Bacterial Endospore Visualization

One droplet of the MCB was heated, fixed on a glass slide, covered with a square of paper towel, saturated with a malachite green stain solution (0.5% w/v in water), and steamed for 5 min over a glass container of boiling water to keep constant moisture. After removal of the paper, samples were washed with distilled water. Subsequently, a safranin stain solution (10 mL of 2.5% w/v in ethanol 95% and 90 mL of distilled water) was added and after 30 seconds, the samples were washed again with distilled water. Images were taken on an inverted microscope. With this staining method, bacterial endospores appear bright green and vegetative cells are stained red.