MICRO-202 Veterinary Microbiology and Mycology

Practical:
BIOCHEMICAL TESTS FOR THE IDENTIFICATION OF
BACTERIAL ISOLATES
Biochemical tests are used to identify bacterial species by differentiating them on the
basis of biochemical activities. The difference in protein and fat metabolism, carbohydrate
metabolism, enzyme production, compound utilization ability, etc. are some factors that aid in
bacterial identification.
All species of bacteria possess a unique set of metabolic activaties which are controlled
by bacterial enzymes. These bacterial enzymes determine the type of test suitable in recognition
of structural differences and metabolic activities.
Intracellular Enzymes
Intracellular enzymes function internally and are responsible for synthesis of new protoplasmic
substances in bacteria. They permit simple substances through the cell membrane and produce
cellular energy that helps in utilization of these cellular substances. Some of tests based on these
enzymes include: Catalase test, oxidase test, Indol test etc.
Extracellular Enzymes
These enzymes are secreted by the bacterial cells and function outside the cells. They are
synthesized inside the cell and assist in breakdown of complex macromolecules into smaller
units after secretion. These smaller weight substances then can permeate the cell membrane and
utilized it with the help of intracellular activities. Some extracellular enzyme test include:
coagulase test etc.
Importance
Biochemical reactions are very important in the identification of bacterial isolates and in
the identification of different bacterial species. These tests depend on the presence of certain
enzymes, such as catalase, oxidase, urease, gelatinase, etc., produced by the bacteria.
Different bacteria produce varying spectra of enzymes. For example, some enzymes are
necessary for the bacterium’s individual metabolism, and some facilitate the bacterium’s ability
to compete with other bacteria or establish an infection. Tests that measure single bacterial
enzymes are simple, rapid, and generally easy to interpret. They can be performed on organisms
already grown in culture and often provide presumptive identification.
MICRO-202 Veterinary Microbiology and Mycology

There are a large number of biochemical test e.g Fermentation of carbohydrates, Citrate
utilization, Decarboxylation of amino acids, gelatin liquefaction, hydrogen sulfide test, indole
test, Methyl red test, nitrate reduction test, Urease test and Voges Proskauer test etc. But the
choice of tests is based on preliminary findings such as gram staining colony and culture
characteristics which give hint that which organism it can be. For example, finding a gram
negative bacilli growing in McConkey agar from stool sample would hint looking at
Enterobacteriaceae group and tests like coagulase (which is used for identification of coagulase
producing staphylococcus species) would of be no use.
Biochemical tests can be classified into 3 groups
1. Universal
These tests are done for almost any isolate and guide the microbiologist to a possible set of
biochemical tests that needs to be done to get a reliable identification.
Examples: Hemolysis pattern, Motility test, Catalase test, Oxidase test
2. Differential
These are a common set of tests that are done to identify the isolate up to species level. The
identification is made based on the results from a combination of tests and individual results by
themselves are not sufficiently informative to make an identification.
Examples: IMViC tests, Triple Sugar Iron test (TSI), Sugar fermentation tests
3. Specific
These are tests that are specific to a particular set of species or for sub-typing a species. These
tests are usually performed to confirm or identify at the subspecies level. The individual tests are
informative by themselves in this case.
Example: γ-Glutamyl aminopeptidase test to differentiate between Neisseria and related species
isolated on selective medium.
MICRO-202 Veterinary Microbiology and Mycology

Catalase Test

This test demonstrate the presence of catalase, an enzyme that catalyses the release of oxygen
from hydrogen peroxide (H2O2). It is used to differentiate those bacteria that produces an
enzyme catalase, such as staphylococci, from non-catalase producing bacteria such as
streptococci. Normally 3% H2O2 is used for the routine culture while 15% H2O2 is used for
detection of catalase in anaerobes.
Objective
To determine whether the bacteria produce catalase enzyme
Principle
The enzyme catalase produced by some bacteria breaks down hydrogen peroxide and oxygen.
This results in the visible formation of bubbles of oxygen.

