar Triple sugar iron agar (TSI) Lysine iron agar Lead acetate agar Kigler iron agar Deoxycholate citrate agar Hydrogen Sulphide Production Test 121 Method Filter paper method • Prepare a Whatman No.1 filter paper impregnated with lead acetate (10% solution) • Expose the filter paper above culture by placing filter paper between the cotton plug and test tube Result Blackening of the lead paper H2 S positive (Citrobacter, Salmonella No blackening of the lead acetate paper H2S negative (e.g Esch coli, Klebsiella) 30 Urease Test ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ PRINCIPLE Some bacteria, particularly those growing naturally in an environment exposed to urine, may decompose urea by enzyme urease and liberate ammonia Urea →Ammonia + CO2 + Water The production of the enzyme urease is detected by growing the organisms in the presence of urea and testing for alkali (NH3) production by means of a suitbale pH indicator (Fig 30.1) Requirements Medium – Christensen’s urea agar Peptone - gm NaCl - gm K2HPO4 - gm Phenol Red (1 in – 500 aqueous solution) - ml Agar - 20 gm Distilled water - litre Urease Test 123 Figure 30.1: Urease test (For colour version, see Plate 8) Glucose 10% solution - 10 ml Urea 20% solution - 100 ml Sterilise the glucose and urea solutions by filteration Prepare the basal medium without glucose or urea, adjust the pH to 6.8-6.9, sterilised by autoclaving, cool to about 50oC, add the glucose and urea and tube the medium as deep slopes Method • Inoculate the medium heavily over the entire slope surface with the test organism culture • Incubate at 37 oC Overnight incubation Result Colour of the medium changes to purple pink Urease positive (e.g Proteus, Klebsiella) No colour change Urease negative (e.g Esch.coli) 31 Citrate Utilisation Test ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ PRINCIPLE Some bacteria can utilise simple organic salts like sodium citrate or sodium acetate as the sole source of carbon and energy source for growth and an ammonium salt as the sole source of nitrogen During such utilisation CO2 is liberated which makes the medium alkaline Koser’s liquid citrate medium or Simmon’s citrate agar may be used Requirements Medium: Simmon’s citrate agar medium (This is a modification of Koser’s medium with agar and an indicator added) (Fig 31.1) Sodium chloride gm Magnesium sulphate 0.2 gm NH4H2PO4 gm gm KH2PO4 Sodium citrate gm Distilled water litre Agar 20 gm Bromothymol blue 0.2% 40 ml Citrate Utilisation Test 125 Figure 31.1: Simmons’s citrate media (For colour version, see Plate 8) Adjust the pH to 6.8 Sterilise by autoclaving at 121oC for 15 Allow to set as slopes in test tubes Method • Inoculate the slope with a wire loop from a saline suspension of the organism to be tested • Incubate at 37oC for 96 hours Result Citrate utilised Growth with deep blue slant (e.g Klebsiella, Proteus) Citrate not utilised Original green colour and No growth (e.g Esch.coli, Shigella spp 32 Voges–Proskauer Test (VP Test) ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ PRINCIPLE Some bacteria ferment carbohydrates with the production of acetyl methyl carbinol or its reduction product 2, butylene glycol This breakdown product when reacts with Alpha-Naphthol in alcohol in the presence of alkali gives a cherry red colour This test is usually done in conjunction with the methyl red test since the production of acetyl methyl carbinol or butylene glycol usually results in insufficient acid accumulation during fermentation to give a methyl red positive reaction An organism of the Enterobacteriaceae family is usually either MR positive and VP negative or MR negative and VP Positive (Fig 32.1) Requirements Medium : Glucose phosphate peptone water (as for the MR Test) 40% potassium hydroxide 5% solution of alpha –naphthol in absolute ethanol Voges-Proskauer Test (VP Test) 127 Figure 32.1: VP test (For colour version, see Plate 9) Method • • • • Inoculate the liquid medium Incubate at 37% for 48 hours Add 1ml of 40% potassium hydroxide and ml of alpha