Practical Mice |. CULTURE MEDIA Salient features: 1. Uses: (i) For growing, pathogenic organism, (ii) For preparation of agar (iii) To determine anii sensitivity 2. Composition: Refer Chapter 3 (page ; 3. Sterilisation: By autoclaving. Fig. 8.1 Nutrient agar Salient features 1. Uses: (i) For growing most of i pathogenic organisms (ii) To differentiate hae lytic colonies from » haemolytic colonies (iii) In the _ preparation potassium tellurite blow agar 2. Classified as enriched medium. Fig. 8.2 Blood agar 3. Composition: Refer Chapter 3 (pas* 4. Sterilisation: By autoclaving. Salient features: « testif 1. Uses: (i) For growing int" organisms . act? (ii) To differentiate” fermenter (LF) col") non-lactose fermen'e" " colonies 2. Classified as differential medium. adicate® 3. Contains neutral red as an indie’ Fig. 8.3 MacConkey's agar 4. Composition: Refer Chapter 3 (P* 5. Sterilisation: By autoclaving: 1 or indictFig. 8.4 Chocolate agar Salient features: 1. Uses: For growing organisms such as Neisseria, pneumococcus and haemo- philus 2. Classified as enriched medium. 3. It is prepared by heating the blood agar at 55°C for 2 hours. 4. Chocolate in colour. 5. Sterilisation: By autoclaving. Salient features: 1. Uses: (i) For growing C. diphtheriae and other corynebacteria (ii) For demonstration of meta- chromatic granules. Classified as enriched medium. 2 3. It is a solid medium without agar. 4. Serum is used in its preparation as a solidifying agent. 5. Sterilisation: All the constituents of this medium are sterilised separately and then mixed. Solidification of the medium is done by inspissation. Salient features: 1. Uses: (i) For growing M. tuberculosis and atypical mycobacteria (ii) For antibiotic susceptibility testing of M.tuberculosis 2 Asolid medium without agar. 3. Egg is used in its Preparation as solidifying agent. 4. Malachite green is selective agent. 5. Green colour of the medium is due to malachite 6. M: i: - Mtuberculosis takes 3 to 6 weeks to g70w on this medium.iy! ee Fig. 8.7 Cooked meat broth (CMB) Glucose broth, Taurocholate broth Fig. 8.8 Blood cutture set Fig. 8.9 Automated blood cut Pin Practical Mirin, | Salient features: 1. Uses: (i) For _ Browing organisms (ii) For preservation of Sto cultures of aerobic Organisms 2. It contains cooked meat, t 3. Sterilisation: By autoclaving. | 4. It is also named as Robertson cooked | meat (RCM) broth | Anaerop;. Salient features: 1. Uses: For blood culture in pyrexia of unknown origin (PUO). 2. Itis Specially useful in enteric fever and septicaemia. 3. In each bottle 50 ml of liquid mediun is inoculated with 5 ml of blood under aseptic conditions, Salient features. “ine Teadymade culture medium usel it automated blood Culture system such @s BACTEC system, * Is used for Specimens such as bloot ascitic fluid etc,Salient features: 1. It contains both liquid medium ep Fig. 8.10 Castaneda medium as well as the solid medium. Used for diagnosis of Any growth in liquid subcultured over the sol tilting the bottle in such liquid medium comes: in the conta surface of solid medium. The above method of subculture reduces chances of infection of hazardous bacteria to laboratory personnel. brucellosis. medium can be lid medium by a manner that ct of Salient features: 1. 2. 3. Fig. 8, 11 Sabouraud's dextrose agar (SDA) Uses: For growing fungi. aS medium has acidic pH. ‘ungi can take weeks to grow but ye: ‘ asts grow in 24-48 hours on this mediurnFig. 8.12 Peptone water 2) Fig. 8.13 Glucose broth Salient features: 1. Uses: (i) For growing most Pathogen organisms ie (ii) For making hanging 4 preparation (iii) For testing indole production (iv) For preparation of sug. media (v) Growing bacteria for ang. biotic susceptibility testing 2. Composition: Refer Chapter 3 (page 10, | 3. Sterilisation: By autoclaving. | top | Salient features: 1. Uses: (i) For — growing fastidious organisms such as Str. pyoge* and enterococci. (ii) For growing above bactet for antibiotic _susceptiili testing. 2. Composition: Refer Chapter 3 (pag 3. Sterilisation: By autoclaving. e 10)Il, CULTURE MEDIA WITH GROWTH Salient features: 1. Most common causative agent for throat. 2. Seen as beta haemolytic colonies. 3. Gram staining: Gram positive cocct chains. 