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Cells of Bacillus, Desulfotomaculum and Clostridium (and several other, lesser-known genera--see Bergey's Manual) may, as a response to nutrient limitations, develop endospores that possess remarkable resistance to heat, dryness, irradiation and many chemical agents. Each cell can produce only one endospore. It is therefore not a reproductive spore as seen for some organisms such as Streptomyces and most molds. The endospore is essentially a specialized cell, containing a full complement of DNA and many proteins, but little water. This dehydration contributes to the spores resistance and makes it metabolically inert. The endospore develops in a characteristic position (for its species) in the vegetative cell. Eventually the cell lyses, releasing a free endospore.
Endospore stains require heat to drive the stain into the cells. For a endospore stain to be successful, the temperature of the stain must be near boiling and the stain cannot dry out. Most failed endospore stains occur because the stain was allowed to completely evaporate during the procedure.
Place the heat-fixed slide over a steaming water bath and place a piece of blotting paper over the area of the smear. The blotting paper should completely cover the smear, but should not stick out past the edges of the slide. If it sticks out over the edges stain will flow over the edge of the slide by capillary action and make a mess.
Saturate the blotting paper with the 5-6% solution of malachite green. Allow the steam to heat the slide for five minutes, and replenish the stain if it appears to be drying out.
Cool the slide to room temperature. Rinse thoroughly and carefully with tap water.
Apply safranin for one minute. Rinse thoroughly but briefly with tap water, blot dry and examine. Mature endospores stain green whether free or in the vegetative cell. Vegetative cells stain pink to red.
The above video demonstrates the endospore stain
Figure 3-12 shows a photomicrograph of an endospore stain.
Figure 3.12. The Endospore Stain. A photomicrograph of an enodspore stain. Spores present in the picture stain green, while the vegetative cells stain red. A) Staphylococcus epdiermidis which does not form endospores. B) The endospore-forming rod, Bacillus cereus.