Hi, I'm Cathy with Level Up RN. In this video, I will be talking about differential staining techniques that are used in microbiology, including the Gram stain, acid-fast stain, capsule stain, and then endospore stain. In this video, I will go through the general steps of each of these staining techniques, how to interpret the results, and the rationale or explanation on why you get those results. To be clear, I will not show you how to physically perform these stains. You will learn that in your lab. But I'm really going to be focused on the key points that you are likely to get tested on in your microbiology class. At the end of the video, I'll be giving you guys a little quiz to test your understanding of some of the key facts I'll be covering. So definitely stay tuned for that. And all of the information that I'll be talking about can be found in our Level Up RN microbiology flashcards. So if you have our flashcards, go ahead and pull them out so you can follow along with me. Let's start off by talking about the Gram stain, which is arguably the most important staining technique that you will need to know. So in terms of the procedure, after heat-fixing your slide, you're going to apply the primary stain, which is crystal violet. This is going to give all the cells a purple color. Next, you are going to apply iodine, which is a mordant, to set the stain. During this step, complexes will form between the crystal violet and the iodine. Next, you apply a decolorizing agent, such as ethanol or acetone. In gram-positive cells that have a thick peptidoglycan layer, this decolorizing agent will dehydrate and collapse the layer, which traps those crystal violet and iodine complexes inside the cell. However, in gram-negative cells that have a thin layer of peptidoglycan, the decolorizing agent will disrupt the outer membrane of the cell. And that thin peptidoglycan layer cannot retain those crystal violet and iodine complexes. So the purple dye is going to be washed out of the gram-negative cells. The last step is to apply safranin, which is a counter stain. Safranin will stain the decolorized gram-negative cells pink, but the stain will be less noticeable in the gram-positive cells that retain the crystal violet stain. So in the end, gram-positive cells will appear purple and gram-negative cells will appear pink. One important thing to note is that you always want to use fresh bacterial cultures for Gram staining. If you use old bacteria, then chances are those bacteria will have cell-wall damage. So gram-positive cells that should be able to retain those crystal violet and iodine complexes will be unable to do so because their cell walls are old and janky. So the complexes will be washed out of the cell when the decolorizing agent is applied. And then those gram-positive cells will take up the counter stain, making them appear gram-negative. Next, we have an acid-fast stain, which is a differential staining technique used to differentiate cells that have mycolic acid in their cell walls. There are several different types of acid-fast staining techniques. One common method is the Ziehl-Neelsen method. With this method, the first step is to apply the primary stain, which is carbol fuchsin, to the heat-fixed slide. And then we want to steam the slide for five minutes. Carbol fuchsin is lipid-soluble and contains phenol, which helps the dye to penetrate cell walls that contain mycolic acid. In addition, the steaming process also helps the dye to penetrate this waxy layer. Next, we apply acid alcohol, which is a decolorizing agent. This will remove the red stain from non-acid-fast bacteria, but it does not penetrate cell walls that contain mycolic acid. So the carbol fuchsin will not be removed from the acid-fast cells. And then finally, we apply methylene blue, which is the counter stain. So the decolorized non-acid-fast cells will take up the counter stain, but the acid-fast cells will not. So in the end, acid-fast bacterial cells will appear pink or red in color, and non-acid-fast bacterial cells will appear blue. Now let's talk about a capsule stain, which is a differential staining technique used to identify bacterial cells that have a capsule. One common method for capsule staining is Anthony's capsule stain. With this method, we are going to prepare our slide and allow it to air dry because heat fixing can destroy the capsules in the specimen. Next, we apply the primary stain, which is crystal violet, and we allow it to remain on the slide for two minutes. Crystal violet will stain both the cell and the capsule. But because the capsule is non-ionic, unlike the rest of the cell, the stain will adhere to the capsule, but it won't bind to the capsule. Next, we gently rinse the slide with 20% copper sulfate, which acts as both a decolorizing agent and a counter stain. So copper sulfate will remove the crystal violet from the capsule, and the capsule will absorb the copper sulfate. After that, you want to let the slide air dry. You don't want to blot the slide. And then you could observe the slide using a microscope with an oil immersion lens. With Anthony's capsule stain, the cells and background will appear violet, and the capsules around the cells will appear as white or light-blue halos. So the image on the screen is using a different capsule-staining technique with different dyes, but you can still see the white halos around the cells, which indicate that those cells have capsules. Lastly, let's talk about an endospore stain, which is a differential staining technique that allows for visualization of endospores in bacterial cells. There are several different types of endospore-staining techniques. One common method is the Schaeffer-Fulton method. With this method, you want to prepare and heat-fix your slide. You would then apply the primary stain, which is malachite green, and steam the slide for five minutes. Steaming the slide allows the malachite green to penetrate the spore wall and bind to the peptidoglycan. After allowing the slide to cool, the slide is rinsed with water, which acts as a decolorizing agent in this procedure. So after cooling, the spore walls will become less permeable. So they are not going to be decolorized by water. However, the vegetative cells in the specimen will be decolorized. Next, we're going to apply our counter stain, which is safranin. So the vegetative cells that were decolorized with water will take up this counter stain, but the endospores will not. So when we observe the slide using a microscope with an oil immersion lens, the vegetative cells will appear pink, but the endospores will appear green. All right. It's quiz time, and I have four questions for you. Question number one: after performing a Gram stain, what color will the gram-negative cells appear? The answer is pink. Question number two: the acid-fast staining technique is used to differentiate cells that contain blank in their cell wall. The answer is mycolic acid. Question number three: why don't you want to heat-fix the slide before performing a capsule stain? The answer is: heat can destroy the capsule. Question number four: after performing a capsule stain, how will cells that have capsules appear when viewing the slide with a microscope? The answer is: capsules will appear as white or light-blue halos around the cells. All right. That's it for this video. I hope it was helpful. Take care, and good luck with studying. [BLOOPERS] Capsule stain and endospores spain-- stain. And bind to the peptosco-- fix the slide before performing-- [inaudible]. 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