The gram stain is a commonly used technique in the microbiology laboratory to help differentiate bacteria into gram positive and gram negative organisms. The principle of this test is the differences in the bacterial cell wall lead to differences in their ability to hold dyes that we apply to them. In the case of gram positives, which you can see on the left hand side of the diagram, there's a very thick layer of peptidic glycan. In gram negative bacteria, that peptidic glycan layer is quite thin. When a primary stain, crystal violet and gram iodine is applied to gram positive organisms, the crystal complexes of dye get bound up in the interlocking layers of peptidic glycan and are not removed in the decolorization or acetone step of the staining procedure. With gram negative bacteria, the thinner peptidic glycan layer allows that stain complex to be removed and leaves the organisms susceptible for counterstaining with saffronine or basic fusion of our secondary stain. There's four basic reagents required for our gram stain, crystal violet and gram iodine for our primary stain, acetone for decolorization and basic fusion of saffronine for our secondary stain. The first step of the procedure is to prepare our glass slide. Remember to place your slide frosted side up. We're next going to draw a circle on the slide using a grease pencil, which will help us to contain the materials that we're staining and also to orient yourself when visualizing under a microscope. Use a calibrated loop to collect a small amount of water, which we're going to deposit into that circle by gently tapping that loop. Next you're going to use a sterile loop to select a colony in which you'd like to visualize. Remember just touch the tip of the colony. There's billions of bacteria there and you only need a little bit. Spread those around, get a nice even layer and wait for your slide to dry. The next step is to heat fix our slide, passing it through the flame several times. We're fixing it, we're not cooking the bacteria, so you want it to be warm to the touch, but not overly hot. Now you're ready for staining. We're going to start with our crystal violet, flood the slide with the stain, and we're going to let it incubate at room temperature for one minute. Crystal violet is a stain that will stain absolutely everything, so it's a really good idea to wear gloves and to be really careful with your clothes not to get any of this stain on you. After a minute, we rinse off the stain in the sink, and we apply our Graham's iodine, which helps to form some crystal complexes of dye within the cell wall. Just like the crystal violet, we leave our Graham's iodine on for one minute, incubating at room temperature, and rinse in the sink as we did before. Now the next step is decolorization, and this is a really critical time in the Graham stain. What you want to do is flood the slide with acetone and rinse off quickly. You want to do this until the water runs clear, but you have to be careful not to over-decolourize your cell, or Graham positive organisms can look Graham negative. Our final step is to apply our counter stain, in this case basic fusion. This is what's going to make Graham negative bacteria look pink. We flood the slide with the stain, let it incubate for 30 seconds, and then rinse it off as we've done in the past. Once this is done, once the slide is rinsed, you can blot it dry, and you're ready to visualize your specimen under the microscope.