naphthol Shake well and keep it in the rack for 10 minutes Result Pink or cherry red colour Yellow colour VP Positive (e.g Klebsiella) VP negative (e.g Escherichia coli) Spotters 259 It consists of 2% peptone, lactose, neutral red indicator, and sodium taurocholate Lactose fermenters appear pink in colour and non-lactose fermenters almost colourless or paler CLOSTRIDIUM TETANI WITH TERMINAL SPORES Gram positive bacilli Terminal spores give drum stick appearance to bacilli Spores not take up the stain SUGAR MEDIA TO TEST FOR CARBOHYDRATE FERMENTATION Sugar media are used to test the biochemical reactions of various bacteria These media consist of 1% desired sugar in peptone water, along with appropriate indicator like, Andrade’s indicator A small tube (DURHAM’S TUBE) is kept inverted in the sugar tube to detect gas production Development of pink colour in the medium indicates acid production Bacteria producing Acid only: a Staphylococci b Salmonella Typhi Bacteria producing Acid and Gas: a Escherichia Coli b Klebsiella Non-fermenting bacteria: a Moraxella b Alkaligenes GRAM NEGATIVE BACILLI Pinkish, rod-shaped bacteria Short and straight with a discrete arrangement Ex: a Klebsiella b E.coli c Salmonella, etc 260 Practical Manual of Medical Microbiology RABBIT Uses For the production of immune sera such as agglutinating and neutralising sera by intravenous inoculation of antigens In Pyrogen tests For the maintenance of pathogenic strains of Treponema pallidum intheirtesticles In Paul’s test, i.e Production of keratitis by inoculation of Small pox virus into the scarified rabbit cornea Rabbit blood is used in the preparation of blood agar MICE Uses Used extensively for isolation of Toxoplasma gondii Suckling mice are used for isolation of Herpes simplex, Enteric viruses and Arbo-viruses To test the toxigencity of teatanus becci.24 day-old cooked deat culture is inoculated into the root of the tail of a mouse If the strain is toxigenic, there will be stiffness of the tall GUINEA PIG Uses In the diagnosis of Weil’s disease for the isolation of Leptospira icterohaemorrhagiae For the diagnosis of murine typhus, i.e to demonstrate NeillMosser reacton Guinea pig serum is used extensively in complement fixation test as a source of complement To determine the virulence of diphtheria bacilli by intravenous inoculation VDRL (VENEREAL DISEASE RESEARCH LABORATORY) TEST SLIDE This is a paraffin-ringed slide used to perform VDRL test 0.05 ml of inactivated patient’s serum is taken in each ring and a drop of Spotters 261 VDRL antigen is added The slide is rotated at 120 rotations per minute in a VDRL rotator for minutes Then it is examined under the microscope Formation of Clumps indicates that the serum is reactive VDRL test is a rapid, serological slide flocculation test for syphilis WIDAL TEST Uses An agglutination test used in the diagnosis of enteric fever caused by Salmonella spp Antigens used : a ‘O’ or somatic antigen b ‘H’ or flagellar antigen The diagnostic titre is 1: 160 and more Positive agglutination is observed in the IInd week of infection Demonstration of a rise in titre of antibodies, by testing two or more serum samples, is more meaningful ANTIBIOTIC SENSITIVITY PLATE This is a method for testing the sensitivity of different bacteria to various antibiotics It consists of inoculated nutrient agar medium containing different filter paper discs impregnated with known concentrations of various antibiotics After the incubation at 37oC for 18-24 hours Inhibition zones are observed around those antibiotic discs to which the bacteria are sensitive No zones are noticed if the bacteria are resistant to antibiotics CORYNEBACTERIUM DIPHTHERIAE Gram positive bacilli The bacilli are arranged in characteristic Chinese Letter or Cuneiform arrangement, resembling the letters V, N, L, T, etc Non-sporing non-capsulated and pleomorphic 262 Practical Manual of Medical Microbiology UREASE POSITIVE AND UREASE NEGATIVE REACTIONS The production of the enzyme urease by bacteria can be tested by growing the