Endospore Staining
The name "endospore" is suggestive
of a spore or seed-like form (endomeans within).
Resting structures formed by some
bacteria for survival during adverse
environmental conditions (nutrient
limitation or extreme environments).
Spores are resistant to heat and chemicals because of a tough
outer covering made of the protein keratin. The keratin also
resists staining, so extreme measures must be taken to stain the
spore.
 Endospores can remain dormant indefinitely (not
reproductive), but germinate quickly when the
appropriate trigger is applied.
 Metabolically inactive.
 Stable for years.
 Endospores differ significantly from the vegetative ,
or normally functioning, cells.
 Formed by Gram-positive bacteria.
Endospore Location
The position of the endospore
differs among bacterial species
and is useful in identification.
When describing spore-forming
bacteria, the location of the
endospore is usually stated as
central, terminal, or subterminal
Endospore Staining
(Schaeffer-fulton Method)
1) Make smears of each organism on separate slides.
2) Allow the slides to air dry, and then heat fix.
3) Apply a few drops of malachite green to the bacteria. Place the
slides directly on a pre-warmed hot plate set on low for 2-3
minutes. Do not allow the stain to evaporate.Apply additional
stain, if necessary.
4) Remove the slides from the hot plate and allow them to cool.
5) Gently rinse the slides with water.
6) Apply enough safranin to the slides to cover the bacteria.
Allow it to set for 30 seconds.
7) Gently rinse the slides
with water.
8) Blot (don't wipe) the
slides with bibulous
paper. Allow the slides
to air dry.
9) Examine the slides under oil immersion. The bacteria
should appear pink; the spores should appear green.
Capsular Staining
Capsules are structures that lay outside of an organism's cell
wall and thus are in direct contact with the environment.
Many, perhaps most, bacteria
produce capsules under the right
conditions.
Some capsules are composed of
Carbohydrates or Glycoprotein.
Capsules Functions
1) Protect the cell from desiccation (drying).
2) Protect the cell from phagocytes (being engulfed by white
blood cells).
3) Provide a food reserve when certain organic compounds
are in excess.
4) A virulence determinant of pathogenic microbes.
5) They serve as binding or adhesion agents for sticking cells
together and/or to a surface such as a rock in flowing
stream or a tooth.
Theory Behind Capsule Stain
Bacterial capsules are non-ionic, so neither acidic nor basic
stains will adhere to their surfaces
Because most capsule materials are water soluble, simple
stains will not adhere to them.
Older cultures are more likely to exhibit capsule production.
When performing a capsule stain on your unknown, be sure
the culture you take your sample from is at least five days old.
Capsular Stain Methods
Gin’s Method
In this stain we use acidic and basic dyes:
Acidic dye as India Ink and Nigrosen use to stain the
background of the slide but basic dye as methylene blue and
crystal violet use to stain the cell .
The India ink gives a semi opaque background against which
the clear capsules can be easily visualized.
Gin’s Method Procedure
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Use an inoculating needle to suspend the organism in a
drop of India Ink at one end of the slide.
Place the short end of a clean microscope slide into the
suspension and spread the mixture across the slide to
form a thin layer.
Allow to air dry. Do not heat fix.
Cover the smear with methylene blue for 2-3 minutes.
Rinse gently with water and allow to air dry.
Examine with oil immersion.
Diagram the appearance of the organism.
Interpretation
Capsules appear as clear zones (halos) around the
refractile organism.
Examples:
Bacteria with capsules: Streptococcus pneumoniae,
Klebsiella pneumoniae, Pseudomonas .
 Anthony’s Method
 Anthony’s procedure employs two reagents. The
primary stain is crystal violet, which gives the
bacterial cell and its capsular material a dark purple
color. Unlike the cell, the capsule is nonionic and
the primary stain cannot adhere.
 Copper sulfate is the decolorizing agent. It removes excess
primary stain as well as color from the capsule.
 In this procedure, smears should not be heat-fixed since
shrinkage is likely to occur and create a clear zone around the
bacterium, which can be mistaken for a capsule.
Anthony’s Method Procedure
1) Aseptically transfer a loopful of culture with an inoculating
loop to the slide. Allow the slide to air dry. Do not heat-fix! .
2) Place the slide on a staining rack. Flood the slide with crystal
violet and let stand for 4 to 7 minutes.
3) Rinse the slide thoroughly with 20% copper sulfate .
4) Blot dry with bibulous paper.
5) Examine under oil immersion (a coverslip is not necessary)
Capsules appear as faint halos around dark cells.