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.