BACTERIAL IDENTIFICATION TESTS

Biochemical Tests
The isolated bacteria are further processed through one or few of the
procedures mentioned below so as to identify the bacteria:
• Staining of the isolated bacteria
• Motility testing
• Biochemical testing
• Serological tests: Detection of antigens
by enzyme or fluorescence immunoassays
• Phage typing: Virus in bacteria-Phage, differentiate between strains/outbreak
• Identification disc testing: Impregnated disc is added to a culture medium-
fermentation-production of acid pH- indicator changes colour (e.g. phenol red
changes from red to orange to yellow).
• Semi-automated and Automated identification systems: Microscan walkaway
system, Vivtek system, Sensititre Gram Negative Auto identification system, the
Phoenix system
• Molecular techniques: G+C % content, DNA-DNA hybridisation and DNA base
sequencing. Amplification techniques like Polymerase chain reaction, ligase
chain reaction, strand displacement amplification, and nucleic acid sequence
Biochemical testing
( IMViC- I- is for indole; M- is for methyl red; V- is for Voges-Proskauer, and
C- is for citrate, lowercase i- is added for the ease of pronunciation

• (a) Catalase test

• (b) Coagulase test
• (c) Oxidase test
• (d) Sugar fermentation test
• (e) Indole test
• (f) Citrate test
• (g) Urease test
(a) Catalase test
• Purpose
• Facilitates the detection of the enzyme catalase in bacteria.
• Essential for differentiating catalase-positive (Micrococcaceae) from catalase
negative (Streptococcaceae).
• Differentiate between genera, certain gram positives such as Aerococcus urinae
(positive) from Aerococcus viridians (negative).
• Procedure:
• Place a microscope slide inside a petri dish.
• Keep the petri dish cover available.
• Using a sterile inoculating loop or wooden applicator stick, collect a small amount
of organism from a well-isolated 18- to 24-hour colony and place it onto the
microscope slide.
• Be careful not to pick up any agar.
• This is particularly important if the colony isolate was grown on agar
containing red blood cells.
• Carryover of red blood cells into the test may result in a false-positive
reaction.
• Using a dropper or Pasteur pipette, place 1 drop of 3% H2O2 onto the
organism on the microscope slide.
• Do not mix.
• Immediately cover the petri dish with a lid to limit aerosols and observe for
immediate bubble formation (O2 + water = bubbles).
• Observing for the formation of bubbles against a dark background
enhances readability.
Catalase positive bacteria: Staphylococcus spp
Catalase negative bacteria: Streptococcus spp
(b) Coagulase test
Purpose
• Differentiates strains of Staphylococcus aureus from other coagulase-
negative species.
• S. aureus strains are capable of coagulating plasma in the tube test and will
produce clumps of cells in the slide test.
• The coagulase test can be performed using two different procedures - Slide
test and tube test.
• The slide test is simple, giving results within 10 seconds, but it can give false
negatives.
• The tube test is the definitive test, it can take up to 24 hours to complete.
• For both tests, clumping or clots of any size indicate a positive response.
• S. aureus is the most commonly isolated coagulase positive organism.
Procedure
• The slide test is performed by preparing a suspension of bacterial
cells mixed into a drop of rabbit plasma on a microscope slide.
• If bound coagulase is present on the bacterial cells, then the presence
of plasma will cause the bacterial cells to clump.
• The clumping will occur because the clumping factor is an adhesin,
which causes the cells to bind to fibrinogen in the plasma.
• This will result in visible clumping of bacterial cells on the microscope
slide.
• Figure given below illustrates the visible clumping of cells on the
microscope slide.
• mixing bacterial cells into a larger volume of plasma in a small test
tube.
• As the bacteria multiply in the plasma, they secrete
staphylocoagulase.
• Staphylocoagulase initiates blood coagulation by activating
prothrombin.
• Staphylocoagulase adheres to fibrinogen, forming a complex that
cleaves fibrinogen into fibrin, bypassing the blood clotting cascade
