Bacterial Cell Culture Methods
509
in low oxygen tensions. These are called
microaerophilic bacteria.
Anaerobic forms occur in several fam-
ilies of bacteria. They include several
pathogens, for example,
Clostridium tetani
,
certain autotrophic bacteria, for example,
the purple sulfur bacteria, and some other
soil or water bacteria, such as
Desulfovibrio
and
Clostridium.
During growth, aerobic bacteria tend to
utilize all available oxygen and so reduce
the medium. Thus, in mixed cultures the
oxidation–reduction potential (Eh) of the
medium may become low enough to allow
anaerobes to develop. This property has
also been utilized to grow pure cultures
of anaerobic bacteria. Thus, double petri
dishes have been cemented together, so
that the aerobe in one half uses the oxygen,
allowing the anaerobe to develop in the
other half.
Pure cultures of anaerobes are usually
grown making use of other methods.
Steps must be taken to ensure that access
of oxygen to cultures is prevented or
reducing substances must be added to
the medium. Media to be used for the
cultivation of anaerobes must be heated at
100
C for 10 min to drive off the dissolved
oxygen and cooled quickly, immediately
before use.
2.5
Methods for Anaerobic Cultures
Stab and shake cultures.
Many anaerobes
cangrowindeepstaborshakeculturesin
glucose agar. This method is particularly
useful for microaerophilic species. A seal
of liquid parafFn or Vaseline is sometimes
advocated
to
help
maintain
anaerobic
conditions, for example, the Hugh &
Leifson fermentation test.
Anaerobe jar culture.
If surface cultures
are required, the plates or slopes should
be placed in an anaerobe jar. This is
a glass or plastic jar with a lid that
can be clamped down Frmly to form an
effective seal. Usually a Gas-Pak method
is used in which a sachet (to which
water has been added) is placed in the
jar. The chemical reaction uses oxygen
and generates both hydrogen and carbon
dioxide. A strip of methylene blue paper
acts as an indicator.
Robertson’s cooked meat broth.
1kg of
lean
meat
is
minced
and
simmered
with 1 L of water plus 1.5 mL of 1 M
NaOH for 20 min, strained and washed
very thoroughly with distilled water and
partially
dried.
It
is
distributed
into
tubes
to
form
a
layer
at
least
1-cm
deep. Nutrient broth is then added to a
dep
tho
fabou
t8cmandthemed
iumis
sterilized. The sterilized tissue contains
reducing
substances
that
are
effective
in maintaining anaerobic conditions at
the bottom of the tube. The reducing
activity of the meat is shown by the
pink
hematin
at
the
bottom
of
the
tube.
Thioglycollate medium.
This is prepared
by adding 0.1% (up to 0.4%) thioglycollic
acid to the nutrient broth before adjusting
the pH. 1% glucose must also be added.
Although the medium may be solidiFed
with agar, it is more usual to use a
semisolid medium, that is, 0.5% agar. The
increased viscosity of the medium prevents
the distribution of oxygen (dissolved at the
exposed surface), by convection currents.
Methylene blue is added to act as an
indicator of reducing conditions. A blue-
green layer at the surface shows the depth
to which oxygen has diffused into the
medium. Inocula should be introduced
carefully by means of a Fne pipette, to the
bottom of the tube.
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