# Acetate Utilization Test:-

Principle: Acetate Utilization Test is used to determine if an organism can use acetate as the sole source
of carbon. (Another similar test in which organisms ability to utilize Citrate as a sole source of carbon is
useful for differentiating the members of Enterobacteriaceae family, this test is called Citrate Utilization
test.) If so, breakdown of the sodium acetate causes the pH of the medium to shift toward the alkaline
range, turning the indicator from green to blue.

Acetate Utilization Test A. Positive B. Negative

Acetate Utilization Test

A. Positive

B. Negative

Method:

1-With a straight inoculating needle, inoculate acetate slant lightly from an 18-24 hour culture. Do not
inoculate from a broth culture, because the growth will be too heavy.

2-Incubate at 35oC for up to 7 days

Expected results:

Positive: Medium becomes alkalinized (blue) because of the growth of the organism.

Negative: No growth or growth with no indicator change to blue

# Indole Test:-
Principle,

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 or Ehrlich’s reagent which contains 4
(p)-dimethylamino benzaldehyde, this reacts with indole to produce a red coloured compound.

Indole Test Reaction

Indole Test Reaction

Indole test is a commonly used biochemical test (e.g. in IMVIC test, SIM test etc). Indole test helps to
differentiate Enterobacteriaceae and other genera.

Two methods are in use;

1-a conventional tube method requiring overnight incubation, which identifies weak indole producing
organisms and

2-a spot indole test, which detects rapid indole producing organisms

Procedure of Conventional Tube method for Indole Test

a. Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the test organism in
tryptophan broth.

b. Incubate at 37°C for 24-28 hours in ambient air.

c. Add 0.5 ml of Kovac’s reagent to the broth culture.

Photo Courtesy: Karen M. Kiser

Expected results:

Positive: Pink colored rink after addition of appropriate reagent

Negative: No color change even after the addition of appropriate reagent. e.g. Klebsiella pneumoniae

# Methyl Red Test:-

Principle,

Some bacteria have ability to perform mixed acid fermentation of glucose in MR-VP medium. The
products of mixed-acid fermentation are a complex mixture of acids, particularly lactate, acetate,
succinate and formate as well as ethanol and equal amounts of H2 and CO2. This causes the medium to
acquire an acidic pH. Methyl Red is a pH indicator, which remains red in color at a pH of 4.4 or less.

Procedure

1-By using sterile inoculating loop, inoculate the unknown microorganism into the fresh, sterile medium.

2-Leave the other broth uninoculated (this will be a control).

3-Incubate the inoculated tube at 35-37C for two to five days.

4-After incubation, obtain the broths from the incubator and add 5 drops of Methyl Red reagent to the
broth

Observe the color.

Interpretation

methyl-red-test

. Development of red color is taken as Positive.

. Development of yellow color (No color change) after addition of reagent is taken as Negative.

Examples

MR Positive Organisms : Escherichia, Salmonella, Proteus

MR Negative Organisms : Enterobacter, Klebsiella

# Nitrate reduction:-
principle:

Principle:Heavy inoculum of test organism is incubated in nitrate broth. After 4 hrs incubation, the broth
is tested for reduction of nitrate (NO3–) to nitrite (NO2–) by adding sulfanilic acid reagent and α-
naphthylamine.

If the organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid. When
sulfanilic acid is added, it will react with the nitrous acid to produce diazotized sulfanilic acid. This reacts
with the α-naphthylamine to form a red-colored compound. Therefore, if the medium turns red after
the addition of the nitrate reagents, it is considered a positive result for nitrate reduction.
nitrate reducation test result

If the medium does not turn red after the addition of the reagents, it can mean that the organism was
unable to reduce the nitrate, or the organism was able to denitrify the nitrate or nitrite to produce
ammonia or molecular nitrogen. Therefore, another step is needed in the test. Add a small amount of
powdered zinc. If the tube turns red after the addition of the zinc, it means that unreduced nitrate was
present*.Therefore, a red color on the second step is a negative result.

