Biochemical Tests
Objectives
Tests to know

Indole

Methyl Red/Voges Proskauer

Citrate

H2S production in SIM

Urea hydrolysis

Motility

Mannitol fermentation

Lactose fermentation
Indole Production Test

The amino acid tryptophan can be broken down by
enzyme tryptophanase to form indole, pyruvic acid
and ammonia as end products. Tryptophanase
differentiates indole-positive enterics, such as E. coli
and P.vulgaris from indole-negative enterics, such as
S. marcescens.

Media and Reagents: SIM with tryptophan and
Kovac’s reagent.

Method: Inoculate medium and incubate at 37°C for
24-48 hours. After incubation, add five drops of
Kovac’s reagent to the surface. Do not stir or shake
the tube.

Expected Results:

Positive test : Kovac’s reagent combines with indole
and turns the surface red.

Negative test: No red color development
Results for indole test
Methyl Red Test

Methyl red test is used to identify enteric bacteria
based on their pattern of glucose metabolism. If they
use mixed acid pathway and produce acidic products,
then they are called methyl-red-positive. If they use
butylene glycol pathway and produce neutral end
products, then they are called methyl-red-negative.

Media and reagents: MR-VP medium and methyl red
indicator

Method: Inoculate broth and incubate at 37°C for 25 days. After incubation, transfer 2.5 ml of inoculate
to another tube and add five drops of methyl red.
Roll between the palms of hands to disperse methyl
red.

Expected results:

Positive test: acids + methyl red = red solution

Negative test: neutral end products + methyl red =
yellow color
Results for Methyl Red Test
Negative
Positive
Voges Proskauer Test

It is used to identify enteric bacteria based on their
pattern of glucose metabolism. The enterics that
produce neutral end-products, such as acetoin are
detected by VP test.

Media and Reagent: MR-VP medium and Barritt’s
Reagent A (contains alpha-naphthol) and Barritt’s
Reagent B (contains KOH).


Method: Inoculate medium and incubate at 37°C for
48 hours. After incubation, transfer 2.5 ml of inoculate
to another tube and add six drops of Barritt’s Reagent
A and two drops of Barritt’s Reagent B. Gently mix
and let it sit for 10-15 minutes to allow time for color
development.

Expected results:

Positive test: acetoin + alpha-naphthol + KOH = red
color

Negative test: alpha-naphthol +KOH = copper color
Results for VP test
Citrate Utilization

Citrate is an organic molecule that can be utilized by
bacteria that produce the enzyme citrase. Citrase is
produced by some bacteria such as E. aerogenes but
not by others like E. Coli

Media and Reagent: Simmon’s Citrate Agar. It has
citrase as the only carbon source and PH indicator
bromothymol blue

Method: Inoculate the slant and incubate at 37°C for
24-48 hours.

Expected results:

Positive test: Growth and color changes to blue

Negative test: No growth and color remains green
Results for Citrate Test
Positive
Negative
H2S Production

Bacteria use enzyme cysteine desulfurase to
hydrolyze the amino acid cysteine, forming
hydrogen sulfide as end-product.

Media and Reagent: SIM with cysteine and
ferrous sulfate (detects H2S)

Method: Inoculate the media and incubate at 37°C
for 24-48 hours.

Expected Results:

Positive Test: H2S production = Black

Negative Test: No H2S production = No blackening of
medium
Results of H2S production
Negative
Positive
Urea Utilization

Some bacteria produce urease, an enzyme capable
of breaking down urea and produce alkaline end
products. This distinguishes Proteus from other
bacteria

Media and Reagent: Urea Broth with phenol red

Method: Inoculate the media with a loop and
incubate at 37°C for 24 hours.

Expected Results:

Positive test: production of alkaline end products =
pinkish red color

Negative test: No color change
Results for Urea Test
Positive
Negative
Motility Test

This is not a biochemical test, but it can distinguish
bacteria. It determines presence of flagella.

Media and reagent: Deep agar

Method: Inoculate deep with a needle and
incubate at 37°C for 24-48 hours.

Expected results:

Positive test: Growth spread away from the line of
inoculation = motile

Negative test: Growth only occurred at the line of
inoculation = Non-motile
Results for Motility Test
Mannitol Fermentation

Mannitol Salt Agar contains 7.5% NaCl, which is
inhibitory to most bacteria. Bacteria that can grow
on this agar can be differentiated based on
mannitol fermentation. Fermentation of mannitol
results in acidic products which turn phenol red pH
indicator from red to yellow.

Media and reagent: MSA and phenol red indicator

Method: Streak MSA plate and incubate at 37°C for
2 days.

Expected results:

Positive test: Mannitol fermentation occurred = growth
and color changed to yellow

Negative test: No mannitol fermentation = may or may
not grow and no color change
Results for mannitol fermentation
No Growth
Negative
Positive
Lactose Fermentation

MacConkey Agar contains bile salts and crystal
violet, both inhibitory to Gram-positive bacteria and
selects Gram-negative bacteria, such as E. Coli.

It also differentiates lactose-fermenting bacteria,
such as E. Coli from non-lactose fermenting
bacteria.

Media and Reagent: MacConkey Agar and neutral
red dye

Method: Streak MAC plate and incubate at
37°C for 2 days.

Expected results:

Positive test: Lactose fermentation = Growth and
color change to pink

Negative test: No lactose fermentation = May or may
not grow and no color change
Results of Lactose Fermentation