MLS 441 by DR VERA

BIOCHEMICAL TESTS FOR

IDENTIFICATION OF BACTERIA
When a culture plate has been grown under the
required temperature and other environmental
factors, the plate reading should be done to know
the organism that grew on the plate using these
steps:-
1. Macroscopic reading of the colonial morphology,
checking for: the colour, texture, topography,
smell of the colony. Hemolysis and pigmentation.

2. Do a gram staining of the colony to know if it is

gram positive or gram negative organism.
3. Then run the necessary biochemical reactions
on the colonies to further differentiate the
organisms.
Biochemical reactions
Tests used to identify Gram Positive
Bacteria

• Catalase Test
• Mannitol salt Agar (MSA)
• Optochin sensitivity testing)
• Bacitracin sensitivity Testing)
• CAMP Test
• Bile Esculin Agar
• Starch hydrolysis test
• Motility Agar
• Coagulase Test
Tests used to identify Gram
Negative Bacteria
Oxidase Test
Carbohydrate utilization test with
Durham tubes
Methyl Red/Voges-Proskauer (MR/VP)
Kliger’s Iron Agar (KIA)
Motility Agar
Simmon’s Citrate Agar
Urease test
Indole Motility Media (IM)
 Carbohydrate fermentation
test (oxidative-
fermentation tests).
 Carbohydrate fermentation is the process
by which the microorganism utilizes any of
the carbohydrate (glucose, xylose,
mannitol, lactose, sucrose, and maltose) to
produce energy in the form of ATP as the
ultimate energy source for the organism.

 Principle: Glucose after entering a cell

can be catabolized either aerobically (in
the presence of O2), (oxidative pathway),
or anaerobically (in absence of O2) in
which inorganic ions can serve as the final
electron acceptor (fermentative pathway).
 The metabolic end products of a carbohydrate
fermentation can either be organic acids (lactic,
formic, acetic acid) and gas (hydrogen or carbon
dioxide).

 A fermentation medium is composed of a basal

medium containing a specific carbohydrate
(glucose, sucrose, or cellulose) along with a pH
indicator (phenol red, Andrade’s indicator, or
bromocresol).

 The gas production as a result of the

fermentation is shown in the Durham tube. This
is a test commonly used when trying to identify
Gram-negative enteric bacteria, all of which are
glucose fermenters but only some of which
 Preparation of
Carbohydrate Fermentation
Broth Sodium chloride,
Weigh and dissolve trypticase,
and Phenol red in 100 ml distilled water and
transfer into conical flasks.
Add 0.5% to 1% of the desired carbohydrate into
all flasks.
Insert inverted Durham tubes into all tubes, the
Durham tubes should be fully filled with broth.

 Sterilize in an autoclave at 115°C for 15 minutes.

(Note: Do not overheat the Phenol red
Carbohydrate fermentation broth. The overheating
will result in breaking down the molecules and
form compounds with a characteristic color and
flavor. The process is known as the caramelization
of sugar (the browning of sugar).
Inoculation of Bacterial
Culture into fermentation
medium tube
Inoculate each tube with 1 drop
of an 18 hour or 24-hour cultural
broth in aseptic condition (keep
uninoculated tubes as control
tubes).
Incubate the tubes at 18-24
hours at 37°C
Examine the tube for acid and
gas production.
Escherichia coli, Proteus mirabilis are
capable of fermenting glucose.
This gas is trapped in the Durham
tube and appears as a bubble at the
top of the tube. Escherichia
coli and Proteus mirabilis (far right)
are both gas producers.

Notice that Shigella dysenteriae (far

left) ferments glucose but does not
produce gas.
Pseudomonas aeruginosa (center) is a
nonfermenter.
Carbohydrate fermentation
test
 1b. The Triple Sugar Iron (TSI)
 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 to solidify at a slanted angle.
 The slanted shape of this medium provides an
array of surfaces that are either exposed to
oxygen-containing air in varying degrees
Objective:-To determine the ability of an
organism to ferment glucose, lactose, and
sucrose, and their ability to produce
hydrogen sulfide.
Method:- . With a straight inoculation needle,
touch the top of a well-isolated colony.

Inoculate TSI by first stabbing through the

center of the medium to the bottom of the
tube and then streaking the surface of the
agar slant.

Leave the cap on loosely and incubate the

tube at 35°-37°C for 18 to 24 hours.
Examine the reaction of medium.
 Uses:-
 The test is used primarily to differentiate members of
the Enterobacteriaceae family from other gram-negative
rods.
 It is also used in the differentiation among
Enterobacteriaceae on the basis of their sugar
fermentation patterns.

 Triple sugar iron agar result:-

 A, Acid slant/acid butt with gas, no H2S (A/A).
 B, Alkaline slant/acid butt, no gas, H2S-positive (K/A H2S+).
 C, Alkaline slant/alkaline butt, no gas, no H2S (K/K).
 GLUCOSE METABOLISM AND ITS
PRODUCT (Methyl red/ Voges-Proskauer
test)
 This test is used to determine which
fermentation pathway is used to utilize glucose.
The mixed acid fermentation pathway, where
glucose is fermented and produces several
organic acids (lactic, acetic, succinic, and formic
acids).

