IN THE NAME OF ALLAH ALMIGHTY
THE MOST COMPASSIONATE
THE MERCIFUL
Biochemical Tests for Identification
of Bacteria
Biochemical tests:

These tests are performed for identification of bacteria.

All of these tests depend upon production of certain enzymes by
the bacteria.
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 H2O2.
2 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- 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.
3- 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.
4- 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, the phenylenediamine
(oxidase reagent) will be oxidized to a deep purple color.
Requirements:
1.Oxidase reagent strips.
2.Stick or glass rod.
Method using an oxidase reagent strip:
 Moisten the strip with a drop of 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
5- Urease Test:
Proteus strains are strong urease producers.
Principle:
 The test organism is cultured in a medium which contains urea
and the indicator phenol red. When the strain is urease
producing, the enzyme will break down the urea to give
ammonia and carbon dioxide.
 With the release of ammonia, the medium becomes alkaline as
shown by a change in colour of the indicator to pink-red.
Method
 Inoculate the media 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.
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.
6- 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 to
the test solution Kovac’s reagent which reacts with the indole
to produce a red colored compound.
SIM agar method (Sulfide-Indole-Motility):
 This agar is used for detection of the organism’s:
1. H2S production.
2. Indole production.
3. Motility.
 Inoculate test organism two-thirds into the medium by stabbing.
 Incubate at 37°C for 18 –24 hours.
 Examine tubes after incubation for motility and H2S production.
 Add 3-4 drops of Kovac’s Reagent after determining motility and H2S
production. Record as indole positive if a pink or red color ring appear, or
as indole negative if there is no color change.
Results
 Motility is indicated by turbidity of the medium or
growth extending from inoculating stab line.
 H2S production is shown by a blackening along the stab
line.
 Indole production is seen as the production of a red
color ring after the addition of Kovac’s Reagent.
Escherichia coli in SIM Medium Motile,
Proteus mirabilis in SIM Medium
indole positive and hydrogen sulfide
Motile, indole negative and hydrogen
negative
sulfide positive
7- Triple Sugar Iron Test (TSI)
In this test we use TSI agar slants (slope media).
Principle:
 Triple Sugar Iron (TSI) agar slants differentiate bacteria on their
ability to ferment glucose, lactose, and/or sucrose and on their
ability to reduce sulfur to hydrogen sulfide gas (H2S).
 This test is used to differentiate among the members of
Enterobacteriaceae i.e. E. coli, Salmonella, Shigella, Klebsiella,
Enterobacter etc.
Composition Of TSI agar:
1. 0.1% glucose.
2. 1% lactose.
3. 1% sucrose.
4. Ferrous sulfate for detection of H2S production.
5. Phenol red as indicator that changes yellow in acidic media.
6. Beef & yeast extract.
7. Low conc. of agar.
 The media is poured in test tubes in the form of slants & deep butt.The color of
the media is red
8- 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 . . . . . . . . . .
. . . . . .Strept. Pneumoniae.
 No clearing of turbidity . . . …….
is not Strept. Pneumoniae.
9- 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.