Biochemical tests used for Enterobacteria identification

According to the general scheme of bacteriological diagnostic, direct diagnostic methods consist
in:

- sampling and transport of samples to the laboratory

- direct examination of the sample

- obtaining isolated colonies and pure culture

- identification of bacteria

- antimicrobial susceptibility testing

Identification of bacteria may be done by different methods:

- biochemical identification

- serological identification

- molecular methods based on DNA analysis etc.

Biochemical identification is very helpful in the diagnostic in Enterobacteria in current practice,

as there are many species that may be often isolated from different pathological samples.

There are several levels of biochemical identification in Enterobacteria:

1. Orientative tests used for the preliminary identification of Enterobacteria (short biochemical
panel )

2. Commercial kits (galeries) for biochemical identification of Enterobacteria

3. Automated equipments for biochemical identification of Enterobacteria

1. Orientative tests used for preliminary identification of Enterobacteria (short

biochemical panel for Enterobacteria identification)
Two multitest media, ureea and Simmons citrate are used currently in many laboratories:
1.a. Triple Sugar Iron (TSI)
1.b. Motility Indol Lysine Phenylalanine (MILPH)
1.c. Urease test
1.d. Citrate utilisation - Ability to use citrate as a sole source of carbon and Ammonia as a sole
source of Nitrogen

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1.a. Triple sugar iron agar (TSI agar) test
TSI slant is a test tube that contains agar, a pH sensitive dye (phenol red), three sugars (lactose 1%,
sucrose 1% and glucose 0.1%, as well as sodium thiosulphate and ferrous sulphate (source of iron)

Helps in identification of Enterobacteria by their specific reactions on the slants; known as multi-test
medium.

Inoculation of TSI tubes (both the butt and the slant have to be inoculated)

Inoculation of TSI tube with the straight microstreaker needle (not with the loop) has to ensure the
contact of bacteria with both the butt (inoculation down the center of the tube to about half the depth
of the medium) and the slant of the medium.

Tubes have to be then incubated at 35-37 0 C ON (over night).

We are checking the following Enterobacteria characters on TSI:

• Fermentation of three sugars (lactose, sucrose, glucose)

• Production of gas

• Production of Hydrogen Sulphide (black colour)

Biochemical reactions:

o 0.1% Glucose: If only glucose is fermented, only enough acid is produced to turn the butt
yellow.  The slant will remain red. Note that all Enterobacteria are glucose fermenters.

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 o 1.0 % lactose/1.0% sucrose:  a large amount of acid turns both butt and slant yellow, thus
indicating the ability of the culture to ferment either lactose or sucrose.
o Iron from Ferrous sulphate will indicate the H2S formation by reacting to it to form ferrous
sulphide.
H2S formation: Some bacteria utilize thiosulphate anion included in the TSI medium as a
terminal electron acceptor, reducing it to sulphide. If this occurs, the newly formed hydrogen
sulfide (H2S) reacts with ferrous sulphate in the medium to form ferrous sulphide, which is
visible as a black precipitate.
Examples of ferrous sulphide-producing bacteria include Salmonella and Proteus.
o Phenol red: Indicator of acidification (It is yellow in acidic condition and red under alkaline
conditions).
Consequently, remember that fermentation of glucose has to be read in the butt of the TSI
multitest medium (positive glucose = yellow butt).

Fermentation of lactose and sucrose has to be read on the slant of the medium. If the bacteria is
fermenting the sugar, the medium pH goes acid, therefore the colour of the pH indicator goes yellow.

Gas production may be noticed as gas accumulation at the bottom of the medium, sometimes
lifting the entire medium column towards the top of the tube, or inside the butt, as gas bubbles.

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 Control Tube Tube 1 Tube 2 Tube 3 Tube 4 Tube 4a Tube 5
(uninoculated
)
Slant - K K K A K A
Butt - - A Unreadable* A A Unreadable*
Gas - - - - G G G
Hs2 - - - + - - +
*Glucose is unreadable because of the black colour of ferrous sulphide; glucose is always positive when
H2S is positive

A = Acid = Positive fermentation result; K= alkaline = Negative fermentation result; G = Gas = Gas production positive

The student will receive a table to compare the results in the given TSI tubes in order to frame the
inoculated bacteria into a bacterial genus.

Bellow there are some expected results for different Enterobacteria and a glucose non-fermentative
Genus (Pseudomonas):

Some examples of Triple Sugar Iron (TSI) Agar Reactions:  

Name of the Slant Butt Gas H2S

organisms (lactose, sucrose) (Glucose)

Escherichia, Acid (A) Acid (A) Pos (+) Neg (-)

Klebsiella,
Enterobacter

Shigella, Alkaline (K) Acid (A) Neg (-) Neg (- )

Serratia

Salmonella, Alkaline (K) Acid (A) Pos (+) Pos (+)

Proteus

Pseudomonas Alkaline (K) Alkaline (K) Neg (-) Neg (-)

 1.b. MILPH (Motility Indole Lysine Phenylalanine) test

Multitest medium MILPH is a semi-solid medium containing: 3 aminoacids (i.e.) tryptophan, lysine,
phenylalanine), glucose, agar and a pH indicator (brom cresol purple). Glucose is needed for causing pH
drop needed to activate lysine decarboxylase.

The pH indicator brom cresol purple is purple at neutral or alkaline/basic pH but turns yellow at pH <5.2.