and directly causing a clot of fibrin to form.
• Formation of a clot will be noted within 24 hours for a positive
response
• Coagulase positive bacteria: Staphylococcus aureus Coagulase
negative bacteria: Staphylococcus epidermis, Staphylococcus
saprophyticus
(c) Oxidase test
Purpose
• Assays for the presence of cytochrome oxidase (indophenol oxidase).
• In the presence of an organism that contains the cytochrome oxidase enzyme, the
reduced colorless reagent becomes an oxidized colored product.
Procedure
• Filter Paper Test Method
• 1. Soak a small piece of filter paper in 1% Kovács oxidase reagent and let dry.
• 2. Use a loop and pick a well-isolated colony from a fresh (18- to 24-hour culture)
bacterial plate and rub onto treated filter paper.
• 3. Observe for color changes.
• 4. Microorganisms are oxidase positive when the color changes to dark purple within 5 to
10 seconds.
• Microorganisms are delayed oxidase positive when the color changes to purple within 60
to 90 seconds.
• Microorganisms are oxidase negative if the color does not change or it takes longer than
2 minutes.
• Oxidase positive bacteria : Pseudomonas, Vibrio cholera
• Oxidase negative bacteria: E. coli, Klebsiell, Salmonella.
(d) Indole test
Purpose
• Ability of an organism to degrade the amino acid tryptophan and produce
indole.
• Tests designed to distinguish among members of the family
Enterobacteriaceae.
• Most strains of E. coli, P. vulgaris, P. rettgeri, M. morganii, and Providencia
species break down the amino acid tryptophan with the release of indole.
Principle
The test organism is cultured in a medium which contains tryptophan.
Indole production is detected by Kovac’s or Ehrlich’s reagent which contains 4
(p)-dimethylamino-benzaldehyde.
This reacts with the indole to produce a red coloured compound.
Procedure
• Inoculate the tube of tryptone broth with
a small amount of a pure culture.
• Incubate at 37°C for 24 to 48 hours.
• Add 5 drops of Kovác's reagent directly to the tube.
• A positive indole test: Pink to red color (“cherryred ring”) in the
reagent layer on top of the medium within seconds of adding the
reagent.
• If a culture is indole negative, the reagent layer will remain yellow or
slightly cloudy.
• Indole positive bacteria : E. coli, Vibrio cholera Indole negative
• bacteria : Klebsiella, Salmonella, Shigella spp.
(e) Citrate Test
Purpose
• The citrate test screens a bacterial isolate for the ability to utilize
citrate as its carbon and energy source.
• A positive diagnostic test rests on the generation of alkaline by-
products of citrate metabolism.
• The subsequent increase in the pH of the medium is demonstrated by
the colour change of a pH indicator.
• Identify gram-negative pathogens and environmental isolates.
Procedure
• Use a fresh (16- to 18-hour) pure culture as an inoculation source.
• Pick a single isolated colony and lightly streak the surface of the slant.
• A needle is the preferred sampling tool in order to limit the amount of
cell material transferred to the agar slant.
• Avoid using liquid cultures as the inoculum source.
• Citrate utilization requires oxygen and thus screw caps, if used, should
be placed loosely on the tube.
• Incubate at 35oC (+/- 2oC) for 18 to 48 hours.
• Some organisms may require up to 7 days of incubation due to their
limited rate of growth on citrate medium.
• Citrate positive: growth will be visible on the slant surface and the
medium will be an intense Prussian blue.
• The alkaline carbonates and bicarbonates produced as by-products of
citrate catabolism raise the pH of the medium to above 7.6, causing the
bromothymol blue to change from the original green color to blue.
• Citrate negative: trace or no growth will be visible.
• No color change will occur; the medium will remain the deep forest green
color of the uninoculated agar.
• Only bacteria that can utilize citrate as the sole carbon and energy source
will be able to grow on the Simmons citrate medium, thus a citrate-
negative test culture will be virtually indistinguishable from an
uninoculated slant.
• Citrate positive bacteria: Klebsiella spp. Citrate negative bacteria: E. coli.
(f) Urease test
Purpose
• The urease test identifies those organisms that are
