Bacterial Cell Culture Methods
519
of
nitrite.
The
production
of
free
nitrogen
may
be
detected
by
al-
lowing
it
to
collect
in
a
Durham
tube.
A second portion of the fluid may
be tested for ammonia with
Nessler’s
reagent
,
but
since
many
organisms
produce ammonia from peptone, the
result must be interpreted with care. [A
sample of uninoculated medium should
also be tested since some peptones give
a positive reaction (i.e. an orange/brown
precipitate) with Nessler’s reagent].
If no nitrite has been detected, the third
portion of the culture should be tested
for residual nitrate for it is possible that
all the nitrate will have been converted
Frst to nitrite and then to nitrogen
or ammonia. To do this, add a small
quantity of zinc dust. This will reduce
the residual nitrate chemically to nitrite
and this can then be tested for as
shown above. The interpretation is as
follows.
If
nitrite
is
then
+
,
it
follows
that
the
bacterium
did
not
reduce
nitrate
to nitrite; rather it was the Zn dust
that
reduced
it.
If
the
test
on
the
third portion is negative for nitrite, it
follows that all the nitrate had been
reduced to another product via nitrite
by bacteria and thus the bacterium will
be recorded as a positive nitrate reducing
organism.
Fermentation
tests – Hugh
and
Leifson’s
method.
Certain carbohydrates that is,
sugars, alcohols, and glucosides are at-
tacked and produce quantities of acid
and/or gas. It is also important to know
which acid and which gas are produced,
that is, 2 keto-gluconic acid, propionic acid,
lactic acid and so on. Generally speaking,
simple tests for detecting the nature of
the acids are lacking and so results are
most frequently recorded as no acid, acid
or acid plus gas. One of the most conve-
nient tests for this purpose is that of Hugh
and Leifson.
A semisolid agar medium is tubed to a
depth of 4 cm, two tubes being required
for each culture and for each sugar
tested. The formula for this medium
is as follows:
Peptone
2.0 g
Sodium chloride
5.0 g
Dipotassium hydrogen
phosphate
0.3 g
Bromothymol blue
(1% aqueous solution)
3.0 mL
Carbohydrate
10.0 g
Water
1.0 L
pH
7.1
The
bromothymol
blue
is
dissolved
in
water
and
3.0 mL
of
a
1%
so-
lution
added
to
each
1000 mL
of
medium. Alcoholic solutions of indi-
cators
should
not
be
used
as
acid
may be produced from the alcohol.
±or critical studies, the carbohydrate
should
be
sterilized
separately
and
added to the otherwise complete sterile
medium.
After
inoculation,
one
of
the
pairs
of
tubes
is
covered
by
a
layer
of
sterile melted Vaseline to a depth of
2cm.
Several types of reaction may be ob-
served.
Fermentative organisms
,w
h
i
c
h
phosphorylate glucose and then split it
into 2 triose molecules before forming
acidwillproduceanacidreactioninboth
tubes (or the sealed tube only).
Oxidative
organisms
, which convert the aldehyde
group to a carboxyl group in glucose to
form gluconic acid will produce an acid
reaction in the open tube only, leaving
the sealed tube unchanged. The acid
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