Effervescence produced by Staphylococcus aureus
The catalase test may also use a tube instead of a slide, though the tube is not used
much because it is time and materials consuming.
Inoculate bacterial strain on an agar slant (TSA). Incubate it at 37 ºC for a day or two.
Add 1-2 ml of hydrogen peroxide to the tube, look for the presence or absence of
bubbles within a minute.  Bubbles mean a positive result.

The superoxol test is analogous to the catalase test, but is performed with a 30%
hydrogen peroxide solution. This reagent is most useful in differentiating between
Neisseria gonorrhoeae (strongly positive), Neisseria cinerea (weakly positive), and
Kingella denitrificans (superoxol negative).

Use culture grown on a blood-free media. Erythrocytes may produce a false-positive
Using a nichrome loop may give a false-positive reaction.
Reaction is more intense when adding on the slide a drop of commercial soap
solution before the hydrogen peroxide.
Some bacteria like
Tolumonas auensis give positive results for catalase when growth
aerobically, while anaerobic cultures are catalase-negative.
Catalase tests for Mycobacterium:

Semiquantitative catalase test
. Add 0.5 ml catalase (containing 1:1 ratio of 10% polysorbate 80 and 30% H2O2) reagent in a
Lowenstein-Jensen medium with mycobacterial growth. The tubes are allowed to stand at room temperature for five minutes and the
column of effervescence above the medium surface is measured.
M. kansasii, M. simiae, most scotochromogens,
nonphotochromogens and rapid growers usually produce a column of more than 45 mm whereas
M. tuberculosis, M. marinum, M.
complex or M. xenopi produce column less than 45 mm. Use M. flavescens ATCC 14474 as positive control and M.
as negative control.

Thermocatalase test. The catalase enzymes of M. tuberculosis  are inactivated at 68 ºC whereas enzymes of some other species are
not. Mycobacterial culture is suspended in 0.5 ml 15 M phosphate buffer (pH 7) and heated to 68 ºC for 20 minutes in a water bath.
After cooling, 0.5 ml of 1:1 Tween80-30% H
2O2 reagent is added and let to stand at room temperature (for 20 minutes) and observed.
Formation of bubbles is a positive test. Use
M. tuberculosis ATCC 15177 as positive control and M. fortuitum ATCC 6841 as negative

1. Helgomar Raducanescu, Valeria Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.
2. Tone Tonjum, 2005. Order IV. Neisseriales ord. nov. In:  Bergey’s Manual of Systematic Bacteriology, Second edition,Vol two, part C
The Alpha-, Beta-, Delta-, and Epsilonproteobacteria, George M. Garrity (Editor-in-Chief), pp 774-863.
3. Shridar Rao. Catalase test. 31st January, 2017 https://www.microrao.com/micronotes/pg/catalase.pdf, retrieved 20.02.2020.
Catalase Test
Hydrogen peroxide and oxygen radicals used in bacterial electron transport chains of aerobic and facultative anaerobic respiration are
toxic compounds. Catalase is an enzyme present in most of the organisms and is involved in decomposition of the hydrogen
peroxyde in H
2O and O2.
If catalase is present, bubbles will form from the oxygen that is made in the reaction:       2H
2O2 + catalase => 2H2O + O2 + catalase.
Because not all bacteria can produce catalase, in bacteriology this test is used together with other tests for bacterial identification.
Prepare a 3% hydrogen peroxide solution and put 1 drop on a slide.
Harvest a well isolated colony and immerse the loop in the solution on the slide.
Observe the quick effervescence for a positive reaction. The absence of bubbling is
considered negative. Record whether bubbles were seen immediately, whether
bubbles were seen with a delay, or if bubbles were not seen within a minute.
As an example, generally Staphylococcus species are  catalase-positive and
Streptococcus species are catalase-negative.
(c) Costin Stoica
Culture media
Biochemical tests
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A demonstration of catalase test is available in video section.