Tube Method
 Pour 1-2 ml of hydrogen peroxide solution into a test tube.
 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
 Use a loop or sterile wooden stick to transfer a small amount of colony growth in the
surface of a clean, dry glass slide.
 Place a drop of 3% H2O2 in the glass slide.
 Observe for the evolution of oxygen bubbles.
Result

Positive = presence of bubbles (oxygen) coming out from the bacterial growth;
Negative = no bubbles observed.
Note: Be careful when taking bacterial colonies from media containing blood. Blood contains the
catalase enzyme, and if this media is carried over with the bacterial colonies, a false-positive
result can occur.
MICRO-202 Veterinary Microbiology and Mycology

Oxidation/ Fermentation Test

Principle
This test is used to determine the oxidative or fermentative metabolism of a carbohydrate by the
bacterium in a semi-solid medium which contains glucose as a test sugar and bromothymol blue
as a pH indicator.
Whether an organism is oxidative or fermentative can be determined by using Hugh and
Leifson’s medium, commonly called as OF medium which contain tryptone and bromothymol
blue (an indicator). One of the sugars, such as glucose, xylose, mannitol, lactose, sucrose, and
maltose is added to the medium which serves as the fermentable carbohydrate.
An organism is inoculated to two tubes of each OF Medium. Once inoculated, one tube is
overlaid with mineral oil or melted paraffin producing an anaerobic environment. The other tube
is left open to the air. Growth of microorganisms in this medium is either by utilizing the
tryptone which results in an alkaline reaction (dark blue colour) or by utilizing glucose, which
results in the production of acid (turning bromothymol blue to yellow).
Oxidative utilization of the carbohydrate will result in acid production (yellow) in the open tube
only. Fermentative utilization of the carbohydrate will result in acid production (yellow) in both
the open and closed tubes. Acidic changes in the overlaid tubes are considered to be a result of
true fermentation, while acidic development in the open tubes are due to oxidative utilization of
the carbohydrate present. Asaccharolytic organisms will not produce acid in either tube.
Objective
To detect the oxidation or fermentation of carbohydrates by bacteria.
Procedure
a) Take two test tubes of (oxidation fermentation) OF medium and heat in a beaker of boiling
water immediately before use to remove the any dissolved oxygen.
b) Cool the tubes rapidly under cold running water.
c) Stab inoculates both tubes with the bacteria to be tested.
d) On the top of one tube, cover with sterile paraffin oil to about 1 cm to stop oxygen to go in the
tube (anaerobic environment).
e) Incubate at 37oC and examine in 24 hours and then daily for up to 14 days.
Result Open tube covered tube with paraffin oil
Unreactive (Negative) Green Green
Oxidation (Positive) Yellow Green
Fermentation (Positive) Yellow Yellow
MICRO-202 Veterinary Microbiology and Mycology

 Positive: A positive carbohydrate utilization test is indicated by the development of a yellow

color in the medium.
 Oxidative: Development of a yellow colouration in the open tube only.
 Fermentative: Development of a yellow colouration in both open and closed tubes.
 Negative: A negative carbohydrate utilization test is indicated by the absence of a yellow
color (media remains green or turns blue). Non-oxidizer/Non-fermenter
Uses
 It aids in the identification of gram-negative bacteria on the basis of their ability to
oxidize or ferment a specific carbohydrate.
 It is used to determine whether an organism uses carbohydrate substrates to produce acid
byproducts.
 Non fermentative bacteria are routinely tested for their ability to produce acid from six
carbohydrates (glucose, xylose, mannitol, lactose, sucrose, and maltose).
Limitations
 It is recommended that biochemical, immunological, molecular, or mass spectrometry
testing be performed on colonies from pure culture for complete identification.
 Slow-growing organisms may not produce results for several days.
 Some microorganisms do not grow in OF Medium. It may be necessary to use another
basal medium containing dextrose to confirm the negative reaction.
 Some mineral oils are acidic and may result in erroneous results.
MICRO-202 Veterinary Microbiology and Mycology

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 dihydrochloride) to indophenols, a purple or dark blue color
end product. When the enzyme is not present, the reagent remains reduced and is colorless.

