514
Bacterial Cell Culture Methods
and then transfer a measured volume
to a second tube. Wash the pipette
out with a diluent and add an equal
volume of diluent to this second tube.
Repeat making further 2-fold dilutions
to give a series of 4-fold, 8-fold, 16-fold
dilutions of the original suspension to
the required degree.
A few drops of methylene blue may be
added to the diluent to make the bacteria
more easily seen.
P
laceaNo
.1covers
l
iponthes
l
ideand
then exactly Fll the chamber (not the
ditches) with the suspension using a
Fne pipette.
±ind the grid under the low power,
cutting
down
the
light
intensity
if
necessary, and use the
×
40 objective
for counting. Oil should not be used
since it causes sagging of the coverslip
and a consequent change in volume of
the liquid.
Count the number of organisms per
small square that cover all planes of
focus, in 20 squares taken diagonally
across the grid. Repeat with a second
mount and if time allows, with a third.
At least 1000 cells should be counted.
The more organisms that are counted,
the more accurate is the estimate.
If cells lie on the lines of the grid,
only those on two sides of the square
should be counted. All the component
cells in small clumps of completely
separated cells should be counted as
single individuals.
±rom the number of organisms per
small square, the number per mL of the
suspension is calculated. The volume of
liquid lying over each square is given
on the slide. In a Thoma hemocytome-
ter, the depth of the chamber is 0.01 cm
and the area of each small square is
0.000025 cm
2
, hence, the number of
bacteria per milliliter of the suspen-
sion is the average number per small
square
×
4
×
10
6
×
the dilution factor.
In the Helber cell, the depth of the
chamber is 0.002 cm, and the area
of each small square is 0.000025 cm
2
.
Consequently, the number of cells per
milliliter
is
the
number
per
small
square
×
2
×
10
7
×
the dilution factor.
The
direct
smear
method.
Ak
n
o
w
n
volume of bacteria in suspension is sp-
read over a deFned area of a microscope
slide, stained, and counted. A variety of
differential stains can be used, for example,
methylene blue.
Transfer 0.01 mL of the suspension to a
1cm
2
area of a microscope slide.
Allow
the
slide
to
dry
at
room
temperature.
Stain in a damp chamber for 5 min with
the following solution (1.3 mL, 0.5 M
carbonate–bicarbonate buffer, pH 9.6;
6.0 mL,
0.001 M
phosphate
buffered
saline, pH 7.1; 5.7 mL, 0.85% physio-
logical saline, 5.3 mg crystalline ±luo-
rescein isothiocyanate (±ITC)).
Wash gently but thoroughly in water
and blot dry. Observe with a fluores-
cence microscope.
Viable counts.
These methods aim at
estimating the number of bacteria ca-
pable of growth and multiplication in
the material. Aseptic methods must be
used throughout.
Making dilutions.
In all these methods,
the preparation of dilutions is the Frst step.
A weighed amount or measured volume of
the material being examined is suspended
in a known volume of sterile diluent, mixed
well by shaking and further dilutions are
made from this.
The diluent used is most important.
Sometimes the natural milieu that is,
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