Catalase Test- Principle, Procedure, Types, Results, Uses
What is Catalase Test?
The catalase test is a biochemical test for aerobic organisms that
detects the production of catalase enzyme in the organism.
Catalase enzyme is a common enzyme that is found in all living
beings that survive in oxygen and catalyzes the decomposition of
hydrogen peroxide, releasing water and oxygen.
Catalase is an essential enzyme in pathogenic organisms as it
protects the organism from oxidative damage from the reactive
oxygen species.
The enzyme neutralizes the bactericidal effects of hydrogen
peroxide, and its concentration in bacteria has been correlated with
the pathogenicity of the organism.
The catalase test has been extensively used over the years as it
allows the differentiation of catalase-positive organisms like
staphylococci from catalase-negative species like streptococci.
The catalase test is useful in the presumptive characterization of
most bacteria.
Under the aerobic condition, 3% H2O2 is used, whereas 15% H2O2 is
used under anaerobic conditions.
Objectives of Catalase Test
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.
PRINCIPLES OF CATALASE TEST
The metabolic activity of aerobic and facultative anaerobic
microorganisms produce toxic by-products like hydrogen peroxide
and superoxide radical (O2—).
These products are toxic to the organisms and might even result in
cell lysis if not broken down. In the case of pathogenic organisms,
different mechanisms are found that break down these products to
non-toxic substances.
Bacteria capable of synthesizing the enzyme catalase hydrolyze
hydrogen peroxide into water and gaseous oxygen, which results in
the liberation of gas bubbles.
H2O2 ———–> H2O + O2
catalase
The production of catalase thus protects the organism against the
lethal effect of hydrogen peroxide accumulated at the end of the
aerobic metabolism.
The presence of the catalase enzyme can be demonstrated by
adding hydrogen peroxide to the bacterial inoculum, which results
in the rapid liberation of oxygen bubbles. The lack of enzyme is
demonstrated by the absence of such bubbles.
Microorganism Tested
Young (18 hours old or less, if possible) colonies of bacteria growing
on agar media, preferably Blood Agar.
For anaerobes, the colonies should be exposed to air for 30 minutes
prior to testing.
Used
Hydrogen peroxide reagent
30% H2O2 for Neisseria
15% H2O2 for anaerobes
3% H2O2 for other bacteria (purchase or dilute 30% 1:10 in deionized
water prior to use)
Supplies
Glass slide
Sterile wooden or glass sticks or platinum loops or wires
PROCEDURE
There are more than one method or procedure variations for the
catalase test. These methods include the slide or drop catalase test,
the tube method, the heat-stable catalase used for the
differentiation of Mycobacterium species, the semi quantitative
catalase for the identification of Mycobacterium tuberculosis, and
the capillary tube and coverslip method. The most popular method
of catalase test in clinical bacteriology is the slide or drop catalase
method as it requires a small number of organisms and works on a
relatively uncomplicated technique.
1. Slide Method
A microscope slide is placed inside a petri dish. The use of a petri
dish is optional and is used to limit catalase aerosols, which might
carry viable bacterial cells.
A small amount of organism is collected from a well-isolated 18- to
24-hour colony with a sterile inoculating loop or wooden applicator
stick and placed onto the microscope slide.
However, no agar must be picked up with the colony, especially
when the culture is picked up from blood agar.
A drop of 3% H2O2 onto the organism on the microscope slide by
using a dropper or Pasteur pipette.
The formation of bubbles is observed against a dark background to
enhance readability.
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2. Tube Method
About 4 to 5 drops of 3% H2O2 are added to a test tube.
Using a wooden applicator stick, a small amount of organisms from
a well-isolated 18- to 24-hour colony is collected and placed into
the test tube.
The tube is placed against a dark background and observed for
immediate bubbles.
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Quality Control
As a form of quality control, the following organisms can be used for
positive and negative results:
1. Staphylococcus aureus: Catalase positive.
2. Streptococcus pyogenes: Catalase-negative.
Test
The positive test is demonstrated by the immediate appearance of
bubbles.
The appearance of one or two bubbles represents a weak reaction.
A negative test is represented by no bubbles or a few bubbles after
20 s.
Reporting result
The catalase test separates staphylococci (positive) from
streptococci and enterococci (negative).
Bacillus is catalase-positive, and Clostridium spp. are catalase-
negative.
The test is useful to separate among the fastidious Gram-negative
rods.
Neisseria gonorrhoeae produces an enhanced elaboration of
bubbles not seen with other members of the genus due to
superoxol.
Uses
Catalase test is essential for differentiating catalase-positive
Micrococcaceae and Staphylococcaceae from catalase-negative
Streptococcaceae.
The test also allows differentiating aerobic and obligate anaerobic
organisms.
The value of the test has been found in the presumptive
differentiation among certain Enterobacteriaceae.
This is an important test for the differentiation of aerotolerant
strains of Clostridium, which are catalase-negative,
from Bacillus, which are catalase positive.
This test has been used to differentiate between genera and is also
valuable in the speciation of certain gram-positive organisms such
as Aerococcus and
Limitation
Test
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.
The test should not be tested from Mueller-Hinton agar.
Collecting colonies with metal bacteriological loop materials might
yield false-positive results; however, platinum loops do not yield
false-positive results.
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.
Reversing the order of adding the reagent to the colony might
result in false-negative results.
The reagent and the colony should not be mixed.
Some strains of S. aureus may appear catalase-negative by drop
method so the test should be repeated with the tube method.
30% H2O2 is extremely caustic to the skin. If contact occurs, wash
immediately with 70% ethyl alcohol, not water.