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.
Objective
To detects the presence of cytochrome oxidase..
Material
Reagents:
 Prepare a 1% solution (0.1g in 10 ml of distilled water) of p amino dimetylaniline
monohydrochloride or 1% of tetramethyl-p-phenylenediamine dihydrochloride.
 1% alpha-naphthol in 95% ethanol
Procedure
Method 1 (Filter paper):
a) A piece of filter paper is moistened in a Peri dish with 1% aqueous solution of tetramethyl-p-
phenylenediamine dihydrochloride.
b) Streak the test bacterium firmly across the filter paper with a glass rod.
Result: positive = A dark purple colour along the streak line.

Method 2 (Test tube):

a) Grow a fresh culture (18 to 24 hours) of bacteria in 4.5 ml of nutrient broth (or standard media
that does not contain a high concentration of sugar).
b) Add 0.2 ml of 1% α-naphthol, then add 0.3 ml of 1% p aminodimethylaniline
monohydrochloride.
Observe for color changes.
MICRO-202 Veterinary Microbiology and Mycology

Result:
 Oxidase positive: color changes to blue within 15 to 30 seconds.
 Delayed oxidase-positive: color changes to purple within 2 to 3 minutes.
 Oxidase negative: no change in color
The reagents used in the test should be colorless and stored in a dark bottle at 4oC
Auto-oxidation of the reagents may be retarded by the addition of 1% ascorbic acid.
Overall Result Positive test is indicated by the development of a purple colour.
– Pseudomonas, Vibrio, Neisseriae
– Salmonella, Shigella

Oxidase positive Neisseria sicca (left) and oxidase negative Staphylococcus aureus (right)
MICRO-202 Veterinary Microbiology and Mycology

Motility Test
Using Motility Test Medium
Principle
Motility medium is a colorless, semisolid agar medium dispensed in a test tube that is soft
enough that motile organisms can spread through the agar as they grow.
Procedure
a. With a needle, pick up some bacterial growth.
b. Stab down the center of the tube but do not penetrate all the way down and withdraw in the
same line. Leave about one cm uninoculated.
c. Incubate for 1-2 days.
d. Hold the tube to the light and observe the growth.
Result:
Positive: growth spreads outward from the stab into the medium; Nonmotile = growth remains in
the stab line of inoculation.
MICRO-202 Veterinary Microbiology and Mycology

TRIPLE SUGAR IRON AGAR (TSI)

Most bacteria have the ability to ferment carbohydrates, particularly sugars. Among them, each
bacterium can ferment only some of the sugars, while it cannot ferment the others. Thus, the
sugars, which a bacteria can ferment and the sugars which it cannot, is the characteristic of the
bacteria and thus an important criterion for its identification.
The Triple Sugar Iron (TSI) test is a microbiological test named for its ability to test a
microorganism’s ability to ferment sugars and to produce hydrogen sulfide. An agar slant of a
special medium with multiple sugars constituting a pH-sensitive dye (phenol red), 1% lactose,
1% sucrose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous
ammonium sulfate is used for carrying out the test. All of these ingredients when mixed together
and allowed solidification at an angle (slant) result in a agar test tube at a slanted angle.
Principle
The triple sugar- iron agar test employing Triple Sugar Iron Agar is designed to differentiate
among organisms based on the differences in carbohydrate fermentation patterns and hydrogen
sulfide production. Carbohydrate fermentation is indicated by the production of gas and a change
in the colour of the pH indicator from red to yellow.
To facilitate the observation of carbohydrate utilization patterns, TSI Agar contains three
fermentative sugars, lactose and sucrose in 1% concentrations and glucose in 0.1%
concentration.
 Due to the building of acid during fermentation, the pH falls. The acid base indicator
Phenol red is incorporated for detecting carbohydrate fermentation that is indicated by the
change in color of the carbohydrate medium from orange red to yellow in the presence of
acids.
 In case of oxidative decarboxylation of peptone, alkaline products are built and the pH
rises. This is indicated by the change in colour of the medium from orange red to deep
red.
 Sodium thiosulfate and ferrous ammonium sulfate present in the medium detects the
production of hydrogen sulfide and is indicated by the black color in the bottom of the
tube.
 To facilitate the detection of organisms that only ferment glucose, the glucose
concentration is one-tenth the concentration of lactose or sucrose. The little amount of
acid production in the slant of the tube during glucose fermentation oxidizes rapidly,
causing the medium to remain orange red at the top and the acid formation (yellow) is
maintained in the bottom of the tube since it is under lower oxygen tension.

