بسم الله الرحمن الرحيم 362 micro lab 6 : Endospores

:Bacterial Spores (Endospores) 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.

The location of the endospores

The location of the endospores in bacterial cell 1- Central e.g : Bacillus cereus Diarrhea

The location of the endospores in bacterial cell 2- Terminal e.g : Clostridium tetani tetanus

The location of the endospores in bacterial cell 3- Subterminal e.g : Clostridium botulinum botulism

The location of the endospores in bacterial cell

Endospore structure

Endospore structure

Endospore formation

Endospore formation

Endospore formation

Endospore staining

Aim: 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.

Principle 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.

Endospore stain procedure

Procedure

Procedure 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.

Result Spores will be a light green Vegetative cell walls will pick up the counterstain safranin.

Thanks… shroog