Experiment 9

Bacterial Endospores and Capsules

BACTERIAL ENDOSPORE
Highly differentiated cell within the
vegetative cell
Definitive characteristic of some
microorganisms (e.g. Bacillus, Clostridium)
Thick spore wall
o Components of Spore Wall
(AtoQ1)
1. Exosporium thin protein
covering
2. Spore coat layers of sporespecific proteins
3. Cortex loosely cross-linked
peptidoglycan
4. Core wall
o Impermeable to stains unless heated
What is the significance of endospore
formation? (AtoQ2a)

Necessary for the SURVIVAL of the

microorganism.
o Formed under unfavorable conditions;
i.e. nutrient depletion, extreme
conditions (temperature, chemical,
and radiation)
o Put microorganism in dormancy
Contributes to the pathogenicity of the
microorganism

In what ways are they a nuisance to man?

(AtoQ2b)
Encapsulated bacteria are pathogenic and
pose several health risks in humans. They
are dangerous once they undergo
germination within the human body.
Can endospores be observed under the
microscope without using endospore staining? If
yes, is there a need to use this type of staining?
Why?
Yes. Endospores can still be observed
because of their strong refractile structures
making them distinguishable even without

stain. But still, endospore staining is

important because it serves as a
confirmatory test for the presence or
absence of the endospore. Bacterial cells
contain cell inclusions. The result is
positive, confirming the presence of
endospore, when there is a structure which
is stained green. The result is negative
when no other structure is stained besides
the vegetative cell.
BACTERIAL CAPSULE

Dense, well-defined polymer layer

Composed of polypeptide, glycoprotein and
polysaccharide

How important are capsules in establishing a

disease process? (AtoQ3)
Virulence
- Important in adherence to bacteria and
host cell
- Protective layer of bacterial cell wall
from host immune system. (Capsules
block the immune cells from identifying
the foreign body because it encloses the
cell wall which contains the proteins
recognized by the antibodies/immune
cells.)
- Biofilm formation
Protect cell from desiccation
Why is heat not used in capsule staining
procedure? (AtoQ6)
Capsules are made of polysaccharides which
contain high amounts of water (about 95%).
Heat fixation will result to capsule shrinkage or
damage.

METHODOLOGY
A. Endospore Staining
Smear of Bacillus megaterium
- Fixed
- Addition of Malachite green (thru heating
over alcohol lamp)
*DO NOT LET STAIN BOIL AND DRY UP.
- Rinse with water
- Addition of 0.5% safranin (1 min)
- Rinse again
- Air dry

Endospore
Observation under microscope (HPO, OIO)
B. Negative Staining for Capsule
Staphylococcus saprophyticus
- To be mixed in a drop of nigrosin
placed on one side of clean side
- Using another clean slide, the
nigrosin dye will be spread all
throughout the slide
- Air dry
- Counterstain with safranin for 30
seconds
- Rinse with water
Capsule (stained)
Observation under microscope (HPO, OIO)

RESULTS
Endospore Staining
- The endospore was stained green and the
vegetative cell, red.
- Endospore can be stained using other
stains, however, cell inclusions can be
mistaken as endospore that is why
endospore staining is necessary to have a
clearer and more accurate view of the
endospore.
Negative Staining for Capsule
- Used the principle of negative staining
- the capsule will be clear or unstained
- the background will be stained to distinguish
the colorless capsules of the cells
- Nigrosin dye is acidic; it wont be able to
stain the bacterial cell making the
background stained instead. Since the dye is
acidic, its negative charge will be repelled
by the bacterias negative charge.