organism in a medium containing urea Christensons’s Medium is used for this purpose Urease decomposes urea resulting in the formation of ammonia, the production of which is detected by change in colour of the indicator, phenol red present in the medium Deep pink colour indicates the production of urease Urease negative bacteria not produce any colour change in the medium Urease positive bacteria a Proteus species b Klebsiella pneumoniae Urease negative bacteria a Escherichia coli b Salmonella DERMATOPHYTE ON SABOURAOUD’S AGAR Colonies are fluffy, cottony with white mycelia Reverse side of the colony is deep wine or brown in colour They are the common cause of tinea pedis, tinea corporis, tinea unguim, tinea barbae and tinea capitis CANDIDA IN GRAM’S SMEAR Gram positive, oval yeast cells Characteristic buds and pseudomycelia are seen Non-capsulate ASPERGILLUS Responsible for aspergillosis It is a fungus having septate mycelia It has long unbranched condiiophore The tip of the condidiophore is vesicle like and possesses bottle shaped sterigmata giving a brush-like appearance Grows well on Sabouraud’s agar Spotters 263 ASPERGILLUS ON SABOURAUD’S AGAR The common saprophytic, contaminant fungi, occasionally causing opportunistic infection The colonies grow more slowly with characteristic blackish pigmentation of conidial heads, an outstanding feature of Aspergillus niger species MYCOBACTERIUM TUBERCULOSIS ON LOWENSTEINJENSEN MEDIUM EUGONIC (luxuriant) growth observed The colonies are dry, raised rough, tough and buff with wrinkled surface un-like that of M.bovis, where the colonies are moist, flat, smooth and white MYCOBACTERIUM TUBERCULOSIS IN ACID-FAST SMEAR (FIG 13.1) Zeihl-Neelsen’s smear showing reddish-Pink acid-fast bacilli which are found singly, in pairs and small groups Non-sporing and non-capsulated LOWENSTEIN-JENSEN MEDIUM This is a special medium used for the cultivation of human type of the tubercle bacilli It consists of beaten egg Malachite green solution, mineral salt solution and glycerol This medium is sterilised by inspissation OVUM ASCARIS LUMBRICOIDES (ROUNDWORM) (FIG 54.9) Fertilised Unfertilised Round or oval Narrower and longer Bile stained Bile stained Surrounded by Contains thinner shell and very little of a Thick, smooth, transparent shell albuminous coat b Albuminous coat, thrown into folds 264 Practical Manual of Medical Microbiology Contains large unsegmented ovum Contains highly refractile granules Floats in saturated common salt solution Does not float in saturated common salt solution OVUM OF ANCYLOSTOMA DUODENALE (HOOKWORM) (FIG 54.6) Oval or elliptical Not bile-stained Surrounded by transparent hyaline shell-membrane Contains segmented ovum with blastomeres Floats in saturate common salt solution Figure 60.5: Hydatid cyst OVUM OF TRICHURIS TRICHURA (WHIP WORM) (FIG 54.7) Barrel shaped with mucous plug at each pole Has a double shell and bile stained Contains an unsegmented ovum Floats in saturated common salt solution Spotters 265 MALARIAL PARASITE (GAMETOCYTE) End products of asexual cycle capable of sexual function in the body of the definitive host (Mosquito) Male cells are called microgametocytes and female cells as macrogametocytes They are spherical or rounded in P vivax, P Malariae and P Ovale and cresent shaped in P falciparum In stained smear, cytoplasm in bluish with red nucleus which is diffuse and lateral in make and compact and peripheral in female MICROFILARIA Larva of filarial worm Causes filariasis It has got a blunt head and a pointed tail Body is covered with a hyaline sheath Somatic cells extend from the head to the tail end MALARIAL PARASITE (TROPHOZOITE) First stage in the erythrocytic schizogony of malarial parasite In stained smear it has signet ring appearance; cytoplasm is bluish and nucleus appears red Trophozite after a period of growth gives rise to schizont HOOKWORM (ANCYLOSTOMA DUODENALE) (FIG 54.6) Small, greyish white and cylindrical Anterior end is bent dorsally giving hook-like appearance Causes ancylostomiasis