Bacterial Cell Culture Methods
505
1
Cultivation of Bacteria
1.1
Introduction
Much of microbiological work depends
upon the maintenance of pure cultures
of microorganisms. Aseptic technique is
largely a matter of commonsense, but
the following points should be borne
in mind:
Clean air contains many bacterial and
fungal spores, carried on dust parti-
cles or in water droplets; any surface
exposed to the air quickly becomes
contaminated. Instruments that can be
sterilized by heating in a Bunsen flame,
for example, inoculating loops, can be
left exposed, but must be flamed thor-
oughly before use. Items that cannot be
treated in this way, for example, pipettes
should be sterilized in wrappings or
containers.
Microbial contamination in the labo-
ratory is most often due to currents
of unsterile air. The chief merit of
inoculation cabinets therefore lies in
the protection they give from draughts.
This protection can be supplemented
by keeping all windows and doors shut
and by cutting down personal move-
ment within the laboratory.
Prior to identiFcation it is often neces-
sary to obtain pure cultures of bacteria
growing on plates containing a suitable
nutrient medium. Bacteria display many
variations of the major nutritional require-
ments and so the utilizable sources of
energy also vary. However, the media
for the cultivation of bacteria must con-
tain (1) water; (2) some source of energy,
usually as a carbon compound; (3) other
essential elements such as N, S, P, K,
Na,
Mg,
Ca,
±e
and
Cl
in
balanced
proportions. Some bacteria also require
vitamins. In preparing the media, chemi-
cals of deFned composition are preferred
to those of undeFned composition. How-
ever, since the nutritional patterns of all
bacteria are not sufFciently well known,
it is often necessary to use substances
of unknown composition, for example,
peptone. In addition, media are often so-
lidiFed with complex substances like agar.
Most chemicals should be dissolved in dis-
tilled or demineralized water. Tap water
may contain toxic elements, particularly
copper.
Most bacteria can only grow within
a restricted pH range, and are usually
intolerant of acid conditions. The reaction
o
fthemed
iummus
tbead
jus
tedsotha
t
the Fnal pH after sterilization is between
6.8 and 7.2, unless a different reaction
is needed for some special purpose. If
a medium contains substances that may
become more acid during sterilization, it
is usually adjusted to a pH value about 0.2
greater than that required, that is, 7.0 to
7.4. Buffers are often required to facilitate
the growth. This is particularly true of
media composed of simple compounds or
media in which acid-producing bacteria
are cultivated.
1.2
Cultures
Liquid media (broths) are distributed in
flasks or tubes plugged with nonabsorbent
cotton wool or plastic caps. Liquid medium
can be solidiFed with agar (1.0–2.0%).
Agar is an extract of seaweed and is a
sulfate ester of a linear galactan, though
contaminated with other organic impuri-
ties. Generally, agar can be regarded as
inert. Agar gels liquefy between 96 and
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