First Step of the Endospore Stain: Malachite green, the primary stain being applied slides of bacterial smears, heating over a water bath.
This stain is designed to distinguish vegetative cells of bacteria (active, living cells) from endospores. A bacterial smear is first placed over a water bath and the stain malachite green is applied. It heats on the water bath for five minutes. Then the slide is rinsed and a pink counterstain (safranin) is applied for one minute. After the final rinse, when viewing the slide under oil immersion (@1000xTM) with a compound light microscope, vegetative cells will appear pink and endospores are stained a bluish green.
Diagram on white board of bacterial controls used for Gram, Acid-fast-and Endospore differential staining of bacterial smears. Positive control on left, negative control on right, unknown bacteria in center.