Ex. 18: IMViC Reactions

Objectives ??

Indole production testMethyl red testVoges-Proskauer testCitrate utilization test.

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 1

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  

Day 2

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 InterpretationResult 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

Enterobacteraerogenes

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 InterpretationResult 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 TestAfter 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 Interpretation Symbol

Blue (even a small amount) Citrate is utilized +

No growth or weak growth with no color change

Citrate is NOT utilized

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