capable of hydrolyzing urea to produce ammonia and
carbon dioxide.
• It is primarily used to distinguish urease-positive
bacteria from other Enterobacteriaceae.
Procedure
• Use a heavy inoculum from an 18- to 24-hour pure
culture to streak the entire slant surface.
• Do not stab the butt as it will serve as a color control .
• Incubate tubes with loosened caps at 35oC.
• Observe the slant for a color change at 6 hours, 24
hours, and every day for up to 6 days.
• Urease production is indicated by a bright pink (fuchsia) color on the
slant that may extend into the butt.
• Note that any degree of pink is considered a positive reaction.
• Prolonged incubation may result in a false-positive test due to
hydrolysis of proteins in the medium.
• To eliminate protein hydrolysis as the cause of a positive test, a
control medium lacking urea should be used.
• Rapidly urease-positive Proteeae (Proteus spp., Morganella morganii,
and some Providencia stuartii strains) will produce a strong positive
reaction within 1 to 6 hours of incubation.
• Delayed-positive organisms (e.g., Klebsiella or Enterobacter) will
typically produce a weak positive reaction on the slant after 6 hours,
but the reaction will intensify and spread to the butt on prolonged
incubation (up to 6 days).
• The culture medium will remain a yellowish color if the organism is
urease negative.
• Urease positive bacteria : Proteus spp., Morganella morganii Urease
negative bacteria : E. coli,
Introduction
Amino acids are the basic constituents of many proteins that compose living
organisms.
• Some microorganisms degrade amino acids to yield energy in a variety of
end products of ammonia, indole acid, and water.
• Many reactions that involve degradation of amino acids are used for
classifying the entero-bacteriaceae.
Certain amino acids degrading bacteria, like E. coli, have the ability to
degrade the amino acids tryptophan into indole and pyruvic acid.
• This hydrolysis is brought about by the enzyme tryptophanase.
Principle
Indole is a nitrogen-containing compound formed from the degradation of
the amino acid tryptophan.
• The indole test is important because only certain bacteria form indole
• Indole can be easily detected with Kovac’s reagent.
• After the addition of the reagent and mixing the contents, the tube is
allowed to stand.
• The alcohol layer gets separated from this aqueous layer and, upon
standing, the reddening of the alcohol layer shows that Indole is present
in the culture.
• Thus, the formation of the red layer at the top of the culture indicates
the positive test.
• Pure tryptophan is not usually used in the test.
• Tryptone is used as a substrate because it contains tryptophan.
Procedure
Preparation of tryptone broth:
Ingredients: Tryptone – 10 gms
Distilled water – 1000 mL
Distribute 5 mL of the broth into the test tubes and plug with cotton
plugs.
Sterilize it at 121°C for 15 minutes.
• Preparation of Kovac’s reagent:
N-amyl alcohol – 75 mL
Concentrated HCl – 25 mL
P-dimethylaminebenzaldehyde – 5 g.
• Inoculate the tubes with the test bacterial culture.
• Incubate all the tubes for 48 hours at 37°C.
(g) Methyl Red Test
Introduction
Bacteria such as [Link] and Proteus, ferment glucose to produce a large
amount of lactic acid, acetic acids, succinic acid, and formic acid.
Carbon dioxide, hydrogen, and ethyl alcohol are also formed.
When the organisms do not convert the acidic product to neutral product,
the pH of the medium is 5 or less.
Such acids are called mixed-acid fermenters.
Methyl red Voges-Proskauer medium is essentially a glucose broth with
some buffered peptone and dipotassium phosphate.
It is inoculated with test organisms.
Methyl red indicator is yellow at a pH of 5.2 and red at a pH of 4.5 is
added to the culture.
If the methyl red indicator turns red, then it indicates that the organism
is a mixed-acid fermenter—a positive reaction.
The yellow color after the addition of methyl red indicator indicates a
negative reaction.
Procedure
• Preparation of methyl red indicator—Dissolve about 0.2 gm of methyl red in 500
mL of 95% ethyl alcohol and add 500 mL of distilled water and filter.
• Preparation of MRVP media – (glucose phosphate broth)