*Note: The addition of the zinc reduced the nitrate to nitrite, and the nitrite in the medium formed
nitrous acid, which reacted with sulfanilic acid. The diazotized sulfanilic acid that was thereby produced
reacted with the α-naphthylamine to create the red complex.

Nitrate reducation test experiments

If the medium does not turn red after the addition of the zinc powder, then the result is called a positive
complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite present in the
medium, either, that means that denitrification took place and ammonia or molecular nitrogen were
formed.

Requirements:

Media: Nitrate Broth with inverted Durhams tube

Reagents: Sulphalinic acid reagent, Alpha napthylamine reagent, zinc dust

Others: Inoculating loop, burner, dropper

Procedure:

1-Inoculate nitrate broth with a heavy growth of test organism using aseptic technique.

2-Incubate at an appropriate temperature for 24 to 48 hours

3-Add one dropperfull of sulfanilic acid and one dropperfull of a α-naphthylamine to each broth.

4-At this point, a color change to RED indicates a POSITIVE nitrate reduction test. If you get a red color,
then you can stop at this point.
5-No color change indicates the absence of nitrite. This can happen either because nitrate was not
reduced or because nitrate was reduced to nitrite, then nitrite was further reduced to some other
molecule. If you DO NOT get a red color, then you must proceed to the next step.

6-Add a small amount of zinc (a toothpick full) to each broth. Zinc catalyzes the reduction of nitrate to
nitrite.

7-At this point, a color change to RED indicates a NEGATIVE nitrate reduction test because this means
that nitrate must have been present and must have been reduced to form nitrite.

8-No color change means that no nitrate was present. Thus no color change at this point is a POSITIVE
result.

Fig. Diagrammatic representation of the detection of nitrite in medium

Result and Interpretation

Nitrate Reduction Positive: (Red after sulfanilic acid + alpha-naphthylamine; no color after zinc)

Nitrate Reduction Negative: (No color after sulfanilic acid + alpha-naphthylamine followed by Red after
zinc)

Quality Control:

Pseudomonas aeruginosa reduces NO3 (Nitrate) to N2 (Nitrogen).

Escherichia coli reduces NO3 (Nitrate) to NO2 (Nitrite).

# ONPG Test (for β-galactosidase):

Principle,

O-Nitrophenyl-β-D-galactopyranoside (ONPG) is structurally similar to lactose (i.e. ONPG is an analog of

lactose), except that orthonitrophenyl has been substituted for glucose.

On hydrolysis, through the action of the enzyme β-galactosidase, ONPG cleaves into two residues,
galactose and o-nitrophenol. ONPG is colorless compound: O-nitrophenol is yellow, providing visual
evidence of hydrolysis.
ONPG Test

ONPG Test

Lactose fermenting bacteria posses both lactose permease and β-galactosidase, two enzymes required
for the production of acid in the lactose fermentation test. The permease is required for the lactose
molecule to penetrate the bacterial cell where the β-galactosidase can cleave the galactoside bond,
producing glucose and galactose.

Non-lactose fermenting bacteria are devoid of both enzymes and are incapable of producing acid from
lactose.

Some bacterial species appear to be non-lactose fermenters because they lack permease, but do possess
β-galactosidase and give a positive ONPG test. So called late lactose fermenters may be delayed in their
production of acid from lactose because of sluggish permease activity. In these instances, a positive
ONPG test may provide a rapid identification of delayed lactose fermentation.

ONPG Test results Vs. Lactose fermentation:

Lactose fermenter (ONPG Positive): E.coli, Klebsiella spp, Enterobacter spp produce β-galactosidase and
permease

Late lactose fermenter (ONPG Positive): Citrobacter spp, Arizona spp produce only β-galactosidase so
they slowly ferment lactose.

