‫بسم هللا الرحمن الرحيم‬
‫‪362 MICRO‬‬
‫‪6 :‬‬
‫‪LAB‬‬
Highly resistant structures.
Usually formed when environmental
conditions become unfavourable.
Produced inside bacterial cells.
Filamentous bacteria produce spores at
the tip of the aerial mycelium.
1- Central
e.g : Bacillus cereus
Diarrhea
2- Terminal
e.g : Clostridium tetani
tetanus
3- Subterminal
e.g :
Clostridium botulinum
botulism
E NDOSPORE
FORMATION
E NDOSPORE
FORMATION
E NDOSPORE
FORMATION

To differentiate between organisms that can
produce endospores and those that cannot.

Also allows differentiation based on the
position of the endospore in the cell.

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 safranin is used to counterstain.Thus
endospores are stained green, while vegetative
cells are stained red.
1.Prepare smears of organisms to be tested for endospores.
2.Heat fix the smears.
3.Cover the smears with a piece of absorbent paper cut to fit the slide and place the
slide on a wire gauze on a ring stand.
4.Saturate the paper with malachite green and holding the Bunsen burner in the hand
heat the slide until steam can be seen rising from the surface.Remove the heat and
reheat the slide as needed to keep the slide steaming for about three minutes.As
the paper begins to dry add a drop or two of malachite green to keep it moist, but
don't add so much at one time that the temperature is appreciably reduced.DO
NOT OVERHEAT.The process is steaming and not baking.
5.Remove the paper with tweezers and rinse the slide thoroughly with tap water.
6.Drain the slide and counterstain45 seconds with 0.5% safranin.
7.Wash, blot, and examine.
8.The vegetative cells will appear red and the spores will appear green.
Spores will be a light green
Vegetative cell walls will pick up the
counterstain safranin.
T HANKS …
SHROOG