BACTERIAL CATALASE AND CYTOCHROME OXIDASE TESTS—
IDENTIFYING MICROBIAL ABILITY TO USE RESPIRATORY
METABOLISM
Respiratory metabolism results in the conversion of glucose into energy in the
form of ATP by the processes of glycolysis, Kreb’s cycle and oxidative
phosphorylation. In the process of respiratory metabolism, most of the ATP is
generated as a result of the activity of the electron transport system. The electron
transport system is composed of membrane associated molecules (cytochromes or
flavoproteins) that accept and donate electrons. Reduced NADH and FADH from
glycolysis and Kreb’s cycle enter at or near the top of the electron transport chain and
reduce the first molecule in the chain as they are concurrently re-oxidized. As electrons
are passed down the chain of the electron transport system, each molecule in the chain
alternate between reduced and oxidized forms. Cytochrome oxidase is found at the
bottom of this chain, cytochrome oxidase catalyzes the re-oxidation of the last
cytochrome molecule in the chain by molecular oxygen. This final process results in
the reduction of molecular oxygen to water.
At times, the incomplete reduction of oxygen leads to the production of
hydrogen peroxide (H2O2) or the superoxide free radical (O2-) that are both toxic to the
cell. Many aerobic or facultatively anaerobic cells produce enzymes that remove these
toxic byproducts. Superoxide dismutase removes the superoxide free radical in the
following reaction (2H+ + 2 O2- - H2O2 + O2). Catalase removes hydrogen
peroxide in the following reaction (2 H2O2 - 2 H2O + O2). Strict anaerobes usually
lack these enzymes and therefore are restricted to an oxygen free environment. As only a
subset of all the bacterial species produce cytochrome oxidase and catalase, one can test
for the presence of these enzymes in bacteria to determine the oxygen tolerance of an
individual bacterium and also to identify the bacterium.
In this laboratory exercise you will be testing Enterococcus faecalis and
Staphylococcus aureus for the presence of cytochrome oxidase and catalase.
Catalase test: In the previous exercise you isolated Enterococcus faecalis and
Staphylococcus aureus from a mixed culture using the streak plate method.
Divide a clean microscope slide in half using a wax pencil; label one half EF and
the other half SA.
Use a sterile loop to transfer a small amount of culture from the plate to the
microscope slide.
Add one drop of a H2O2 solution to each culture sample and watch for the
production of bubbles in the suspension. The bubbles result from oxygen gas that
is released as a result of catalase activity that has converted the externally
supplied hydrogen peroxide into oxygen and water. If no bubbles are produced
the test is negative for catalase activity.
Cytochrome oxidase test:
Divide a clean piece of filter paper in half using a wax pencil; label one half EF
and the other half SA.
Use a sterile loop to transfer a small amount of culture from the plate to the
microscope slide. Use a sterile wooden applicator to rube the sample into the
paper without tearing the paper. This acts to remove capsule (glycocalx, mucin)
from the surface of the bacteria so the substrate can interact with the bacteria.
Add one drop of tetramethyl-p-phenylenediamine to each sample and record the
results immediately after 20 seconds.
Tetramethyl-p-phenylenediamine is a chromogenic reducing reagent. In
its reduced state tetramethyl-p-phenylenediamine is colorless; in its
oxidized state it is deep purple or blue. Therefore, if the bacteria produce
cytochrome oxidase, the reagent will be oxidized and will turn blue. The
reaction is described below.
In a negative test for cytochrome oxidase the sample will remain clear,
turn yellow or light gray.
[Tetramethyl-p-phenylenediamine (reduced) + 2 Cytochrome oxidase
(oxidized)- Tetramethyl-p-phenylenediamine (oxidized) + 2 Cytochrome
oxidase (reduced)].
As this reagent is unstable in the presence of water and oxidizes quickly you
should observe your results immediately!!!!!
Record your results for the catalase and the cytochrome oxidase tests in the table below.
Using a (+) for a positive test and a (-) for a negative test. What constitutes a positive or
negative reaction for each test?
Organism
Staphylococcus aureus
Enterococcus faecalis
Catalase activity
Cytochrome oxidase activity