Non lactose fermenter (ONPG Negative): Salmonella spp; Shigella spp; Proteus spp; Providencia spp and
Morganella spp do not produce β-galactosidase so can not ferment lactose.

Media and Reagents

Sodium phosphate buffer, 1 M, pH 7.0

O-Nitrophenyl-β-D-galactopyranoside (ONPG), 0.75 M

Physiologic saline
Toulene

Quality control

Positive control: Escherichia coli

Negative control: Proteus vulgaris

Procedure

Bacteria grown in medium containing lactose (to induce the production of the galactosidase enzyme) ,
such as Kligler iron agar (KIA) or Triple sugar Iron (TSI) agar, produces optimal results in ONPG Test.

Note: β-galactosidase enzyme (inducible enzyme) is made ONLY in the presence of the lactose substrate

1-A loopful of bacterial growth is emulsified in 0.05mL of physiologic saline to produce a heavy
suspension

2-One drop of toluene is added to the suspension and vigorously mixed for a few seconds to release the
enzyme for bacterial cells.

3-An equal quantity of buffered ONPG solution is added to the suspension.

4-The mixture is placed in a 37oC water bath

When Using ONPG Tablets

A loopful of bacterial suspension is added directly to the ONPG substrate resulting from adding 1mL of
distilled water to a tablet in a test tube.

This suspension is also placed in a 37oC water bath

Results and Interpretations:

The rate of hydrolysis of ONPG to o-nitrophenol may be rapid for some organisms; producing a visible
yellow color reaction within 5 to 10 minutes.

ONPG Test results

ONPG Test results

Most tests are positive within 1 hour; however, reactions should not be interpreted as negative before 24
hours of incubation.

The yellow color is usually distinct and indicates that the organism has produced o-nitrophenol from the
ONPG substrate through the action of β-galactosidase

# Principle of Catalase Test:-

The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The
presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into
hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by
a lack of or weak bubble production. The culture should not be more than 24 hours old.

Principle

Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated
as an end product of aerobic carbohydrate metabolism.

Purpose or Uses of Catalase Test

The morphologically similar Enterococcusor Streptococcus (catalase negative) and Staphylococcus

(catalase positive) can be differentiated using the catalase test.

Also valuable in differentiating aerobic and obligate anaerobic bacteria.

Semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis.

It is used to differentiate aerotolerant strains of Clostridium, which are catalase negative, from Bacillus
species, which are positive.
Catalase test can be used as an aid to the identification of Enterobacteriaceae.

Procedure of Catalase Test

Tube Method

1-Pour 1-2 ml of hydrogen peroxide solution into a test tube.

2-Using a sterile wooden stick or a glass rod, take several colonies of the 18 to 24 hours test organism
and immerse in the hydrogen peroxide solution.Observe for immediate bubbling.

Slide Method

1-Use a loop or sterile wooden stick to transfer a small amount of colony growth in the surface of a
clean, dry glass slide.

2-Place a drop of 3% H2O2 in the glass slide.

3-Observe for the evolution of oxygen bubbles.

4-Result Interpretation of Catalase Test and Examples

5-Result Interpretation of Catalase Test and Examples

Positive: Copious bubbles produced, active bubbling

Examples: Staphylococci, Micrococci, Listeria, Corynebacterium diphtheriae, Burkholderia cepacia,

Nocardia, the family Enterobacteriaceae (Citrobacter, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia,
Proteus, Salmonella, Serratia), Pseudomonas, Mycobacterium tuberculosis, Aspergillus, Cryptococcus,
and Rhodococcus equi.

Negative: No or very few bubbles produced.

Examples: Streptococcus and Enterococcus spp

Quality Control of Catalase Test

Positive: Staphylococcus aureus– ATCC 33592

Negative: Enterococcus faecalis– ATCC 29212

Precautions of Catalase Test

The test organisms should not be taken from blood agar culture. Red Blood cells contain catalase and
their presence will give a false positive test.

