Visualization of microorganisms in the living state is most difficult, not only
because they are minute but also because they are transparent and practically
colourless when suspended in an aqueous medium.
To study their properties
and to differentiate microorganism into specific groups for diagnostic
purposes, biological stains and staining procedures in conjunction with light
microscopy have become major tools in microbiology. Chemically a stain may
be defined as an organic compound containing a benzene ring, a chromophore
(chemical group that imparts colour to benzene), and an auxochrome (chemical
compound that helps in binding to cells).
After studying and performing this experiment, you should be able to:
• learn the practical and theoretical basis of chemical staining;
• describe manipulative technique of smear preparation;
• explain procedures for simple staining and negative staining; and
• perform differential staining procedures such as the Gram’s staining, acid
fast staining and spore staining.
Staining by various dyes provides contrast between microorganisms and their
background, permitting differentiation among various morphological types and
internal structure such as cell wall, vacuoles or nuclear bodies.
It also enables
the microbiologist to use higher magnifications.
Numerous staining techniques are available for visualization, differentiation
and separation of bacteria in terms of morphological characteristics and
Requirements (Equipment /Machinery/Instrument and Chemicals/
Preparation and fixation of bacteria for staining
stick to the glass slide upon which is to be stained. If a preparation is not fixed,
the film of cells will wash off during the staining procedure. Purpose of
fixation is also to kill the microorganism and coagulate the protoplasm of the
cell so as to fix it on glass surface (Figure 2.2).
preparation. Follow these basic rules meticulously:
microbial smears. Grease or oil from fingers on slides must be removed by
washing the slides with soap and water, followed by a water rinse. After
cleaning dry the slides and place them on laboratory towels until ready for
essential. A good smear is one that, when dried, appears as a thin whitish
layer or film. Those made from broth cultures or cultures from a solid
medium require variations in technique.
applied directly to the glass slide with a sterile inoculating loop and
spread evenly over a small area.
produce thick, dense surface growth and are not amenable to direct
transfer to the glass slide. These cultures must be diluted by placing a loopful of water on the slide in which the cells will then be emulsified.
Suspension is accomplished by spreading the cells in a circular motion
in the drop of water with the needle tip. At this point, the smear must be
allowed to dry completely. Do not blow or wave it in the air.
wash away during the staining procedure. This is avoided by heat fixation,
during which the bacterial proteins are coagulated and fixed to the glass
surface. Heat fixation is performed by the rapid passage of the air-dried
smear two or three times over the flame of the Bunsen burner.
Staining with basic dyes
nucleic acid and certain cell wall components carry a negative charge that
strongly attract and bind to the cationic (negatively charged) chromogen.
purpose of simple staining is to elucidate the morphology and arrangement of
The most commonly used basic stains are methylene blue, crystal violet and
for each of these stains; carbol fuchsin requires 15-30 seconds, crystal violet
20-60 seconds and methylene blue 1-2 minutes for fresh cultures. For old
cultures more time is required for staining.
fixed prior to staining.
this step, hold the slide parallel to the stream of water; in this way you can
reduce the loss of organisms from the preparation.
Negative or indirect staining
in the drop of nigrosin and mix.
front of the bacterial surface, push the mixture to from a thin smear.
Caution: Do not over-decolorize. Add reagent drop by drop until crystal
violet fails to wash from smear.
the preparation to steam for 5 minutes. Caution: Do not allow stain to
evaporate, replenish stain as needed. Also prevent stain from boiling by
adjusting the hot plate to a proper temperature.
for 10-30 seconds, a carbol fuchsin fails to wash from smear.
12 6. Wash with water
7. Counter stain with methylene blue for 2 min.
the preparation to steam for 2-3 minute.
Caution: Do not allow stain to evaporate; replenish stain as needed.
Prevent the stain from boiling by adjusting the hot plate at a proper
(bacilli, cocci, spirilli) and arrangements (chains, clusters, pairs)
surroundings so that they are more readily visible. Certain stains can also be
used to identify certain structures of the cell which would otherwise be unseen.