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: ◦ Tap water. ◦ Spores cannot be decolorized by tap water (remain green). ◦ Vegetative cells become colorless.  Counter stain: ◦ 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.