Culture should be 18 to 24 hours old.

Hydrogen peroxide must be fresh as it is very unstable.

Iron wire loop should not be used.

Some bacteria produce a peroxidase that catalyzes a breakdown of hydrogen peroxide causing the
reaction to be weakly positive; (a few bubbles elaborated slowly). This should not be confused with a
truly positive reaction.

Do not add organism to reagent, particularly if iron-containing inoculating loops are used. Iron containing
loops will cause false positive test results if exposed to hydrogen peroxide.

# Introduction to Coagulase Test

Coagulase test is used to differentiate Staphylococcus aureus (positive) which produce the enzyme
coagulase, from S. epidermis and S. saprophyticus (negative) which do not produce coagulase. i.e
Coagulase Negative Staphylococcus (CONS).

Principle of Coagulase Test

Coagulase is an enzyme-like protein and causes plasma to clot by converting fibrinogen to fibrin.
Staphylococcus aureus produces two forms of coagulase: bound and free.

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Bound coagulase (clumping factor) is bound to the bacterial cell wall and reacts directly with fibrinogen.
This results in an alternation of fibrinogen so that it precipitates on the staphylococcal cell, causing the
cells to clump when a bacterial suspension is mixed with plasma. This doesn’t require coagulase-reacting
factor.

Free coagulase involves the activation of plasma coagulase-reacting factor (CRP), which is a modified or
derived thrombin molecule, to from a coagulase-CRP complex. This complex in turn reacts with
fibrinogen to produce the fibrin clot.

Procedure and Types of Coagulase Test

Slide Test (to detect bound coagulase)

.Place a drop of physiological saline on each end of a slide, or on two separate slides.

.With the loop, straight wire or wodden stick, emulsify a portion of the isolated colony in each drops to
make two thick suspensions.

.Add a drop of human or rabbit plasma to one of the suspensions, and mix gently.

.Look for clumping of the organisms within 10 seconds.

.No plasma is added to the second suspension to differentiate any granular appearance of the organism
from true coagulase clumping.

Tube Test (to detect free coagulase)

.Dilute the plasma 1 in 10 in physiological saline ( mix 0.2 ml of plasma with 1.8 ml of saline).

.Take 3 small test tubes and label as T (Test), P (Positive Control) and N (Negative Control). Test is 18-24
hour broth culture, Positive control is 18-24 hr S. aureus broth culture and Negative control is sterile
broth.

.Pipette 0.5 ml of the diluted plasma into each tube.

.Add 5 drops (0.1 ml) of the Test organisms to the tube labelled “T”, 5 drops of S. aureus culture to the
tube labelled “P” and 5 drops of sterile broth to the tube labelled “N”.

.After mixing, incubate the three tubes at 35-37 Degree Celsius.

.Examine for clotting after 1 hours. If no clotting has occurred, examine at 30 minutes intervals for up to
6 hours.

Interpretation of Coagulase Test

Slide Test (to detect bound coagulase)

Tube Test (to detect free coagulase)

(Picture Source: University of Malta, Msida MSD 2080, Malta)

Fibrin Clot of any size- Positive

No Clot- Negative

Clumping in both drops of slides indicates that the organism auto agglutinates and is unsuitable for the
slide coagulase test. All the negative slide test must be confirmed using the tube test.

During slide test, there may be chance to false positive results in case of citrate utilizing bacteria
( Enterococcus and Pseudomonas). In this case also, tube test should be performed and confirmed.

Examples

Coagulase Positive Organisms: Staphylococcus aureus and other animal host bacteria like S.
pseudintermedius, S. intermedius, S. schleiferi, S. delphini, S. hyicus, S. lutrae, S. hyicus

Coagulase Negative Organisms: Staphylococcus epidermidis, S. saprophyticus, S. warneri, S. hominis, S.

caprae, etc.

