 Hi everybody, Dr. O. 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 in 1884 by a Danish bacteriologist, his name was Hans Christian Gram, as the name implies. So the reason this is so important is because it tells us foremost bacteria, it tells us what we need to know about their cell walls. It tells us, so Grampot, we'll 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 the 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 going to 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 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. All right, 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 going to 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 steam, for example. But what happens here is crystal violet oozes in to your Gram positive or Gram negative bacteria, but it would ooze right back out. So 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 ooze out of the cells. So all the cells, Gram 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, 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% ethanol works fine. So Gram positive cells, the alcohol in the Grams decolorizer shrink wraps them. The cell wall is going to tighten up and actually shrink wrap around the organism, and that's going to 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 going to pop. The alcohol actually pops it, so the purple stain is going to ooze out. So now after the decolorizer, the Gram positive is staying purple and will the whole time. Gram negative cells are now colorless because that purple stain has been allowed to escape. And the last step is what's called a counter stain. So it's actually going to be Saffronin, and Saffronin is a red stain, and it's going to stain everything red, whether you're Gram positive or Gram negative, but you can't stain purple red. So the Gram positive 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 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 going to be the first one you do in almost all situations. I hope this helps. Have a wonderful day. Be blessed.