CAPSULE STAINING

INTRODUCTION

Capsule staining is done by using acidic strain and basic strain to detect the
presence of capsule. Capsules are:-

• Slimy covering which is also called as glycocalyx or extracellular

polymeric substance
• Usually composed of polysaccharide, polypeptide or both
• The ability of an organism to produce a capsule is an inherited property
of the organism
• It is often produced only under specific growth conditions
• Helps bacteria to survive in nature
• Also helps many pathogenic and normal flora bacteria to initially resist
phagocytosis by the host's phagocytic cells
• In soil and water, capsules help prevent bacteria from being engulfed
by protozoans
• Capsules also help many bacteria to adhere to surfaces and thus resist
flushing so not easily stained, this reason negative stain is used
• It also enables many bacteria to form biofilms
o A biofilm consists layers of bacterial populations adhering to
host cells and embedded in a common capsular mass.

PRINCIPLE

• Capsules protect bacteria from the phagocytic action of leukocytes and allow
pathogens to invade the body
• Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere
to their surfaces so an acidic stain is used
• A stain which stains the background against which the uncolored capsule can
be seen.
• Procedure is called the Gin's Method where india ink is used to color the
background and crystal violet to stain the bacterial cell "body"
 • this structure helps the bacterial cell to attach to surfaces and avoid being
phagocyted
• Common example would be Klebsiella pneumoniae

METHODS

Use a loop to mix a drop of water, a drop of india ink and a small
amount of Klebsiella pneumoniae together at the end of a slide

Use another slide to spread the smear like a blood smear. Allow the
smear to air dry.

Flood the smear with crystal violet, 1 minute. Wash with water, blot,
dry, observe.

RESULTS

+ = Positive (clear "halos" around cells when viewed under oil immersion
- = Negative (no clear "halos" around cells when viewed under oil immersion
ENDOSPORE STAINING

INTRODUCTION

The endospore stain is a differential stain used to visualize bacterial endospores.

Endospores are formed by a few genera of bacteria, such as Bacillus . By forming
spores, bacteria can survive in hostile conditions. Spores are resistant to heat,
dessication, chemicals, and radiation. Bacteria can form endospores in
approximately 6 to 8 hours after being exposed to adverse conditions. The normally-
growing cell that forms the endospore is called a vegetative cell. Spores are
metabolically inactive and dehydrated. They can remain viable for thousands of
years. When spores are exposed to favorable conditions, they can germinate into a
vegetative cell within 90 minutes.

Endospores can form within different areas of the vegetative cell. They can be
central, subterminal, or terminal. Central endospores are located within the middle
of the vegetative cell. Terminal endospores are located at the end of the vegetative
cell. Subterminal endospores are located between the middle and the end of the
cell.

Endospores can also be larger or smaller in diameter than the vegetative cell. Those
that are larger in diameter will produce an area of "swelling" in the vegetative cell.
These endospore characteristics are consistent within the spore-forming species and
can be used to identify the organism.
Because of their tough protein coats made of keratin, spores are highly resistant to
normal staining procedures. The primary stain in the endospore stain procedure,
malachite green, is driven into the cells with heat. Since malachite green is water-
soluble and does not adhere well to the cell, and since the vegetative cells have
been disrupted by heat, the malachite green rinses easily from the vegetative cells,
allowing them to readily take up the counterstain.

PRINCIPLE

Endospores do not stain easily but, once stained,they strongly resist decolorization.
This property is the basis of the Schaeffer-Fulton or Wirtz-Conklin method of staining
endospores. Spores have a durable outer coating that is composed of the protein
keratin. This keratin coat resists staining so in order to stain a spore the primary
stain, malchite green, must be heated to drive the stain into the spores. Vegatative
cells are then decolorized with water and 0.5% safranin is used to counterstain.
Thus endospores are stained green, while vegetative cells are stained red.

METHOD

With a wax pencil, the names of the respective bacteria placed on the edge of four clean
glass slide

Aseptically one species of bacterium is transferred with an inoculating loop to each of

the respective slides, air dry (or use a slide warmer), and heat-fix.

the slide to be stained is placed on a hot plate or boiling water bath equipped with a
staining loop or rack. the smear is covered with paper toweling that has been cut the
same size as the microscope slide

the paper is soaked with the malachite green staining solution. Gently heat on the hot
plate (just until the stain steams) for 5 to 6 minutes after the malachite green solution
begins to steam. Replace the malachite green solution as it evaporates so that the
paper remains saturated during heating.
 Remove the paper using forceps, allow the slide to cool, and rinse the slide with water
for 30 seconds.

Counter stain with safranin 60 -90seconds

Rinse the slide with water for 30 seconds.

Blot dry with bibulous paper and examine under oil immersion. A coverslip is not
necessary. The spores, both endospores and free spores, stain green; vegetative cells
stain red.

RESULTS

The stain Shows a bacterium capable of endospore production.

A. Vegetative Cell
B. Endospore