BIOCHEMICAL TESTING

Bile Solubility Test:

 Used to differentiate
S.peumoniae which
is soluble in bile and
bile salts from
viridans streptococci
which are insoluble.
 A heavy inoculum of
the test organism is
emulsified in
physiological saline
to give a turbid
suspension.
 Bile salt is then
added to the cell
suspension.
 Bile salt
dissolves
S.pneumoniae
shown by
clearing of
turbidity within
10 - 15 min.
 Viridans
streptococci are
not dissolved
and there is no
clearing of
turbidity.
 Bile-Aesculin Test
 Used to
differentiate group
D streptococci from
other streptococci.
 Group D
streptococci can
grow in the
presence of 40%
bile and
subsequently
hydrolyze aesculin
to aesculetin and
glucose.
 Aesculetin
combines with ferric
citrate in the
medium to give a
black complex.
 Bile-Aesculin Test
Method

The test organism
is inoculated on a
bile-aesculin agar
and incubated at
37 C for 18 - 24
o
hours and for an
additional day if
the test is
negative.

Blackening of
agar is a positive
test.

Positive - group D
Streptococcus.

Negative -
viridans
Streptococci.
 Catalase Test:
 Used to
differentiate
bacteria that
produce the
enzyme catalase
e.g. staphylococci
from non-catalase
producing
bacteria e.g.
streptococci.
 Catalase breaks
down hydrogen
peroxide to O2
and H2O.
 Catalase Test:
Method
 Bacterial cells
from blood-free
medium are
transferred to a
slide followed by
addition of H2O2 or
the cells are added
to a test tube
containing 3%
H2O2.
 Bubbles of oxygen
are released if the
organism
produces
catalase.
 Citrate Test:
 Used in the
identification of
enterobacteria.
 Test is based on
the ability of an
organism to use
citrate as the
only source of
carbon and
ammomnia as
the only source
of nitrogen.
 Citrate Test:
Method
 Organism is
cultured in medium
which contains
sodium citrate, an
ammonium salt
and pH indicator,
bromo-thymol
blue.
 Change in colour of
pH indicator from
light green to blue,
due to an alkaline
reaction, following
citrate
utilization is a
positive result.
 Citrate Test:
 Klebsiella
pneumoniae
gives a positive
reaction
whereas
Escherichia coli,
Citrobacter and
Enterobacter
species are
negative.
 Coagulase Test:
 Used to differentiate Staphylococcus
aureus, which produces the
coagulase enzyme, from
S.epidermidis and S.saprophyticus
which do not produce coagulase.
 Coagulase causes plasma to clot by
converting fibrinogen to fibrin
 Coagulase Test:
Two Types of Coagulase produced by
S.aureus:

a) Free coagulase - which converts
fibrinogen to fibrin by activating a
coagulase-reacting factor present in
plasma.

Free coagulase is detected by the
appearance of a fibrin clot in the test tube
where S.aureus cells are mixed with
human or rabbit plasma.

b) Bound coagulase (clumping factor) -
which converts fibrinogen directly to fibrin
without requiring a coagulase-reacting
factor.

Can be detected by clumping of bacterial
cells in the rapid slide test where plasma
and bacterial cells are mixed.
 Coagulase Test:
Slide Method:
 Colony of organism
is emulsified in two
drops of
physiological saline
on a glass slide to
make thick
suspensions.
 A drop of plasma is
added to one
suspension and
mixed gently,
clumping of cells
should occur within
10 sec.
 The other
suspension is a
control.
 Coagulase Test:
Test Tube Method:
 Suspension of the test
organism is added to
tube with dilute
plasma.
 The contents are
mixed and the tube is
incubated at 37oC for
at least one hour and
observed for a clot.
 A positive control e.g.
S.aureus and a
negative control e.g.
sterile broth are
carried out.
Deoxyribonuclease
(DNAse) Test:
 Used to differentiate
S.aureus which produces
DNAse enzyme from other
staphylococci.
Method
Test organism is cultured
on a medium containing
DNA.
 After overnight incubation,
colonies are tested for
DNAse production by
flooding the plate with a
weak hydrochloric acid
solution.
 DNAse producing colonies
are surrounded by clear
areas indicating DNA
hydrolysis.
 Acid precipitates
unhydrolysed DNA.
 Hydrogen Sulphide
(H2S) Production:
 Used in the
identification of
enterobacteria.

H2S is produced when
sulphur-containing
amino acids (cystein,
methionine) are
decomposed.
Kligler Iron Agar
(KIA):

H2S is detected by the
ferric citrate
contained in the
medium which reacts
with H2S produced
resulting in
blackening of medium.
 Hydrogen Sulphide
(H2S) Production:
 Salmonella and
Proteus species
are positive
whereas
Shigella, E.coli
and Klebsiella
species are
negative.
 Indole Test:

Used in identification
of enterobacteria e.g.
E.coli and Proteus
vulgaris which break
down the amino acid
tryptophan
with release of indole.
Method

Test organism is
cultured in liquid
medium which
contains tryptophan.

