508
Bacterial Cell Culture Methods
tangential streaks cutting across the frst
streak and covering one halF oF the
plate.
Turn the plate through 90
and con-
tinue the process, flaming and cool-
ing
the
loop
between
each
set
oF
streaks.
AFter incubation, well-separated colonies
should be Found along the streak marks.
BeFore deciding that a culture is pure by
either method, colonies should be picked
oFF, grown and then reseparated, until all
the colonies are the same. Staining must
be used as a check on the purity oF the
fnal isolations.
2.3
Isolation of Bacteria from Natural Sources
The object oF viable counting techniques
is to estimate the number oF all live
organisms in a given sample oF material.
To
do
this,
a
medium
satisFying
the
nutritional
requirements
oF
as
many
bacteria in the sample as possible is
required. Other types oF viable count and
all isolation techniques embody the reverse
principles. Here it is necessary to pick out
and encourage one type oF organism and
to prevent or repress the development oF
the other types. In natural habitats, the
organisms that one is isolating may be
present only in small numbers. The frst
step in such cases is thus to obtain an
enrichment culture by one or more oF
three methods:
1. using selective media,
2. using selective conditions oF incubation,
3. selective pretreatment oF the material.
Several generations oF subcultures on
liquid, or solid media may be necessary,
but the fnal step will consist oF plating out
the organisms.
2.4
Use of Selective Media
A completely selective medium allowing
the growth oF only a single species is not
attainable in practice, but media that will
discourage all but the required species can
be obtained.
Some selective media are also diFFeren-
tial. Certain organisms, when grown on
them, exhibit distinctive biochemical or
morphological characters, which enable
them to be recognized easily. This may
be very important From a medical point
oF view, where the range oF species con-
cerned is small. Where a large range oF
spe
c
ie
si
sinvo
lved
,Fo
rexamp
le
,inso
i
l
,
diFFerential media cannot be used to sep-
arate taxonomic groups, but are useFul to
distinguish groups concerned with some
biochemical process, For example, cellu-
lose decomposition.
Selectivity may be achieved in three
ways,
1. by adding something to the medium to
discourage the growth oF species that
are not required,
2. by altering the pH oF the medium,
3. by omission oF some ingredient re-
quired by most bacteria, but not the
ones required.
Most bacteria are Facultative aerobes,
that is, they can grow under aerobic
or anaerobic conditions, the latter ability
being dependent upon the presence oF
some
substance
that
can
be
utilized
as a hydrogen acceptor by the species
concerned. Some bacteria are obligate
aerobes,
unable
to
use
anything
but
oxygen as a hydrogen acceptor. Others
are obligate anaerobes, which cannot use
oxygen as a hydrogen acceptor. A Few
bacteria are intermediate, growing best
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