Ex. 18: IMViC Reactions Objectives ?? Indole production test Methyl red test Voges-Proskauer test Citrate utilization test. Objectives ??

Day 1 Materials needed per team of two students: 2 tryptone broth tubes 2 MR-VP broth tubes 2 Simmons citrate slants Pure cultures needed per table: Escherichia coli Enterobacter aerogenes Work as a team of two. Each team member will work with one bacterial species

Day 2 Materials needed per team of two: 2 clean tubes and two 1 ml transfer pipettes Materials needed per table: Small biohazard bag Kovac’s reagent Methyl red indicator reagent VP reagents A and B Work as a team of two. Each team member will work with one bacterial species  

Indole Test Checks for Tryptophanase Medium: 1% Tryptone broth Test for indole with Kovac’s reagent dimethylamino-benzaldehyde (DMABA) in DMABA in Kovac’s reagent combines with indole and forms red band at the top of the medium

Table 18-1: Indole Production Results and Interpretation Symbol Red in the alcohol layer of Kovac’s reagent Tryptophan is broken down into indole and pyruvate + Reagent color is unchanged Tryptophan is not broken down into indole and pyruvate  Enterobacter aerogenes E. coli

Methyl Red and Voges-Proskauer Tests Physiologically related tests, use same medium (MRVP broth), NB with glucose MR test checks for large amounts of acid endproducts from glucose fermentation Do not confuse methyl red indicator with phenol red indicator!!! VP test checks for production of acetoin, a neutral fermentation end product

Table 18-2: Methyl Red Test Results and Interpretation Result Symbol Red Mixed acid fermentation + No color change No mixed acid fermentation  Table 18-3: Voges-Proskauer Test Results and Interpretation Result Interpretation Symbol Red Acetoin produced + No color change Acetoin is not produced 

MRVP after incubation and before adding test reagents

MR test After addition of 3 -4 drops of methyl red indicator Explain the meaning of this outcome

VP Test After addition of 15 drops of VP reagent A, and 5 drops of VP reagent B E. a. E. c. Explain the meaning of this outcome

Citrate Utilization Test Checks if organism has citrate permease Simmons citrate agar also contains ammonium phosphate as sole N-source and bromothymol blue as indicator. If bacterium is able to use citrate it will also convert ammonium phosphate to NH3 and NH4OH

Table 18-4: Citrate Test Results and Interpretation Symbol Blue (even a small amount) Citrate is utilized + No growth or weak growth with no color change Citrate is NOT utilized 

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Could an organism be both MR and VP positive? Explain. The End