Biochemical Tests for

Identification of Bacteria

Dr. Chinthika Gunasekara

Department of Microbiology
USJP
Biochemical tests:
 These tests are performed for identification of bacteria.

 Bacteria have various biochemical properties.

 These include production of enzymes,

 utilization of specific carbon and nitrogen sources,

 oxidative or fermentative metabolism.

Laboratory identification of Bacteria
Microscopy
Culture techniques
Biochemical tests

Demonstration of specific antigen or antibody

Demonstration of specific DNA or RNA
Doing a Biochemical test
Use overnight cultured colonies of bacteria or
inoculated broths.
Ensure the colony or broth is a pure culture and not a
mixed growth.
 Purity plate test to check for contamination
Use of a known positive control organism and
negative control organism.
Observing the result within the specified time.
Correct incubation time and temperature.
1. Catalase test
 This test is used to differentiate those bacteria that
produce the enzyme catalase, such as staphylococci,
from non-catalase producing bacteria such as
streptococci.
 This enzyme converts hydrogen peroxide into water
and oxygen.
 Principle:

Catalase producing bacteria will produce O2 when mixed with

H2O22. H2O2 -------------> 2 H2O and O2 (Bubbles)

Methods:

1. Slide Test

2. Tube Test

Slide Method:

 Take a drop of 3% H2O2 on a glass slide.

 Mix it with a small quantity of test bacteria.

Results:
Active bubbling . . . . . . . . . . . . Positive catalase test.
No bubbles . . . . . . . . . . . . . . Negative catalase test.
Caution: Performing the test on a slide is not

recommended because of the risk of contamination

from active bubbling.

When the rapid slide technique is used, the hydrogen

peroxide solution should be added to the organism

suspension after placing the slide in a petridish. The
dish should then be covered immediately, and the
preparation observed for bubbling through the lid.
 Tube Method
Pour 2–3 ml of the hydrogen peroxide solution into a test

tube.
Using a sterile wooden stick or a glass rod (not a nichrome

wire loop), remove several colonies of the test organism and

immerse in the hydrogen peroxide solution.
Important: Care must be taken when testing an organism

cultured on a medium containing blood because catalase is

present in red cells & a false positive reaction may occur.
Look for immediate bubbling.
Results
•Active bubbling . . . . . . . . . . . . Positive catalase test.
•No bubbles . . . . . . . . . . . . . . Negative catalase test.
 2- Coagulase Test:
This test is used to identify Staph. aureus, which produces coagulase enzyme.

Principle:
Coagulase causes plasma to clot by converting fibrinogen to fibrin. Two

types of coagulase are produced by most strains of Staph. aureus:

Free coagulase which converts fibrinogen to fibrin by activating a

coagulase-reacting factor present in plasma. Free coagulase is detected by

clotting in the tube test.
Bound coagulase(clumping factor) which converts fibrinogen directly to

fibrin without requiring a coagulase reacting factor. It can be detected by the

clumping of bacterial cells in the rapid slide test.
A tube test must always be performed when the result of a slide

test is not clear, or when the slide test is negative and

Staphylococcus has been isolated from a serious infection.

Requirements
Anticoagulated human plasma or rabbit plasma ( by EDTA,

oxalate or heparin).
The plasma should be allowed to warm to room temperature

before being used.

Do not use citrated plasma because citrate-utilizing bacteria

e.g. enterococci & Pseudomonas may cause clotting of the

plasma (in tube test).
1- Slide test method (detects bound coagulase):
Place a drop of distilled water on each end of a slide or on two
separate slides.
Emulsify a colony of the test organism in each of the drops to
make two thick suspensions.
Add a loopful (not more) of plasma to one of the suspensions,
and mix gently.
Look for clumping of
the organisms within 10 seconds.
Results
Clumping within 10 sec
. . . . . . Staph. aureus.
No clumping within 10 sec
. . . . . No bound Coagulase.
2- Tube test method (detects free coagulase):
Label two small test tubes one as: Test organism (18–24 h broth

culture) and the other as: Control (sterile broth).

