BIOCHEMICAL
TESTS
BACTERIAL IDENTIFICATION
1. Catalase test
2. Coagulase test
3. Oxidase test
4. Indole production
5. Citrate utilization
6. Methyl Red test
7. Voges Prausker test
8. Hydrogen sulphide production
CATALASE TEST
➢ Testis performed to detect the production of enzyme
Catalase by some bacteria
➢ Hydrogen Peroxide (H2O2) is formed as metabolic end
product & is toxic to the bacteria when accumulated .
➢The enzyme acts on H2O2 to form water and oxygen
CATALASE
H 2O 2 2H2O + O2
Ø A small amount of
bacterial colony is
taken using a sterile
capillary tube
Ø It is dipped in 3%
H2O2 .
Ø If catalase is
produced , then
oxygen formed is
released as
effervescence ,
indicating a positive
test
COAGULASE TEST
•Helps to differentiate S. aureus from all other species
( COPS & CONS) – based on production of
COAGULASE enzyme
•The enzyme clots human and rabbit plasma
•Two methods – Slide method ( bound coagulase ) and
Tube method (free coagulase )
•Slide coagulase - clumping factor , surface protein .
S. aureus suspension in saline + drop of Human
plasma shows clumping on the slide .
TUBE COAGULASE : Coagulase
enzyme in the presence of CRF ,
binds to prothrombin and convert
fibrinogen to fibrin .
TEST : a.) 0.5ml human plasma is
mixed with 0.1ml of broth culture of
Staphylococcus in a test tube.
b.) The tubes are incubated at 370C
for 3 – 6hrs .
c.) If positive , plasma clot is formed
which does not flow when the tube
is tilted .
Positive
Negative
OXIDASE TEST
➢ Thetest detects the presence of the enzyme Cytochrome
C Oxidase
➢ It catalyses the reaction of terminal electron acceptor in
the electron transport chain, resulting in oxidation
➢The substrate used in the test is Tetramethyl p phenylene
diamine dihydrochloride, which on oxidation forms a purple
colour
Tetramethyl paraphenylene
diamine dihydrochloride
(Kovac’s reagent)
ENZYME : CYTOCHROME C
OXIDASE
INDOLE TEST
➢Bacteria breakdown Tryptophan (aa) to produce
Indole in the presence of the enzyme
Tryptophanase
INDOLE
➢Indole reacts with aldehydes to give a red coloured
complex RED
COLOURED
COMPLEX
ALDEHYDE
24 hour broth culture of the test
organism is taken
5 drops of p –
dimethylaminobenzaldehyde is added
( dissolved in isoamyl alcohol)
The alcohol extracts the indole , which
reacts with the aldehyde forming a red
ring on top of the broth
METHYL RED TEST
➢ This test helps to detect the ability of bacteria to produce and maintain stable acid end
products of glucose fermentation
➢Test is done with MRVP broth
➢Bacterial culture is inoculated into the broth
➢The tubes are incubated at 370C for 48 hours
➢Few drops of Methyl red indicator are added
➢Positive – red colour , Negative – pale .
VOGES PRAUSKER TEST
➢This test helps to detect ability of organisms to form Acetyl methyl carbinol (Acetoin) as
a product on prolonged fermentation of glucose.
➢The test is done with MRVP broth , after 48 hours of incubation .
➢To the test broth , α napthol and 40% KOH are added in the same order .
➢Acetoin combines with α napthol and KOH to form Diacetyl giving a pinkish red polymer
CITRATE UTILIZATION
➢ Test is done to identify if bacteria can utilize citrate as the sole source of carbon.
➢Media contains sodium citrate and ammonium salts .
➢End product of the citrate and ammonium salts breakdown is alkaline – pH
➢The pH indicator in the medium changes colour accordingly
➢Commonly used media is Simmon’s Citrate Agar , pH indicator is Bromothymol blue
Media incubated at 370C /24hrs
Media
turns
BLUE
Citrate
breakdown
(alkaline pH)
Simmons
citrate
media Bacteria inoculated on slant by stroke method
(Green)
HYDROGEN
SULPHIDE TEST
➢ This test is performed to
detect the production of H2S by
bacteria , due to breakdown of
sulphur compounds
➢H2S combines with iron to form
a black precipitate - Ferric
Sulphide (Triple Sugar Iron
Agar , TSI )
POSITIVE
HYDROGEN SULPHIDE
TEST
➢Test can also be done using Lead
Acetate papers – H2S gas combines
with lead acetate forming lead
sulphide ( black residue)
GRAM POSITIVE COCCI
CATALASE TEST
+ -
STAPHYLOCOCCUS STREPTOCOCCUS
COAGULASE TEST
SPECIATION BASED ON
HEMOLYSIS AND
+ - BIOCHEMICAL REACTIONS **
S. AUREUS
CONS
(COPS)
GRAM NEGATIVE BACILLI
MA AGAR
LACTOSE FERMENTING NON LACTOSE
COLONIES FERMENTING COLONIES
Assignment
•List two examples for all biochemical reactions (2 each
for positive and negative )
•Write a flow chart to explain steps for identification of
gram-positive cocci . Mention the tests** to differentiate
Streptococcus species.