characterised chiefly by anaemia ROUND WORM (ASCARIS LUMBRICOIDES) (FIG 54.9) Longest intestinal nematode Resembles earthworm Causes ascariasis (round worm infection) Round or cylindrical 266 Practical Manual of Medical Microbiology Male: i ii Female: i ii Tail end curved ventrally Vulvar waist absent Tail end straight Vulvar waist present at the junction of anterior 1/3, and the middle 1/3, of the body WHIP WORM (TRICHURIS TRICHURA) (FIG 54.7) Anterior 3/5 is thin and posterior 2/5 is thick giving a whip like appearance In male, caudal extremity is ventrally coiled Found in vermiform appendix IT causes trichuriasis HYDATID CYST (FIG 60.5) Larva of Echinococcus granulosus is responsible for the development of hydatid cyst This is found in the intermediate host (man) The cyst wall consists of layers of ectocyst (cuticular layer) and endocyst (germinal layer) The ectocyst has the appearance of the white of hard boiled egg and curl when cut Contains hydatid fluid which is responsible for the anaphylactic symptoms in hydatid disease TAPE WORM (FIG 60.6) Flat, long, ribbon like worm with segmented body Two species Taenia solium (pork tape worm) and Taenia saginata (beef tape worm) Body is divided into scolex, neck and strobila Figure 60.6: Beef tapeworm Index ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ A Accidents and spills Accidents in laboratory management Acid-fast staining 41 Albert’s stain 56 method 57 observation 57 requirement 57 Anaerobic cultivation 84 classifications 84 obligate aerobes 84 obligate anaerobes 84 facultative anaerobes 84 microaerophilic organisms 85 McIntosh and Filde’s anaerobic jar 87 source 85 methods of creating anaerobiosis 85 Anti-streptolysin 150 principle 150 procedure 150 requirements 150 B Babes-Ernst granules 56 Bacterial motility 40 Bacterial pathogens and diseases 88 Bacteriological examination of water 233 Eijkman test 238 method of analysis 236 procedure 236 reporting 238 standards 238 water sampling 233 dug well 235 reservoir 235 tap or pump outlet 235 Bacteriological media 69 preparation and checking 70 blood agar 74 blood tellurite agar 75 buffered glycerol saline 75 chocolate agar 74 glucose broth 71 Loeffler serum medium 75 nutrient agar 71 nutrient broth 71 types 69 differential and selective media 70 enriched media 69 ordinary culture media 69 Bile solubility test 133 principle 133 procedure 134 requirements 133 result 134 Biochemical tests 106 Biohazard waste management 33 Biological safety cabinets Brownian movement 40 Brucella agglutination test 148 materials required 148 principle 148 procedure 148 Burial 36 268 Practical Manual of Medical Microbiology C Capillary tube 40 Catalase test 110 method 110 principle 110 requirements 110 result 111 Cell wall theory 52 Citrate utilisation test 124 method 125 principle 124 requirements 124 result 125 Cleaning of glassware 62 chromic acid cleaning 64 cleaning of pipettes 66 new glass wares 62 used glasswares 62 Clinical materials for microbiological investigations 95 blood for serological tests 101 materials for mycological investigations 102 hairs 102 materials from systemic fungal infections 102 nail 102 skin scrapings 102 swabs from mouth and vagina 102 materials for sterility tests 105 specimen for bacteriological investigations 96 blood for culture 96 cervical and vaginal swabs 100 conjunctival swab 100 CSF 99 ear swab 99 nasal, nasopharyngeal and oral swabs 99 pus 100 rectal swab 99 serous fluid 100 sputum 99 stool samples 99 throat swab 99 urethral swab and prostatic fluid 100 urine 96 specimens for acid-fast bacilli 100 CSF 101 faeces 101 gastric lavage 101 laryngeal swab 101 pleural and peritoneal fluids 101 pus from cold abscess 101 sputum 100 tissues 101 tissues smear for lepra bacilli 101 urine for tubercle bacilli 100 specimens for anaerobic culture 101 blood 101 prostatic fluid 101 pus 101 serous fluid 101 urine 101 specimens for parasitological examination 102 blood for microfilaria 102 faeces 102 specimens for virological investigation 103 Coagulase test 135 principle 135 