Frequently Asked Questions

Lowenstein-Jensen (LJ) Medium is gold standard for Mycobacterium tuberculosis cultivation from clinical specimens. Egg-based medium contains: malachite green (inhibits non-mycobacteria), asparagine, glycerol. Prepare by: inspissation (solidification by heating at 85°C for 50 min rather than autoclaving). M. tuberculosis grows slowly (3-8 weeks), producing buff/cream rough colonies. Used for: TB diagnosis, drug susceptibility testing, mycobacterial identification. Slopes stored in screw-cap tubes. For isolation and cultivation of Mycobacterium species from clinical samples.

EMB Agar should NOT be autoclaved because: (1) Excessive heat precipitates eosin Y and methylene blue dyes, (2) Overheating reduces selectivity, (3) Can inhibit organism growth, (4) Color reactions may be obscured. Proper preparation: Heat to dissolve completely (boiling), cool immediately to 45-50°C, pour plates. Brief boiling is sufficient for sterilization. Autoclaving EMB results in: dark precipitate in medium, poor selectivity, false-negative metallic sheen. This is why many labs prefer RTU EMB plates - eliminates preparation errors.

Suspend 50g of MacConkey Agar powder in 1000mL purified water. Heat to boiling to dissolve completely (do NOT autoclave). Cool to 45-50°C. Mix well and pour into sterile petri dishes (approximately 20mL per 90mm plate). Final pH 7.1±0.2. Medium should be pinkish-red (from neutral red indicator). Do not overheat or autoclave MacConkey - excessive heating destroys bile salts and crystal violet reducing selectivity. Once poured and solidified, store plates inverted at 2-8°C. Use within 1-2 weeks or as validated.

Antibiotic Assay Medium No.1 is used for microbiological assay of antibiotics (penicillin, streptomycin, etc.) in pharmaceutical products per USP. Standardized formulation ensures consistent results. Procedure: (1) Seed medium with test organism (Bacillus subtilis, Staph. aureus), (2) Pour into plates, (3) Place antibiotic standards and samples in wells/disks, (4) Incubate, (5) Measure inhibition zones, (6) Calculate potency vs. standards. pH and nutrient composition optimized for reproducible zone diameters. Essential for antibiotic potency testing in pharmaceutical QC.

Luria Bertani (LB) Agar is standard medium for cultivation of E. coli and other Enterobacteriaceae in molecular biology and genetic engineering. Simple formulation (tryptone, yeast extract, NaCl) supports rapid E. coli growth. Used for: (1) Transformation experiments (with antibiotic selection), (2) Maintaining recombinant strains, (3) Plasmid preparation, (4) Blue-white screening (with X-Gal, IPTG), (5) Cloning procedures. Can add ampicillin, kanamycin, or other antibiotics for selection of transformed colonies. Incubate 16-18 hours at 37°C.

Triple Sugar Iron (TSI) Agar differentiates Enterobacteriaceae based on: (1) Glucose fermentation, (2) Lactose/sucrose fermentation, (3) H2S production, (4) Gas production. Contains glucose (0.1%), lactose (1%), sucrose (1%), ferric ammonium citrate, sodium thiosulfate. Inoculate by stabbing butt and streaking slant. Results interpreted by butt/slant colors and gas/H2S. E. coli: yellow/yellow (ferments all). Salmonella: red/yellow with black (glucose only + H2S). Shigella: red/yellow (glucose only, no H2S). Pseudomonas: red/red (none). Classic identification tool.

Detects ammonia production from peptone, Pale yellow to a dark brown precipitate is indicated by nesslers reagent as a positive reaction. Used in some identification schemes.

Two-part reagent (alpha-naphthol + KOH) for VP test. Add to VP broth after incubation. Cherry /red indicates acetoin production (VP positive).

Methyl red solution for MR test. Add after 48h incubation in MR-VP broth. Red = MR positive (mixed acid fermentation).

Detects indole from tryptophan breakdown. Add to indole broth after incubation. Red ring = indole positive (E. coli).

Tests cytochrome c oxidase. Purple color = positive (Pseudomonas, Neisseria). Differentiates oxidase-positive from Enterobacteriaceae (negative).

3% hydrogen peroxide tests catalase enzyme. Bubbles = positive (Staph). No bubbles = negative (Strep). Critical differentiation test.

For detection of esculin hydrolysis in the presence of bile, for differentiating Group D streptococci from other Streptococcal groups.

Used for cultivation and differentiation of the enterococcal group D streptococci from nonenterococcal group D streptococci based on salt tolerance.

Bromocresol purple changes yellow with acid production. Alternative to phenol red. Used in some lactose fermentation tests. Recommended for identification of Escherichia coli and coliform bacteria from water samples.