Bacterial Cell Culture Methods
513
however, unless translated into terms of
cell concentration (viable or total counts),
but the relationship between turbidity
and cell concentration is different for
obtained for a series of dilutions of a
known number of organisms should be
plotted as a calibration graph.
4.3
Theory of Bacterial Growth
Logarithmic
growth
.
The
growth
of
a
bacterial culture undergoing binary Fssion
is expressed by the equation,
x
=
x
o
2
n
where
x
=
the Fnal number of organisms,
x
o
the initial number and
n
is the number
of generations.
If
n
generations are produced in time
t
,then
g
=
t
n
and
n
=
t
g
where
g
=
the generation or doubling
time. Thus
x
=
x
o
2
t
/
g
and
ln
x
=
ln
x
o
+
ln 2
t
g
This is usually written in the form
ln
x
=
ln
x
o
+
µ
t
where
µ
=
ln 2/
g
=
0
.
6931
/
g
.
µ
represents
the
number
of
e-fold
increases
in
cell
numbers
that
have
occurred in time
t
.
If the equation
ln
x
=
ln
x
o
+
µ
t
is differentiated, it becomes the familiar
growth equation
d
x
d
t
=
µ
x
which may be stated as the rate of growth
of the population is proportional to the
initial number of organisms present.
4.4
Counting Methods
Counts may be used to determine the
number of organisms in a sample and
these counts can be converted to estimates
of biomass if suitable conversion factors
are known.
Total counts.
Total counts include the
various methods of estimating the total
number of bacteria per unit volume or
weight of a sample, without distinguishing
between viable and nonviable organisms.
Counting chambers.
The number of bac-
t
e
r
i
ap
e
rm
Lo
fas
u
s
p
e
n
s
i
o
nc
a
nb
e
determined by means of a counting cham-
ber. The best form of a counting chamber
is the Helber cell, but a hemocytometer is
still useful. Suspensions of pathogenic or
motile bacteria should be inactivated be-
fore they are counted, by heating for 1 h at
56 to 60
C.
Prepare a dilution that will give an
average count of about 20 bacteria per
small square, if necessary, examining a
drop of undiluted suspension to decide
the
dilution.
The
following
method
is
suitable
unless
extreme
accuracy
is
needed.
Measure
out
0.5 mL
of
suspension into a small tube and add an
equal volume of diluent (formol saline;
0.5% formalin in 0.9% sodium chloride
solution is a suitable diluent) to make
a twofold dilution. Mix well by drawing
up and down several times in the pipette