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The Use of Stains in Microscopy
Stains and the Prokaryotic Envelope
Prokaryotes are difficult to see in wet
mounts because they are translucent. Wet
mounts of live cells are useful in observing
motility (swimming), the bacteria lack contrast
against the white background. While special
types of light microscopy have been invented to
create better contrast stains are more frequently
used to visualize cells in the lab. Stains or
dyes colorize the cells or the background so
they can be more clearly observed.
The prokaryotic cell wall and
membrane is composed of phospholipid
bilayers, proteins, peptidoglycan,
lipopolysaccharides (in gram positive bacteria),
and teichoic acids (in gram negative bacteria).
Lipopolysaccharides and teichoic acids in
particular carry negative charges and are
responsible for giving the bacterial envelope an
overall negative charge.
Stains or dyes used to colorize cells in
microbiology are usually ionic compounds that
disassociate into ions when mixed with water.
The ion of the compound responsible for the
color is known as the chromatophore. Stains
are divided into basic and acidic stains based
on the charge of the chromatophoric ion.
Basic Stains
Basic stains have positively charged
chromatophores that are attracted to and bind to
the negatively charged bacterial cell envelope.
Since basic stains produce colorized cells
against a white background (after rinsing), they
are also called direct stains. Common basic
stains used in microbiology labs include
methylene blue, crystal violet, safranin, and
carbofuschin.
Basic stains must be used with bacterial
smears that are fixed to a microscope slide so
that unbound stain can be rinsed away without
fear of losing the specimen. The fixing of
bacteria to the slide often involves heating or
treatment with alcohol, both of which
dehydrate and slightly distort the shape of the
bacterial cells. For this reason, using basic
stains may not be the best way to discern the
cell morphology (shape).
Acidic Stains
Acidic stains have negatively charged
chromatophores that are repelled by the
bacterial cell envelope. They are usually mixed
together with a small amount of liquid culture
and spread or "painted" across a microscope
slide. The slide is then viewed after the stain
has dried to prevent getting stain on the
microscope lenses.
Note that in preparing a negatively
stained specimen, the cells are not distorted by
heat or fixing chemicals. This makes the
negative stain a better procedure for observing
cell morphology.
Acidic stains produce clear, white
bacterial cells against a dark field. This is the
reverse of direct staining, which produces dark
or colored cells against a white field. For this
reason, acidic stains are also known as
negative stains. The most common negative
stains are india ink and nigrosin.
Staining Singly or in Combination
When a single stain is used to visualize
cell morphology, the procedure is said to
involve a simple stain. But staining
procedures have also been designed to reveal
differences in the characteristics between
bacteria. Procedures which often involve
several stains are called differential stains.
Differential staining procedures include the
gram stain, acid-fast stain, endospore stain, and
flagellar stain.