Catalase Test: Atlas, p. 50-51
Purpose: assays for the presence of the enzyme catalase. This test is normally used to help identify Gram positive
cocci, but we'll use it somewhat inappropriately on your unknown because we will
not be running all the microbial tests.
Catalase is needed by cells that produce hydrogen peroxide and oxygen
radicals from passing electrons in their electron transport chains of aerobic
and facultative anaerobic respiration. Otherwise, these toxic compounds
would damage the cell, especially the DNA. If
catalase is present, bubbles will form from the oxygen that is made in the reaction:
2H2O2 + catalase ----> 2H2O
+ O2 + catalase.
Slide Procedure and Results:
- Obtain your unknown bacteria, a slide, and hydrogen peroxide (H2O2).
The H2O2 may be in the refrigerator if its not
- Aseptically, put a lot of fresh bacteria on your slide and add a large
drop or two of H2O2. Do not allow the bacteria
to dry out.
- The bacteria should be from fresh growth, but often this is not required.
- If your cells have catalase, you will see bubbles within a minute.
Usually, there are so many immediate bubbles that it is easy to tell a positive
result, but a few species only have a little catalase so that only one or
two small bubbles form after a minute.
- It is a good idea to test your H2O2 on a known
positive control to ensure that the H2O2 is
still good, especially if you do not get bubbles (E. coli, S. epidermidis
& B. subtilis are all positive controls for catalase).
(There is another procedure to test for catalase, the Tube Procedure.
We will not perform this procedure, it is only described here for your benefit.)
- (The catalase test may also use a tube instead of a slide, though the tube
is not used much because of its greater cost and time.
- Aseptically, inoculate your unknown bacteria on an agar slant (TSA or
NA). Label the tube.
- Incubate it at 35 degrees C for a day or two.
- Add about 2 ml of hydrogen peroxide to the tube, look for the presence of
bubbles within a minute. Bubbles mean a positive result.)