Catalase Test

The catalase test is a type of biochemical test which is used to detect the production of catalase enzymes in the organism.

• The catalase enzyme serves to neutralize the bactericidal effects of hydrogen peroxide. Catalase expedites the breakdown of hydrogen peroxide (H2O2) into water and oxygen (2H2O2 + Catalase → 2H2O + O2). This reaction is evident by the rapid formation of bubbles (2, 7).
• Catalase enzymes can be found in all types of living beings, which endure in oxygen and catalyzes the breakdown of hydrogen peroxide, releasing water and oxygen.
• In pathogenic organisms, the catalase enzyme protects them from oxidative damage from the reactive oxygen species. It neutralizes the bactericidal effects of hydrogen peroxide within the bacterial cell, as it is correlated with the pathogenicity of the organism.
• Over the years this test is performed to differentiate between catalase-positive organisms like staphylococci and catalase-negative species like streptococci.
• 3% H2O2 is used under the aerobic condition, while 15% H2O2 is used under anaerobic conditions.
• The strict anaerobes are unable to produce catalase, peroxidase, or superoxide dismutase, that may explain why oxygen is poisonous to these microorganisms. In the absence of these enzymes, the toxic concentration of H2O2 cannot be degraded when these organisms are cultivated in the presence of oxygen.
• It is a tetramer which contains four polypeptide chains that are usually  500 amino acids long. It also contains four porphyrin heme groups(ie., iron groups)  that will allow the enzyme to react with the hydrogen peroxide.
• The enzyme catalase is present in most cytochrome-containing aerobic and facultative anaerobic bacteria. 
• Catalase is the enzyme which contains one of the highest turnover numbers compared to all other enzymes; one molecule of catalase has the ability to convert millions of  molecules of hydrogen peroxide to water and oxygen in each second.

Catalase test purpose

• To detect the enzyme catalase in bacteria.
• To differentiate catalase-positive Micrococcaceae from catalase-negative Streptococcaceae. 

Principle of Catalase Test

Some bacteria has flavoproteins that reduce the oxygen (O2), as a result, it leads to the formation of hydrogen peroxide (H2O2) and, in some cases, it produces an extremely toxic product known as superoxide (O2–). These are powerful oxidizing agents which can destroy cellular constituents very rapidly, on the accumulation of these agents can lead to the death of the organism until they are enzymatically degraded.

These toxic agents are formed when the aerobes, facultative anaerobes, and microaerophiles utilize the aerobic respiratory pathway. Bacterial cells has developed such defense mechanisms to protect themselves against such O2 products or it will be killed. Many of them contain enzymes that can afford protection against toxic O2 products.

Facultative anaerobes and Obligate aerobes  usually contain the enzymes superoxide dismutase, which has the ability to  catalyze the destruction of superoxide, and either catalase or peroxidase, which catalyze the destruction of hydrogen peroxide as follows:

The detection of Catalase production and activity can be done by adding the substrate H2O2 to an appropriately incubated (18- to 24-hour) tryptic soy agar slant culture. As a result, the Catalase producing organisms will break down the hydrogen peroxide and will form O2 which will release as bubbles in the reagent drop, indicating a positive test. 

The organisms lacking the ability to produce Catalase will be unable to break down hydrogen peroxide, into O2 and water and are catalase-negative. 

Catalase activity is very useful in differentiating between groups of bacteria. For example, the morphologically similar Enterococcus (catalase negative) and Staphylococcus (catalase positive) can be differentiated using the catalase test.

Material Required

• Hydrogen peroxide reagent: 3% H2O2 for other bacteria, 15% H2O2 for Identification of anaerobic bacteria, 30% H2O2 In the superoxol catalase (used for the presumptive speciation of certain Neisseria sps).
• Equipment: Bunsen burner, Inoculating loop, Test tubes, Test tube rack, Microscopic slides.

Procedure of Catalase Test

The catalase test can be performed by two methods such as;

1. Slant method/Tube Method
2. Slide method

Slant method/Tube Method of Catalase Test

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.

Slide Method of Catalase Test

1. Divide a clean glass slide into two sections with a 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 a control.
7. Observe the fluid over the smears for the appearance of gas bubbles.
8. Discard the slide in a jar of disinfectant.

Quality Control of Catalase Test

The following two organisms can be used Quality Control of Catalase Test;

• Staphylococcus aureus for Catalase positive test.
• Streptococcus pyogenes for Catalase-negative test.

Catalase Test Results

• Positive catalase test: Indicated by the immediate appearance of bubbles.
• Negative catalase test: Indicated by no bubbles or a few bubbles after 20 s.

Note: The appearance of one or two bubbles represents a weak reaction.

Application of Catalase Test

• This test is used to differentiate catalase-negative Streptococcaceae from catalase-positive Micrococcaceae and Staphylococcaceae.
• Used to differentiate between aerobic and obligate anaerobic organisms.
• Used to differentiate among certain Enterobacteriaceae.
• Used for identification of aerotolerant strains of Clostridium, which are catalase-negative, from Bacillus, which are catalase positive.
• It is used to differentiate between genera and is also valuable in the speciation of certain gram-positive organisms such as Aerococcus.

Limitations of Catalase Test

• Hydrogen peroxide is unstable and should undergo a control check daily prior to use.
• 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.
• 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.
• A positive catalase reaction with anaerobic organisms may be delayed for up to a minute after the addition of the reagent.
• A weak catalase or pseudo catalase reaction may be produced by some strains of Aerococcus species and Enterococcus species.
• Don’t pick up blood agar with the colony, it can lead to the false-positive result because RBCs contain catalase.
• Don’t use Mueller-Hinton agar.
• The use of metal bacteriological loop materials can lead to false-positive results; however, platinum loops do not yield false-positive results.
• Avoid using colonies that are older than 24 hours it may give false-negative results.
• Reversing the order of adding the reagent to the colony might result in false-negative results.
• Don’t mix the reagent and the colony.
• Some strains of S. aureus may appear catalase-negative by drop method so the test should be repeated with the tube method.
• Avoid direct contact with the 30% H2O2, it is extremely caustic to the skin. If contact occurs accidentally, wash it immediately with 70% ethyl alcohol, not water.

Precautions of Catalase Test

• Dispose the hydrogen peroxide slides in the appropriate container filled with disinfectant. 
• Nichrome wire should be used when testing the organism. Platinum wires may cause a false-positive reaction.
• 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. 
• 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.
• Always use fresh hydrogen peroxide, since it is unstable.
• 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.

References

• https://en.wikipedia.org/wiki/Catalase
• https://microbenotes.com/catalase-test-principle-procedure-and-result-interpretation/
• https://microbiologyinfo.com/catalase-test-principle-uses-procedure-result-interpretation-with-precautions/
• https://asm.org/getattachment/72a871fc-ba92-4128-a194-6f1bab5c3ab7/Catalase-Test-Protocol.pdf
• https://microbeonline.com/catalase-test-principle-uses-procedure-results/
• https://vlab.amrita.edu/?sub=3&brch=73&sim=703&cnt=2
• https://himedialabs.com/TD/K044.pdf