Bacterial Cell Culture Methods
511
should be small, it should be spread
evenly, and allowed to air-dry.
Because of their small size, bacteria dry
without great distortion and so the only
Fxation required is to pass the slide
quickly two or three times through the
Bunsen flame at this stage.
General staining principles
.Dyesmaybe
of the acidic or basic type, the former
owing their colored properties to an anion,
the latter to a cation. Basic dyes have
greatest afFnity for the nuclei of cells
because of the acidic nature of the nuclear
material. Acid dyes have greatest afFnity
for the cytoplasm.
The chemicals used in the preparation of
staining solutions are frequently variable
in composition. Variations of this type and
those of technique make it essential to
use proper controls. Smears of known
positive and negative organisms should
always be prepared.
Differential stains
. There are a number
of
more
complex
staining
procedures
designed to demonstrate different parts
of the bacterial cell, and different types
of bacteria.
Gram’s method.
Some bacteria when
treated
with
para-rosaniline
dyes
and
iodine retain the stain when subsequently
treated
with
decolorizing
agents
such
as
alcohol
or
acetone.
Other
bacteria
lose the stain. This property is used to
distinguish two types of bacteria, gram-
positive and gram-negative forms. Gram-
negative forms, which are those that lose
the stain on decolorization, can be made
visible by using a suitable counterstain. It
is known that the stain plus the iodine react
with the RNA fraction of the cytoplasm and
form a colored, alcohol-soluble substance.
Gram-positive forms have cell walls (
95%
peptidoglycan) that prevent the ‘‘leaching
out’’ of the stain from the cytoplasm
with alcohol while the gram-negative ones
(
5% peptidoglycan) do not. A useful
form of the gram’s stain is Hucker’s
modiFcation.
The smears are stained with ammonium
oxalate crystal violet solution for 1 min.
Ammonium oxylate crystal violet is pre-
pared using ammonium oxylate 0.8 g,
crystal violet 2.0 g, ethyl alcohol 20 mL
and distilled water 80 mL.
The slides are washed in tap water
for 2 to 3 s and then immersed for
a
further
minute
in
Lugol’s
iodine
solution. Lugol’s iodine is prepared by
adding iodine 1.0 g, potassium iodide
2.0 g, distilled water 300.0 mL.
The slides are washed again and blot-
ted dry.
This is followed by treatment with
95%
ethanol
for 30 s. The slide should be
gently rocked during this step.
After further quick washing with water
and blotting, the slides are counter-
stained with
safranin solution
for 10
to 30 s. Safranin solution is prepared
by adding safranin, (2.5% solution in
95% ethanol), 10 mL and distilled water
100 mL.
The preparation is washed, blotted, and
examined under the oil immersion lens.
Gram-positive organisms will be stained
purple and gram-negative organisms
will be stained red. Endospores remain
unstained. Organisms with known reac-
tions should be used as controls on the
same slide.
Acid-fast staining (ModiFed Ziehl–Neilsen
method).
Certain bacteria, for exam-
ple,
Mycobacterium
,
cannot
be
stained
by ordinary aniline dyes except by us-
ing strong solutions with phenol and
possibly heat. Once stained, these bacte-
ria resist decolorization by acids and are
called
acid-fast bacteria
. This stain is used
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