Bacterial Cell Culture Methods
517
for the delimitation of species, although
in certain cases, it is possible to assign
the
organisms
to
families
and
some-
times to genera. Other characters have
been used therefore, to enable more pre-
cise classiFcations of organisms to be
made. Perhaps the most widely used tests,
however, have been those designed to
demonstrate the biochemical and physio-
logical characteristics of microorganisms.
The biochemical methods used fall into
four groups:
1. Mineral growth requirements and sub-
strate utilization.
2. Detection of metabolic end products.
3. IdentiFcation
of
characteristic
com-
pounds, for example, enzymes, gums,
and toxins.
4. Resistance and sensitivity to chemicals,
for example, antibiotics.
5.1.1
Minimal Growth Requirements and
Substrate Utilization
Nutritional studies.
It is often useful to
know something of the nutrition of a par-
ticular bacterium, from the point of view
of both taxonomic and cultural studies.
It is important to obtain information on
the relation of bacteria to nitrogen sources
and growth factors. Growth of bacteria
with an inorganic nitrogen source can be
detected in the Ayer, Rupp, and John-
son’s medium: NH
4
H
2
PO
4
,1
.
0g
;KC
l
,
0.2 g; MgSO
4
7H
2
O, 0.2 g; glucose, 10.0 g;
water, 1 L. The pH should be adjusted
to about 7.0. Several serial subcultures
should be attempted in this medium be-
fore recording growth as positive since
the inoculum may carry over organic
nitrogen and growth factors. ±urther nu-
tritional studies can be made by replacing
sources of nitrogen or growth factors in
this medium with others. If growth is
obtained in the basal medium, all sub-
sequent tests can be carried out using this
medium with the particular substances
under investigation substituted, for exam-
ple, in fermentation tests. If no growth is
recorded, peptone media must be used as
a basis for growth.
Effects of environment.
The relation of
organisms to free oxygen and pH also
fall into this group of tests. These rela-
tionships can be elucidated by methods
outlined earlier.
Utilization of organic acids.
Some bac-
teria are able to utilize salts of certain
organic acids as sole carbon sources.
Pathogenic species, which are nutrition-
ally exacting, are less likely to be able to
do this, while many of the less exacting
soil and water forms can. Thus, while the
soil coliforms can utilize citrate, intestinal
coliforms cannot, for example,
Escherichia
coli
, a commensal in the animal intestine.
Koser’s
citrate
medium
is
a
mineral
medium with 0.2% anhydrous sodium
citrate as the sole carbon source. Bro-
mothymol blue is usually added to assist
in detecting slight growth, by the al-
kaline reaction produced. Other organic
acids can be detected in a similar way,
but a lower concentration, for example,
0.1% should be used for the more toxic
acids, that is, formic and oxalic acids. The
mineral medium most frequently used
is KH
2
PO
4
,1
.
0g
;M
g
SO
4
·
7H
2
O, 0.2 g;
Na(NH
4
)HPO
4
·
4H
2
O, 1.5 g; distilled wa-
ter, 1 L.
5.1.2
Detection of Metabolic End Products
Indole.
Indole is produced by some
bacteria from the amino acid tryptophan.
A tube of peptone water is inoculated
and samples of the culture are tested
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