LAB 02

Lecture # 02
Biochemical Identification Tests
Topics:
1) Oxidase Test
2) Urease Test
3) IMViC Tests
Oxidase Test
PRINCIPLE
• Organisms that produce oxidase enzyme ‘’Cytochrome oxidase’’ also
known as indophenol that is a hemoprotein, carry cytochrome c in their
respiratory chain
• This enzyme is responsible for oxidation of cytochrome c by transferring
of electrons from NADH to oxygen usually in ETC.
• It also oxidizes the reagent (TMPD) tetra methyl para phenylene-diamine
dichloride used in this test that gives characteristic purplish blue color.
• This system of cytochrome is present in aerobes only because they have
oxygen to accept hydrogen as final acceptor therefore end product is
either H2O2 or simple H2O.
PURPOSE
• Oxidase test is used to check whether the organism carries
cytochrome oxidase enzyme or not and also help to differentiate
pseudomonas from other related species.
PROCEDURE
• A strip of filter paper is soaked with a little freshly made 1% solution
of the Kovac's reagen.
• A colony from culture is rubbed on it with a platinum loop and wait
for change in color of filter paper.
• A quality control was also run for oxidase positive (Pseudomonas) and
negative organism (E.coli).
RESULTS
• A positive reaction is indicated by an intense deep-purple color,
appearing within 5-10 seconds, a “delayed positive” reaction by
colouration in 10-60 seconds, and a negative reaction by absence of
colouration or by colouration after 60 seconds.
Urease Test
PRINCIPLE
• This test is performed to detect the presence of urease enzyme in
bacteria.
• If bacteria are urease positive, they consecutively produce Urease
which is responsible for hydrolysis of urea into ammonia and carbon
dioxide.
• Phenol red in urea broth is indicator therefore when urease hydrolyze,
ammonia is produced that result in increase in pH as a result change
in color of indicator from light orange to pink color is observed. pH
changes from 6.8 to 8.1.
• This test is used to differentiate various Enterobacteriaceae.
PROCEDURE
• Prepare urea agar, sterile it and then dispensed into the test tubes.
• Keep test tubes tilted for some time in laminar flow hood for the
formation of slants.
• Use autoclaved toothpicks to pick isolated colonies of test organisms
from the fresh streaked plate not more than 24 hrs old.
• Streak slants very gently with toothpick and plug test tubes with
sterilized cotton plugs and then incubate for twenty-four hours at
37°C.
• After incubation of 24 hrs. observe test tubes for change in color.
Results
• Positive: Change of color from yellow to pink. Pink color on the slant
that may extend into the butt
• Negative: No change in color for urease.
IMViC Tests
• The IMViC series is a group of four individual tests that are commonly
used to identify bacterial species, especially coliforms.
• A coliform is a gram negative, aerobic anaerobic rod which produces
gas from lactose within 48 hours. The presence of some coliforms
indicate fecal contamination.
• Each of the letters in “IMViC” stands for one of these tests.
• “I” is for indole;
• “M” is for methyl red;
• “V” is for Voges-Proskauer, and
• “C” is for citrate, lowercase “i” is added for the ease of pronunciation.
• IMViC is an acronym that stands for four different tests
IMViC Tests
• To obtain the results of these four tests three test tubes are inoculated
• Indole test – tryptone broth
• Methyl red test – methyl red (MR-VP broth)
• Voges-Proskauer test – Voges Proskauer broth
• Citrate utilization test – Simmons citrate agar
• IMViC tests are employed in the identification/differentiation of
members of family enterobacteriaceae.
• General procedure for performing IMViC Tests and their
interpretations:
• Cultures of any members of enterobacteriaceae have to grow for 24 to
48 hours at 37°C and the respective tests can be performed
IMViC Tests
• IMViC tests of Escherichia coli
• Indole: Positive
• Methyl-Red: Positive
• Voges-Proskauer test: Negative
• Citrate test: Negative
• IMViC tests of Enterobacter aerogenes
• Indole: Negative
• Methyl-Red: Negative
• Voges-Proskauer test: Positive
• Citrate test: Positive
• IMViC tests of Citrobacter freundii
• Indole: Negative
• Methyl-Red: Positive
• Voges-Proskauer test: Negative
• Citrate test: Positive