hi in this short video we'll talk about endospore staining now many bacteria especially that of bacillus genus forms endospore in harsh environments and endospores are resistant to heat and harsh environmental situations and they can survive in this situation for very long and this particular endospore staining method actually determines bacteria that forms endospores because of their tough protein code made up of keratin spores are highly resistant to any type of staining method so they won't take up gram staining so the primary stain that is used for in endospore staining is malachite green which is highly penetrant so at the end of the staining what you would be seeing that these bacteria that has produced endospore would be stained in green whereas the other vegetative bacteria that doesn't form endospore would appear something pinkish in this video it would be clear what is the basis of staining and why these kind of coloration is generated so the step one is as usual smearing on the slide you would take some amount of the culture and put it on the slide and smear it then in order to fix them on the slide you can heat it quickly and briefly and all of these bacteria would be now immobilized on this slide it would be easier in staining procedure then we have to add the primary stain in this case the primary stain is malachite green alternatively in some cases people also use carbol fusion which is used for acid fast staining anyway the point of using carbol fusion or malachite green is both of them are highly highly penetrant an endospore wall is almost impermeable to anything so in order to penetrate we need to use a stain that is highly penetrant such as carbol fusion or malachite green anyway carbol fusing would easily penetrate in the cell walls or in this endospore wall and once we add malachite green we wait for some time for incubation such that the stain gets in but honestly this stain would not get in very easily that's why sometimes we have to put these slides on steam steam would help these stain to get inside the endospore wall more efficiently so we have to steam it then the step is decolorization again we'll be using a decolorizing agent here to wash these stains now in this particular situation the bacteria that had that that didn't have endospore they would not retain the stain the decolorizing agent would actually wash away all those malachite green from those bacteria and it is actually soluble in water so it would be easily wiped out but the endospore staining would be retained because once the stain has entered that endospore wall it's very hard to wipe that stain out and take it out so while counter staining with safranin only the vegetative portion of the cells would be colored as pink or like reddish color that is why we are going to see this characteristic appearance in our microscope we are going to see the vegetative cells in red and generally we are going to see this spherical staining of endospore in that so all the notes are provided in the description and you can find more notes and flash cards in my facebook page don't forget to like share and subscribe you can support me on patreon my courses are also present in an academy you can use my code ip10 to get a 10 percent discount thank you