BIOCHEMICAL TESTING
PART TWO
Distinguishing Enterobacteriaceae
•Large family of bacteria
•Gram negative rods
•Capable of fermenting various sugars
•Many found in the intestines of human or other mammals
•Varrying pathogenicity:
•Commensals, opportunists or pathogens
•Some found in the environment
Example species:
Escherichia coli
Klebsiella pneumoniae
Citrobacter freundii
Enterobacter aerogenes
Proteus mirabilis
Salmonella typhi
Shigella dysenteriae
Yersinia enterocolitica
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Previous Experience with
Enterobacteriaceae: EMB Media
Large amounts of acid
from lactose fermentation
cause the dyes to
precipitate on the colony
surface, producing a black
center or a “green metallic
sheen” (E. coli)
Smaller amounts of acid
production result in pink
coloration of the growth
(E. aerogenes)
Nonfermenting enterics do
not produce acid so their
colonies remain colorless
or take on the color of the
media (P. vulgaris)
Biochemical Testing
Triple Sugar Iron (TSI): fermentation of sugars, sulfur reduction
IMViC:
Indole: Break down the amino acid Tryptophan
Methyl Red: Glucose oxidation
Voges-Proskauer: Production of neutral end products
Citrate: Citrate fermentation
Urease: Hydrolyzation of Urea
Phenylalanine Deaminase: converts the amino acid phenylalanine
to phenylpyruvic acid
Nitrate Reductase: Reduction of nitrate (NO3) to nitrite (NO2)
TRIPLE SUGAR IRON TEST
(TSI)

Used to differentiate among the different
groups of Enterobacteriaceae
based on their ability to ferment glucose,
lactose and/or sucrose
Also differentiates between groups capable of
reducing sulfur to hydrogen sulfide
(Sodium Thiosulfate -> Hydrogen sulfide)
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TSI Results:
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Red slant/Red butt = no fermentation
Red slant/Yellow butt = only glucose
fermentation
Yellow slant/yellow butt = lactose
and/or
sucrose
fermentation
Dark color: Hydrogen Sulfide produced
Sodium thiosulfate reduced
P 190
IMViC TESTS

A series of four tests consisting of:
Indole: Break down the amino acid Tryptophan
Methyl Red: Glucose oxidation
Voges-Proskauer: Production of neutral end products
Citrate: Citrate fermentation
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Used to differentiate the Enterobacteriaceae
We will look at each test individually
Indole Test
(SIM: Sulfide,Indole,Motility)
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Identifies bacteria capable of producing indole
Some bacteria are capable of converting tryptophan
(an amino acid) to indole and pyruvic acid by using
the enzyme tryptophanase
Pyruvic acid can be converted to energy or used to
synthesize other compounds required by the cell
Tryptophan
Indole Ring
Pyruvic Acid Ammonia
Procedure:
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Obtain 4 SIM Deep tubes
Inoculate by the stab method with the
following organisms:
E.coli,
P.vulgaris,
E. aerogenes K. pneumoniae
Indole Test Results:
If indole is produced, upon addition of
Kovac’s Reagent (10 drops), a “cherry-red”
band forms on the surface of the media
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Motility (if present) can be seen as
growth of the bacteria away from the
stab line
Sulfur in the media may be reduced
to hydrogen sulfide (H2S); this
appears as a “blackening” within the
media
Methyl Red Test
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Used to determine the ability of a bacteria to
oxidize glucose and produce stable acid end
products
Methyl red is a pH indicator (red at pH less
than 4.4 and yellow at a pH greater than 6)
The combination medium used for this test is
the MR-VP (methyl red/Voges-Proskauer)
broth
Acid production: positive methyl red
End products of neutral pH : positive Voges-Proskauer
Procedure:
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Obtain 3 MR-VP broth tubes
Inoculate (using a loop) with the
following organisms:
E.coli
K.pneumoniae
E.aerogenes
Results:
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From the 3 MR-VP broths that you inoculated,
transfer 2 mLs from each and place into 3
separate clean tubes (set aside these aliquots
for the VP test)
To the remaining, original tubes that you
inoculated add 5 drops of methyl red
indicator
A red color indicates that glucose has been
oxidized
Methyl Red Test Results:
A red color indicates that
glucose has been oxidized.
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Methyl red positive
tube on the right
Methyl red negative
tube on the left
Voges-Proskauer Test
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Used to determine the ability of microbes to
produce nonacidic or neutral end products
Remember that the MR-VP broth is a
combined medium used for two tests—
Methyl Red and Voges-Proskauer
You have already inoculated the 3 MR-VP
broth tubes from the previous procedure
(Methyl Red Test) with
E.coli
K.pneumoniae
E.aerogenes
Voges-Proskauer Procedure:
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To the aliquots of each broth culture
separated during the methyl red test, add:
10 drops of Barritt’s Reagent A; shake
10 drops of Barritt’s Reagent B; shake
Reshake the culture every 3 to 4 min.
It can take as long as 15 min. for a color
change to occur
Voges-Proskauer Results:
The presence of a deep rose color after
15 minutes is indicative of non-acidic /
neutral metabolic end products and a
positive VP test result.
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Voges-Proskauer
positive on the right
Voges-Proskauer
negative on the left
Citrate Utilization Test
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Used to determine if an organism is
capable of fermenting citrate and using
that citrate as its sole carbon source
The ability of an organism to utilize
citrate occurs via the enzyme citrase
Procedure:
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Obtain 3 Simmons Citrate agar slants
Inoculate these slants using the stab
and streak method (the same way you
inoculated the TSI media using a
needle) with the following organisms:
E.coli
K.pneumoniae
E.aerogenes
Citrate Test Results:
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Simmon’s Citrate agar utilizes
sodium citrate as its sole carbon
source
Bromthymol blue is included as a
pH indicator; the medium initially is
green
Organisms capable of using citrate
as a carbon source turn the media
“Prussian blue”.
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+
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+
Summary of IMViC Reactions
Page 199
Urease Test
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Used to differentiate organisms based on
their ability to hydrolyze urea with the
enzyme urease
The pH indicator, phenol red, is used to
detect the breakdown of urea and the
production of ammonia which is used by
bacteria to produce amino acids and
nucleotides
Procedure:
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Obtain 2 urea broth tubes
Inoculate with the following organisms:
E.coli
P.vulgaris
Urease Test Results:
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Urinary tract pathogens from the genus
Proteus may be distinguished from other
enterics
urease
Urea + H2O
CO2 + H20 + NH3
As the alkaline end products build, phenol red
turns from yellowish gold to pink—a positive
result
Urease Test Results
As the alkaline end products build, phenol red turns
from yellowish gold to pink—a positive result
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Urease positive
organism on the
right
Urease negative
organism on the left
Phenylalanine Deaminase Test
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Used to identify bacteria possessing the
enzyme phenylalanine deaminase
Phenylalanine deaminase converts the amino
acid phenylalanine to phenylpyruvic acid + NH3
Procedure:
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Obtain 2 phenylalanine agar slants
Inoculate (with a loop on the surface)
with the following organisms:
E.coli
P.vulgaris
Results
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Phenylpyruvic acid produced by some
organisms is colorless
After inoculation and incubation, 10% ferric
chloride, an oxidizing agent, is added to the
surface of the slants
Ferric chloride (FeCl3) reacts with the
phenylpyruvic acid (if present) and changes
color from yellow to green—a positive result
Phenylalanine Deaminase Results:
Positive
Ferric chloride (FeCl3) reacts with the
phenylpyruvic acid (if present) and
changes color from yellow to
green — a positive result
Positive
Negative
Negative
Nitrate Reductase Test

