substances, including meat and yeast extract, and as a result are less well defined,
since their precise composition is largely unknown. Such media are, however, rich in
nutrients and therefore generally suitable for culturing fastidious organisms that
require a mixture of nutrients for growth. Defined media, by contrast, are relatively
simple. These are usually designed to the specific needs of the bacterial species to be
cultivated and as a result are made up of known components put together in the
required amounts. This flexibility is usually exploited to select or eliminate certain
species by taking advantage of their distinguishing nutritional requirements. For
instance, bile salts may be included in media when selective cultivation of enteric
bacteria (rod-shaped Gram-negative bacteria such asSalmonellaor Shigella)is
required, since growth of most other Gram-positive and Gram-negative bacteria
will be inhibited.2.7.4 Culture procedures for bacterial cells
Bacteria can be cultured in the laboratory using either liquid or solid media. Liquid
media are normally dispensed into flasks and inoculated with an aliquot of the
organism to be grown. This is then agitated continuously on a shaker that rotates
in an orbital manner, mixing and ensuring that cultures are kept in suspension. For
such cultures, sufficient space should be allowed above the medium to facilitate
adequate diffusion of oxygen into the solution. Thus, as a rule of thumb, the volume
of medium added to the flasks should not exceed more than 20% of the total volume
of the flask. This is particularly important for aerobic bacteria and less so for
anaerobic microorganisms.
In large-scale culture,fermentersorbioreactorsequipped with stirring devices for
improved mixing and gas exchange may be used. The device (Fig. 2.13) is usually
fitted with probes that monitor changes in pH, oxygen concentration and temperature.
In addition most systems are surrounded by a water jacket with fast-flowing cold
water to reduce the heat generated during fermentation. Outlets are also included to
release CO 2 and other gases produced by cell metabolism.
When fermenters are used, precautions should be taken to reduce potential con-
tamination with airborne microorganisms when air is bubbled through the cultures.
Sterilisation of the air may therefore be necessary and can be achieved by introducing
a filter (pore size of approximately 0.2 mm) at the point of entry of the air flow into
the chamber.
Solid medium is usually prepared by solidifying the selected medium with 1–2% of
the seaweed extract agar, which, although organic, is not degraded by most microbes
thereby providing an inert gelling medium on which bacteria can grow. Solid agar
media are widely used to separate mixed cultures and form the basis for isolation of
pure cultures of bacteria. This is achieved by streaking diluted cultures of bacteria
onto the surface of an agar plate by using a sterile inoculating loop. Cells streaked
across the plate will eventually grow into a colony, each colony being the product of a
single cell and thus of a single species.
Once isolated, cells can be cultivated either inbatchorcontinuous cultures.Of
these, batch cultures are the most commonly used for routine liquid growth and entail69 2.7 Bacterial cell culture