Today we're going to be taking a look at gram staining as part of the microbiology series. To start this topic, we first have to understand that bacteria can broadly be categorized into two main groups. The first group is referred to as gram positive and the second type is referred to as gram negative. And the main difference between both groups is that they both have different cell structures and more specifically different outer cell structures that are unique to each group. If we take a closer look at these outer cell structures, we can see the differences in more detail. First of all, you can see that both gram positive and gram negative cells have an inner cell membrane which is composed of phospholipid billayer. The next layer up is referred to as the peptidoglycin cell wall. And you can see that in gram positive cells, this wall is much more thick and compact compared to the gram negative cells. And this is important when thinking about gram staining. As well as this, gram negative cells also have an outer cell membrane which basically encapsulates the cell wall while this is missing in gram positive cells. Finally, both groups also have an outer slime capsule which basically helps to provide an extra layer of covering. Based on this you can see that the main differences between gram positive and gram negative cell types is that a gram positive cell has a thick peptidoglycan wall and only one inner cell membrane while the gram negative cells have thin peptidoglycan cell walls and both inner and outer cell membranes. Let's now take a look at the process of gram staining which is a technique used to differentiate between gram positive and gram negative cells under a microscope. This process involves a few different steps and we're going to be taking a look at each of these steps individually. Starting with fixing first. This is basically a preliminary step where we take a sample of the bacteria from a petry dish and then apply the sample onto a glass slide so it can be viewed under a microscope. We also apply some heat which helps to fix the bacteria to the slide. If you were to take a look at the sample under a microscope at this point, we would see multiple undifferiated bacterial cells and the remainder of the gram staining process basically involves adding different reagents to distinguish between the different types of bacteria. The first reagent we add in this process is a substance called crystal violet. And to understand how these substances work, we're going to split the slide into gram positive and gram negative cells. When we add crystal violet, it diffuses across all of the outer layers and enters inside the cell. And this happens in both gram positive and gram negative types. As the name suggests, crystal violet has quite a heavy purple color. So if you looked under a microscope at this point, all the cells would be stained purple. The next step involves adding another substance known as iodine. And just like the crystal violet, this iodine is able to diffuse across all the outer layers and enter the cell. Once inside, these iodine molecules bind to the crystal violet to form complexes. And the entire purpose of this step is to basically fix the crystal violet inside the cells. Again, if you looked under a microscope at this point, the cells would remain purple because the crystal violet is still retained. Following this, we add our next reagent, which is ethanol or acetone. And this is perhaps the most important step in differentiation. If we take a look at the gram negative cells first, when we add the ethanol, it ends up dissolving away the outer membrane and causing the peptidoglycen cell wall to become quite shriveled and shrunk. As a result of these changes, the crystal violet iodine complexes diffuse across the cell membrane and the thin peptidoglycen cell wall essentially getting washed out and they eventually leave the cell entirely. If we compare this to gram positive cells when we add the ethanol in this case it again diffuses across and hits the cell wall causing it to become quite shriveled and shrunk. However, because the cell wall was quite thick to begin with, it's still strong enough to retain the crystal violet and it prevents the crystal violet iodine complexes from being washed out. You can see that at the end of this step, the gram positive cells have retained the violet iodine complexes while the gram negative cells have lost them. And if we looked under the microscope here, we would find a range of purple cells representing the gram positive types. and a range of colorless cells representing the gram negative. To visualize the gram negative cells more clearly, we have a final step which involves adding a counter stain known as saffronin. And this saffronin is a red colored substance which again diffuses across all of the layers and enters inside both cell types. In the gram positive cells, the color of the crystal violet complexes far outweighs the color of saffronin. and these cells remain purple. However, in the gram negative cells, the saffronin is the only dye remaining and therefore these cells appear red or pink in color. If you were to take a final look under the microscope, we would see that the gram positive cells would appear purple while the gram negative cells would appear red. Just to take a look at some more scientifically accurate pictures, we have a couple images comparing the gram positive and gram negative cells under microscopy after the gram staining process is complete. To finish off, we have a summary slide here which outlines the key steps involved in the gram staining technique. And we also have a textual summary here. I hope you found this video helpful and I'll see you in the next one.