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 spore-

specific 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.

- 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

- 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

METHODOLOGY

A. Endospore Staining

B. Negative Staining for Capsule

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 won’t 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 bacteria’s negative charge.

Smear of Bacillus megaterium

Endospore

Observation under microscope (HPO, OIO)

Staphylococcus saprophyticus

Capsule (stained)

Observation under microscope (HPO, OIO)