• Dipotassium hydrogen phosphate (K2HPO4) – 5 gms

• Peptone – 5 gms Glucose – 5 gms

• Distilled water – 1000 mL

• Suspend all the ingredients in distilled water and gently warm. Do not alter the
pH. 5 mL of media that is distributed in plugged test tubes. Sterilize at 121°C for
15 minutes.
• Innoculate the tubes with the test bacterial culture (except for the control tube.)
• Incubate all the tubes at 37°C for 48 hrs. 5. Add 3-4 drops of MR indicator into
each tube.
• A distinct red color indicates the positive test; yellow color indicates a negative
test.
Discussion
Studies in recent years have emphasized the complexity of the coliform
group.
Classification is based on the results of the 4 tests—indole, MR test, VP test,
and sodium citrate test.
Enterobacter cultures ferment lactose with the formation of acids, and the
pH becomes less than 5.
Klebsiella do not produce 2,3-butylene glycol, but others do.
[Link], Bacillus, Proteus, and Staphylococcus a positive result as they oxidize
the glucose to organic acid and stabilized the organic acid concentration.
Klebsiella produces a negative result.
(h) Voges-Proskauer Test
• All species of Enterobacter and Serratia, as well as some species of
Bacillus and Aeromonas produce a lot of 2,3-butanediol and ethyl
alcohol instead of acids.
• These organisms are called butanediol fermenters.
• The production of these non-acid end products results in less
lowering of the pH in methyl red Voges- Proskauer media and, thus,
the test is negative.

Principles
Butanediol fermenters produces 2,3-butanediol, for which there is no
satisfactory test.
 Acetyl methyl carbinol or acetoin yield 2,3-
butanediol which can be easily detected
with Barrit’s reagent.
 This test is valid, since acetoin and 2,3-
butanediol are present simultaneously.
 In the VP test, the formation of acetyl
methyl carbinol is from glucose.
 In the presence of alkali, atmospheric
oxygen and Barrit’s reagent, small amounts
of acetyl methyl carbinol present in the
medium are oxidized to diacetyl, which
produces a red color with guanidine
residue in the media.
 Thus the formation of the red or pink color
indicates the positive VP reaction.
Procedure
(a) Preparation of MRVP medium or glucose phosphate broth:

• Dipotassium hydrogen phosphate (K2HPO4) - 5 gms

- Peptone - 5 gms
- Glucose - 5 gms
- Distilled water - 1000 mL
Suspend all the ingredients in distilled water and gently warm. Do not alter
the pH. 3 mL of the media are distributed into test tubes, which are
plugged. Sterilized at 121°C.
(b) Preparation of Barrit’s reagent:
Barrit’s reagent consists of 2 solutions, i.e., solutions A and B.

• Solution A is prepared by dissolving 6 gms of alpha naphthol in 100 mL of

95% ethyl alcohol.
• Solution B is prepared by dissolving 16 gms of potassium hydroxide in 100
mL of water.
• Inoculate the tubes with the bacterial culture.
• Incubate for 48 hours at 37°C.
• Pipette 1 mL from each culture tube into clean separate tubes. Use
separate pipettes for each tubes.
• Add 18 drops (0.5 mL) of Barrit’s solution A (a-naphthol) to each tube that
contains the media. Add an equal amount of solution B into the same tube.
• Shake the tubes vigorously every 30 seconds. A positive reaction is
indicated by the development of a pink color, which turns red in 1–2 hours,
after vigorous shaking. It is a very important step to achieve complete
aeration.
Discussion
The addition of KOH to the cultures of organisms of the hemorrhagic
septicemia by the Pasteurella group resulted in the development of a
pinkish-red color, if allowed to stand for 24 hours or longer.
The fermentation of glucose by the 2 organisms yielded varying
amounts of products like acids, alcohol, carbon dioxide, etc.
It was due to the formation of 2,3-butylene glycol and acetyl methyl
carbinol by Klebsiella, but not by [Link].
Acetyl methyl carbinol in the presence of KOH and air is further
oxidized to diacetyl, which in the presence of peptone produces an
eosin-like color.
This color is due to the guanidine nucleus of amino acids present in it.
Thus, this test is of considerable significance in testing various samples,
because it disintegrates to a high degree between related
enterobacters.
TRIPLE SUGAR IRON TEST (TSI)

