Endospore Staining procedure: Primary stain, malachite green, being applied to slide over a water bath.

Endospore Stain Procedure Lab Notes - P2
Page last updated: 2/2016

Challenges Interpreting the Endospore Stain
If the malachite green stain is allowed to dry out when the slide is on the water bath, a green crust of dye will obscure your specimen. If this has happened, gently tap the crusted dye with your gloved finger while rinsing. This helps dislodge the dried dye.

It should be noted that any debris on the slide can also take up and hold the green stain. Everything that ends up green on the slide is not necessarily an endospore. 

Bacterial smear being stained with malachite green over a steaming water bath. 
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How to Prepare a Bacterial Smear for Endospore Staining
How to Do an Endospore Stain
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Prokaryotic Cell, Mariana Ruiz
Acid-fast Bacteria and the Endospore Stain
Acid-fast cells, such as members of Mycobacterium  and Nocardia have waxy molecules in their cell wall that will take up and retain the malachite green stain when subjected to the endospore staining process. 

If the unknown that you have stained has a uniformly green appearance after endospore staining, it may be an acid-fast bacteria. This doesn’t mean that they produce endospores. These are vegetative cells that have taken up color from the heat driving malachite green into their waxy cell wall. With this specimen, a student should also do an acid-fast stain to confirm identification.

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Endospores are small, typically oval and you should see numerous uniform examples on your slide. Large or irregular globs of green on the slide may be artifacts.