الاختبارات البايوكيميائية لفحص بكتريا الحليب

الاختبارات البايوكيميائية لفحص بكتريا الحليب

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IMViC
Enterobacteriaeae (enterics) are Gram-negative bacteria that grow in the intestinal tract of humans and other animals.
The IMViC tests are frequently employed for identification of this group
of microbes which includes such organisms as Klebsiella, Enterobacter,
and Escherichia coli.
The presence of E. coli is used by public health officials as an
indicator of fecal contamination of food and water supplies. While
Enterobacter and Klebsiella resemble E.coli in being lactose fermenters,
their presence does not necessarily indicate fecal contamination
because they are widespread in soil and grass.
The IMViC tests can be used to differentiate these three organisms.
IMViC is an acronym that stands for indole , methyl red, Voges-Proskauer , and citrate .
To obtain the results of these four tests, three test tubes are
inoculated: tryptone broth (indole test), methyl red - Voges Proskauer
broth (MR-VP broth), and citrate.

The Indole Test
The test organism is inoculated into tryptone broth, a rich source of
the amino acid tryptophan. Indole positive bacteria such as Escherichia
coli produce tryptophanase, an enzyme that cleaves tryptophan, producing
indole and other products. When Kovac's reagent
(p-dimethylaminobenzaldehyde) is added to a broth with indole in it, a
dark pink color develops.
The indole test must be read by 48 hours of incubation because the
indole can be further degraded if prolonged incubation occurs.
The acidic pH produced by Escherichia coli limits its growth.

The Methyl Red and Voges-Proskauer Tests
The methyl red (MR) and Voges-Proskauer (VP) tests are read from a
single inoculated tube of MR-VP broth. After 24-48 hours of incubation
the MR-VP broth is split into two tubes. One tube is used for the MR
test; the other is used for the VP test.

MR-VP media contains glucose and peptone. All enterics oxidize glucose
for energy; however the end products vary depending on bacterial
enzymes.
Both the MR and VP tests are used to determine what end products result when the test organism degrades glucose.
E. coli is one of the bacteria that produces acids, causing the pH to
drop below 4.4. When the pH indicator methyl red is added to this acidic
broth it will be cherry red (a positive MR test).
Klebsiella and Enterobacter produce more neutral products from glucose
(e.g. ethyl alcohol, acetyl methyl carbinol). In this neutral pH the
growth of the bacteria is not inhibited. The bacteria thus begin to
attack the peptone in the broth, causing the pH to rise above 6.2. At
this pH, methyl red indicator is a yellow color (a negative MR test).

The reagents used for the VP test are Barritt's A (alpha-napthol) and
Barritt's B (potassium hydroxide). When these reagents are added to a
broth in which acetyl methyl carbinol is present, they turn a
pink-burgundy color (a positive VP test). This color may take 20 to 30
minutes to develop.
E. coli does not produce acetyl methyl carbinol, but Enterobacter and Klebsiella do.

The Citrate Test
The citrate test utilizes Simmon's citrate media to determine if a
bacterium can grow utilizing citrate as its sole carbon and energy
source. Simmon's media contains bromthymol blue, a pH indicator with a
range of 6.0 to 7.6. Bromthymol blue is yellow at acidic pH's (around
6), and gradually changes to blue at more alkaline pH's (around 7.6).
Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an
intermediate green color. Growth of bacteria in the media leads to
development of a Prussian blue color (positive citrate). Enterobacter
and Klebsiella are citrate positive while E.coli is negative.

Thus E.coli gives ++-- results on the IMViC tests, while Enterobacter and Klebsiella give the reverse: --++

1. indole 2. methyl red 3. Voges-Proskauer 4. citrate