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Biochemical tests
are the tests used for the identification of bacteria species
based on the differences in the biochemical activities of
different bacteria.
MSc. Sarah Ahmed
Catalase test
Catalase is an enzyme, which is produced by microorganisms
that live in oxygenated environments to neutralize toxic forms
of oxygen metabolites; H2O2.
The catalase enzyme
neutralizes the bactericidal effects of hydrogen peroxide and
protects them. Anaerobes generally lack the catalase enzyme.
Catalase mediates the breakdown of hydrogen peroxide H2O2
into oxygen and water. To find out if a particular bacterial
isolate is able to produce catalase enzyme, small inoculum of
bacterial isolate is mixed into hydrogen peroxide solution (3%)
and the rapid elaboration of oxygen bubbles occurs. The lack
of catalase is evident by a lack of or weak bubble production.
Procedure of catalase test (Slide Test)
1.
Transfer a small amount of bacterial colony to a surface of clean,
dry glass slide using a loop or sterile wooden stick
2.
Place a drop of 3% H2O2 on to the slide and mix.
3.
A positive result is the rapid evolution of oxygen (within 5-10 sec.)
as evidenced by bubbling.
4.
A negative result is no bubbles or only a few scattered bubbles.
5. Dispose of your slide in the biohazard glass disposal container.
Tube Catalase Test-Procedure and Results
1.
Add 4 to 5 drops of 3% H2O2 (Hydrogen peroxide) to in a test tube
2.
Using a wooden applicator stick, collect a small amount of
organism from a well-isolated 18- to 24-hour colony and place into the test
tube (Note: Be careful not to pick up any agar (esp if using Blood Agar
WHY?
3.
Place the tube against a dark background and observe for
immediate bubble formation (O2 + water = bubbles) at the end of the
wooden applicator stick.
Results:
•Catalase Positive reactions: (bubble formation)
•Catalase Negative reaction: No bubble formation
Oxidase test
The oxidase test is used to identify bacteria that
produce cytochrome c oxidase, an enzyme of the
bacterial electron transport chain. When present, the
cytochrome c oxidase oxidizes the reagent
(tetramethyl-p-phenylenediamine dihydrochloride)
to (indophenols) purple color end product. When the
enzyme is not present, the reagent remains reduced
and is colorless.
Procedure of Oxidase test:
1.
Take a filter paper soaked with the substrate
tetramethyl-p-phenylenediamine dihydrochloride
2.
Pick the colony to be tested with wooden or
platinum loop and smear in the filter paper
3- Observe inoculated area of paper for a color change
to deep blue or purple within 10 seconds
Result
1. Positive: Development of dark purple color
(indophenols) within 10 seconds
2.
Negative: Absence of color
Procedure for urease test
1.
The broth medium is inoculated with a loopful of a pure culture
of the test organism
2.
incubate the test tube at 35 °C in ambient air for 18 to 24
hours.
Organisms that hydrolyze urea rapidly (e.g. Proteus spp) may produce
positive reactions within 1 or 2 hours; less active species (e.g. Klebsiella
spp) may require 3 or more days. In routine diagnostic laboratories the
urease test result is read within 24 hours.
THE END