Biochemical Tests Procedure Quiz

Biochemical Tests Procedure

• Motility Test: Media used are SIM media and MIO media.

IMVIC Reaction

• Indole Test: Detects the ability of an organism to split indole from tryptophan. Use tryptophan broth or peptone broth, or SIM/MIO media. Reagent: Kovac's. Procedure: Inoculate tryptophan broth, incubate at 35-37°C for 24 hours, add Kovac's reagent, and read immediately. Positive result is a pink or red ring.

• Methyl Red Test: Determines an organism's ability to produce acid as final products from glucose fermentation. Use MR/VP broth. Reagent: Methyl red. Procedure: Inoculate MR/VP broth, incubate at 35-37°C for 48 hours, add methyl red reagent, and read immediately. Positive result is red; negative result is yellow.

• Voges-Proskauer Test: Determines if an organism produces acetoin. Use MR/VP broth, 1-alpha naphthol, 2-40% KOH. Inoculate MR/VP broth and incubate at 35-37°C for 48 hours. Add reagents, observe for 15-45 minutes for a positive result. Positive result is a red ring; negative result is yellow.

• Citrate Test: Determines if an organism can utilize citrate as its sole carbon source. Use Simmons' citrate agar. Indicator: bromothymol blue. Procedure: Inoculate citrate agar as a fishtail. Incubate at 35-37°C for 1-4 days. Monitor for a color change. A deep blue color indicates a positive result; no change (green) indicates a negative result.

KIA (Kligler Iron Agar) Test

• Principle: Determines carbohydrate fermentation (glucose and lactose), gas production (CO2), and hydrogen sulfide (H2S) gas production. Uses glucose and lactose.

• Procedure: Inoculate KIA tubes with an organism by stabbing the butt and streaking the slant. Incubate at 35-37°C for 24 hours. Observe the results immediately following the incubation period.

Urease Test

• Principle: Determines the presence of urease enzyme.

• Procedure: Inoculate urea broth media or urea agar/slant with the organism. Incubate at 35-37°C for 24-72 hours. Observe the results following incubation. Positive result is a red color; negative result is no change (yellow).

Oxidase Test

• Principle: Detects the presence of cytochrome oxidase enzyme.

• Procedure: Pick up one or two isolated colonies using a wooden applicator stick and rub directly on filter paper impregnated with TPD reagent. Read the result within 10 seconds. A dark purple or blue color indicates a positive result; no change indicates a negative result.

Enterobacteriaceae Family

• Characteristics: Gram-negative, rod-shaped bacteria. Many are motile. Aerobic or facultative anaerobic. Ferment glucose. Oxidase negative. Catalase positive.

Other tests

• Salmonella Shigella Agar: Selective medium for isolating Salmonella and Shigella. Inhibitor factors: Brilliant green, bile salts, sodium thiosulfate, sodium citrate. pH indicator: neutral red. Source of carbohydrate: lactose.
• Other Bacteria: Specific tests and characteristics are provided for each bacterial species including E. coli, Enterobacter aerogenes, Klebsiella, Citrobacter, Proteus, Morganella, Providencia, Seratia, Yersinia pestis, Shigella.