Endospore staining

Endospore is domanet strucure which have no or less metabolic activities. Bactiers form endospre when environemntal conditions are  unfavorable to them such as  high oxygen concentraion, temperature, heat and desssication. Endospore cannot be killed by just boilling at 100◦C but at 121◦C. Bacillus, Clostridium and Sporalactobacillus are examples for endospore froming bacteria.

                             The outermost layer of the endopsore is proteinaceous coat. Bneeth coat, there is a a very thick layer of peptidoglycan which is called as the cortex. Under cortex, grem cell wall reside. After endospire germination, grem cell wall become the cell wall fo bacterium. Bneath gren cell wall, inner membrane is founsd. It act as adverse premeability barrier. The core exsits in the center of the endopore. The core is the dehydreted DNA of the bacterium cell.

Endospore  staining is a differentiate staining technique which is used to differentiate bacteria into two groups such as endospore forming baetria and non endospore forming bateria. Endospore is stained in green while vegetative cell is stained in pink.

Position of endospore: The position of endospore inside the vegetative cell varies from one bacterium species to another as follows. 

Procedure- A loopful from a broth or agar plate is taken into a glass slide. Then the smear is prepared by heat fixing at bunson flame. As the proteins of the cell wall are denatured by heat, it supports the bacterium cells to adhere to the slide. The smear is stained by flooding with Malachite green. It is allowed Malachite green to act for 5 minutes.  The underside of the slide is heated by using alcohol deep cotton wool.  It is allowed the slide to cool. Stained smear is decolorized by using water and air dry. Safranin is added as the counterstain. It is allowed Safranin to react for 30seconds. Excess Safranin is washed away by using water and air dry. Endospore and it vegetative cell are observed under oil immersion objectives.

Observations:

Theory:

1)    At the room temperature, the spore wall which mostly contain keratin resist to dyes. But heating make the spore wall more permeable to malachite green and allow it to bind to peptidoglycan layer.

2)    So both vegetative cell and endospore are stained in green color.

3)    After cooling, spore wall seals again and trap malachite green inside the wall.

4)    At the decolonization step, malachite green rinse only forms the vegetative cells.

5)    Counter stain, safranin I only able to stain the vegetative cell because stain is unable to penetrate the spore wall due to the less permeability.