 Welcome to the video Gram Stain Procedure. In this video, we'll view and discuss video clips demonstrating Gram Staining Procedure and make distinctions between the stained slides shown. Gram Stain Procedure. Creating the smear. The first step in making a smear is to take a small loop full of water and put some water on the slide. Flame your loop to make sure it's sterile. You'll pick up your culture, cool your loop, and take one small colony from this plate, an isolated colony, and make a thin smear on the slide, spreading it out so that it doesn't clump, but that there are enough organisms that you can see. Then always sterilize your loop before you set it down. The smear that I just made has air dried, and now I will heat fix the slide by touching it briefly to the burner. And now I am ready to Gram Stain. For the Gram Stain, the first step is to take crystal violet and put it over the smear I just made, leave it on the slide for 30 seconds. The crystal violet stains all cells on the smear. If you looked at the smear under the microscope at this point, Gram Positive and Gram Negative cells would stain dark blue. Let's return to the video. When 30 seconds has passed, you take distilled water and rinse the slide off, keeping the back clean. The second step in the Gram Stain is to put on Gram's iodine. Again, put this over the smear that you made, leave Gram's iodine on the slide for one minute. Gram's iodine acts as a mordant and causes the dye to form crystals within the peptidoglycan of the cell wall. The Gram Positive layer is much thicker, so much more dye is trapped in the wall. If you looked at the smear under the microscope at this point, Gram Positive and Gram Negative cells would stain dark blue. Let's return to the video. After one minute, you rinse the slide with distilled water. Now is the decolorization step. At this step, you should get the decolorizer and the saffronin ready. You will hold the slide, decolorize until the blue stops running off or about two quick squirts of the decolorizer. The decolorizer is ethanol, and it dissolves the lipids in the outer membrane of the Gram Negative cell and removes the dye from the thin layer of peptidoglycan. The Gram Negative cells are now colorless. The Gram Positive cells retain the dye because of the thicker peptidoglycan layer. If you looked at the smear under the microscope at this point, Gram Positive cells would still be dark blue or purple, and the Gram Negative cells would be colorless. After 30 seconds has passed, you pour off the stain, rinse the slide, again keep the back of the slide clean. Then you can wipe the back of the slide and you will blot the front so that you will be able to view it under oil immersion of the microscope. The slide is now ready to look at. If you looked at the smear under the microscope at this point, Gram Positive cells would be dark blue or purple, and the Gram Negative cells would be red. Okay, now we'll look at Gram Stain results. Gram Stain results must include the Gram Reaction, Gram Positive or Gram Negative, the shape of the cells, coxies or rods, sometimes curved or spirals, and the arrangement of cells on the slide. Chains, clusters, packets, pairs, singles, or random. Gram Reaction. Gram Positive cells are dark blue. Gram Negative cells are red. Shape. Cells that are round are called coxies. Cells that are elongated are called rods or bacilli. Arrangement. Cells may be arranged in chains, clusters, or palisades, Xs, Ys, and Vs. Now, let's discuss how to report Gram Stain results. When reporting Gram Stain results include all three, Gram Reaction, Shape, and Arrangement. Let's take a look. Gram Positive coxies, chains and pairs. Gram Negative coxies, clusters and pairs. Gram Positive rods and chains. Gram Negative rods, clusters, chains, and singles. Gram Positive rods, palisades, and singles. Now we'll go through a series of questions that ask you to identify the correct term for each Gram Stain element. In this set of questions, we'll be looking for the correct term for each type of Gram Stain reaction. Feel free to pause the video if you need more time to think about your answer. Which cells stain red at the end of the staining procedure? Gram Negative. Which cells stain dark blue at the end of the procedure? Gram Positive. Which cells are colorless after the ethanol step in the procedure? Gram Negative. Which cells are dark blue after the ethanol step in the procedure? Gram Positive. And which cells are dark blue after the Gram's iodine step has been completed? Gram Negative and Gram Positive. Now in this set of questions, choose the correct term for the corresponding shape of each Gram Stain cell. Which cells have the thick peptidoglycan layer? Gram Positive. Which cells have an outer membrane? Gram Negative. And which cells have the crystal violet rinsed out of their cell wall during the Gram Stain procedure? Gram Negative. And lastly, in this set of questions, you'll choose the correct description on the left that matches its corresponding Gram Stain structure on the slides on the right. Again, you can always pause the video if you need more time to think about it. Gram Positive coxi in clusters. Gram Negative coxi in pairs. Gram Negative rods in clusters and singles. Gram Positive rods in chains. And now, let's recap. The Gram Stain procedure is the most common staining procedure done in the microbiology laboratory. Accurate Gram Stain results are necessary to determine the tests that should be done to identify the organism. The Gram Stain results are also necessary to determine the antibiotics that should be used in sensitivity testing. Congratulations, you've completed the video, Gram Stain procedure. Thanks for watching. If you want to see more videos like this, subscribe to our channel. We'll notify you when we have something new for you to learn.