Objective
To study different properties of a bacterium – sugar fermentation, gas production and H2S
production.
Materials: TSI agar, test bacteria, two types of inoculating needles (straight and wire loop)
Procedure
MICRO-202 Veterinary Microbiology and Mycology

a) Inoculate the bottom of the TSI agar with the straight needles (Fig a).
b) Using the wire loop, streak the slant by stabbing to the bottom (Fig b) and then inoculating the
surface of the slant as you withdraw the needle.
c) Cap very loosely and incubate overnight at 37C for 24 hours and write your observations after
24 hours.
Result
Yellow – Acid (A)
Pink (red) – Alkaline (K)
 Yellow slant / Yellow bottom (A/A) – Lactose fermenters ( E.coli, Klebsiella)
 Pink slant / Yellow buttom (K/A) – Non lactose fermenters (Salmonella, Shigella)
 Pink slant / no colour change (K/K) – Non fermenters
 Black colour – H2S production. (Proteus)
 Gas bubbles or crack in the medium – gas production.
MICRO-202 Veterinary Microbiology and Mycology

Urease Test (Urea hydrolysis test)

Principle:
Organisms that possess the enzyme urease, hydrolyze urea to form ammonia.
Urea is the product of decarboxylation of amino acids. Hydrolysis of urea produces ammonia
and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the
color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid
urease-positive organisms turn the entire medium pink within 24 hours.
Weakly positive organisms may take several days, and negative organisms produce no color
change or yellow as a result of acid production.

Procedure
1. Streak the slant portion of a urea agar (Christensen’s medium) slant
2. Cap loosely.
3. Incubate at 37oC for 24 hrs.
Result:
Positive: Pink color, often starting at the slant. Rapid urea splitters such as
Proteus produce a positive reaction in 1-2 hours. Slow splitters take 24-48 hours.
Negative: the agar remains yellow
MICRO-202 Veterinary Microbiology and Mycology

IMViC tests

The IMViC tests are a group of individual tests used in microbiology lab testing to identify an
organism in the coliform group. A coliform is a gram negative, aerobic, or facultative anaerobic
rod, which produces gas from lactose within 48 hours. The presence of some coliforms indicate
fecal contamination.
The term "IMViC" is an acronym for each of these tests. "I" is for indole test; "M" is for methyl
red test; "V" is for Voges-Proskauer test, and "C" is for citrate test. The lower case "i" is merely
for "in" as the Citrate test requires coliform samples to be placed "in Citrate"

INDOLE TEST

This test demonstrates the ability of certain bacteria to decompose the amino acid tryptophane to
indole, which accumulates in the medium. Indole production test is important in the
identification of Enterobacteria. Most strains of E. coli and proteus species break down the
amino acid tryptophan with the release of indole.
Reagents:
Kovac′s reagent is prepared by mixing p-dimethylaminobenzaldehyde, isoamyl alcohol and
concentrated hydrochloric acid.
p-dimethylamino-benzaldehyde 20.0g,
Iso-amyl alcohol: 300.0 ml,
Concentrated hydrochloric acid 100.0 ml
The aldehyde is dissolved in the alcohol with gentle heat. The acid is added after cooling. The
reagent is stored in a dark bottle at 4oC.

Principle
Tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that
express tryptophanase enzyme. Tryptophanase catalyzes the deamination reaction, during which
the amine (-NH2) group of the tryptophan molecule is removed. Final products of the reaction
are indole, pyruvic acid, ammonium (NH4+) and energy.

Figure: Indole Test Reaction

MICRO-202 Veterinary Microbiology and Mycology

When indole is combined with Kovac’s Reagent (which contains hydrochloric acid and p-
dimethylaminobenzaldehyde in amyl alcohol) the solution turns from yellow to cherry red.
Because amyl alcohol is not water soluble, the red coloration will form in an oily layer at the top
of the broth.

Procedure
Method :
a) Incubate the organism in trypticase soy broth containing tryptophan
b) After overnight incubation, a few drops of indole reagent (Kovac’s reagent) are added.

