INSTRUMENTS CULTURE MEDIA METHODS REACTIONS.pptx

Microbiology stoll exam

1. INSTRUMENTS, MEDIA, METHODS & BIOCHEMICAL REACTIONS DR GEETA S H PROF MICROBIOLOGY BLOCK 1 - PRACTICALS

2. McINTOSH -FILDES JAR Commonly known as Anaerobic jar Consists of glass/metal jar with a metal lid. 2 tubes in the lid- gas inlet & outlet.; & 2 terminals for electric supply; alk. Methylene blue- indicator. A layer of palladinised asbestos / alumina pellets is placed. Inoculated culture plates are kept inside & lid is clamped tight. For anaerobiosis, air inside the jar is evacuated completely & filled with hydrogen gas. Alumina pellets- catalyst-removes residual oxygen.

3. INOCULATING TOOLS Made of Nichrome/ Platinum wire. Used to inoculate specimens, bacterial/fungal colony into respective media. Standard loop- inoculation of specimens & bacterial colony. Straight wire- inoculation of bacterial colony. Spud- inoculation of Fungal colony.

4. TUBERCULIN SYRINGE Syringe made of plastic/ glass. Capacity- 1ml; fitted with 26” needle. Graduated upto 0.01ml. Used to inoculate antigens like tuberculin (Mantoux test), etc through intra- dermal route.

5. CANDLE FILTER Made up of kaolin/ sand/ diatomaceous earth (Berkefield filter). Different grade of porosity. Sterilized by autoclaving. Used to sterilize water in household or in large scale in industries.

6. VDRL SLIDE Multiple depression slide usually with 6-12 concavities . Diameter of the concavity- 14mm. Used for V.D.R.L.(Venereal Disease Research Laboratory) test; a slide flocculation test done for serodiagnosis of Syphilis.

7. PASTEUR PIPETTE Named after Louis Pasteur. Elongated glass tubes with narrow long nozzle fitted on the other end with rubber teat. Used to transfer fluids like sera or culture suspensions following sterile precautions.

8. STERILE SWAB Made of absorbent cotton bud at one tip of wooden/ plastic stick place in a clean, dry test tube plugged with non-absorbent cotton/plastic screw cap. The entire unit can be sterilized in a hot-air oven. Used to collect clinical samples from various sites such as: nose, throat, ear, or pus samples.

9. MICROTITRE PLATE Made of plastic or polyvinyl with 96 microwells; 12 horizontal & 8 vertical. Used for serological tests like: ELISA, Haemagglutination etc.

10. BLOOD CULTURE BOTTLE Enrichment medium -Brain-Heart infusion broth (Ox brain & Heart infusion). Used for direct inoculation of whole blood, bone marrow & body fluids. Adults- 10-20ml ; Children- 1-5ml (blood inoculation). Cultivation of organisms like- Salmonella sps., Brucella sps.

11. ROBERTSON’S COOKED MEAT MEDIUM Widely employed fluid medium for cultivation of Anaerobes; eg: Clostridium sps . Consists of fat free minced meat particles & beef infusion broth. Indicate saccharolytic (meat-pink colour) or proteolytic (meat- black colour) property of the organism.

12. Nutrient Agar Basal medium. Uses – 1. cultivation of non-fastidious organisms, 2. observation of pigment production, 3. maintaining stock cultures, 4. obtaining pure culture for slide agglutination.

13. Blood Agar Enriched media. Contains 5-10% defibrinated sheep/horse/rabbit blood. Used for growth of fastidious bacteria like Streptococcus spp., Neisseria spp.

14. Chocolate Agar Enriched medium. Contains lysed RBC releasing intracellular nutrients such as X & V factors. Used for the growth of fastidious organisms like Haemophilus, Meningococci, Gonococci.

15. MacConkey Agar Selective/ Differential medium. Used for primary isolation for GNB, identification & differentiation especially Enterobacteriaceae. Contains bile salts for the inhibition of Gram positive organisms.