procedure 136 result 136 Compound microscope 18 illuminating parts 21 electric bulb 22 filter holder 21 iris diaphragm 21 mirror 21 sub-stage condenser 21 Index 269 magnifying parts 20 eyepieces 20 objectives 20 mechanical parts 18 adjustment knobs 19 base 18 limb 19 revolving nose piece 20 stage 19 CRP screen latex agglutination slide test 152 interpretation of results 154 qualitative estimation 153, 154 semiquantitative estimation 154 principle 152 procedure 153 quality control 154 requirements 153 specimen 153 Culture of fungi 179 culture media 179 Cytoplasmic theory 52 Czapek’s dox agar 180 D Diseases of 103, 104 central nervous system 103 eye 104 respiratory tract 104 skin 104 Disposal options 35 Durhams’s tube 259 E ELISA 165, 169, 170 antibody detection 165 procedure 167 regents 166 antigen detection 169 procedure 169 reagents 169 types 170 competitive 171 inhibition 171 stick 171 Experimental animals 172 guinea pigs 173 mice 174 rabbits 172 rats 174 F First aid box 10 G Germ tube test 195 method 196 observation 196 requirements 195 Gram’s stain 41, 49 bacilli 51 observation 51 procedure 49 requirement 41, 49 H Hand washing 12 method 12 procedures 13 Hanging drop preparation 37 observation 39 procedure 38 requirements 37 Heat fixed smear 44 Hugh and Leifson’s test 108 method 109 requirements 108 Hydrogen sulphide production test 120 list of media used for H2S detection 120 method 120 method 121 270 Practical Manual of Medical Microbiology principle 120 result 121 Hypodermic syringes and needles I Identification of bacteria 106 Identification of fungal isolates 184 methods 184 macroscopic examination 184 microscopic examination 185 morphology of identification 185 aspergillus 191 Candida albicans 186 Cryptococcus neoformans 185 epidermophyton floccosum 190 microsporum canis 189 mucor 194 pencillium 193 rhizopus 193 trichophyton rubrum 187 Indian ink staining 47 observation 47 staining procedure 47 Indole test 130 method 132 principle 130 requirements 130 result 132 Inoculation of culture media 77 aseptic technique 77 incubation of cultures 82 inoculation of fluid media 82 inoculation of media in petri dishes 79 inoculation of slopes 81 inoculation of stab media 82 labelling of inoculated media 82 temperature of incubation 83 wire loop 79 L Laboratory access Laboratory biosafety levels Laboratory clathing Laboratory facilities in BSL-2 Lactophenol cotton blue mount 177 method 178 requirements 177 Leishman’s stain 59 method 59 observation 61 requirements 59 M Malaria 201 lab diagnosis 201 blood smear 201 examination of stained slides 205 examination of thick film 206 examination of thin film 206 Giemsa staining 205 malaria parasite 206 thick smear 202 thin smear 205 Medical entomology 220 cyclops 231 housefly 226 diseases transmitted 226 morphology 227 itch mite 229 morphology 230 louse 228 morphology 229 mosquitoes 221 aedes mosquitoes 223 anopheles mosquitoes 222 culex mosquitoes 223 mansonia mosquitoes 224 Index 271 rat flea 227 diseases transmitted 227 sandyfly 225 morphology 225 ticks 230 hard ticks 230 soft ticks 231 tsetse flies 226 morphology 226 Methyl red test 128 method 129 principle 128 requirement 128 result 129 Methylene blue staining 45 requirements 45 staining method 45 uses 46 Microbiology of milk 239 common test 239 mehtylene blue reduction test 239 phosphatase test 240 test for colifrom bacteria 239 turbidity test 240 viable count 239 Micrometry 24 calibration of eyepiece micrometer 25 measuring object 26 method 24 requirement 24 eyepiece micrometer 24 stage micrometer 24 Microscope 17 types 17 compound 17 dark ground 17 electron 17 fluorescent 17 inverted 17 phase contrast 17 polarizing 17 simple 17 ultraviolet 17 Modified Kirby-Bauer method 140 quality assurance 144 requirements 140 antibiotic discs 140 Mueller-Hinton agar 140 swabs 141 turbidity standard 141 results 143 Motility of anaerobic bacteria 40 N Neisser’s staining 56 Nitrate reduction test 118 method 119 principle 118 requirement 118 result 119 O Oxidase test 112 methods 112 dry filter paper