Ex. 18: IMViC Reactions 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 Materials needed per table: two: Small biohazard bag 2 clean tubes and two 1 ml Kovac’s reagent Methyl red indicator reagent transfer pipettes 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 • 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 Result 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 Interpretation 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 Explain the meaning of this outcome E. a. E. c. 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 Result Blue (even a small amount) No growth or weak growth with no color change Interpretation Symbol Citrate is utilized + Citrate is NOT utilized Could an organism be both MR and VP positive? Explain.