The spore was neutralized before stain removal insued.
Heat is the mordant used in the spore stain, it fixes the primary stain.
spore is a dormant structure and necessary to stain because in this we use malachite green which will retain in spore
Green
Because stains can't penetrate the spore covering. You have to heat the spore to open it and let the stain in.
the counter stain is safranin 0.5%
A negative result for the spores stain indicate that the gram-negative organism is present. A positive result for a spore stain indicates that a gram positive organism is present.
Gram positive spore forming rods
In the spore stain malachite green is used as a primary stain. This is driven into the cell by heat because of the impermeability of the spore. The stain is allowed to sit for 30 mins to make sure it gets in to the endospores.The stain is then washed and counterstained with safranin red. The endospores retain the green colour from malachite green and of course appear green under the microscope. Whereas the vegetative cells will appear red.
Endospores are impermeable to most stains so heat is usually applied to drive the stain into the endospore.
because they cannot be differentiated from inclusions of stored material without a special stain .
Depends if heat is used