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Endospore Stain

Background and Introduction

spore lifecycle diagram

The most important endospore-forming bacteria are members of the genera Bacillus and Clostridium, both of which are Gram-positive rods. An endospore is a dense, multilayered structure that contains the genetic material of the bacterial cell. Endospores are formed within a vegetative bacterial cell when the environmental conditions no longer support cell growth. As the vegetative cell dies, the endospore is released into the environment where it can survive indefinitely in the presence of many environmental stresses, such as desiccation, extremes in temperature, radiation, and lack of nutrients. When more favorable conditions arise the endospore germinates, again forming a viable vegetative cell. 

The presence of endospores in a bacterial culture can be detected by staining with malachite green. Because the endospore coat is so tough, steam is used to enable dye penetration. After washing, only the endospores will retain the primary stain Malachite green. Safranin is then used as a counterstain for vegetative cells. The endospore stain is a differential stain because it differentiates spore-formers from non spore-formers. 

Note: Formation of an endospore. The spore stains green and the vegetative cells stain red to orange. 


Students will work individually.

  1. Prepare a smear of Bacillus subtilis using a darkly colored colony grown on nutrient sporulation media (NSM).

  2. Air dry the smear and heat fix it.

  3. Cover the smear with the primary stain malachite green. Place a paper towel on top of the dye. Steam the slide for five minutes on the steam bath in the fume hood.

  4. After the slide is cool, rinse both the top and the bottom well with water.

  5. Counterstain with safranin for 30 seconds.

  6. Rinse both the top and bottom of the slide well with water. Clean the bottom of the slide with a paper tower and blot dry.

  7. Allow the preparation to air dry. Focus on the smear using the 10X objective and then observe the sample using the oil immersion. Record your observations in the results section of this experiment.


endospore stainendospore stainendospore stain

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