It is heated.
The crystal violet method and the Schaeffer-Fulton method
Bacterial endospores stain green in the Schaeffer-Fulton spore stain due to the malachite green dye used. This technique is commonly used to visualize endospores in bacteria.
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.
The negative spore stain color is pink or red, indicating that the spores are colorless or only weakly stained compared to the rest of the cell. This is in contrast to the positive spore stain, where the spores appear green due to the malachite green staining.
You can try using soap and warm water to wash your hands thoroughly, or rubbing alcohol to help break down the stain. You can also use a mixture of baking soda and water as a gentle abrasive to help lift the stain off your skin.
Malachite green
Applying heat during endospore staining helps in the penetration of the primary stain, usually malachite green, into the endospore wall. Heat acts as a mordant that allows the stain to bind more effectively to the endospore, enhancing its visibility under the microscope. This technique improves the contrast between the endospore and the rest of the cell, aiding in their identification and study.
The endospore stain uses malachite green, but this dye is rinsed off the cell during the staining procedure. The endospore itself retains the green color due to its resistance to decolorization, making it appear green against a contrasting counterstain like safranin.
the purpose of boiling of smear in malachite green is to forces a stain to penetrate the endospore wall, it is necessary to heat the slide and the stain to prod the wall to allow the stain to enter.
The Dorner endospore stain is a technique that involves using malachite green and safranin dyes to identify endospores in bacterial cells. Endospores are a dormant form of certain bacteria that are resistant to harsh conditions. The stain helps visualize endospores as green structures against a pink background.
Malachite green is commonly used to stain endospores in the Schaeffer-Fulton staining technique. This dye is applied to the heat-fixed smear and heated to drive the dye into the endospores. The spores appear green under the microscope while the surrounding cells are counterstained red.
Heating malachite green helps break down the compound into its active form, which is more effective in treating certain types of infections and parasites. Additionally, heating can help improve the solubility of the compound for better absorption in the target organisms.
Acid fast Mycobacterium have a waxy molecule in their cell wall that will take up and retain the malachite green stain when subjected to the endospore staining process. The uniformly green appearance of endospore stained acid fast cells doesn't mean they produce endospores. These are vegetative cells that have taken up color from the heat driving malachite green into their waxy cell wall.
Gram-staining does not stain the endospore due to the tough, resistant water-proof structure. It appears as an unstained area in a vegetative cell. Malachite green must be forced into the endospore with heat to stain it.
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The crystal violet method and the Schaeffer-Fulton method
One thing that endospore stains have in common with the acid fast stain is that heat primary stain penetration. Another thing that endospore stains have in common with acid fast stains are counterstain.