506
Bacterial Cell Culture Methods
100
C, but do not set until their tempera-
ture has dropped to 42
C.
Occasionally, it may be necessary to
exclude all organic material (including
agar) from the media. In this case, silica
gel can be used to solidify the medium.
Na
2
SiO
3
·
9H
2
O (13.4 g) is dissolved in
100 mL distilled water. The pH must be
adjusted to 10.0 using cation-exchange
resin AG50 WX-8 (Bio-Rad Laboratories)
under constant mixing. After 30 min, the
pH of the resin must be readjusted to
10.0. The resin must then be removed
by Fltration using Whatman No. 1 Flter
paper. After the addition of appropriate
nutrients for bacterial growth, sterilization
is achieved by Fltration through a sterile
membrane Flter (0
.
45
µ
m). The pH of the
solution is then adjusted to 7.0 using phos-
phoric acid (5 M). ±inally the medium is
pouredandallowedtogel(40minatroom
temperature).
So
l
idm
ed
i
ac
anb
eu
s
edinav
a
r
i
e
t
y
of ways:
Slope cultures.
Tubes or small bottles
containing 6 mL of medium are heated
to melt the agar. This is allowed to set
in a sloping position, thus increasing the
surface area of the medium for the growth
of bacteria.
Deep stab cultures.
Tubes or bottles con-
taining 10 to 15 mL of solid medium
are
heated
and
the
molten
medium
is
allowed
to
cool
to
45
C.
While
still liquid, the medium is inoculated,
mixed
well
by
rotating
the
tube
be-
tween the hands, and then allowed to
set.
Plate cultures.
About 15 to 20 mL of
solid medium are needed for each 9-cm
petri dish. The medium is melted and
cooled to 45
C and poured into the dish.
Hot media must never be poured since
water may condense inside the lid. The
medium can be inoculated in several
ways:
1. The inoculum is Frst placed in the petri
dish and then the medium is poured
over. To ensure even distribution of
the
inoculum,
the
plate
is
moved
gently with a rotating movement. This
method is used when all the bacteria
in a measured inoculum are required
to grow into separate colonies, for
example, a dilution-plate count.
2. Alternatively, the agar can be inoculated
by the poured plate or the streak plate
methods, which are described below.
1.3
Culture Incubations
Cultures should be incubated at the opti-
mum temperature for growth or for the
speciFc activity being studied. Pathogens
and commensal species grow best at body
temperature, that is, 37
C. Soil organisms
and plant pathogens are incubated at 20 to
25
C.
1.4
Types of Media Used for Growing Selected
Bacteria
Bacteriological media are frequently clas-
siFed as complex or deFned according
to their chemical composition. A com-
plex medium is one that contains such
ill-deFned substances as yeast or beef ex-
tract, while a deFned medium can have 10
to 20 different chemicals present, but the
identity of each is known. All of the most
widely used media are available from a
range of supplies, some of which are listed
in Appendix 1.
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