Catalase Test: Objective, Principle, Procedure, Types, Results, Uses

By Prof Jeremiah Seni

Introduction

The Catalase test is used to differentiate staphylococci (catalase-positive) from streptococci (catalase-negative). The enzyme catalase, is produced by bacteria that respire using oxygen and protects them from the toxic by-product of oxygen metabolism. Catalase-positive bacteria include strict aerobes as well as facultative anaerobes, although they all have ability to respire using oxygen as a terminal electron acceptor. Catalase-negative bacteria may be anaerobes or they may be facultative anaerobes that only ferment and do not respire using oxygen as a terminal electron acceptor (i.e Streptococci).

Objectives

• To detect the ability of organisms to produce the catalase enzyme.
• To differentiate catalase-positive organisms like micrococci and staphylococci from catalase-negative organisms like streptococci.

Principle

Catalase is an enzyme that breaks Hydrogen peroxide (H₂O₂), a toxic metabolic byproducts of aerobic and facultative anaerobic bacteria into non toxic products water (H₂O) and Oxygen (O₂). H₂O₂ is toxic to cells. It is highly reactive molecule that damage cell components. So the bacteria living in presence of Oxygen produces enzyme catalase that breakdown H₂O₂ into H₂O and O₂

H₂O₂ ————catalase—–>H₂O      +    O₂

Catalase is an inevitable enzyme for the aerobic organisms. Hydrogen peroxide (H2O2) is produced as the result of non-enzymatic transfer of electrons from reduced flavoprotein to Oxygen as well as by the action of superoxide dismutase. The enzyme catalase degrades hydrogen peroxide in cell before it can do any damage to cell. Aerobic and facultative anaerobes have catalase enzyme to convert H₂O₂ into non toxic forms while obligate anaerobes so not have such enzyme, therefore they cannot tolerate O₂.

FP H₂( reduced flavoprotein) + O₂—————->FP (oxidized flavoprotein)+ H₂O₂

2H+      +   2 O₂–   ——————–>  H₂O₂  +     O₂

Most microorganisms that are aerobic and facultative anaerobic produce the enzyme catalase except Streptococcus spp, lactobacillus spp, Leuconostoc spp, Clostridium spp, Mycoplasma. Other bacteria that do not produce catalase enzyme are Obligate anaerobes or microaerophiles. Absence of enzyme catalase is the reason why oxygen is poisonous to anaerobes.

Experiment

Reagent And Materials Required

• Slides
• Bibulous paper
• Inoculating loop
• Bunsen burner
• Laminar flow chamber
• Hydrogen peroxide (3%), isolated colonies or pure cultures of bacteria.

Method 1: Slide Method

Procedure

1. Take a clean, dry and grease free microscopic glass slide and place a drop of 3% H2O2 onto the slide.
2. At one time you can easily perform Catalase test of 2 or 3 specimens on a single slide so divide the space accordingly and Place the H2O2 drop in the center of the divided portions of the slide.
3. Now, transfer a portion of the isolated bacterial colony to the drop of hydrogen peroxide on to the glass slide using a sterile inoculating stick.
4. Mix well the specimen and H2O2 drop on the slide.
5. Carefully observe for the evolution of Bubbled in the mixture on the Glass slide.

Method 2:Tube Method

Procedure

1. Take a Clean, Dry and sterilized Laboratory test tube and add 5-7 drops of hydrogen peroxide (H2O2) Solution in it.
2. Now, pick a small portion of the isolated bacterial colony using a sterile wooden applicator stick and carefully place it into the test tube.
3. Finally, observe the tube against a dark background for immediate bubble formation at the end of the wooden stick in the test tube.

Observation And Results Interpretation

• Catalase Positive Reaction: Effervescence (bubble formation).
• Catalase Negative Reaction: No Effervescence (bubble formation)-No catalase enzyme to hydrolyze hydrogen peroxide.

Examples Of Catalase Positive Bacteria

Staphylococci, Micrococci, Listeria, Corynebacterium diphtheriae, Burkholderia cepacia, Nocardia, the family Enterobacteriaceae (Citrobacter, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella, Serratia),Pseudomonas, Mycobacterium tuberculosis, Aspergillus, Cryptococcus, and Rhodococcus equi.

Examples Of Catalase Negative Bacteria

Streptococcus species, Enterococcus species etc.

Precautions Of Catalase Test

• Hydrogen peroxide must be fresh as it is very unstable.
• Don’t  use Iron wire loop for this test.
• Some bacteria produce a peroxidase that catalyzes a breakdown of hydrogen peroxide causing the reaction to be weakly positive; (a few bubbles elaborated slowly). This should not be confused with a truly positive reaction.
• Do not add organism to reagent but reagent to organism, particularly if iron-containing inoculating loops are used. Iron containing loops will cause false positive test results if exposed to hydrogen peroxide.
• The test organisms should not be taken from blood agar culture. Red Blood cells contain catalase and their presence will give a false positive test.Culture should be 18 to 24 hours old.
• Dispose of your test slides and inoculating stick/loops in the respective biohazard disposal containers as per the norms.

Uses Of Catalase Test

• The catalase test is primarily used to distinguish among Gram-positive cocci: Members of the genus Staphylococcus are catalase-positive and members of genera Streptococcus and Enterococcus are catalase-negative.
• Catalase test is used to differentiate aerotolerant strains of Clostridium, which are catalase negative, from Bacillus species, which are positive.
• Semiquantitative catalase test is used for the identification of Mycobacterium tuberculosis.
• Catalase test can be used as an aid to the identification of Enterobacteriaceae. Members of Enterobacteriaceae family are catalase positive.

Limitations Of Catalase Test

1. The reagent and the colony should not be mixed.
2. Some strains of S. aureus may appear catalase-negative by drop method so the test should be repeated with the tube method.
3. 30% H₂O₂ is extremely caustic to the skin. If contact occurs, wash immediately with 70% ethyl alcohol, not water.
4. RBCs contain catalase, and thus, in order to avoid false-positive results, blood agar should not be picked up with the colony. If a colony is difficult to pick up or doesn’t grow well, the test can be repeated from the culture on a different media.
5. The test should not be tested from Mueller-Hinton agar.
6. Collecting colonies with metal bacteriological loop materials might yield false-positive results; however, platinum loops do not yield false-positive results.
7. Because the enzyme is present in viable cells only, colonies that are older than 24 hours should not be used. Older cultures may give false-negative results.
8. Reversing the order of adding the reagent to the colony might result in false-negative results.