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The Endospore stain is considered a structural stain. Some bacteria, notably the genera Bacillus, Clostridium, and Sporosarcina produce an internal spore during periods of environmental stress. In the presence of plenty of water and nutrients these bacteria reproduce vegetative cells via binary fission and may not produce spores. As they age the bacteria may readily produce spores as a survival mechanism, especially in a closed system like a petri dish or test tube where nutrients are depleted over time. Endospore production is not a part of reproduction. The spore is a structure for the preservation of the DNA while conditions are poor; once conditions improve the spores will germinate back into vegetative cells, which will then divide normally. The endospore is released as the vegetative cell breaks down, and can survive long periods of time-- some claim millions of years! The spore has a very thick cortex and spore coat that enables it to be resistant to heat, chemicals, UV radiation, desiccation, etc. This can include disinfectants, which makes pathogens that produce endospores to be very problematic. You may have heard of the disease “C-diff” which causes repeated episodes of diarrhea and is transmitted in feces. C-diff is caused by the bacterium Clostridium difficile, an endospore former and thus not readily killed, and easily transmitted in healthcare facilities.
The Endospore stain was developed because of the difficulty, due to the structural resistance, in staining spores with traditional techniques. An endospore can be seen in simple and Gram stains, as a clear or empty space in a vegetative cell. In addition, endospores can be seen in wet mounts of live cells using phase contrast microscopy where they appear as bright refractile bodies inside vegetative cells. However, the endospore stain is important for confirmation of the presence of spores.
The procedure requires heating of the primary stain, Malachite Green, while it covers the smear. As the stain heats up it will dry out and the stain can precipitate onto the smear. To prevent this, a small piece of filter paper is placed over the smear and is kept moist with additional stain during the heating process (5-10 minutes). The heating process makes the spore coat more permeable to the stain. Once stained the smear is rinsed with DI water. This removes the stain from any vegetative cells, but is not enough of a decolorizer to remove the stain from the spore (as the spore cools it becomes less permeable and the Malachite Green remains “locked” in the spore). The vegetative cells can be counterstained with Safranin (pink). The Safranin cannot enter the spore. Therefore the spores appear green, while vegetative cells appear pink.
Contributors and Attributions
Kelly C. Burke (College of the Canyons)