CATALASE TEST: RATIONALE, TECHNIQUE AND USES - BIOLOGY - 2024

The catalase test is a methodology used in bacteriology laboratories to reveal the presence of the enzyme catalase in those bacteria that possess it. Together with the Gram stain, they are the main tests that should be performed on newly isolated microorganisms. These tests guide the microbiologist on the steps to follow for the definitive identification of the microorganism in question.

In general, bacteria containing cytochrome have the enzyme catalase, that is, aerobic and facultative anaerobic bacteria should have it. However, there are exceptions, such as Streptococcus, which despite being facultative anaerobic microorganisms, do not possess the enzyme catalase.

Running the Catalase Test, showing a positive reaction. Source: No machine-readable author provided. Nase assumed (based on copyright claims).

This is why the catalase test is used primarily to distinguish the Staphylococaceae and Micrococaceae families (both catalase positive) from the Streptococaceae family (catalase negative).

Likewise, the genus Bacillus (catalase positive) is distinguished from the genus Clostridium (catalase negative), among others.

Basis

Catalase is an enzyme classified as a hydroperoxidase, this means that they use hydrogen peroxide (H ₂ O ₂) as a substrate.

It is also considered an oxidoreductase, since in the reaction where it participates there is an element that serves as an electron donor (reducing substance) and another as an electron receptor (oxidizing substance).

Catalase is a protein that contains a proseric group with four trivalent iron atoms (Fe ⁺⁺⁺), therefore it is a homoprotein. The ferric ion remains oxidized during the reaction.

It can be said that catalase is a detoxifying enzyme, as its function is to eliminate substances that are produced during bacterial metabolism that are toxic to bacteria. Among these substances is hydrogen peroxide.

Hydrogen peroxide is formed from the breakdown of sugars aerobically. This process occurs as follows:

The superoxide ion (O ₂ ^-) (free radical) is formed as the end product of the assimilation of glucose by the aerobic route. This is toxic and is eliminated by the enzyme superoxide dismutase that transforms it into gaseous oxygen and hydrogen peroxide.

Hydrogen peroxide is also toxic to bacteria and must be removed. The enzyme catalase breaks down hydrogen peroxide into water and oxygen.

Catalase can act on substrates other than hydrogen peroxide, such as alcohols, aldehydes, acids, aromatic amines and phenols. However, hydrogen peroxide can also be used by catalase to oxidize other toxic compounds such as methyl and ethyl alcohol.

Likewise, catalase is present in phagocytic cells, protecting it from the toxic action of hydrogen peroxide.

Routine Technique for Catalase Test

-Slide method

materials

3% hydrogen peroxide (10 volumes).

Microscope slide

Disposable plastic handle or wooden toothpick.

Process

Take enough of the colony to study without touching the agar from which it came. The colony must be fresh, that is, from a culture of 18 to 24 hours.

Place the colony on the dry slide and add a drop of 3% hydrogen peroxide to it (30% H ₂ O ₂ can also be used). Observe immediately whether or not bubbles are released.

Interpretation

Positive reaction: evolution of gas, evidenced by the formation of bubbles (strong bubbling).

Negative reaction: no bubble formation.

-Direct method in pure culture

Place 1 ml of 3% H ₂ O ₂ on a pure plate or wedge culture that does not contain blood (preferably nutrient agar). Observe whether or not there is bubble formation immediately. 30% H ₂ O ₂ can also be used.

It is interpreted the same as the porta object method.

-Method with capillary tube or Fung and Petrishko

Fill a 67 mm capillary tube to a height of 20 mm with 3% hydrogen peroxide by capillarity.

Touch the isolated colony to be studied with the capillary filled with 3% H ₂ O ₂ . Observe if the capillary fills with bubbles in approximately 10 seconds. This method allows semi-quantification of the reaction in crosses:

Without crosses there are no bubbles (negative reaction).

+ ---- Few bubbles (weak or delayed reaction).

