 Dr. O. In this video I want to review the differential stains. So sometimes you'll hear me call these diagnostic stains because what I like about these is you know there are simple stains that just color something, stain something to make it easier to see. Differential stains, they answer questions, they tell you something as well so that's why I like to use the word diagnostic stains but differential stains is just fine. So I'm going to jump right in here and cover all of these so let's go ahead and dive in. The first one is the Gram Stain. I did a separate video on the Gram Stain because it is so important but the Gram Stain is generally the first stain you're going to use on any organism, any unknown organism because it'll help you determine what kind of cell wall it has. So gram positive cells are going to have big thick peptidoglycan cell walls, gram negative cells are going to have a thin peptidoglycan cell wall with a lipid outer membrane that can make them a lot harder to deal with in a lot of cases. So I don't want to spend any more time on that because just watch the separate video on the Gram Stain. So here we see just a close up of the purple organisms would be gram positive, the red or pinkish organisms would be gram negative here. The acid fast stain is very important so now the key thing I want you to know is which types of organisms we're looking for when we do a stain like this. Now with the Gram Stain I do expect you to know the actual stains you use and the order you use them in but that's the only one. So acid fast organisms are going to be two types and that is the mycobacterium and the nocardia. So here you see an example of mycobacterium tuberculosis obviously the causative agent of tuberculosis. The other big mycobacterium is mycobacterium lepre which causes Hansen's disease or leprosy. So acid fast stain is used to determine if you're looking at a mycobacterium, a nocardia or anything else. You see how there Gram Stain tells you something about almost every organism. The acid fast stain is not going to do that. So if you want to know the real quick basics of the of the acid fast stain you use here it says to use basic fusion that's fine but I prefer Kinyun's Carbofusion. I feel like it's a more effective and then you use acid alcohol as your decolorizing agent and then you add the counter stain Methylene Blue. So here we see a close up of that organism. So the red organisms would be acid fast positive and the blue organisms would be acid fast negative. So what it's looking for specifically is what organisms have a waxy material in their cell wall called mycolic acid and that would be your mycobacterium and your nocardia. So that's the acid fast stain. Next we have the spore stain or the endo spore stain. So when would you use this? Well, you'd use this anytime you're concerned that you're dealing with a spore forming organism and that would be the clostridium and the bacillus. So bacillus you think like bacillus anthracis, the causative agent of anthrax. Bacillus serious, bacillus subtillus. These can sometimes cause foodborne illness. But the clostridium are to me the really big ones. You have clostridium difficile or C. diff. Anyone that works in healthcare knows that's a big concern. Clostridium tetanus, the causative agent of tetanus. Clostridium botulinum, the causative agent of botulism. So spores are very, very scary, much harder to kill than vegetative cells. But the spore stain will highlight them. So there are multiple staining techniques to look for spores. But primarily what you're going to use when you're looking for spores is you're going to use malachite green as the stain that should stain the actual spores green. And then whatever technique you use, you're going to counter stain with saffronin, the same saffronin used in the gram stain. So here we see a close up. So you see the green spores inside the regular bacterial cells. Then you see some vegetative bacterial cells that don't have spores. So that's the spore stain. Just a reminder, it's useful when you're looking for a clostridium or a bacillus as far as the two genus here. All right, next we have the flagella stain. So like the name implies, the flagella stain is going to try to identify organisms or help you see flagella if they're there. So this would be an example of a mordant. I don't care if you know what the mordants are, but mordants are generally used to attach stain to an organism. And the flagella stain is where we need a mordant to actually thicken. So it makes the flagella more visible by layering the mordant, the chemical compounds on them. So the flagella stain is useful if you are trying to identify organisms that have flagella, which were used for movement. We cover that in a separate video as far as bacterial anatomy. Here you see a more close up of a really awesome one. Obviously, when you think of flagella with human cells, you think of that kind of one whip-like flagella on a human male sperm. But the flagella is a very powerful, very complex system in bacteria. Pretty awesome, so we'll cover that in other places. And then lastly, we have the capsule stain. So here's the key with the capsule stain. This is actually not a great image, but capsules can't be stained. So a capsule stain is an example of a negative stain. So very important to understand that term. A negative stain is where you stain the background. You stain everything else but what you are trying to identify, which is the capsule. So we can go a little bit closer here. Actually, we have two images here now. The one on the right is better. So notice the white, the clear areas around those microorganisms. Those are the capsule. So you stain the organism inside the capsule. You stain the environment outside the capsule, but you couldn't stain the capsule itself. That's why it's called a negative stain. You stain the background. And what you see is what isn't stained. So all right, so that is the capsule stain.