# Deoxyribonuclease (DNase) Test-

This test is presumptively used to differentiate Staphylococcus aureus which produces the enzyme
deoxyribonuclease from other Staphylococci which do not produce deoxyribonuclease (DNase).
Staphylococcus aureus possesses a heat-stable enzyme, a thermonuclease. To detect this enzyme, first
the organisms are destroyed by heat and then the free DNase reacts with the medium. This test is also
given positive by Vibrio, Helicobacter, Moraxella, Serratia, and Aeromonas.
Principle

This test determines the ability of an organism that produce DNase. DNase are extracellular
endonucleases that cleave DNA and release free nucleotides and phosphate. To detect these enzymes,
DNase agar using no indicators or various indicators (toluidine blue or methyl green) are used to detect
the hydrolysis of DNA.

In DNase agar without indicator, the hydrolysis of DNA is observed by a clearing of the agar after addition
of HCL (oligonucleotides dissolves in acid but DNA salts are insoluble). The acid precipitates
unhydrolyzed DNA making the medium opaque. Therefore, DNase producing colonies hydrolyze DNA
and produce a clear zone around the growth.

In case of DNase agar with methyl green, DNA combines with methyl green (act as cation) to produce
mint green color. When the DNA is hydrolyzed, the complex is released and the free methyl green is
colorless at pH 7.5. So the clear halo is appeared around the areas where DNase producing organism
grow.

When toluidine blue O (TBO) is added to the DNase agar, a complex is formed with the DNA, which
changes structure when DNA is hydrolyzed, resulting in a bright pink color.

Uses

Used to determine the ability of an organism to hydrolyze deoxyribonucleic acid.

Used to differentiate Staphylococcus aureus which produces the enzyme deoxyribonuclease from other
Staphylococci which do not produce DNase.

Particularly useful if plasma is not available to perform coagulase test or when the result of coagulase
tests are difficult to interpret.

Procedure

A. DNase test method

. Using a sterile loop, several colonies from an 18-24 hours culture is picked.
. Inoculate the test and control organism in each test area.

. Incubate the plate at 35-37°C for 24 hours.

. After incubation observe the color change in DNase with methyl green.

. In DNase agar without indicators:

. Flood the surface of agar with 1N HCL solution. Tip off the excess acid.

. Allow the reagent to absorb into the plate.

. Observe for clear zone around the colonies within 5 minutes.

B. Thermonuclease method

. Pick several well-isolated staphylococcal colonies using sterile needle and inoculate into BHI.

. Incubate at 35-37°C for 18-24 hours.

Place broth in boiling heat block for 15 minute.

. Cool to room temperature.

. Make a hole in Toluidine Blue O (TBO) agar with Pasteur glass pipette.

. Fill the well with 2 drops of cooled broth culture.

. Incubate at 35-37°C for 3 hour.

. Observe for color change.

Result Interpretation

DNase test

A. DNase test method

Positive

Development of clear halo around the colony.

Negative

No clear zone in the medium. Agar remains green due to no degradation of DNA.
B. Thermonuclease method

Positive

Development of pink or red halo around the colony.

Negative

No change in the royal blue color.

Quality Control

Organisms

Positive control

Staphylococcus aureus ATCC 25923

Serratia marcescens ATCC 13880

Negative control

Staphylococcus epidermidis ATCC 12228

Escherichia coli ATCC 25922

Examples

DNase positive organism

Staphylococcus aureus, Streptococcus pyogenes, Moraxella catarrhalis, Serratia species, Aeromonas,

Vibrio.

DNase negative organism

Staphylococcus epidermidis, Klebsiella, Enterobacter, E.coli, Serratia fonticola, Plesiomonas shigelloides.

Limitations

1N HCL is bactericidal. Hence if the HCL has been applied the test must be read within 5 minutes.

Methyl green medium is better for gram negative rods that first grow on the medium and then
demonstrate a positive test.

Some MRSA strain do not give positive result and some strain of coagulase negative Staphylococci such
as Staphylococcus capitis give weak positive reaction.

