Identification of unknown bacteria

Biochemical tests IMViC REACTIONS IMViC reactions are a set of four useful reactions that are commonly employed in the identification of members of family enterobacteriaceae. The four reactions are: Indole test, Methyl Red test, Voges Proskauer test and Citrate utilization test.

INDOLE TEST: Principle: Some bacteria can produce indole from amino acid tryptophan using the enzyme typtophanase. Usage medium is peptone water Production of indole is detected using Ehrlich’s reagent or Kovac’s reagent. Indole reacts with the aldehyde in the reagent to give a red color. An alcoholic layer concentrates the red color as a ring at the top.

Results of Indole test Example: (A) Escherichia coli (B) Klebsiella pneumoniae proteus vulgaris Entrobacter spp.

SIM: Indole test, H2S, and motility

METHYL RED (MR) TEST: This is to detect the ability of an organism to produce and maintain stable acid end products from glucose fermentation. : medium: MR-VP medium Reagent: methyl red reagent (Red)+ve result (Eschericihia coli ) ( proteus vulgaris ) (Brown –yellow) -ve result (Klebsiella pneumoniae) (Enterobacter spp.)

VOGES PROSKAUER (VP) TEST: VP test detects butylene glycol producers. In this test two reagents, 40% KOH and alpha-naphthol are added to test broth

+ve –ve Enterobacter spp. E. coli Klebsiella pneumoniae Citrobacter spp.

CITRATE UTILIZATION TEST: This test detects the ability of an organism to utilize citrate as the sole source of carbon and energy. Simmon citrate agar = media Bromothymol blue= reagent enzyme citritase, Examples: Escherichia coli: P. vulgaris-= Negative; Klebsiella pneumoniae: Enterobacter spp.: Citrobacter spp.: Positive

Citrate Left positive right negative

Motility Hanging drop slide • Motility test medium

Motility Test How to Perform Test: Stab motility media with inoculating needle. Property it tests for: This test is done to help differentiate species of bacteria that are motile. Media and Reagents Used: Motility media contains tryptose, sodium chloride, agar, and a color indicator. Reading Results: If bacteria is motile, there will be growth going out away from the stab line, and test is positive. If bacteria is not motile, there will only be growth along the stab line. A colored indicator can be used to make the results easier to see.

From left to right: + – +