Introduction to Lab Ex. 16 Diagnostic Media

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Introduction to Lab Ex. 16 Diagnostic
Media Urea broth Sulfide Indole
Motility Citrate
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Introduction to Lab Ex. 16 Diagnostic Media
Urea, SIM, Citrate Urease Urea is a protein
compound. Bacteria that are able to break down
the molecule have access to a lot of protein
nutrients. Bacteria that have the enzyme urease
are able to use the urea.   Urea-------------?
CO2 NH3   The reaction is made observable by
including a pH indicator in the medium.
Phenol red is the indicator (is peach color at
neutral pH, turns yellow below 6.8 and pink
above 8.4). The release of ammonia by the
breakdown of urea results in the pH of the
medium becoming alkaline turning the medium
pink. Thus pink color indicates production
of urease by the organisms and no pink color
indicates a negative test for urease.
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S.I.M. Agar This test tests 3 different
characteristics of the bacterium (combination
test). The 3 characteristics tested are
hydrogen sulfide production, indole production
and motility Hydrogen sulfide gas may be
produced by bacteria that are able to
metabolize certain sulfur containing compounds
(amino acids/ thiosulfate compounds). There
are at least 2 different enzymes bacteria may use
for sulfide production The amino acid
Cysteine contains sulfur in its structure and
may be broken down by bacteria that are able to
produce the enzyme cysteine desulfurase.
This results in the production of hydrogen
sulfide gas, which will get trapped in the
agar. The bacteria that are able to produce
another enzyme thiosulfate reductase metabolize
thiosulfates with the production of hydrogen
sulfide gas.
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In order to visualize the presence of H2S in the
medium an indicator is included iron salts
will react with H2S and form Fe2S, which is a
black precipitate. Hence the black discoloration
of the medium indicates H2S production by the
bacterium.
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Indole is a compound that is produced by bacteria
when they metabolize the amino acid tryptophan.
Those bacteria able to produce the enzyme
tryptophanase break down the tryptophan and
produce indole. Indole can be visualized by its
reaction with a reagent Kovacs reagent forming
a red color. After incubation, a few drops of
the reagent is added to look for red
coloration.
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Motility is a characteristic seen in some
bacteria that have flagella (motility
organelle- prokaryotic flagella are very
different in structure and mode of action from
the eukaryotic flagella). Presence of motility
is tested by the use of a semi-solid medium
(reduced concentration of agar in the medium
0.9). Motile bacteria show dispersed growth
(seen as turbidity or cloudiness) in contrast
to non-motile bacteria that are restricted in
their growth to the stab line.
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Citrate The Simmons Citrate medium tests the
ability of the bacteria culture to be able to
use citrate as the sole C source. Bacteria that
are able to produce the enzyme citrase are able
to transport the citrate into the cell and use
it as a source of C. Since the medium does not
contain any other source for C, only those
bacteria that can produce citrase are able to
grow in this medium. When cultures are able to
use the citrate they break it down, producing
sodium bicarbonate, which changes the pH of
the medium to alkaline. The pH indicator in the
medium (bromothymol blue) changes to a blue
color from its original green color.