Pipette 0.2 ml of plasma into each tube.

Add 0.8 ml of the test broth culture to “Test” tube .

Add 0.8 ml of sterile broth to “Control”

After mixing gently, incubate the tubes at 35–370C for 6-12 hours

and examine hourly.

If the test is still negative, leave the tube at room temperature

overnight and examine again.

Note: When looking for clotting, tilt each tube gently.
Results
Clotting of tube contents . . . . . . . . . . . Staph. aureus.
No clotting ………………. Not Staph. aureus.
 3- Oxidase Test:
The oxidase test is used to assist in the identification of

Pseudomonas, Neisseria, Vibrio, Brucella, and Pasteurella

species, all of which produce the enzyme cytochrome oxidase.
Principle
When the organism is oxidase-producing, ie. It produces the

enzyme cytochrome oxidase it can be detected by adding the

reduced tertra methyl –p- phenylenediamine dihydrochloride
(oxidase reagent) which will be will be oxidized by
molecular oxygen to a deep purple color.
Requirements:
1.Oxidase reagent
2.Stick or glass rod.
Method using an oxidase reagent strip:
Moisten the strip of sterile filter paper with oxidase reagent

dissolved in sterile water.

Using a piece of stick or glass rod (not an oxidized wire loop)

remove a colony of the test organism and rub it on the strip.

Look for a deep purple color within 20 seconds.

Results
Deep purple color. . . . . . . . positive oxidase test
 4- Urease Test:
Proteus strains are strong urease producers.

Principle:
The test detects the ability of an organism to produce

urease enzyme. The enzyme converts urea into ammonia

and carbon dioxide.
With the release of ammonia, the medium becomes

alkaline as shown by a change in colour of the pH

indicator phenol red to pink-red.
 Method
Inoculate the media Christensen’s

Medium with the tested

organism.
Incubate at 37°C for

18 –24 hours.

Results:
Change of the color into pink red ……………. Urease producing

“Proteus”
No change of the color …………… non urease producing organism.

(Salmonella , Shigella)
The tube on the left is a positive reaction; the tube in the middle is a negative
reaction and the tube on the right in an un-inoculated control.
IMViC test
This is a useful method for identification of

Escherichia coli and Klebsiella of the

Enterobacteriaceae family. It has 4 main tests:

Indole test

Methyl Red test

Voges-Proskauer test

Citrate utilization test

5- Indole production test
• Testing for indole production is important in the
identification of enterobacteria.

Principle:
 To determine the ability of an organism to produce enzyme
tryptophanase that splits amino acid tryptophan into indole.
 Indole production is detected by adding Kovac’s reagent (4
(p) dimethylaminobenzaldehyde to the test solution.
Kovac’s reagent reacts with the indole to produce a red
colored compound.
o Method
- The organism is inoculated in peptone water and after
incubation at 37 degree for 24 hours, Kovac’s reagent
is added.

o Result
- If a pink ring is produced, the organism is indole +Ve
(E. coli).
- If a yellow ring is produced, the organsim is indole –
Ve (Klebsiella).
6. Methyl Red test
Principle
Some organisms produce acid from the metabolism
of glucose (fermentation) in a sufficient quantity
to alter the pH of the media to about 4.4. These
are stable acids and are not further metabolized.

Methyl red indicator is used to detect the presence

of these acids in the MRVP broth medium.
Methyl red indicator at this pH (~ 4.4) changes to
a bright cherry red color.
Method
Inoculate the organism in 2ml glucose phosphate broth
and incubate overnight at 37oC.
Add 5 drops of Methyl Red Solution.
Mix and read immediately.