4. It is catalase negative (pages 55 and 58) which differentiates it from staphylococcus sore fo. 8.14 Streptococcus pyogenes 7 on blood agar Salient features; 1. Seen as beta haemolytic colonies with golden-yellow pigment. 2. Gram staining: Gram positive cocci in clusters. 3, It is catalase positive (page 55) which differentiates it from streptococcus. 4. Confirmatory test: Coagulase positive (page 56). 8.15 Staphylococcus aureus on blood agar Salient features: 1. Shows swarming on blood agar. 2. Gram negative, motile bacilli. 3. Two species are Pry, is Pr.mirabilis. a 4. PPA and urease tests are positi co Positive Fig. 8.16 Proteus sp. on blood agarSalient features: 1, Bacterial growth shows green diffu pigment. le 2. Gram negative, motile bacillus. 3, Oxidase positive (page 54). 4, Common cause of nosocomial infection, Fig. 8.17 Pseudomonas aeruginosa on nutrient agar Salient features: 1. Shows lactose fermenter, non-mucoid colonies. 2. Gram negative, motile bacilli. 3. Indole positive, urease negative (page 90). 4. Causative agent of urinary tract infection (UTI) and diarrhoea. Salient features: 1. Shows lactose _ fermenter, mucoi colonies. cd 2. Gram negative, non-motile caps rae bacilli. 3. ae positive, indole negative ge 90). 4, Catwativg agent of urinary tract infec" Pneumonia. Fig. 8.19 Klebsiella sp. on MacConkey's agar |ed Salient features: 1. Non-lactose fer! 2. These may be motile/ negative bacilli (refer menter (NLF) colonies: non-motile, Gram Appendix III). fig 6.20 Non-lactose fermenter (NLF) * colonies on MacConkey’s agar Salient features: 1. Seen as black coloured colonies. 2 Potassium tellurite blood agar selective medium for C. diphtheriae. 3, Other bacteria which can grow as black colonies on this medium - Staphylococcus aureus. is a Fig. 8.21 C. diphtheriae on potassium tellurite blood agar Salient features: ‘Mycobacterium tuberculosis is an acid-fast bacillus (AFB). It takes 3 to 6 weeks to grow on this medium. Colonies are rough, tough and buff coloured. Growth is eugonic. . Causative agent of tuberculosis. -e oN os » Fig. 8.22 Mycobacterium tuberculosis 0” Lowenstein-Jensen (LJ) mediumeS UCal Mic, Salient features: 1, It is a Gram positive yeast. 2. Confirmtory test: Germ tube positiy. 3. Opportunistic fungus. 4, Causative agent of oral tha, vaginitis. Fig. 8.23 Candida albicans on sabouraud’s dextrose agar (SDA) Salient Features: 1. Aspergillus fumigatus is an opportunistic fungus. | 2. This fungus grows as green coloured | colonies on SDA medium. | 3. Diseases: (i) Aspergillus asthma (ii) Bronchopulmonary aspergillosis (iii) Aspergilloma | (iv) Invasive aspergillosis | (v) Mycotic keratitis (vi) Sinusitis 4, Since Aspergillus fumigatis is laboratory —_ contaminant, isolations may be of help to pathogenic role. a common" repeated acertain its Fig. 8.24 SDA with growth of Aspergillus fumigatus89 Salient Features 1. Aspergillus niger is an opportunistic fungus. 2. This fungus grows as black coloured colonies on SDA medium. 3. Diseases; (i) Otomycosis (i) Mycotic keratitis 4. Since Aspergillus niger is a common laboratory contaminant, repeated isolations may be of help to acertain its pathogenic role. Fig. 8.25 SDA with growth of Aspergillus niger ll, BIOCHEMICAL REACTIONS Salient features: 1. Pink colour of the solution is due to acid production by fermentation of glucose. . Durham’s tube shows gas production if present. Indicator used: Andrade’s indicator. Examples: All. ~— members — of Enterobacteriaceae are glucose fermenters with/without gas production. vn ee Fig. 8.26 Glucose with Durham's tube— Salient features 1. Principle: It is used to J ability of an organism to th oa” ific carbohydrate to a ee and gas. i 2. Indicator used: Andrade’s indicatoy solution of acid fuchsin to hig, © added sodium hydroxide). © * 3. Pink colour of the solution is due 4, acid production by fermentation og carbohydrates. Durham's tube shoys gas production, if present. ‘ 4. This test is used to identi Fe 827 Cartonydate ementshon GSS negative bacilli such one Klebsiella sp., Salmonella sp., Proteus etc. Salient features: 1. Principle: To determine the ability of an organism to produce an enzyme ureast which splits urea to ammonia. 