Tests to know

Lactose fermentation
Indole
Motility
H
2
S production in SIM
Methyl Red/Voges Proskauer
Citrate
Oxidase test


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 37C 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
Enteric Gram-negative rods
Lactose-fermenters (LFs)
Escherichia coli
Klebsiella pneumoniae
Non-lactose fermenters
(NLFs)
Salmonella enterica
Shigella spp.
Proteus mirabilis
Pseudomonas aeruginosa
Pink on
Macconkey
Agar
Pale on
Macconkey
Agar

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 Kovacs
reagent.
Method: Inoculate medium and incubate at 37C for 24-48
hours. After incubation, add five drops of Kovacs reagent to
the surface. Do not stir or shake the tube.
Expected Results:
Positive test : Kovacs reagent combines with indole and turns the
surface red.
Negative test: No red color development
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 37C 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
SIM Medium
(Sulfur Reduction Test, Indole Production, Motility)
Indole Production
Purpose: The indole test
is used to identify
bacteria capable of
producing indole using
the enzyme
tryptophanase.
Kovacs Reagent
2
Positive
Negative
Sulfur Reduction
Purpose: This test is used
to identify those bacteria
capable of reducing
sulfur.
Motile
Positive for H
2
S production
. SIM Tube Results
A. Indole positive and
hydrogen sulfide
positive
B. Hydrogen sulfide
positive
C. Indole positive and
motility positive (note
fuzzy growth away
from stab line)
D. Negative Control
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 37C for 2-5 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



Positive Negative
Methyl Red (MR) Test
Purpose: Used
to identify
bacteria that
produce stable
acid end
products by
means of mixed
acid
fermentation of
glucose
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 Barritts Reagent
A (contains alpha-naphthol) and Barritts Reagent B
(contains KOH).
Method: Inoculate medium and incubate at 37C for 48
hours. After incubation, transfer 2.5 ml of inoculate to
another tube and add six drops of Barritts Reagent A and
two drops of Barritts 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

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: Simmons Citrate Agar. It has citrase
as the only carbon source and PH indicator bromothymol
blue
Method: Inoculate the slant and incubate at 37C for 24-48
hours.
Expected results:
Positive test: Growth and color changes to blue
Negative test: No growth and color remains green
SIMMONS CITRATE
Purpose: The
citrate utilization
test is used to
determine the
ability of an
organism, using
the enzyme
citrase, to use
citrate as its sole
carbon source





Escherichia coli
Enterobacter aerogenes

IMViC Reactions
I M Vi C
Escherichia coli + +
Proteus vulgaris + +
Klebsiella pneumoniae + +
Enterobacter spp. + +
Serratia marcescens + +
Citrobacter freundii + +
Citrobacter koseri + + +



This test is used to identify microorganisms containing the enzyme
cytochrome oxidase (important in the electron transport chain). It is
commonly used to distinguish between oxidase negative Enterobacteriaceae
and oxidase positive Pseudomadaceae.
Cytochrome oxidase transfers electrons from the electron transport chain to
oxygen (the final electron acceptor) and reduces it to water. In the oxidase
test, artificial electron donors and acceptors are provided. When the electron
donor is oxidized by cytochrome oxidase it turns a dark purple. This is
considered a positive result. In the picture below the organism on the right
(Pseudomonas aeruginosa) is oxidase positive.
TOP
Oxidase Test

Oxidase test
Oxidase positive Oxidase negative
Campylobacter spp.
Bordatella parapertussis

Brucella spp.
Kingella kingae
Acinetobacter spp.

Moraxella spp. Enterobacteriaceae
Neisseria spp.
Pseudomonas spp.