16. MACONKEY AGAR UNINOCULATED; MACONKEY AGAR WITH LF GROWTH

17. Thiosulphate Citrate Bile Sucrose Agar (TCBS) Partially differential/selective medium for Vibrios. Differentiates sucrose fermenters & non-fermenters. Bile salts & alkaline pH (8.6) – inhibits - Gram positives & coliforms. Indicator - Bromothymol Blue & Thymol blue. Vibrio cholerae - yellow convex colonies (sucrose fermenter). V. parahaemolyticus - green colonies (sucrose non-fermenters).

18. Wilson & Blair Medium Selective medium for Isolation of Salmonella spp . Brilliant green – indicator – inhibits intestinal flora. Salmonella Typhi produces black colonies with metallic sheen (H2S +)

19. Tellurite Blood Agar Selective media for isolation of Corynebacterium diphtheriae. Contains 0.04% tellurite. Diphtheria bacilli reduces Tellurite to Metallic tellurium producing Jet Black colonies.

20. Lowenstein – Jensens medium Enriched/Selective medium used for Mycobacterium tuberculosis Contains egg, mineral salt solution and malachite green as indicator & inhibitory substance. Sterilized by inspissation.

21. LJ MEDIUM WITH MYCOBACTERIA Picture shows colonies of Mycobacterium tuberculosis on Lowenstein Jensen medium. Growth is seen after 2-8 weeks of incubation at 37⁰C. Colonies are dry, rough, raised, irregular, with a wrinkled surface, and buff colored (Rough – Tough – Buff).

22. Sabouraud Dextrose Agar (SDA) Used for the growth of fungi. Low pH (5.4) and high dextrose content with gentamicin & Chloramphenicol to inhibit the growth of bacteria.

23. NUTRIENT AGAR WITH SWARMING OF PROTEUS Cultures of Proteus mirabilis/vulgaris swarm on solid culture media like nutrient agar. Motile cells spreads on the surface of the plate in successive waves to form a thin filmy layer in concentric circles. Inhibition of swarming : Increased concentration of agar (6%), Incorporation of chloral hydrate (1:500), sodium azide (1:500), alcohol (5-6%) sulphonamide surface active agents boric acid (1:1000) Inoculation into MacConkey agar.

24. ANTIBIOGRAM Medium used- Mueller Hinton agar. Method- Kirby- Bauer method. Inoculation of organism- As Lawn culture. Antibiotic discs are placed on it (6 discs used- single 9 cm petri dish) & incubated overnight (18 hrs) at 37⁰C. Zone size interpretation- based on CLSI guidelines.

25. ANTIBIOTIC SENSITIVITY TESTING (AST) PLATE

26. Loeffler’s Serum Slope Enriched media for luxuriant growth of Corynebacterium diphtheriae. Contains Glucose & Serum, Sterilized by Inspissation. Growth seen in 6-8 hours of incubation with Granule production.

27. Triple Sugar Iron (TSI) Reactions

28. Urease Test Ability of the organism to hydrolyze urea to produce ammonia by the enzyme urease. Phenol red – Indicator. Ammonia – Alkaline pH – Pink colour – Positive Reaction. + -

29. Indole Test Ability of the organism to degrade tryptophan to produce indole by the enzyme tryptophanase. Production of red complex (indole) with addition of Kovac/Ehrlich reagents – Positive.

30. CITRATE TEST Simmon’s citrate medium Detects ability of an organism to use citrate as sole source of carbon. Indicator used – Bromothymol blue Used in differentiation of Enterobacteriaceae & in identification other bacteria. Example:- Citrate positive (blue ): Klebsiella sps . Citrate negative (no change): E.col i. - +

31. Methyl Red test Quantitative Test for acid production. Ability of an organism to produce enough strong acids from glucose to overcome the buffering capacity of the medium (eg: Glucose phosphate broth). Methyl red – Used to indicate the low pH.

32. Voges – Proskauer Test Ability of the organism to produce Acetyl methyl carbinol (acetoin) from pyruvic acid (end product of glucose metabolism). Addition of 40% KOH converts acetoin to diacetyl ;& α -naphthol serves as a catalyst to produce a red complex.

33. Coagulase Test Free coagulase – thrombin – like substance – culture filtrates. Coagulase producing organism + Coagulase Reacting Factor (plasma) fibrinogen Fibrin Clot. Coagulase Pos – Staphylococcus aureus Coagulase Neg – Staphylococcus epidermidis.