2. Name of the medium: Christensen’s urease medium. 3. Indicator used: Phenol red. 4, Ammonia produced from urea makes the medium alkaline and thus pheno! ted indicator changes to pink/red # colour. 5. Positive urease test: Pink colour. Negative urease test: Pale yellow colou! 6. Examples of urease positive id Klebsiella sp, Proteus sp, Y°™ enterocolitica, Helicobacter pylori. Negative Postive Fig. 8.28 Urease test Fig. 8.31||; | aac Fig. 8.27 Carbohydrate fermentation tests Negative Positive Fig. 8.28 Urease test pr » “stele, Salient features: 1. Principle: It is used to. deter, ability of an organism to jen specific carbohydrate to Produce" # | or acid and gas, iy Indicator used: Andrade’s in solution of acid fuchsin to added sodium hydroxide), 3. Pink colour of the solution is dy. acid production by fermentation ‘ carbohydrates. Durham's tubs shows gas production, if present, 4 This test is used to identify. son, Gram negative bacilli such as Esc.) Klebsiella sp., Salmonella sp., Proteus y etc. N icatoy Which 5, Salient features: 1. Principle: To determine the ability of an organism to produce an enzyme ures which splits urea to ammonia. Name of the medium: Christensen’s urea: medium. . Indicator used: Phenol red. Ammonia produced from urea make the medium alkaline and thus phen ted indicator changes to pink/red colour, 5. Positive urease test: Pink colour. Negative urease test: Pale yellow oh 6. Examples of urease positive ae, Klebsiella sp, Proteus sp, Ye! enterocolitica, Helicobacter pylori. P owSalient features: 1. Principle: It is used to determine the ability of an organism to decompose aminoacid tryptophan into indole. 2. (i) Indole positive test: A red coloured ring near the surface of the medium appears. (ii) iol negative test: Yellow coloured ring near the surface of the medium. 3. Examples of indole positive bacteria: Esch. coli, Proteus vulgaris, Edwardsiella sp- Negative Positive Fig. 8.29 Indole test Salient features: 1. Principle: It is the ability of an organism to utilise citrate as the sole source of carbon for its growth, with resulting alkalinity. 2. Name of the medium: Simmonr’s citrate. 3, Indicator used: Bromothymol blue. 4. (i) Positive citrate test: Bacterial growth with an intense blue colour on the slant. (ii) Negative citrate test: No growth with no change in colour (green). 5. ne of citrate positive bacteria: Klebsiella sp., Salmonella . S.typhi), Citrobacter Sp. (except Negative Positive Fig. 8.30 Citrate utilisation testFig. 6.31 Phenyloyruvie acid (PPA) test Control Positive Fig. 8.32 Triple sugar iron (TS!) agar Salient features: 1. Principle: It is used to des ability of an organism to phenylalanine to phenylp (PPA). 2. (i) Positive PPA test: Green, (i) Negtioe PPA est: No colouring, 3, Examples of postive PPA ts Prag Morganella sp. and Providenia sp, ¥ y Salient features: 1. Principle: Tis used to determine the abit an organism to attack specific carbohydrate incorporated in a growth medium, with without the production of gas, the determination of possible hydrogen sulphide (H,S) production. = Three carbohydrates i.e, glucose, and sucrose are present in this It also contains ferric salts for testing HS production. This medium is in the form of a butt and slant in the test tube. Indicator used: Phenol red. Yellow colour occurs due to of carbohydrate, while bubbles in show that gas is also produced fermentation process. a HS production can be blackening of the medium. . Possible combinations of different reactions: Remember that the slant is followed by butt reaction: = (i) K/A (red/yellow) ~ Glucose o#lY fermented. ao (i) A/A (yellow/yellow) - fermented, lactose and/or sua fermented. (iil) K/K (red/red) - Neither lactose, nor sucrose (K - Alkaline; A - Acidic) 2 gos < = Fig. 8.33 Universal conte Fig, 8.34 Bijou bottle Fig 8.95 Medical flat bottle or Me= IV. GLASSWARE Salient features: 1. Uses: (i) Specimens collection. (ii) For preparation of media such as LJ medium, RCM broth. 2. Sterilisation: By hot-air oven. 3. Capacity: 30ml Fig. 8.33 Universal container Salient features: 1. Uses: (i) Specimens collection, such as CSE. (ii) For preparation of media, such as LSS medium. (iii) For preparation of urease medium. 