 The stable production of enough acid to

overcome the phosphate buffer will result in a
pH of below 4.4. If the pH indicator (methyl red)
is added to an aliquot of the culture broth and
the pH is below 4.4, a red color will appear (first
picture, tube on the left).


 Voges-Proskauer test detects the presence of
acetoin, a precursor of 2,3butanediol.

 If the culture is positive for acetoin, it will turn

“brownish-red to pink” (tube on the left in the
second picture). If the culture is negative for
acetoin, it will turn “brownish-green to yellow”
(tube on the left in the second picture). Note: A
culture will usually only be positive for one
pathway: either MR+ or VP+. Escherichia coli is
MR+ and VP- .

 In contrast, Enterobacter aerogenes and Klebsiella

pneumoniae are MR- and VP+. Pseudomonas
aeruginosa is a glucose nonfermenter and is thus
MR- and VP-.
Positive and Positive and
Negative results Negative results for
for MR test VP test
 2b. Kliger’s Iron Agar (KIA)
 Thisis a differential medium. It tests for
organisms’ abilities to ferment glucose and
lactose to acid and acid plus gas end products. It
also allows for identification of sulfur reducers.

 This media is commonly used to separate lactose

fermenting members of family
Enterobacteriaceae (e.g. Escherichia coli) from
members that do not ferment lactose,
like Shigella dysenteriae.

 These lactose non fermenting enterics generally

tend to be the more serious pathogens of the
gastrointestinal tract. KIA tubes are also capable
of detecting the production of H2S.
KIA tubes test
 AMINO ACID DECARBOXYLASE
TEST :- (Decarboxylase test)

 Objective:-To differentiate decarboxylase producing

Enterobacteriaceae from other gram negative rods.

 Principle: Amino acids are metabolized variably by

gram negative aerobic and facultatively anaerobic
bacteria as well as gram positive cocci.

 These amino acids are decarboxylated, hydrolysed or

deaminated depending on the organism and the
amino acid in question.

 In decarboxylation, the enzymes break the bond

holding the carboxylic (-COOH) group to the rest of
the amino acid.
There are three decarboxylase enzymes
that is routinely tested for – arginine
decarboxylase, ornithine
decarboxylase, and lysine
decarboxylase.

The production of lysine, arginine,

ornithine decarboxylase by various
members of Enterobacteriaceae offers
an important parameter to other
biochemical tests for differentiating
bacteria within closely related groups.
Decarboxylase Test.
 Phenylalanine deaminase test:
 This
is 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.

 Phenylalanine Deaminase Test is a procedure used to

find out the capability of Proteus species to
deaminate phenylalanine to phenylpyruvic acid with
the aid of an enzyme.

 Specifically,
it is used to differentiate Proteus and
Providencia organisms from the other
Enterobacteriaceae.
Phenylalanine
deaminase test
OTHER BIOCHEMICAL TEST
Nitrate Reduction Tests
Uses: In Differentiating
Mycobacterium species.
Identifying Corynebacterium and
Neisseria and separating them
from Moraxella and Kingella species.
The nitrate reduction test is a critical
test for differentiating between
N.gonorrhoeae and differentiating
Mycobacterium species.
 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.
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)
 NB;-Pseudomonas aeruginosa reduces
NO3 (Nitrate) to N2 (Nitrogen).
Escherichia coli reduces NO3 (Nitrate) to
NO2 (Nitrite).
Bile Esculin Agar
ESCULIN TEST
This is a medium that is both selective and
differential. It tests the ability of
organisms to hydrolyze esculin in the
presence of bile. It is commonly used to
identify members of the
genus Enterococcus (E faecalis and E.
faecium).
The first selective ingredient in this agar is
Bile, which inhibits the growth of Gram-
positives other than enterococci and some
streptococci species. The second selective
ingredient is Sodium azide. This chemical
inhibits the growth of Gram-negatives.
The differential ingredient is esculin.
If an organism can hydrolyze esculin in
the presence of bile, the product
esculetin is formed. Esculetin reacts
with ferric citrate (in the medium),
forming a phenolic iron complex which
turns the entire slant dark brown to
black.
The tube on the far right was
inoculated with E. faecalis (positive).
The tube in the center was inoculated
with a biie esculin negative organism
and the tube on the left was
uninoculated.
Catalase Test:
This test is used to identify organisms
that produce the enzyme, catalase. This
enzyme detoxifies hydrogen peroxide by
breaking it down into water and oxygen
The bubbles resulting from
production of oxygen gas clearly
indicate a catalase positive
result. The sample on the right
below is catalase positive.
The Staphylococcus spp. and
the Micrococcus spp. are catalase
positive.
The Streptococcus and Enterococ
cus spp. are catalase negative.
 Oxidase Test:
 This test is used to identify microorganisms containing the
enzyme cytochrome oxidase (important in the electron
transport chain). It is commonly used to distinguish
between oxidase negative Enterobacteriaceae and
oxidase positive Pseudomadaceae.