Positive test :Bright red

colour
Negative test : Yellow
colour

Positive control : E. coli

Negative control :
Klebsiella pneumoniae
7. Voges-Proskauer (V-P Test)
Principle
Some organisms produce acetoin (acetylmethyl carbinol) a
neutral reacting end product as the main end product of glucose
fermentation. Acetoin is oxidized into diacetyl in the presence of
atmospheric oxygen and alkaline condition which forms a pink
compound with alpha napthol.
Procedure
Inoculate test organism into 2ml glucose phosphate broth.
Incubate at 37oC for 24 hours.
Add 1 ml KOH and 3ml 5% alpha napthol and shake well for
maximum aeration.
Remove bottle cap and leave at room temperature for one hour.
Positive test : Pink
colour which becomes
crimson after 30
minutes
Negative test : Yellow
colour
Positive control :
Klebsiella
Negative control : E.
coli
 8- Citrate Utilization Test:
• This test is one of several techniques used occasionally to assist in the
identification of Enterobacteria.

Principle:
 The test is based on ability of an organism to use citrate as its only source of carbon.

Citrate method using Simmon’s citrate agar (Green color)

 Prepare slopes of the medium .

 Using a sterile straight wire, first streak the slope with a saline suspension of the test

organism and then stab the butt.

 Incubate at 350C for 48 hours.

 Look for a bright blue color in the medium.

e.g. Enterobacter and Klebsiella are citrate positive while E. coli is negative.
Results
Bright blue . . . . . . Positive citrate test.
No change in color. . . . . Negative citrate test.
 9- Bile solubility test
Principle
This helps to differentiate Strep. pneumoniae, which is

soluble in bile and bile salts, from other alpha haemolytic

streptococci (viridans streptococci) which are insoluble.

Requirements
Sodium deoxycholate reagent.

Sterile physiological saline.

 Method
Emulsify several colonies of the test organism in a tube containing 2

ml sterile physiological saline, to give a turbid suspension.

Divide the organism suspension between two tubes.

To one tube, add 2 drops of the sodium deoxycholate reagent and

mix.
To the other tube (negative control), add 2 drops of sterile distilled

water and mix.

Leave both tubes for 10–15 minutes at 35–37 0C.

Look for a clearing of turbidity in the tube containing the sodium

deoxycholate.
Results
Clearing of

turbidity . . . . . . . . . . . . . . . .Stre
pt. Pneumoniae.
No clearing of turbidity . . .

……. is not Strept. Pneumoniae.

 10 - DNA-ase test
 This test is used to help in the identification of Staph. aureus which produces

deoxyribonuclease (DNAase) enzymes.

 The DNA-ase test is particularly useful when plasma is not available to perform

a coagulase test or when the results of a coagulase test are difficult to interpret.

Principle
 Deoxyribonuclease hydrolyzes deoxyribonucleic acid (DNA).

 The test organism is cultured on a medium which contains DNA.

 After overnight incubation, the colonies are tested for DNA-ase production by

flooding the plate with a weak hydrochloric acid solution.

 The acid precipitates unhydrolyzed DNA.

 DNA-ase-producing colonies are therefore surrounded by clear areas due to

DNA hydrolysis.
Requirements
DNA-ase agar.
Hydrochloric acid solution 1 mol/l (1N).

Method
Using a sterile loop or swab, inoculate the test and control
organisms.
Incubate the plate at 35–37 0C overnight.
Cover the surface of the plate with 1 mol/l hydrochloric acid
solution.
Look for clearing around the colonies within 5 minutes of
adding the acid.
Results
Clearing around the colonies . . . . . . .

. . . DNA-ase positive strain

Staphylococcus aureus.
No clearing …………

Negative DNA-ase Staphylococcus

epidermidis.
API systemAnalytical profile index
(API):
o Principle:
- It is composed of a plastic strip with cupules
containing dehydrated substances. Each
cupule has a small hole at the top.
o Procedure:
- A saline suspension of the test organism is
dropped in the capsules.
- The strip is covered with a lid and placed in a
humidified plastic chamber and incubated at
37 degree for 24-48 hours.
o Interpretation:
- Biochemical profiles are determined by reading the
color change and interpret according to the available
charts.
- These are then converted to numerical codes which
will be read from a profile index to identify the
bacteria.
THANK YOU