Used to detect the ability of an
organism to reduce nitrate (NO3) to
nitrite (NO2) or some other nitrogenous
compound, such as molecular nitrogen
(N3) using the enzyme nitrate reductase
-
NO3
Nitrate Reductase
-
NO2
Forms red color once
solutions A and B are added.
Procedure:
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Obtain 3 Nitrate broth tubes
Inoculate (with a loop) those tubes with
the following organisms:
E.coli
A.faecalis
P.aeruginosa
Results:
(Pay close attention to this
test; its one of the hardest test to read)
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After inoculation and incubation, the ability of
an organism to reduce nitrate to nitrite (or
molecular nitrogen) is detected by adding two
reagents:
Solution A (sulfanilic acid)
Solution B (α-naphthylamine)
If a red color appears after addition of solution
A and B, this is considered a positive result
-
NO3
Nitrate Reductase
-
NO2
Forms red color once
solutions A and B are added.
Results continued:
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1)
If there is no color change occurs after
additions of solutions A & B, two
possibilities must be considered:
nitrates were not reduced by the organism
NO3-
2)
NO3-
the organism possessed such potent nitrate
reductase enzymes that nitrates were
reduced beyond nitrites to ammonia or even
molecular nitrogen
-
NO3
Nitrate Reductase
-
NO2
N2 (Nitrogen Gas)
NH3+ (Ammonia)
P 219
Results Continued:
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Next, solution C (zinc) is added to the tubes that showed
no color change after addition of Nitrate A & B
Zinc, a reducing agent, is capable of converting nitrate to
nitrite; the development of a red color after addition of
Nitrate C means that nitrates were present and reduced by
the zinc not the organism; this is a negative result
NO3-
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NO3-
If the addition of zinc does not produce a color change, the
nitrates were reduced beyond nitrites to ammonia or
nitrogen gas; this is a positive reaction
-
NO3
Nitrate Reductase
-
NO2
N2 (Nitrogen Gas)
NH3+ (Ammonia)
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Solutions A and B
have been added to
these tubes
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Solution C has been
added to these tubes
C. xerosis - Reductase Negative
NO3-
NO3-
Unreactive
tubes
P. aeruginosa
Reductase Positive
E. coli - Reductase Positive
NO3-
Nitrate Reductase
NO2-
-
NO3
Nitrate
Reductase
-
NO2
N2 (Nitrogen Gas)
NH3+ (Ammonia)