Result:
Indole Positive: Red (pink) ring at the interface within seconds.
Indole test Negative - yellow ring

Indole positive – E.coli

Indole negative – Klebsiella, Salmonella.
MICRO-202 Veterinary Microbiology and Mycology

Citrate Utilization test

Principle:
The purpose of the citrate test is to determine if an organism is capable of utilizing sodium citrate
as a sole source of carbon. If sodium citrate is utilized, alkaline products including NaOH are
produced. Simmons Citrate agar is used to test an organism’s ability to utilize citrate as a source
of energy. The medium contains citrate as the sole carbon source and inorganic ammonium salts
(NH4H2PO4) as the sole source of nitrogen.
Bacteria that can grow on this medium produce an enzyme, citrate-permease/citrase, capable of
converting citrate to pyruvate in the cell. Pyruvate can then enter the organism’s metabolic cycle
for the production of energy. Growth is indicative of utilization of citrate, an intermediate
metabolite in the Krebs cycle.
When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which
increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from
green to blue above pH 7.6.

Procedure
1. Inoculate the surface of Simmons citrate slant in a single streak.
2. Cap loosely and incubate 24 hours.
Result: Positive test shows development of deep blue color or visible growth along with the
streak.
Negative test: No colour change
MICRO-202 Veterinary Microbiology and Mycology
MICRO-202 Veterinary Microbiology and Mycology

Methyl Red (MR) test

Principle
Some bacteria have the ability to utilize glucose and convert it to a stable acid like lactic acid,
acetic acid or formic acid as the end product.
These bacteria initially metabolise glucose to pyruvic acid, which is further metabolized through
the ‘mixed acid pathway to produce the stable acid. The type of acid produced differs from
species to species and depends on the specific enzymatic pathways present in the bacteria. The
acid so produced decreases the pH to 4.5 or below, which is indicated by a change in the colour
of methyl red from yellow to red.
In the methyl red test (MR test), the test bacteria is grown in a broth medium containing glucose.
If the bacteria has the ability to utilize glucose with production of a stable acid, the colour of the
methyl red changes from yellow to red, when added into the broth culture.

Methyl red solution, 0.02%

a. Dissolve 0.1 g of methyl red in 300 ml of ethyl alcohol, 95%.
b. Add sufficient distilled water to make 500 ml.
c. Store at 4 to 8oC in a brown bottle. Solution is stable for 1 year.
Procedure of Methyl Red (MR) Test
1. Incubate the organism in broth medium for an 24 hours.
2. Take 1 ml of broth in another tube and add 2 to 3 drops of methyl red indicator to it.
3. Observe for red color immediately.
Results:
MR positive – red colour (E. coli, Yersinia sps)
MR negative – yellow colour (Enterobacter aerogenes, Klebsiella pneumoniae, etc.)
MICRO-202 Veterinary Microbiology and Mycology

Voges-Proskauer (VP) Test

The VP test determines the capability of some organisms to produce nanacidic or neutral end
products such as acetylmethyl carbinol (acetoin) from the organic acids that result from glucose
metabolism.
Principle:
The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethyl
carbinol from glucose fermentation. If present, acetylmethyl carbinol is converted to diacetyl in
the presence of ∝- naphthol, strong alkali (40% KOH), and atmospheric oxygen.
The diacetyl and guanidine (creatine) containing compounds found in the peptones of the broth
then condense to form a pinkish red polymer.

Reagent:
Barritt’s reagent solution A: is prepared by dissolving 6 grams of a-naphtholin in 100 ml of 95%
ethyl alcohol.
Barritt’s reagent solution B: is prepared by dissolving 16 grams of potassium hydroxide in 100
ml of water.

Procedure
1. Take 18-24 hour pure broth culture and aliquot 2 ml of the broth to a clean test tube.
4. Add 6 drops of 5% alpha-naphthol, and mix well to aerate.
5. Add 2 drops of 40% potassium hydroxide, and mix well to aerate.
2. A bright orange red color develops and gradually extends throughout the broth if
acetylmethyl-carbinol has been produced.
3. The color development takes 30 minutes or longer.
Note: Be extremely careful with the KOH. It is caustic and may cause burns if it gets on your
skin.