For Moraxella and Gram-positive cocci with TBO testing, a low inoculum can result in a false-negative
test, since these organisms may not grow well on the medium.

# Oxidase test:
Principle

The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the
bacterial electron transport chain. When present, the cytochrome c oxidase oxidizes the reagent
(tetramethyl-p-phenylenediamine) to (indophenols) purple color end product. When the enzyme is not
present, the reagent remains reduced and is colorless.

Note: All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron
acceptor in respiration. This does NOT mean that they are strict aerobes. Bacteria that are oxidase-
negative may be anaerobic, aerobic, or facultative; the oxidase negative result just means that these
organisms do not have the cytochrome c oxidase that oxidizes the test reagent. They may respire using
other oxidases in electron transport.

Test requirements for Oxidase test: Moist filter paper with the substrate (1% tetramethyl-p-
phenylenediamine dihydrochloride), or commercially prepared paper disk, wooden wire or platinum
wire.

Expected results of Oxidase test

Positive: Development of dark purple color (indophenols) within 10 seconds

Negative: Absence of color

Related posts: Modified Oxidase Test (Microdase): Principle, Procedure and Uses

Procedure of Oxidase test:

. Take a filter paper soaked with the substrate tetramethyl-p-phenylenediamine dihydrochloride

. Moisten the paper with a sterile distilled water

. Pick the colony to be tested with wooden or platinum loop and smear in the filter paper

. Observe inoculated area of paper for a color change to deep blue or purple within 10-30 seconds

. Precaution to be taken while performing oxidase test:

. Do not use Nickel-base alloy wires containing chromium and iron (nichrome) to pick the colony and
make smear as this may give false positive results

. Interpret the results within 10 seconds, timing is critical

Note:

The oxidase test must be performed from 5% Sheep blood agar or another medium without a
fermentable sugar . Fermentation of a carbohydrate results in acidification of the medium (e.g., lactose
in MacConkey Agar or Sucrose in TCBS), and a false negative oxidase test may result if the surrounding
pH is below 5.1.

During identification of suspected Vibrio cholerae isolate, it is not possible to perform an oxidase test
directly from a TCBS culture because the acid produced by the sucrose fermenting colonies will inhibit
the oxidase reaction.

Oxidase test results

Bacterial genera characterized as oxidase positive include Neisseria and Pseudomonas. Genera of the
Enterobacteriaceae family are characterized as oxidase negative.

Name of Oxidase positive bacteria are: Mneomoics for Oxidase Positive Organisms- PVNCH ( Its just an
acronyms inspired by the famous mneomonic for Urease Positive organisms-PUNCH)
P: Pseudomonas spp

V: Vibrio cholerae

N: Neisseria spp

C: Campylobacter spp

H: Helicobacter spp/ Haemophilus spp.

Aeromonas spp

Alcaligens

# Urease test:
Medium used for urease test: Any urea medium, agar or broth (It can be a sole medium or part of panel
like motility indole urease (MIU) test)

Indicator used in urease test: Phenol red

Color change:

Urea

Original: orange yellow color

Final color (in positive test): Bright pink

Urease test principle

Urea is a diamide of carbonic acid. It is hydrolyzed with the release of ammonia and carbon dioxide.

Many organisms especially those that infect the urinary tract, have an urease enzyme which is able to
split urea in the presence of water to release ammonia and carbon dioxide. The ammonia combines with
carbon dioxide and water to form ammonium carbonate which turns the medium alkaline, turning the
indicator phenol red from its original orange yellow color to bright pink.

Principle-of-Urease-Test

Procedure for urease test

. The broth medium is inoculated with a loopful of a pure culture of the test organism; the surface of the
agar slant is streaked with the test organism.
. Leave the cap on loosely and incubate the test tube at 35 °C in ambient air for 18 to 24 hours; unless
specified for longer incubation.