2. Sterilisation: By hot-air oven. 3. Capacity: 6ml. Salient features: 1. Uses: (i) For preparation of glucose broth and taurocholate broth used in blood culture (Also refer Fig. 8.8) (ii) For preparation of Castaneda medium used for diagnosis of brucellosis (Also refer Fig- 8.9) (ii) For collection and storage of sterile solutions in the laboratory (av) Capacity: 120 ml 2. Sterilisation: By hot-air oven Fig 8.35 Medical flat bottle oF Me cartney bottleoem} Fig. 8.36 Glass syringe | Fig. 8.37 Tuberculin syringe foo Fig. 8.38 Petriish Salient features: 1. Uses: () For collection oF ogy venepuncture, Ye (i) To collect ids and py, (i) To collect blood fro (sheep, rabbit), which used for preparation of bi agar. a (iv) For injecting medicine , patients. 2. Sterilisation: By hot-air oven, Salient features: 1. One ml syringe (glass/ plastic) 2. Uses: (i) In tuberculin test. (ii) Lepromin test. (iii) BCG vaccination. (iv) To inject a given dose of insulin, (¥) To inject small amount of et material into animals. 3. Sterilisation: (i) Glass syringe by hota oven. (ii) Plastic syringe by gamm radiations. Salient features: 1. Use: For preparation of culture med such as nutrient agar, blood #8 MacConkey’s agar. 2. Sterilisation: By hot-air oven. Fig. 8.39Salient features: 1 Use: For measuring the quantity of fluid used in serological tests or other tests. 2, Sterilisation: By hot-air oven. Fig, 8.39 Graduated pipette|| =e Pratl Micro, Salient features: 1. Use: To deliver solutions oF reageny, test tubes, containers etc. during various procedures. 2. Sterilisation: By hot-air oven, Fig. 8.40 Pasteur pipette V. DISPOSABLE ITEMS game. Salient features: i 1. Uses: For specimen collection such i urine, pus, body fluids etc. 4 2. For one time use only and then dispos 4 of. 3. Sterilisation: (i) By ethylene oxide. (ii) By gamma radiations Fig 8.41 Pre-sterilised disposable containerSalient features: 1. Uses: (i) For collection of blood via venepuncture. (ii) To collect fluids and pus. (iii) To collect blood from animals (sheep, rabbit), which may be used for preparation of blood \ sg agar. (iv) For injecting medicine to fy 842 Pre-sterlised disposable syringe patients, 2. For one time use only and then disposed of. 3. Sterilisation: (i) By ethylene oxide. (i) By gamma radiations. Salient features: 1. Uses; For preparation of various cell lines for growing viruses. 2, Pre-sterilised one-time use disposable bottle. 3, Sterilisation: By gamma radiations. 4 eae Fig 8.43 Tissue culture bottle98 Salient features: ' 1. Uses: (i) For collection of from throat, cery,, lesions. SPecimer, (ii) For preparing lawn Cultus, of bacterial Srowth as antibiotic sensitivity testin 2. It is available commercially.” 3. Sterilisation: By gamma radiations Fig. 8.44 Pre-sterilised disposable swab VI. EQUIPMENTS/INSTRUMENTS Salient features: ® 1. Use: For incubating cul liquid media at specified temperati”® for growth of bacteria, ert 2. Temperature used for growinS bac! a is 37°C. Fig. 8.45 Incubator ture platesFig, 8.46 Hot-air oven Fig. 8.47 Autoclave Salient features: 1. Use: For sterilisation of glasswares such as glass syringes, petridishes, flasks, Pipettes and test tubes. . Sterilisation principle: Dry _ heat sterilisation method. . Operation requirements: At 160°C for two hours holding time ie., after attaining 160°C. 4. Precautions: (i) It should not be overloaded. (ii) The material should be arranged in a manner which allows free circulation of air. (iii) The oven must be allowed to cool before opening the doors since the glassware may crack by sudden cooling. Nn we Salient features: 1. Use: For sterilisation of culture media, rubber material, gloves, gowns, dressing etc. 2. Sterilisation principle: Moist heat sterilisation method. 3. Operation requirements: 121°C at 15 pound pressure for a period of 15 minutes. 4, Precautions: () The air must be allowed to escape from the chamber as temperature of air steam mixture is lower than that of pure steam. (ji) Materials should be arranged in such a manner as to ensure free circulation of steam inside the chamber.Salient features: Ll (i) For inspissation (80°C fg, minutes daily for three consecyy), iy days). (ii) Locffler’s serum slope (155) 4, Lowenst J) mein prilised by inspissation .. is strument. 