hey everybody dr. up in this video we're going to talk about the Gram stain which to me is by far the most important differential or diagnostic stain that we use in microbiology it was developed 1884 by a Danish bacteriologist his name was Hans Christian Graham as the name implies so the reason this is so important is because it tells us for most bacteria it tells us what we need to know about their cell walls it tells us oh grandpa will do several videos about the cell walls but gram-positive cells have really big thick peptidoglycan cell walls gram-negative cells have a really thin cell wall but outside of it they have a lipid outer membrane that has a toxin in it called lipid a and it's very very important to know if you're dealing with a gram positive or gram-negative bacteria because it helps us figure out how to kill them if we need to like for example penicillin and the cephalosporins they're gonna be useful against the gram positives but not so much against the gram negatives both of them can be super dangerous there are some gram positive organisms that are super scary right like your flesh-eating organisms and stuff but in general we are much more concerned about our future looking at the gram negative bacteria I would say that from everything I've seen about 95% of the research funding being done right now is dealing with drug-resistant gram-negative bacteria let's go ahead and look at the actual staining technique though one last thing as far as I'll do a separate video on smear preparation and and the bacteria involved in making smears and staining them but the Gram stain process does work much better with young freshly growing bacteria if you use older bacteria you are more likely to get strange results gram positive bacteria that actually stain gram-negative for example alright so the first step we'll go through all four steps here the first step is what's called your primary stain and this is going to be crystal violet so crystal violet is gonna stain all of the cells whether they're gram positive or gram negative they're going to be stained purple so after this step everything's going to be purple that's our primary stain the second step is iodine so iodine is our mordant a mordant is something that actually helps attach the stain to the organism it doesn't always have to be a chemical in this case it's iodine but it can even be steamed for example but what happens here is crystal violet losses into your grand positive gram-negative bacteria but what is right back out iodine forms a complex with crystal violet called the crystal violet iodine complex so iodine is small crystal violet is small when they fuse together inside the cell they're now large enough where they're not just going to use out of the cells so all the cells Grande positive or gram-negative are still purple at this stage but now we're ready to actually see what happens so the third step which would be the grams decolorizer this is the most important step in the Gram stain I know we need all of them yeah so maybe that's a little strange to say that but this is truly where gram-positive cells show themselves as gram positive and gram negative cells show themselves as gram negative so that's why I say the decolorizing step is the most important step so here's what happens we use grams decolorizer which is alcohol some use alcohol mixed with acetone but I find that just 95 percent ethanol works fine so gram positive cells the alcohol in the grams decolorizer shrink wraps them thus out the the cell walls gonna tighten up and actually shrink wrap around the organism and that's gonna trap the purple the crystal violet in the gram positive cells with the gram negative cells that lipid outer membrane i told you about is gonna pop the alcohol actually pops it so that so the purple stain is going to lose out so now after the decolorizer the gram positive is staying purple and we'll the whole time gram negative cells are now colorless because that purple stain has been allowed to escape then the last step is what's called a counter stain so it's actually gonna be safranin and safranin is a red stain and it's gonna stain everything red whether you're grande positive or gram negative but you can't stain purple red so the gram positive cells cells stay purple the gram negative cells that were made colorless in the last step the grams decolorizer will now stain red or pink let's go ahead here I have an image you can look at so here we see a mixture of both gram positive and gram negative cells so you do see that the gram positive cells stain purple the gram negative cells are going to stain pink some would say rim but red but usually it's pretty light like this all right so that's the Gram stain very very important staining technique in a lab it's usually gonna be the first one you do in all all situations I hope this helps have a wonderful day be blessed