There may be different types of oxidase enzymes produced by bacteria. The colorless redox reagent,
tetramethyl-p-phenylenediamine dihydrochloride (or dimethyl can be used)
used in the test will detect the presence of the enzyme oxidase and, reacting with oxygen, turn a color.
How do you make oxidase reagent?
Prepare a 1.0% Kovac’s oxidase reagent
by dissolving 0.1 g of tetramethyl-p- phenylenediamine dihydrochloride into 10 ml of sterile distilled water
. 2. Mix well and then let stand for 15 minutes. The solution should be made fresh daily and the unused portion should be discarded.
What is the reagent for the oxidase test?
The oxidase test often uses a reagent,
tetra-methyl- p-phenylenediamine dihydrochloride
, as an artificial electron donor for cytochrome c. When the reagent is oxidized by cytochrome c, it changes from colorless to a dark blue or purple compound, indophenol
What happens to the reagent if the oxidase is present?
In the presence of an organism that contains the cytochrome oxidase enzyme, the
reduced colorless reagent becomes an oxidized colored product
(2, 4, 9).
How is oxidase test done?
Use the
dropper to add a drop of oxidase reagent to the bacteria on the filter paper
, and look for the appearance of blue or purple spots – a positive result. After 15 seconds, if no color appears, the test is negative for the presence of cytochrome oxidase.
What is oxidase test used for?
The oxidase test is used to
identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain
. (note: All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron acceptor in respiration.
What is the principle of indole test?
Principle of Indole Test
Tryptophanase catalyzes the deamination reaction, during which the amine (-NH2) group of the tryptophan molecule is removed
. Final products of the reaction are indole, pyruvic acid, ammonium (NH4+) and energy. Pyridoxal phosphate is required as a coenzyme.
What does a positive catalase test look like?
Add one drop of hydrogen peroxide
and look for bubbles
. Bubbles are a positive result for the presence of catalase. If no bubbles form, it is a negative result; this suggests that the organism does not produce catalase.
Is E coli catalase positive or negative?
| Characteristics E. coli | Catalase Positive (+ve) | Oxidase Negative (-ve) | MR Positive (+ve) | VP Negative (-ve) |
|---|
What type of bacteria are catalase negative?
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 (ie. Streptococci).
What does a positive indole test mean?
A positive indole test is indicated by
the formation of a red color in the reagent layer on top of the agar deep within seconds of adding the reagent
. If a culture is indole negative, the reagent layer will remain yellow or be slightly cloudy.
What is a positive catalase reaction?
This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas.
The bubbles resulting from production of oxygen gas
clearly indicate a catalase positive result.
Where is catalase produced in the cell?
Catalase is usually located in
a cellular organelle called the peroxisome
. Peroxisomes in plant cells are involved in photorespiration (the use of oxygen and production of carbon dioxide) and symbiotic nitrogen fixation (the breaking apart of diatomic nitrogen (N
2
) to reactive nitrogen atoms).
Why is E coli oxidase negative?
Bacteria that are oxidase-negative may be anaerobic, aerobic, or facultative; the oxidase negative result just means that these
organisms do not have the cytochrome c oxidase that oxidizes the test reagent
. They may respire using other oxidases in electron transport.
Are all Enterobacteriaceae oxidase negative?
Human Pathogenic Enterobacteriaceae
Nearly all are facultative anaerobes. They ferment glucose, reduce nitrates to nitrites, and are oxidase negative. With the exceptions of Shigella and Klebsiella which are nonmotile, these bacteria have peritrichous flagella.
What is the purpose of catalase test?
The catalase test is a particularly important test
used to determine whether a gram-positive cocci is a staphylococci or a streptococci
. Catalase is an enzyme that converts hydrogen peroxide to water and oxygen gas. The test is easy to perform; bacteria are simply mixed with H
2
O
2
.