method 113 plate methods 113 wet filter paper method 113 principle 112 requirements 112 P Parasitological examination of faeces 210 collection of faecal sample 210 macroscopic examination 211 microscopic examination 212 concentration techniques 212 procedure 212 saturated salt flotation technique 215 transportation of samples 211 pH in microbiology 67 comparater and capillator methods 68 methods used for measurement 67 pH indicator papers 68 272 Practical Manual of Medical Microbiology Pipetting Plastic pasteur pipettes 65 Potassium hydroxide wet mount 175 method 175 observation 176 requirements 175 slide KOH mount 176 Preparation and fixation of smears 41 requirements 41 procedure 42 drying of smears 42 fixation of smears 43 labeling of slides 44 making of smears 42 Preparation of glass pasteur pipette 65 Protozoan parasites 37 Q Qualitative serum test 158 Quantitative serum test 158 R Riddle’s method 182 method 183 observation 183 requirements 182 Ryle’s tube 101 S Sabouraud’s dextrose agar 180 Simmons’s citrate media 125 Sperm motility 37 Spotters 253 agar agar 255 antibiotic sensitivity plate 261 aspergillus 262 aspergillus on Sabouraud’s agar 263 blood agar 255 candida in Gram’s smear 262 candle jar 253 centrifuge 254 corynebacterium diphtheriae 261 dermatophyte on Sabouraoud’s agar 262 gelatin 255 glucose broth 254 gram negative bacilli 259 gram-positive bacilli 257 guinea pig 260 haemolysis on blood agar 256 hookworm 265 hydatid cyst 266 Lowenstein-Jensen medium 263 MacConkey’s agar 258 malarial parasite 265 McIntosh and Filde’s jar 257 mice 260 microfilaria 265 Neisseria gonorrhoeae 257 nutrient agar 255 ovum of ancylostoma duodenale 264 ovum of Trichuris trichura 264 platinum wire loop 253 rabbit 260 Robertson’s cooked meat medium 257 round worm 265 Sabouraud’s glucose agar 255 seitzfilter 254 sugar media to test for carbohydrate fermentation 259 tape worm 266 testing of autoclaves 253 thioglycollate medium 257 throat swab 254 urease positive and urease negative reactions 262 VDRL 260 whip worm 266 widal test 261 Sterilization 27 autoclave 27 Index 273 gravity displacement 30 pressure-cooker type 28 boiling 32 disinfection 32 heat 27 hot air oven 31 operating instructions 31 Sugar fermentation test 115 method 116 principle 115 requirements 115 result 117 T Treponema pallidum haemagglutination assay 159 observation 160 principle 159 quantitative test 160 results 160 Tuberculosis 241 lab diagnosis 241 biosafety 246 collection of sputum sample 242 culture 244 grading of microscopy smears 243 microscopy of sputum 241 morphology 241 preparation of smear and Ziehl-Neelsen staining 242 storage and transportation of specimens 242 Tumbling motility 40 U Units 14 SI units 14 prefixes 15 Urease test 122 method 123 principle 122 requirements 122 result 123 Urinary tract infection 247 causative organisms 247 culture 250 screening procedures 250 specimen collection 248 infant and young children 249 men 248 women 249 specimen transport 249 V VDRL test 156 preparation of antigen 157 preparation of serum 157 requirements 156 Virus infections 197 diagnosis 197 embryonated egg inoculation 198 serology 198 tissue culture 198 Voges-Proskauer test 126 method 127 principle 126 requirements 126 result 127 W Widal test 162 observation 163 principle 162 procedure 163 requirement 162 result 163 Z Ziehl-Neelsen’s stain 53 requirements 53 procedure 53 observation 54 ... Trimethoprim 25 sulfamethoxazole 20 -26 27 -35 22 -28 29 -37 19 -26 22 -30 24 -30 22 -30 19 -27 18 -22 17 -28 18 -24 26 -37 19 -28 19 -29 19 -26 24 - 32 E.coli (ATCC 25 922 ) P aeruginosa (ATCC 27 853) 19 -26 16 -22 29 -35 15 -21 ... 29 -35 15 -21 21 -27 30-40 19 -26 22 -28 20 -25 28 -35 24 -30 18 -25 18 -26 21 -28 24 - 32 18 -26 17 -23 25 -33 16 -21 25 -33 19 -25 - Quality Assurance in Susceptibility Test The precision and accuracy of the test... negatives Staphylococci 14-16 14-16 20 -22 10-13 18 -20 11- 12 11- 12 18 -20 15-19 >17 >29 >17 >16 >23 >14 >16 >21 >13 >13 >29 >15 >18 >21 >15 >20 COMBINATIONS 20 /10 µg 20 20 /10 µg 10/10 µg