Indole production is
detected by addition
of Kovac's reagent (P-
dimethylaminobenzal
dehyde) which reacts
with indole to
produce a red
coloured compound.
 Oxidase Test (Cytochrome
Oxidase):
 Used in the
identification of
Pseudomonas,
Neisseria,
Vibrio,
Aeromonas,
Pasteurella,
Campylobacter,
which produce
Cytochrome C
oxidase enzymes.
 Oxidase Test (Cytochrome
Oxidase):
Method
 A piece of filter
paper is soaked
with a few drops of
the oxidase
reagent.
 Colony of organism
is smeared on
moistened filter
paper.
 Oxidase-producing
organism oxidizes
the
phenylenediamine
in reagent to a
deep purple colour.
 Urease Test:

Used to differentiate
enterobacteria.
Method

Test organism is
cultured in a
medium containing
urea and phenol red
(pH indicator).

Urease-producing
organisms hydrolyse
urea to give
ammonia and CO2.

Medium becomes
alkaline after
release of ammonia
as shown by change
in colour of indicator
to pink red.
 Urease Test:
 Proteus and
Klebsiella
species are
urease positive
whereas
Shigella and
Salmonella are
urease negative.
Other Biochemical Tests
 Hippurate hydrolysis test.
 Voges-Proskauer test.
 Methyl Red test.
 IMViC Tests
 SENSITIVITY AND
RESISTANCE TO
CHEMICALS:
Optochin Test:
 Used to identify

Streptococcus
pneumoniae
which is
sensitive to
optochin.
 Most other

streptococci e.g.
Viridans
streptococci are
resistant to
optochin.
 Optochin Test:
Method
 Chocolate agar plate

is inoculated with
test organism to
have confluent
growth.
 Optochin disc is

placed on inoculated
agar medium and
then incubated.
 Zone of inhibition

around the optochin

disc indicates
that the organism is
sensitive to
optochin.
 Bacitracin Sensitivity
Test:
 Used to identify
Streptococcus
pyogenes (Group
A).
 The other β-
haemolytic
streptococci are
resistant to
bacitracin e.g.
Streptococcus
agalactiae (group
B),
Enterococci (Group
D).
 Bacitracin Sensitivity
Test:
Method
 Blood agar plate is

inoculated with test

organism to have
confluent growth.
 Bacitracin disc is

placed on
inoculated BA plate.
 Zone of inhibition

around the
bacitracin disc after
incubation indicates
that the organism is
sensitive to the
antibiotic.
Bacitracin Sensitivity
Test:
GROWTH FACTOR
REQUIREMENTS:
X and V Factors:
 Used to identify Haemophilus species which
are fastidious organisms.
 X factor - porphyrin (haematin) is required
for respiratory activities when bacteria are
grown aerobically.
 V factor - NAD or NADP.
 Blood agar has mainly X factor available but
both X and V factors are present in
Chocolate agar.
 X and V Factors:
Method

Test organism is
inoculated on
Mueller Hinton agar
to have confluent
growth.

Discs with X, V and
XV factors are placed
on inoculated plate.

Growth of organism
around disc indicates
requirement for
growth factor.

H.influenzae and
H.aegyptius require
both X and V factors.

H.parainfluenzae
requires V factor.

H.ducreyi requires X
factor.
 Satellitism Test:
 Used to identify
Haemophilus species.
Method
 Test organism is
inoculated on Mueller
Hinton agar and Blood
agar in order to have
confluent growth.
 A pure culture of
Staphylococcus aureus
is streaked across
each plate or on a
spot and the plate is
incubated.
 Satellitism Test:
 H.influenzae grows
as satellite colonies
around S.aureus
culture on BA and
not on Mueller
Hinton agar because
S.aureus produces
only the V factor.
 Growth on both
plates suggests
H.parainfluenzae
which requires only
the V factor.
 CAMP Test:

Used to identify
Streptococcus
agalactiae (Group B).
S.agalactiae
produces
extracellular protein
(CAMP factor) which
interact with
staphylococcal β-
haemolysin on sheep
or ox rbc.
Method

A staphylococcal
culture is streaked
across a BA plate and
then inoculated with
the test organism at
right angles to it
without touching the
staphylococcal
inoculum.
CAMP Test:
 CAMP Test:
 The inoculated plate
is incubated
aerobically at 37oC
for 18 – 24 hours.
 Interaction of test
organism with
staphylococcal
haemolysin, showing
an arrowhead of
haemolysis after
overnight incubation,
indicates that the
test organism is
S.agalactiae.
 Motility Test:
 Used to identify
enterobacteria.
Method
 Test organism is
inoculated by stabbing
in a semisolid medium
with 0.5% agar.
 After incubation,
motile organisms
produce growth
throughout the
medium whereas non-
motile organisms grow
only along the line of
inoculum.
 E.coli, Salmonella,
Proteus sp. are motile;
Shigella and Klebsiella
sp are non-motile.
THE END