 Coagulase test:
 Coagulase is an enzyme that clots blood plasma. This test is
performed on Gram-positive, catalase positive species to
identify the coagulase positive Staphylococcus
aureus. Coagulase is a virulence factor of S. aureus. The
formation of clot around an infection caused by this bacteria
likely protects it from phagocytosis. This test
differentiates Staphylococcus aureus from other coagulase
negative Staphylococcus species
 coagulase

 Bacitracin sensitivity testing:

 This is a differential test used to distinguish between
organisms sensitive to the antibiotic bacitracin and those not.
Bacitracin is a peptide antibiotic produced by Bacillus subtilis.
It inhibits cell wall synthesis and disrupts the cell membrane.
This test is commonly used to distinguish between the-
hemolytic streptococci: Streptococcus agalactiae (bacitracin
resistant) and Streptococcus pyogenes (bacitracin sensitive).
The plate below was streaked with Streptococcus pyogenes;
notice the large zone of inhibition surrounding the disk.
 Optochin sensitivity testing
 This is a differential test used to distinguish
between organisms sensitive to the antibiotic
optochin and those not. This test is used to
distinguish Streptococcus pneumoniae (optochin
sensitive (pictured on the right below) from
other hemolytic streptococci (optochin resistant
Streptococcus mitis is pictured on the left below).
 Simmon’s Citrate Agar
 This is a defined medium used to determine if an organism can
use citrate as its sole carbon source. It is often used to
differentiate between members of Enterobacteriaceae. In
organisms capable of utilizing citrate as a carbon source, the
enzyme citrase hydrolyzes citrate into oxaoloacetic acid and
acetic acid. The oxaloacetic acid is then hydrolyzed into
pyruvic acid and CO2. If CO2 is produced, it reacts with
components of the medium to produce an alkaline compound
(e.g. Na2CO3). The alkaline pH turns the pH indicator
(bromthymol blue) from green to blue. This is a positive result
(the tube on the right is citrate
positive). Klebsiellapneumoniae and Proteus mirabilis are
examples of citrate positive organisms. Escherichia
coli and Shigelladysenteriae are citrate negative.
Citrate test
 Urease test
 This test is used to identify bacteria capable of hydrolyzing
urea using the enzyme urease. It is commonly used to
distinguish the genus Proteus from other enteric bacteria.
The media is called Christensen’s Urea Agar (pH.4 to 5).The
organism hydrolyzes the urea and forms a weak base,
ammonia, as one of its products. This weak base raises the
pH of the media above 8.4 and the pH indicator, phenol red,
turns from yellow to pink. Proteus mirabilis is a rapid
hydrolyzer of urea (center tube pictured here). The tube on
the far right was inoculated with a urease negative organism
and the tube on the far left was uninoculated.
 Urease test

 Motility agar: is a differential medium used to determine

whether an organism is equipped with flagella and thus
capable of swimming away from a stab mark. The results of
motility agar are often difficult to interpret. Generally, if the
entire tube is turbid, this indicates that the bacteria have
moved away from the stab mark (are motile). The organisms in
the two tubes pictured on the right are motile. If, however, the
stab mark is clearly visible and the rest of the tube is not
turbid, the organism is likely nonmotile (tube pictured on the
left).
 Indole test
 Purpose Theindole test screens for the ability of an organism
to degrade the amino acid tryptophan and produce indole. It
is used as part of the IMViC (indole, MR-Vp Citrate)
procedures, a battery of tests designed to distinguish among
members of the family Enterobacteriaceae. Procedure
Inoculate the tube of tryptone broth with a small amount of a
pure culture. Incubate at 37°C for 24 to 48 hours.
 To test for indole production, add 5 drops of Kovác's reagent
directly to the tube. A positive indole test is indicated by the
formation of a 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 be slightly cloudy. Indole positive
bacteria : E. coli, Vibrio cholera Indole negative bacteria :
Klebsiella, Salmonella, Shigella spp.
 DNase Test:-
 Principle:
 The test is used to determine the ability of an organism to
hydrolyze DNA. DNase agar is a differential medium that
tests the ability of an organism to produce an exo-enzyme,
called deoxyribonuclease. DNaseare extracellular
endonucleases that cleave DNA and release free nucleotides
and phosphate. DNase agar contains nutrients for the
bacteria, DNA, and mostly methyl green as an indicator.
Methyl green is a cation which binds to the negatively-
charged DNA.
 Uses: it is 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.
Gelatin liquefaction or
hydrolysis test:-
Principle

of Gelatin hydrolysis test:
Gelatin hydrolysis test is used to detect the ability of
an organism to produce gelatinases that liquefy
gelatin. This process takes place in two sequential
reactions. In the first reaction, gelatinases degrade
gelatin to polypeptides. Then, the polypeptides are
further converted into amino acids. The bacterial cells
can then take up these amino acids and use them in
their metabolic processes. Organisms that hydrolizes
or liquifies the gelatin are Bacillus subtilis
 Clostridium perfringens, Staphylococcus aureus whlile
E. coli does not.