IN THE NAME OF ALLAH ALMIGHTY

THE MOST COMPASSIONATE

THE MERCIFUL

Biochemical Tests for Biochemical Tests for Identification of BacteriaIdentification of Bacteria

Biochemical tests:Biochemical tests:

These tests are performed for identification of

bacteria.

All of these tests depend upon production of certain

enzymes by the bacteria.

1.1. Catalase testCatalase 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:Principle:

Catalase producing bacteria will produce O2 when

mixed with H2O2.

Methods: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.

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

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:2- Citrate Utilization Test:

• This test is one of several techniques used occasionally to assist

in the identification of Enterobacteria.

Principle: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:3- Coagulase Test:This test is used to identify Staph. aureus, which produces

coagulase enzyme.

Principle: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- 1- Slide test method (detects bound Slide test method (detects bound coagulase)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. ResultsClumping within 10 sec . . . . . . Staph. aureus.No clumping within 10 sec. . . . . No bound Coagulase.

2- 2- Tube test method (detects free Tube test method (detects free

coagulase)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: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.

PrinciplePrinciple

When the organism is oxidase-producing, the

phenylenediamine (oxidase reagent) will be

oxidized to a deep purple color.

RequirementsRequirements::

1.Oxidase reagent strips.

2.Stick or glass rod.

Method using an oxidase reagent stripMethod 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.

ResultsResults

Deep purple color. . . . . . . . positive oxidase test

5- Urease 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.

MethodMethod

Inoculate the media with the tested

organism.

Incubate at 37°C for

18 –24 hours.

Results: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 test6- Indole production test

• Testing for indole production is important in the

identification of enterobacteria.

Principle: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 (SIM agar method (Sulfide-Indole-Motility)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.

ResultsResults

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,

indole positive and hydrogen sulfide

negative

Proteus mirabilis in SIM Medium

Motile, indole negative and hydrogen

sulfide positive

7- Triple Sugar Iron Test 7- Triple Sugar Iron Test (TSI)(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 test8- Bile solubility test

PrinciplePrinciple

This helps to differentiate Strep. pneumoniae,

which is soluble in bile and bile salts, from other

alpha haemolytic streptococci (viridans

streptococci) which are insoluble.

RequirementsRequirements

Sodium deoxycholate reagent.

Sterile physiological saline.

MethodMethodEmulsify 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.

ResultsResults

Clearing of turbidity . . . .

. . . . . . . . . . . .Strept.

Pneumoniae.

No clearing of

turbidity . . . ……. is not

Strept. Pneumoniae.

9- DNA-ase test9- 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.

PrinciplePrinciple

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.

RequirementsRequirementsDNA-ase agar.Hydrochloric acid solution 1 mol/l (1N).

MethodMethodUsing 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.

ResultsResults

Clearing around the colonies .

. . . . . . . . . DNA-ase positive

strain Staphylococcus aureus.

No clearing …………

Negative DNA-ase

Staphylococcus epidermidis.