• It has three sugar (Lactose, Sucrose, and Glucose) and

also iron; and it contains Agar Agar as solidifying
agent (TSI is a semi solid media having slant and butt).
• 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 gas (H2S)
• Composition of Triple Sugar Iron Agar (TSI)
Lactose, Sucrose and Glucose in the concentration of [Link] (i.e. 10 part
Lactose (1%), 10 part Sucrose (1%) and 1 part Glucose (0.1%)).
• TSI is similar to Kligler’s iron agar (KIA), except that Kligler’s iron agar
contains only two carbohydrates: glucose (0.1%) and lactose (1%).
• 0.1% Glucose: If only glucose is fermented, only enough acid is produced to
turn the butt yellow. The slant will remain red
• 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.
• Iron: Ferrous sulfate: Indicator of H2S formation
• Phenol red: Indicator of acidification (It is yellow in acidic condition and
red under alkaline conditions).
• It also contains Peptone which acts as source of nitrogen. (Remember that
when ever peptone is utilized under aerobic condition ammonia is
produced)
Why Sugar is added to TSI?
Addition of sucrose in TSI Agar permits earlier detection of
coliform bacteria that ferment sucrose more rapidly than
lactose. Adding sucrose also aids the identification of
certain gram-negative bacteria that could ferment sucrose
but not lactose. Other basic understanding is TSI Tube
contains butt (poorly oxygenated area on the
bottom) slant (angled well oxygenated area on the top).
Procedure for Triple Sugar Iron Agar (TSI) Test

• With a sterilized straight inoculation needle touch the top of a well-

isolated colony
• Inoculate TSI Agar by first stabbing through the center of the medium
to the bottom of the tube and then streaking on the surface of the
agar slant.
• Leave the cap on loosely and incubate the tube at 35°C in ambient air
for 18 to 24 hours
Interpretation of Triple Sugar Iron Agar Test
• If lactose (or sucrose) is fermented, a large amount of acid is produced,
which turns the phenol red indicator yellow both in butt and in the
slant. Some organisms generate gases, which produces bubbles/cracks on
the medium.
• If lactose is not fermented but the small amount of glucose is, the oxygen
deficient butt will be yellow (remember that butt comparatively have
more glucose compared to slant i.e. more media more glucose), but on the
slant the acid (less acid as media in slant is very less) will be oxidized to
carbondioxide and water by the organism and the slant will be
red (alkaline or neutral pH).
• If neither lactose/sucrose nor glucose is fermented, both the butt and the
slant will be red. The slant can become a deeper red-purple (more
alkaline) as a result of production of ammonia from the oxidative
deamination of amino acids (peoptone is a major constitutents).
• if H2S is produced, the black color of ferrous sulfide is seen.
TSI Agar Test results
• Alkaline slant/no change in butt (K/NC) i.e Red/Red = glucose, lactose
and sucrose non-fermenter
• Alkaline slant/Alkaline butt (K/K) i.e Red/Red = glucose, lactose and
sucrose non-fermenter
• Alkaline slant/acidic butt (K/A); Red/Yellow = glucose fermentation
only, gas (+ or -), H2s (+ or -)
• Acidic slant/acidic butt (A/A); Yellow/Yellow = glucose, lactose and/or
sucrose fermenter gas (+ or -), H2s (+ or -).
IMViC Test results of Some
Genera of
Enterobacteriaceae: [Link] tests of Proteus vulgaris
[Link] tests of Escherichia coli [Link]: Positive
[Link]: Positive [Link]-Red: Positive
[Link]-Red: Positive [Link]-Proskauer test:
[Link]-Proskauer test: Negative
Negative [Link] test: Negative
[Link] test: Negative [Link] tests of Citrobacter freundii
2. IMViC tests of Enterobacter [Link]: Negative
aerogenes [Link]-Red: Positive
[Link]: Negative [Link]-Proskauer test:
[Link]-Red: Negative Negative
[Link]-Proskauer test: Positive [Link] test: Positive
[Link] test: Positive