510
Bacterial Cell Culture Methods
3
Microscopic Examination of Bacteria
3.1
Introduction
Since most bacteria are not more than 2 to
4
µ
minlengthand0.3to1.0
µ
minwidth,
any examination of bacteria involves the
use of high magniFcations. To attain these
magniFcations use is made of oil immer-
sion lenses with focal lengths of 2 mm
or less. Care must be taken when using
them as the working distance between the
lens and object is very small. Always locate
bacteria on a slide with the
×
10 and
×
40
lenses before preparing to examine them
under oil.
3.2
The Examination of Living Unstained
Bacteria
The most useful and frequently adopted
method of examining live bacteria is the
‘‘hanging drop’’ preparation.
A small piece of plasticine should be
rolled out until its length equals the
circumference of the cover slip.
The plasticine is placed on the slide in
the form of a ring or a square.
With a wire loop, a small drop of the
bacterial suspension is placed on a cover
slip laid on the bench.
The slide is then inverted, the plasticine
pressed gently on the cover slip and the
slide quickly turned over again, leaving
the suspension as a ‘‘hanging drop.’’
The slide should then be examined un-
der the
×
10 objective, and with the edge
of the drop focused. Swing the
×
40
objective lens into position and adjust
the microscope. The method is used to
detect motility, but this should be distin-
guished from the Brownian movement
that is exhibited by any small particles
suspended in a liquid. Examination of
these preparations under oil is difFcult
since currents of liquid are set up dur-
ing focusing.
Phase contrast microscopy is the easi-
est method for examining hanging drops
or even ordinary cover slip preparations.
Bacteria appear as dark objectives against
a pale background. The method, utiliz-
ing special objectives and condensers,
depends upon the change of phase of the
white light as it passes through objects
with different refractive indices.
3.3
The Measurement of Bacteria
The measurement of bacteria is achieved
by using a stage micrometer, together
with
a
micrometer
eyepiece.
On
the
stage micrometer, a scale graduated in
hundredths of a millimeter is engraved. In
the micrometer, the eyepiece is a second
graduated scale, which may be focused
with a movable eyepiece lens.
The micrometer eyepiece is inserted
into
the
microscope
and
the
stage
micrometer is focused.
The number of divisions of the eyepiece
scale corresponding to a deFnite num-
ber of stage micrometer scale divisions
is determined.
The stage micrometer is removed, the
object to be measured is focused, and
the number of divisions of the eyepiece
scale, which cover the object, is noted.
3.4
Methods of Staining Bacteria
The preparation of Flms for staining
A drop of the
bacterial suspension
should be transferred to a clean grease-
free slide with a sterile loop. The drop
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