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THE STAIN

Procedure:
1) Smear the organism and heat fix to a slide
2) Place the slide over a steam bath and cover with Malachite Green
3) Keep the stain over the bath for 3 - 5 minutes, recovering the slide with Malachite Green if some evaporates
4) Dump the Malachite Green off and allow to cool
5) Rinse the slide with water to remove excess stain
6) Cover the smear with Safranin for two minutes
7) Rinse the slide with water to remove excess stain
8) Blot dry the stain and view under a microscope


"Smear Diagram" (7)

Step Finished Color of Vegetative Cell Color of Endospore
Smear Colorless Colorless
Malachite Green Green Green
Cool/Wash Colorless Green
Safranin Safranin Green

Why This Stain:
The Endospore stain is a special stain that is used to see specialized cell structures. The Endospore stain is used to determine if certain bacterium cells contain highly resistant spores within their vegetative cells.

How it Works:
It is difficult to get a dye into an endospore because of its low penetrability and high degree of resistance due to multiple coats surrounding the spore. Therefore, during the primary stain, the smeared culture is heated over a steam bath to soften the sturdy and durable outer layer of the cell and allow the Malachite Green to bind within the spore.
The most common staining procedure used is the Schaeffer-Fulton. This procedure uses Malachite Green as the primary stain for the spores. This stain can not be decolorized from the spores once they are removed from the heat; however, water can remove the Malachite Green from the vegetative cells, making them again colorless. The vegetative cell is counterstained with Safranin. This causes the surrounding sporangium to appear red/pink.
When the smear is removed from the heat and allowed to dry, the spores harden/reseal their outer layer and trap the primary stain, Malachite Green, inside. At this point, the vegetative cells are also green. When the smear is washed, the primary stain is removed from the vegetative cells. Safranin, the counter stain, is then added and only taken up by the vegetative cells. This is because the counter stain is applied at room temperature and the spores are tightly closed and full of Malachite Green. Once completely dried, the vegetative cells are red/pink and the spores are green.


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