++ --– Abundant bubbles (moderate reaction).

+++ --Bubbles reach the opposite end (strong reaction).

-Taylor and Achanzar method for catalase tests that give questionable

Place an isolated colony on a clean, dry slide, then place a drop of 0.5% H ₂ O ₂ and cover with a coverslip. Observe whether or not there is formation of trapped bubbles.

Interpretation: the presence of bubbles indicates a positive reaction. No bubbles, it is interpreted as a negative reaction.

Catalase test for Mycobacterium species

This technique needs to be done by controlling the pH and temperature. It must be carried out under a laminar flow hood, since the manipulation of the different Mycobacterium species is dangerous.

-Materials

Hydrogen peroxide 30% or 110 volumes (superoxal).

Phosphate buffer pH 7

10% Tween 80

Mycobacterium wedge culture for 3 to 4 weeks

-Preparation

Phosphate buffer pH 7

To weigh:

1.361 g of anhydrous monopotassium phosphate (KH ₂ PO ₄).

1.420 g of anhydrous disodium (Na2HPO3) phosphate.

Dissolve both salts in a little sterile distilled water and make up to 1000 ml with water.

10% Tween 80

Make a 1:10 dilution to the Tween 80 that is commercially concentrated, to do this proceed as follows:

Take 1 ml of Tween 80 and place it in a little distilled water, dissolve and then make up the volume with water to 10 ml.

Final reagent

Mix a quantity of phosphate buffer with a quantity of 10% Tween 80 (equal parts). Define in the laboratory how much you want to prepare.

-Process

Place 5 ml of phosphate buffer in a sterile screw-capped test tube (Bakelite).

With an inoculation loop, take enough colony of a Mycobacterium growth seeded in wedges and dissolve in the phosphate buffer.

Cap the tube without over-tightening the thread. Place in a water bath at 68 ° C for 20 to 30 minutes. Take out and let cool to 22-25 ° C

Measure 0.5 ml of the final reagent (mix) and add it to the tube with the cold solution. Observe the formation or not of bubbles.

It is interpreted the same as the previous techniques.

Use

When colony growth is obtained in enriched media, a Gram stain and a catalase test should be performed on the colonies obtained. This will guide the microbiologist on the procedures to follow for definitive identification.

Source: Prepared by the author MSc. Marielsa gil

QA

To evaluate the good performance of the hydrogen peroxide reagent use freshly cultured control strains, such as Staphylococcus aureus as a positive control and Streptococcus sp strains as a negative control.

Another alternative that serves as a positive control is to place a drop of hydrogen peroxide on the blood agar, the erythrocytes have catalase, therefore, there will be a bubbling if the reagent is in good condition.

A chocolate agar can be used as a negative control, here the erythrocytes are already lysed and the test is negative.

Limitations

-Do not use old cultures for the test, as this can cause false negatives.

-Avoid taking colonies from cultures on blood agar, if you are careful not to touch the agar; This procedure can lead to false positives, as red blood cells contain catalase.

-If you take the colony with a platinum handle, do not reverse the order of the procedure because this can generate false positives. This is because platinum is able to react with hydrogen peroxide, causing a bubbling.

-Do not use the hydrogen peroxide reagent if it is very old, as the reagent is very unstable and tends to break down over time.

-Keep the hydrogen peroxide reagent protected from light and refrigerated to prevent damage.

-Perform a quality control of the hydrogen peroxide reagent each time it is used.

-Take into account that if 30% H ₂ O ₂ is used, the reactions are stronger than those carried out with 3% H ₂ O ₂.

References

1. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.
2. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.
3. Mac Faddin J. (2003). Biochemical tests for the identification of bacteria of clinical importance. 3rd ed. Editorial Panamericana. Buenos Aires. Argentina.
4. BD Laboratories. Catalase-Gotario Reagent. Available at:
5. Vadequímica Laboratories. Peroxide. Equivalence between volumes and percentage. Available at: vadequimica.com