Enterobacteriaceae, found in the intestinal tract of humans and some animals, are short, gram-negative, and non-spore producing. The IMViC (indole, methyl red, Voges-Proskauer, and citrate utilization) test can help differentiate groups of Enterobacteriaceae.
Indole production test. Indole (C8H7N) is a 6-benzene ring fused to a 5-benzene ring, and is a foul smelling crystalline structure. The essential amino acid, tryptophan, may be hydrolyzed via tryptophanase into indole, pyruvic acid, and ammonia. SIM agar deep tubes with tryptophan are inoculated. After inoculation and incubation, 10 drops of Kovac’s reagent is added. A cherry-red top-layer indicates a positive test for indole production.
Methyl red test. Glucose fermentors often produce quite acidic by-products detectable using the methyl red pH indicator. E. coli will produce and maintain a very acidic environment. E. aerogenes will start producing some acidic by-products but then converts these into nonacidic products (e.g. 2,3-butanediol and acetoin/acetylmethylcarbinol) making the environment more basic (pH 6). Methyl red indicator turns red for an acidic environment (positive), and is yellow for a negative result.
Voges-Proskauer test. Some organisms produce nonacidic (or even more “neutral” types) products (e.g. acetylmethylcarbinol) like the aforementioned E. aerogenes. Barrit’s reagent (alpha-naphthol and 40% potassium hydroxide) oxidizes acetylmethylcarbinol into diacetyl and Guanidine. A deep rose color after the addition of Barrit’s indicates a positive result (presence of acetylmethylcarbinol).
Citrate test. Some organisms can use citrate as a carbon source instead of glucose/lactose. These organisms can make citrate permease which breaks down citrate into oxaloacetic acid and acetate which get converted to pyruvic acid and carbon dioxide. The carbon dioxide combines with sodium and water to for sodium carbonate which is alkaline and changes the bromthymol blue indicator in the medium from green (negative) to deep blue (positive).
Clinical significance.
Reference
Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual.
