Structure Stains (Spore & Capsule Stains)
Abdelraheem BA

Some bacteria have two forms of life:
◦ Active vegetative cells.
 Under normal conditions.
◦ Inactive cells called spores.
 When environmental conditions become unfavorable.

Examples of such bacteria:
◦ Anaerobic genus Clostridium.
◦ Aerobic genus Bacillus.

Under unfavorable conditions cells undergo
sporogenesis;
◦ Giving rise to an extra intracellular structure
(Endospore).
◦ Endospore is surrounded be three layers:
 Outermost spore coat.
 Intermediate spore wall.
 Innermost spore membrane.
◦ Endospore surrounds the matrix (DNA, RNA,
ribosomes… etc.)

As conditions worsen;
◦ Endospore is released, becoming independent
spore.

Such spores are resistant to the damaging
effect of:
◦
◦
◦
◦
Excessive heat.
Freezing.
Radiations.
Chemical agents (Including stains).

When environmental conditions become
favorable;
◦ The free spore germinates to a vegetative cell by
the rupture of spores.
◦ This process is called germination.

NOTE:
◦ Germination and sporogenesis are not means of
reproduction.
◦ They are means of survival.

Shape:
◦ Spherical Vs Oval.
◦ Swollen.

Location:
◦ Terminal, sub-terminal or central.

Figure:






(a) Oval & Terminal.
(b) Spherical & Sub-terminal.
(c) Oval & Central.
(d) Spherical & Terminal (Swollen).
(e) Oval & Terminal (Swollen).
(f) Oval & Central (Swollen).

C. botulinum :
◦ Swollen oval sub-terminal endospore.

C. perfringens :
◦ Large oval central or sub-terminal endospore.

C. tetani :
◦ Swollen spherical terminal “drumstick” endospore.

Bacillus spp.
◦ Including B. anthracis, B. subtilis, B. cereus).
◦ All have non-swollen oval central endospores.

Primary stain:
◦ Malachite green.
◦ Due to their impervious coats, heat must be applied.
 Don’t allow the stain to evaporate, so add stain as needed.
◦ Now: Both, spores and vegetative cells are green.

Decolorizer:

Counter stain:
◦ Tap water.
◦ Spores cannot be decolorized by tap water (remain
green).
◦ Vegetative cells become colorless.
◦ Safranin.
◦ Spores remain green.
◦ Vegetative cells become red.

Definition:
◦ A gelatinous outer layer secreted by the cell,
surrounding and adhering to cell wall.

Function:
◦ Protection of bacteria against phagocytic activity of
host cells.

Chemical composition:
◦ Polysaccharide.
◦ Glycoprotein, or
◦ Polypeptide.

Principle:
◦ Indirect stain.
◦ Using an acidic stain; like Eosin or Nigrosin.

Procedure:
 Place a small drop of eosin close to one end of a glass
slide.
 Using a sterile technique, place a loopful of bacteria
into the drop of eosin, mix well.
 With the edge of a second slide (held at 30º), push the
mixture to form a bacterial smear.
 Air dry (Don’t heat).
 Examine under microscope.