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.