Heat is necessary when staining spores because the application of heat disrupts vegetative cells and causes green malachite to be rinsed from them, which then allows the counterstain into the cells. This is a required step in certain spores that are resistant to traditional methods of spore staining.
According to Austin Community College, staining is a technique used by scientists to identify the spores in a bacteria sample. One of the most important steps in spore staining is to anticipate the properties of the spore that is being analyzed, because some of them are resistant to traditional dying processes. Often, this is a result of their tough exterior layer of protein, known as keratin. For endospores of this nature, it is necessary to utilize a heat-staining technique in order to stain the spores with their primary stain, malachite green.
The application of heat weakens the wall of a cell and allows it to be rinsed and permeated by dyes. The original cell that forms an endospore is called a vegetative cell. These cells are more susceptible to heat than the endospores themselves, and can be rinsed and dyed with malachite green. Based on the swelling and reactions of the endospores, this dyeing process can then be used to identify spores that have tough, protective layers.