MILPH multitest medium is helping us to check 4 characters of the inoculated bacteria:

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- Motility

- Use of the amino acid tryptophan as a source of carbon and energy for growth (delivery of indole)

- Use of the amino acid lysine as a source of carbon and energy for growth (presence of lysine
decarboxylase)

- Use of the amino acid phenylalanine as a source of carbon and energy for growth (presence of
phenylalaninedeaminase)

Inoculation of MILPH medium has to be done with a straight microstreaker needl (not with the loop),
making a single stab down the center of the tube to about half the depth of the medium.

The inoculated tube is incubated at 35-37 C for 24 hours and the preliminary results are determined.
Final results are read at 48 hours.

Motility

In semi-solid agar media, motile bacteria ‘swarm’ and give a diffuse spreading growth that is easily
recognized by the naked eye.

Reading of results: Hold the tube up to the light and look at the stab line to determine motility.

1. Non-motile bacteria generally give growths that are confined to the stab-line, have
sharply defined margins and leave the surrounding medium clearly transparent.
2. Motile Bacteria typically give diffuse, hazy growths that spread throughout the medium
rendering it slightly opaque.

Use of tryptophan (Indole test)

Indole test is used to determine the ability of an organism to split amino acid tryptophan to
form the compound indole. 
Tryptophan is hydrolysed by tryptophanase to produce three possible end products – one of
which is indole.
Indole production is detected by Kovac’s  reagent which contains 4 (p)-dimethylamino
benzaldehyde, this reacts with indole to produce a red coloured compound.
For determining the delivery of indole in the MILPH multitest tube, we use Kovac’s reagent
impregnated on filter paper strips, that are placed on the top of the MILPH tube and fixed with
the tube cap before incubation.

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 Positive indole
reaction – indole strip

Indole positive organisms: Most strains of E.coli and Proteus vulgaris are indole positive.

Use of lysine

The microbe must first use the glucose present to cause the pH to drop. This is indicated by a change
from purple to yellow. Once the medium has been acidified, the enzyme lysine decarboxylase is
activated. The culture is incubated an additional 24 hours at 35-37 C to allow the microbe to now use
the lysine. The color must be noted at both 24 and 48 hours to determine the result.
Final results: Change back to purple from yellow indicates a positive test for lysine decarboxylase.
Failure to turn yellow at 24 hours or to revert back to purple at 48 hours indicates a negative result.

Klebsiella pneumoniae –
UnInoculated medium nonmotile, positive Escherichia coli – motile,
lysindecarboxylase positive lysindecarboxylase

Use of phenylalanine

Phenylalanine deaminase test also known as phenylpyruvic acid (PPA) test is used to test the ability of
an organism to produce enzyme deaminase.  This enzyme removes the amine group from the amino
acid phenylalanine and produces phenylpyruvic acid (PPA) and ammonia i.e. oxidative deamination of
phenylalanine.

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In oder to detect production of the phenolpyruvic acid we have to add 4-5 drops of 10% aqueous ferric
chloride (FeCl3) solution to the slant.

Interpretation

1. Positive test: Production of green colour (Phenylpyruvic acid thus formed reacts with ferric
chloride producing a green colored compound thus turning the medium dark green). Proteus sp.
give positive PPA test.
2. Negative: No colour change (no PPA to react with ferric chloride).

Phenylalaine deaminase test is used to differentiate members of the genera Proteus etc., (+ve)  from
other members of Enterobacteriaceae which give negative results.

1.c. Urease test

Medium used for urease test: Any urea medium, agar or broth
Urease test principle
Urea is a diamide of carbonic acid.  

Urea molecule

It is hydrolyzed with the release of ammonia and carbon dioxide by bacterial urease.

Indicator used in urease test: Phenol red

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Delivery of ammonia turns the medium alkaline, which is turning the indicator phenol red from its
original orange yellow color to bright pink.

Color change:
 Original: orange yellow color
 Final color (in alkaline positive test medium): Bright pink
Positive test Negative test

Urease test on urease broth medium

Enterobacteria producing urease (urease positive): Klebsiella, Proteus

1.d. Citrate utilisation - Ability to use citrate as a sole source of carbon and
Ammonia as a sole source of Nitrogen

Simmons citrate medium contains citrate and Ammonia and no other source of carbon and
nitrogen. Not all bacteria are able to grow in this conditions.

The pH indicator is brom thymol blau, which is of a deep forest green color at neutral pH.  With an
increase in medium pH to above 7.6, bromo thymol blue changes to blue.

Principle: 

When an organic acid such as citrate is used as a carbon and energy source, alkaline
carbonates and bicarbonates are produced ultimately.  
In addition, ammonium hydroxide is produced when the ammonium salts in the medium are
used as the sole nitrogen source.

Growth usually results in the bromothymol blue indicator, turning from green to blue (positive
reaction).

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Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate
medium.
Bacteria which gives positive citrate utilization test: Klebsiella pneumonia, Salmonella other than Typhi
and Paratyphi A

2. Commercial kits (galleries):

E.g. API kits – may contain different number of tests (e.g. API 20 E, API 32 E); the precision of the result
increases with the number of tests.API 20E

API 32 E

3. Automated equipments for biochemical identification of Enterobacteria:

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There are several automated equipments for biochemical identification and antimicrobial susceptibility
testing .

Examples:

VITEK2 COMPACT system Phoenix system

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