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Catalase Test Procedure and Materials

This document describes the catalase test procedure used to identify bacteria. The catalase test detects the presence of the enzyme catalase in bacteria by observing if bubbles form when hydrogen peroxide is added. There are two methods - the slant method where bacteria are grown on an agar slant and hydrogen peroxide is added, and the slide method where a bacterial sample is placed on a slide with hydrogen peroxide. Bubbling or foaming indicates a positive catalase result. Precautions are needed as hydrogen peroxide is unstable and contaminants can cause false results.

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Md Ahsanul Haque
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134 views4 pages

Catalase Test Procedure and Materials

This document describes the catalase test procedure used to identify bacteria. The catalase test detects the presence of the enzyme catalase in bacteria by observing if bubbles form when hydrogen peroxide is added. There are two methods - the slant method where bacteria are grown on an agar slant and hydrogen peroxide is added, and the slide method where a bacterial sample is placed on a slide with hydrogen peroxide. Bubbling or foaming indicates a positive catalase result. Precautions are needed as hydrogen peroxide is unstable and contaminants can cause false results.

Uploaded by

Md Ahsanul Haque
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd

I) Catalase Test

Materials Required:
Cultures:
24-48 hour tryptic soy broth cultures of bacteria

Media:
Tryptic soy agar

Reagent:
3% hydrogen peroxide
(Storage:-Upon receipt, store at 2-8˚C away from direct light. Reagents should not be used if there
are signs of deterioration or if the expiration date has passed.)

Equipments:
 Bunsen burner
 Inoculating loop
 Test tubes
 Test tube rack
 Microscopic slides

Procedure:
The test can be done by two methods.
a)    Slant method
b)    Slide method

a)    Slant Method:


1. Using a sterile technique, inoculate each experimental organism into its appropriately labeled
tube by means of a streak inoculation.
2. Incubate all cultures for 24-48 hours at 37˚C.
3. Allow three or four drops of the 3% hydrogen peroxide to flow over the entire surface of each
slant culture.
4. Examine each culture for the presence or absence of bubbling or foaming.
Negative            Positive

b)    Slide Method:


1. Divide a clean glass slide into two sections with grease pencil. One should be labeled as “test”
and the other as “control”.
2. Place a small drop of normal saline on each area.
3. With a sterilized and cooled inoculating loop, pick up a small amount of the culture from the
nutrient agar slant or Petri plate.
4. Emulsify one or two colonies on each drop to make a smooth suspension. The smear should be
about the size of a pea.
5. With a Pasteur pipette, place one drop of hydrogen peroxide over the test smear. Be careful
not to run the drops together.
6. Do not put anything in the other drop that serves as control.
7. Observe the fluid over the smears for the appearance of gas bubbles.
8. Discard the slide in a jar of disinfectant.

 
Limitations:
 
1. Hydrogen peroxide is unstable and should undergo a control check daily prior to use.
2. Growth for catalase testing must be taken from an 18-24 hour culture. Organisms lose their
catalase activity with age, resulting in a false-negative reaction.
3. Catalase activity is a function of aerobic process. Organisms incubated anaerobically must be
exposed to atmospheric oxygen for a minimum of 30 minutes before a catalase test is performed.
Failure to complete this step may produce false-negative results.
4. A positive catalase reaction with anaerobic organisms may be delayed for up to a minute after
addition of the reagent.
5. A weak catalase or pseudocatalase reaction may be produced by some strains
of Aerococcus species and Enterococcus species.
 

Hints and Precautions:


 
1. Dispose the hydrogen peroxide slides in the appropriate container filled with disinfectant. 
2. Nichrome wire should be used when testing the organism. Platinum wires may cause a false-
positive reaction.
3. When using a slant for other purposes in the same laboratory period, it is possible to save
material by adding H2O2 to the slant after finishing with it. 
4. Extreme care must be taken if a colony is taken from a blood agar plate. Erythrocytes contain
catalase, and a transfer of only a few blood cells can give a false-positive reaction.
5. Always use fresh hydrogen peroxide, since it is unstable.
6. Do not add organism to reagent, particularly if iron-containing inoculating loops are used. Iron
containing loops will cause false positive test results if exposed to hydrogen peroxide.
 
 
 
Md Ahsanul Haque
[Link] in Microbiology(BATCH-37th)
GONO BISHWABIDYALAY

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