Positive Reaction:
A pink-red color at the surface
Examples: Viridans group streptococci (except Streptococcus vestibularis), Listeria,
Enterobacter, Klebsiella, Serratia marcescens, Hafnia alvei, Vibrio eltor, Vibrio alginolyticus, etc.
MICRO-202 Veterinary Microbiology and Mycology

Negative Reaction: A lack of a pink-red color

Examples: Streptococcus mitis, Citrobacter sp., Shigella, Yersinia, Edwardsiella, Salmonella,
Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio parahaemolyticus etc.
MICRO-202 Veterinary Microbiology and Mycology

Carbohydrate Fermentation Test

The ability of bacteria to form organic compounds by metabolizing certain carbohydrates and
related compounds is a widely used method for the identification of microorganisms. Different
fermentation media are used to differentiate organisms based on their ability to ferment
carbohydrates incorporated into the basal medium.
 Phenol Red Broth Medium with various added carbohydrates (Sugars) serves as a differential
medium by aiding in differentiation of various species and genera by their ability to ferment
the specific carbohydrate, with the production of acid or acid and gas.
 The carbohydrate source can vary based on test requirements. The common broth media used
are:
 Phenol Red Glucose Broth
 Phenol Red Lactose Broth
Objective

To determine the fermentation reactions of pure cultures of microorganisms.

Principle

 The principle of carbohydrate fermentation states that the action of organism on a

carbohydrate substrate results in acidification of the medium, detected by a pH indicator
dye.
 Carbohydrate fermentation is the process microorganisms use to produce energy. Most
microorganisms convert glucose to pyruvate during glycolysis; however, some organisms
use alternate pathways.
 A fermentation medium consists of a basal medium containing a single carbohydrate
(glucose, lactose, sucrose, mannitol etc.) for fermentation.
 However, the medium also contains various pH indicators.
 In addition to a pH indicator to detect the production of acid from fermentation, a
Durham tube is placed in each tube to capture gas produced by metabolism.
 The carbohydrate fermentation patterns shown by different organisms are useful in
differentiating among bacterial groups or species.
 Phenol Red Broth is a general-purpose differential test medium typically used to
differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH
indicator), a Durham tube, and one carbohydrate (glucose, lactose, or sucrose). Phenol
red is a pH indicator which turns yellow below a pH of 6.8 and purplish above a pH of
7.4.
 If the organism is able to utilize the carbohydrate, an acid by-product is created, which
turns the media yellow.
 If the organism is unable to utilize the carbohydrate but does use the peptone, the by-
product is ammonia, which raises the pH of the media and turns it purple.
 When the organism is able to use the carbohydrate, a gas by-product may be produced. If
gas is produced an air bubble will be trapped inside the Durham tube. If the organism is
unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be
formed.
MICRO-202 Veterinary Microbiology and Mycology

Figure showing results of Sugar fermentation test

Method

1. Aseptically inoculate each test tube with the test microorganism using an inoculating
needle or loop.
2. Incubate tubes at 35-37°C for 18-24 hours.
3. Check for color changes or formation of gas.
Result Interpretation

I. Acid production:
 Positive: After incubation the liquid in the tube turns yellow (indicated by the change in
the color of the phenol red indicator). It indicates that there is drop in the pH because of
the production of the acid by the fermentation of the carbohydrate (sugar) present in the
media.
 Negative:The tube containing medium will remain red, indicating the bacteria cannot
ferment that particular carbohydrate source present in the media.
II. Gas Production
 Positive:A bubble (small or big depending up the amount of gas produced) seen in the
inverted Durham tube.
 Negative: No bubble in the inverted Durham tube i.e. bacteria does not produce gas from
the fermentation of that particular carbohydrate present in the media i.e. anaerogenic
organism.
MICRO-202 Veterinary Microbiology and Mycology

Figures showing gas bubble in the durhum tube.

Uses

 It is recommended to determine the fermentation reaction of carbohydrates for the

differentiation of microorganisms.
 It is useful in identifying Gram negative bacilli, especially Enterobacteriaceae.
Limitations

 Phenol Red Broth test is not intended for use in the diagnosis of disease or other
conditions in humans.
 Due to nutritional variation, some strains may be encountered that grow poorly or fail to
grow on this medium.
 The addition of some carbohydrates to the basal medium may result in an acid reaction.
To ensure accuracy of interpretation, uninoculated control tubes and/or inoculated Phenol
Red Broth Base control tubes should be run in parallel with the fermentation tests.