Bacterial Cell Culture Methods
515
seawater or river water is used, but more
often
sterile
quarter-strength
Ringer’s
solution is recommended (NaCl, 9 g; KCl,
0.42 g; CaCl
2
,0
.
4
8g
;N
aH
2
CO
3
,0
.
2g
;
water, 4 L). A 0.1% peptone solution may
also be used as a diluent.
The required number of tubes con-
taining 9 mL diluent are prepared with
a 1-mL sterile blow-out pipette, 1 mL
o
ft
h
es
am
p
l
ei
sa
d
d
e
dt
ot
h
eF
r
s
t
tube.
With a fresh pipette tip, the liquid is
mixed and 1 mL of this 1/10 dilution
transferred to a second 9 mL of dilu-
ent.
Repeat
until
the
sample
is
diluted
sufFciently. The number of dilutions
needed will depend on the material
concerned, only the two or three highest
being used.
The dilution-plate method and the spread
plate
method.
Tenfold
dilutions
are
prepared as described above.
With the pipette used for mixing the
dilution, 1 mL of this dilution is placed
in each of three or more sterile petri
dishes;
then
after
making the
next
dilutions, 1 mL of this is treated in the
same manner and so on until a range of
dilutions is plated (usually 3).
After mixing the dilutions, 15 or 20 mL
of an agar medium, previously melted
and
cooled
to
45
C,
is
added
to
each plate and an even distribution
is ensured.
When set, the plates are incubated at the
required temperature. After incubation,
dilutions where the plates show more
than 300 or fewer than 30 colonies are
discarded. Errors due to overcrowding
or due to sampling make these counts
unreliable. Thus only one set of plates
will
actually
be
counted
if
tenfold
dilutions are used.
The inoculum may, as an alternative,
be spread over the surface of prepared
plates of agar. Usually an inoculum
of 0.1 mL is then used and the agar
is dried to remove surface water. The
inoculum is spread with a sterile glass
spreader.
The membrane flter method.
This method
is used for water samples in which only
a few organisms are present. It depends
upon the concentration of the inoculum
rather than the dilution.
Sterile
cellulose
acetate
membranes
(pore size
0.45
µ
m) are placed in a
sterilized Flter holder.
A known volume of the fluid is passed
through the Flter.
T
h
i
sF
l
t
e
ri
st
h
e
nl
a
i
do
nat
h
i
n
layer of the medium in a petri dish
and incubated. Bacteria held in the
Flter develop into colonies that can be
counted. DifFculty is often experienced
since
the
even
distribution
of
the
colonies cannot be ensured. By using
suitable media, particular groups of
organisms can be selected.
The ‘‘Most Probable Number’’ method.
As
well as being economical in time and
materials, this method can be used where
the
plate
count
cannot,
for
example,
with materials that contain species that
form ‘‘spreading’’ colonies on agar or
where it is desired to determine numbers
of a particular group that is sparsely
represented in material with a mixed
bacterial flora.
A series of tenfold dilutions are pre-
pared, so that when 1 mL samples of the
highest dilution are inoculated into a suit-
able medium, very few if any bacteria are
likely to grow. Thus, if a suspension con-
tains 5
×
10
9
bacteria per mL, 1 mL of a
10
9
dilution would contain 5 organisms,
and most or all tubes of medium would
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