Result and Interpretation

Organisms that hydrolyze urea rapidly (e.g. Proteus spp) may produce positive reactions within 1 or 2
hours; less active species (e.g. Klebsiella spp) may require 3 or more days. In routine diagnostic
laboratories the urease test result is read within 24 hours.

If organism produces urease enzyme, the color of the slant changes from light orange to magenta.

If organism do not produce urease the agar slant and butt remain light orange (medium retains original
color).

urease test

Urease Test Results

Diagnostic utility of Urease test

Urease test is used for the presumptive evidence of the presence of Helicobacter pylori in tissue biopsy
material. This is done by placing a portion of crushed tissue biopsy material directly into urease broth. A
positive urease test is considered presence of Helicobacter pylori. Commercially available urease agar
kits are also available.

Rapid Urease test is can be used to differentiate between the yeasts, Candida albicans and Cryptococcus
neoformans. A presumptive identification of C. neoformans may be based on rapid urease production,
whereas Candida albicans do not.

Urea breath test: A common noninvasive test to detect Helicobacter pylori also based on urease activity.
This is highly sensitive and specific test.

Principle of Urea Breath Test:

Patient ingests radioactively labeled (13C or 14C) Urea. If infection is present, the urease produced by
Helicobacter pylori hydrolyzes the urea to form ammonia and labeled bicarbonate that is exhaled as
CO2. The labeled CO2 is detected either by a scintillation counter or a special spectrometer.
Urease test is useful test:

This test can be used as part of the identification of several genera and species of Enterobacteriaceae
including Proteus and Klebsiella. It is also useful to identify Cryptococcus species, Brucella, Helicobacter
pylori.

Name of urease positive organisms (Bacteria)

Proteus spp

Cryptococcus spp

Corynebacterium spp

Helicobacter pylori

Brucella spp

Note: Mneomonics to remember urease positive organisms: PUNCH (Similar mneomonic for oxidase
positive organism is PVNC

# Voges Proskauer (VP) Test:

Principle

Voges-Proskauer is a double eponym, named after two microbiologists working at the beginning of the
20th century. They first observed the red color reaction produced by appropriate culture media after
treatment with potassium hydroxide. It was later discovered that the active product in the medium
formed by bacterial metabolism is acetyl methyl carbinol, a product of the butylenes glycol pathway.

Pyruvic acid, the pivotal compound in the fermentative degradation of glucose, is further metabolized
through various metabolic pathways, depending on the enzyme systems possessed by different bacteria.
One such pathways result in the production of acetion (acetyl methyl carbinol), a neutral-reacting end
product.

Results of Voges-Proskauer (VP) Test

Results of Voges-Proskauer (VP) Test

Organisms such as members of the Klebsiella-Enterobacter-Hafnia-Serratia group produce acetoin as the

chief end product of glucose metabolism and form smaller quantities of mixed acids. In the presence of
atmospheric oxygen and 40% potassium hydroxide, acetoin is converted to diacetyl, and alpha-naphthol
serves as a catalyst to bring out a red complex.
Media and Reagents

Media: The medium is MR/VP broth

Reagents:

Alpha-naphthol, 5% color intensifier

Alpha Naphthol-5g

Absolute ethyl alcohol- 100 mL

Potassium Hydrooxide, 40%, oxidizing agent

Potassium hydroxide 40g

Distilled water to: 100 mL

Quality Control

Positive and negative controls should be run after preparation of each lot of medium and after making
each batch of reagent. Suggested controls include the following:

Positive Control: Enterobacter aerogenes

Negative Control: Escherichia coli

Procedure of Voges Proskauer Test

Inoculate a tube of MR/VP broth with a pure culture of the test organism.

Incubate for 24 hours at 35oC

At the end of this time, aliquot 1 mL of broth to clean test tube.

Add 0.6mL of 5% alpha naphthol, followed by 0.2 mL of 40% KOH. (Note: It is essential that the reagents
be added in this order.)
Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain
undisturbed for 10 to 15 minutes.

