Introduction
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).
Objectives
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
cellular structures.
Requirements (Equipment /Machinery/Instrument and Chemicals/
Material)
• Bunsen Burner
• Microscope
• Test tube shaker
• Inoculating needle
• Cover slips
• Glass slides
• Sterilized test tubes
• Wash bottles
• Microbial cultures
• Distilled water
• Stains
• Immersion oil
• Tissue paper
Preparation and fixation of bacteria for staining
Prior to staining, you must “fix” the material to be observed that is make it
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).
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).
The fixing technique, although not difficult, requires adequate care in its
preparation. Follow these basic rules meticulously:
preparation. Follow these basic rules meticulously:
1. Preparation of glass slides: Clean slides are essential for preparation of
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
use.
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
use.
2. Preparation of smear: Avoidance of thick, dense smears is absolutely
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.
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.
• Broth cultures: One or two loopful of suspended cells should be
applied directly to the glass slide with a sterile inoculating loop and
spread evenly over a small area.
applied directly to the glass slide with a sterile inoculating loop and
spread evenly over a small area.
• Cultures from a solid media: Organisms cultured in a solid medium
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.
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.
3. Heat fixation: Unless fixed on the glass slide, the bacterial smear will
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.
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
Herein, the bacterial smear is stained with a single basic stain. The bacterial
nucleic acid and certain cell wall components carry a negative charge that
strongly attract and bind to the cationic (negatively charged) chromogen.
nucleic acid and certain cell wall components carry a negative charge that
strongly attract and bind to the cationic (negatively charged) chromogen.
The
purpose of simple staining is to elucidate the morphology and arrangement of
bacteria.
The most commonly used basic stains are methylene blue, crystal violet and
carbol fuchsin.
purpose of simple staining is to elucidate the morphology and arrangement of
bacteria.
The most commonly used basic stains are methylene blue, crystal violet and
carbol fuchsin.
Note that exposure time for staining cells to these dyes differs
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.
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.
General staining
Procedure for staining with different dyes:
1. Prepare bacterial smear of the organisms. Note: All smears must be heat
fixed prior to staining.
fixed prior to staining.
2. Flood the smear with any one of the stains, using the appropriate exposure
time.
time.
3. Wash the stained preparation with tap water to remove excess stain. During
this step, hold the slide parallel to the stream of water; in this way you can
reduce the loss of organisms from the preparation.
this step, hold the slide parallel to the stream of water; in this way you can
reduce the loss of organisms from the preparation.
4. Dry the slide using blotting paper.
5. Examine the stained preparation under the oil-immersion objective of the
microscope.
microscope.
Observe closely for significant difference in cell size, shape and arrangements.
Negative or indirect staining
1. Place a small drop of nigrosin close to one end of a clean slide.
2. Using sterile technique, place a loopful of inoculum from the mixed culture
in the drop of nigrosin and mix.
in the drop of nigrosin and mix.
3. With the edge of the second slide held at above 30° angle and placed in
front of the bacterial surface, push the mixture to from a thin smear.
front of the bacterial surface, push the mixture to from a thin smear.
4. Air dry. Do not heat fixed slide.
5. Examine the slide under oil-immersion objective of the microscope.
Differential staining
1. Prepare smear of the bacterial culture. Air-dry and fix these preparations
with heat.
with heat.
2. Flood smear with crystal violet and let stained for 30 seconds.
3. Rinse with water.
4. Cover the film with Gram’s Iodine instantly and let stained for 1 min.
5. Wash with tap water.
6. Decolorize with 95% alcohol. For a thin smear, 10-20 second is long
enough.
Caution: Do not over-decolorize. Add reagent drop by drop until crystal
violet fails to wash from smear.
enough.
Caution: Do not over-decolorize. Add reagent drop by drop until crystal
violet fails to wash from smear.
7. Rinse with water.
8. Counter stain with safranine for 20-30 seconds.
9. Rinse with water and blot dry.
10. Examine under the oil-immersion objective.
Steps in the gram’s stain
Acid fast stain
1. Prepare a smear of bacterial culture.
2. Allow to air dry and heat fix in usual manner.
3. Flood smear with carbol fuchsin and place on a warm hot plate, allowing
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.
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.
4. Wash with tap water. Heated slides must be cooled prior to washing.
5. Decolorize with acidic alcohol (95% ethyl alcohol containing 2.5% HNO3)
for 10-30 seconds, a carbol fuchsin fails to wash from smear.
12 6. Wash with water
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.
8. Wash smear with tap water and blot dry.
9. Examine under the oil immersion objective.
Structural stain
1. Prepare smear, air dry and fix with heat.
2. Flood smear with malachite green and place on a warm hot plate, allowing
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
temperature.
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
temperature.
3. Cool slide and wash with water.
4. Counter stain with safranine for 30 min.
5. Wash with water and blot dry.
6. Examine under oil immersion objective.
Observations
1. Draw a representative field for each organism
2. Describe the morphology of the organism with reference to their shape
(bacilli, cocci, spirilli) and arrangements (chains, clusters, pairs)
(bacilli, cocci, spirilli) and arrangements (chains, clusters, pairs)
Staining the microorganisms makes them contrast in colour with their
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.
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.