2. Principle The first day heating destroys thy vegetative forms of bacteria while the spores (which are germinated) are killed by subsequent heating. Fig. 8.48 Inspissator Salient features: 1. Single channel micropipette can deliver @ single volume of liquid with accuracy «! fraction of microliter (ul). . Multichannel micropipette can deliver multiple samples of a single volume in’ multiple wells. 3. Micropipettes of different capaciti available. 4. Uses: ' | | (i) Commonly used for performins 1% | ELISA technique. . eto l (ii) Can be used for other techni : J icroliter volumes manner werSiveted such as Polyme's= chain reaction (PCR). N jes are Fig. 8.49 Micropipettes A.Single channel B. MultichannelVil. SWABS Salient features: 1. Uses: (i) For collection of specimens from throat, cervix, local lesions. (ji) For preparing lawn culture of bacterial growth as in antibiotic sensitivity testing. 2. Sterilisation: By hot-air oven. 3. Precaution: Unplugged swab tubes should never be used. Fig. 8.50 Sterile swab Salient features: 1. Use: To collect a sample for demonstration of eggs of Enterobius vermicularis from the anal region. Fig, 8.51 NIH swab Mee ep102 Practical Micro Vill, SPECIMENS OF ADULT PARASITES Salient features: 1. Scientific name: Ascaris lumbricoides, 2, Tailend of male wormis curved ventral in the form of a hook, while in femal, worm it is conical and straight. 3. Route of infection: By ingestion. 4. Infective form: Embryonated ogg. Fig. 8.52 Roundworm Salient features: 1. Causative agent: Echinococcus granulosus. 2. The cyst wall consists of two layers i¢ ectocyst and endocyst. . 3. Hydatid fluid is used for casoni test €) diagnose patients of hydatid cyst. 4. Man is the intermediate host. 5. Infective agent: Eggs, in dog’s faeces- s 6. Mode of infection: By ingestion of en, 7. The most common organ involve = liver. Fig. 8.53 Hydatid cyst> . 103 XX. MICROSCOPIC SLIDES Salient features: 1. Violet in colour. 2. Cocci may occur either in clusters, chains or in pairs. Examples: Staph. aureus (in clusters), Strept. pyogenes (in chains), Strept. pneumoniae (in pairs). ‘Also refer Appendix V. » ca Fig, 8.54 Gram-positive cocc! Salient features: 1. Red in colour. 2. Examples: Esch. coli, Klebsiella sp., Proteus sp. etc. 3, Also refer Appendix III. J. 8.55 Gram-negative bacilli Salient features: 1. Red in colour and are found in pairs. 2. Present intracellularly but some diplococci may also be present extra- cellularly. 3. Examples: Neisseria gonorrhoeae, Neisseria meningitidis, Gram-negative intracellular diplococe!Salient features: 1. Acid-fast bacilli (AFB). 2. Causative agent of tuberculosis. 3. Commonly used medium for its culture is LJ medium. Tt takes 3-6 weeks to grow on L/ medium. > fig. 860 Mycobacterium tuberculosis Salient features: 1. Acid-fast bacilli (AFB). 2.5% sulphuric acid is used for ZN staining, 3. Causative agent of leprosy. 4. It cannot be grown on inanimate media. 8.61 Mycobacterium leprae Salient features: 1. Cytoplasm opposite the nucleus is thicker in early trophozoite or ring form. 2. Young erythrocytes are infected and these are enlarged. 2 Ring stage of Plasmodium vivax : 3. Causative agent for malaria.°8 Rhinosporidiosis: sporangium with numerous endospores 107 Salient features: 1. Leishman-Donovan is aflagellar {amastigote) stage of Leishmania donovani. It is round or oval body (2 to 4 um). . Present in the cells of reticuloendothelial system of man suffering from Kala-azar or visceral leishmaniasis. 4 Insect vector for Kala-azar is sandfly (Also refer Fig. 8.85) eR Salient features 1 Microfilaria bancrofti is an embryo of Wuchereria bancrofti. 2, Tt measures 290 jum x 6-7 wm in size. 3. Covered by a hyaline sheath which is much longer than the embryo. The somatic cells or nuclei extend from the head to the tail end. The nuclei do not extend up to the tip of the tail, a distinguishing feature of Microfilaria bancrofti > a Salient features: ‘1. Causative agent: Rhinosporidium seeberi. 2. Causes rhinosporidiosis, a chronic granulomatous disease characterised by friable polyps, usually confined to the nose, mouth or eye. . Majority of infections occur in persons who have frequent contact with stagnant water or aquatic life. 2