Results and Interpretation

A positive test is represented by the development of a red color 15 minutes or more after the addition of
the reagents indicating the presence of diacetyl, the oxidation product of acetoin . The test should not
be read after standing for over 1 hour because negative Voges-Proskauer cultures may produce a copper
like color, potentially resulting in a false positive interpretation.

# Citrate utilization test:

Citrate utilization test is commonly employed as part of a group of tests, the IMViC (Indole, Methyl Red,
VP and Citrate) tests, that distinguish between members of the Enterobacteriaceae family based on
their metabolic by-products. Citrate utilization can be used to distinguish between coliforms such as
Enterobacter aerogenes (+ve) which occur naturally in the soil and in aquatic environments and fecal
coliforms such as Escherichia coli (-ve) whose presence would be indicative of fecal contamination.

Citrate Test Right: Negative Left: Positive

Citrate Test

Right: Negative

Left: Positive

Citrate utlilization test is used to determine the ability of bacteria to utilize sodium citrate as its only
carbon source and inorganic (NH4H2PO4) is the sole fixed nitrogen source.

Principle:

When an organic acid such as citrate (remember Krebs cycle) 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.
Utilization of exogenous citrate requires the presence of citrate transport proteins (permeases). Upon
uptake by the cell, citrate is cleaved by citrate lyase to oxaloacetate and acetate. The oxaloacetate is then
metabolized to pyruvate and CO2.

Citrate = oxaloacetate + acetate

oxalacetate = pyruvate + CO2

Citrate Utilization Test

Citrate Utilization Test

Further metabolic breakdown is dependent upon the pH of the medium.

A. Under alkaline conditions, pyruvate is metabolized to acetate and formate.

pyruvate = acetate + formate

B. At pH 7.0 and below, lactate and acetoin are also produced.

pyruvate = acetate + lactate + CO2

pyruvate = acetoin + CO2

The carbon dioxide that is released will subsequently react with water and the sodium ion in the medium
to produce sodium carbonate, an alkaline compound that will raise the pH. 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. The bromothymol
blue pH indicator is a deep forest green at neutral pH. With an increase in medium pH to above 7.6,
bromothymol blue changes to blue

Citrate utilization test A: Negative B: Positive

Citrate utilization test (Image source: Microbelibrary)

A: Negative

B: Positive

Procedure of citrate utilization test:

Inoculate simmons citrate agar lightly on the slant by touching the tip of a needle to a colony that is 18
to 24 hours old.

Incubate at 35oC to 37oC for 18 to 24 hours. Some organisms may require up to 7 days of incubation
due to their limited rate of growth on citrate medium.

Observe the development of blue color; denoting alkalinization.

Expected results in citrate utilization test:

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

Quality Control strains used in citrate utilization test

Positive: Klebsiella pneumoniae

Negative: Escherichia coli

List of Bacteria which gives positive citrate utilization test

Klebsiella pneumoniae
Enterobacter species (minority of strains gives negative result)

Citrobacter freundii

Salmonella other than Typhi and Paratyphi A

Serratia marcescens

Proteus mirabilis (minority of strains gives negative result)

Providencia

Citrate Test: variable (different strains give different results)

Proteus vulgaris

Vibrio cholerae

Vibrio parahaemolyticus

Citrate test: negative

Escherichia coli

Shigella spp

Salmonella Typhi

Salmonella Paratyphi A

Morganella morganii

Yersinia enterocolitica

Although uncommon, natural E. coli variants that are citrate positive have been isolated. Citrate-
negative strains of E. aerogenes have also been found.

Uses of Citrate Utilization Test

Citrate utilization test is often part of a battery of tests used to identify gram-negative pathogens of
Enterobacteriaceae family and environmental isolates. For instance, test kits such as the API-20E and
Enterotube II include citrate utilization medium as one of the diagnostic tests.