518
Bacterial Cell Culture Methods
at intervals with Ehrlich’s reagent (2%
para-dimethylaminobenzaldehyde in 95%
alcohol). 1 mL is added to the culture
andcon
cen
t
ra
tedHC
li
saddedd
ropb
y
drop until a red zone appears between
the alcohol and the peptone solution. No
more than 0.5 mL acid is needed. If the
colored pigment is soluble in amyl alcohol
or chloroform it can be considered to
be indole.
Hydrogen sulfde.
Hydrogen sulFde may
be produced from sulfur-containing amino
acids,
for
example,
hydrogen
cystine
present in peptone. A test for hydrogen
sulFde production can be combined with
the above test. When the peptone water
is inoculated, a Flter paper strip impreg-
nated with a saturated lead acetate solution
is inserted between the plug and the tube,
taking care to keep it dry. If hydrogen
sulFde is produced, the paper will be-
come blackened.
Some bacteria also reduce inorganic
sulfur compounds. A chemically deFned
medium containing a sulfur compound,
for example, sulfate, or thiosulfate, can be
used instead of peptone water. Only organ-
isms that can utilize inorganic nitrogen
canbetestedinthisway.
A number of special media containing
iron salts and a source of sulfate can also
be used, for example, Kligler’s iron agar.
Hydrogen sulFde production is recognized
by blackening of the medium.
Acetyl methyl carbinol – The Vosges–Pros-
kauer (VP) test.
Acetoin or acetylmethyl-
carbinol can be produced from glucose via
pyruvic acid. Two enzymes are necessary
for this to occur, that is, pyruvate enzyme
and acetolactic decarboxylase. Acetoin may
be reduced to 2.3 butanediol and so give
a negative result. Sensitive tests should
therefore be used to detect acetoin.
The normal method of detection is to
oxidize acetoin to diacetyl, which reacts
with guanidine residues in the medium
to give a cherry red color. The medium
used usually contains 0.5% glucose, 0.5%
peptone and 0.5% K
2
HPO
4
in distilled
water. To prevent reducing conditions
from developing, fumarate is sometimes
added as a hydrogen acceptor or else
glucose is replaced by pyruvate.
After 3 days of incubation, a 2 mL sam-
pleoftheculturefluidistestedbyadding
about 5 mL of 40% KOH and a trace of
creatine, shaking vigorously and allow-
ing it to stand for up to 60 min. The
culture is positive if a cherry red color
develops.
Methyl red test.
In the examination of
coliform bacteria, the VP Test is carried
out at the same time as the methyl red
test. The latter determines the hydrogen
ion concentration produced in a deFnite
time and in a speciFc medium. The glu-
cose–phosphate medium described above
must be used and not a modiFcation
of it.
The culture is tested after 3 days at 30
C.
To one part of the culture is added a
0.4% solution of methyl red (1 drop per
mL of culture). A magenta red color is
positive, a yellow color is negative.
Reduction oF nitrates.
Certain organisms
are capable of reducing nitrate to nitrite
and even to nitrogen or ammonia.
The organism under test is grown in
peptone water containing 1% potas-
sium nitrate.
After incubation, the culture fluid is
divided into three portions.
One portion is tested by adding a few
drops of sulfanilic acid reagent fol-
lowed by a few drops of
α
-napthylamine
reagent (Griess’ reagents I and II).
A
red
color
indicates
the
presence
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