my name is Kevin tow cough in this video we're gonna discuss the theory of the Gram stain without really going into the history of the Gram stain the basis is that it's used to differentiate two types of bacteria that you should know about gram positive bacteria and gram-negative bacteria however before we go into the theory of the Gram stain specifically we need to understand structurally the main differences between gram positive and gram negative bacteria gram positive bacteria are shown here on the right in this image the main two things about gram positive bacteria you should know is that they have a thick very thick outer layer of peptidoglycan outside of their single plasma membrane so a they have a single plasma membrane and superficial that outside of it they have a thick peptidoglycan layer in case you don't know peptidoglycan is a structural and protective layer of their cell wall that makes them more resilient to our immune system and other potential threats to them gram-negative bacteria on the other hand which are shown here on the left have two plasma membranes in fact you can see between the two plasma membranes they have a space it's a physical space called the periplasmic space the fluid inside of which is called periplasm and inside the periplasm we have a thin peptidoglycan layer okay and the basis of gram staining is whether or not the peptidoglycan layers the thin one of gram negative or the thick one of gram positive can actually retain a dye known as crystal violet okay now this is something that often confuses students don't ever think of grandpas and grandmas the number of membranes we're only really concerned about whether or not it's a thick layer of peptidoglycan as in gram positive or a thin layer as in gram negative okay now like I said the question is whether or not the peptidoglycan layers will retain the crystal violet dye so here's the way I want you to think about it and then we'll go into the actual specific theory imagine you have two pools so I have one pool over here like the pools you swim in right one over here and adjacent to it another one over here the one over here on the right let's suppose at that that pool is actually not filled with normal water it's actually filled with quicksand quicksand or molasses or something like that something very viscous and over here on the left that pool is just filled with normal water right now suppose you your body you are the crystal violet dye you are the crystal violet dye let's suppose that for one minute and you jump into the pool you jump into this pool and then an identical you jumps into this pool which pool is it going to be easier to get out of well it's gonna be easier for you the crystal violet died to get out of a pool this is water I mean I think we all know how to get out of a pool right however if you the crystal violet died jump into a pool that's nothing but quicksand or molasses or whatever it's gonna be harder for you to get out I think obviously quicksands probably the better analogy there so the basis here the sort of general basis of gram staining is that the crystal violet dye is retained by the thick peptidoglycan layer of gram positive bacteria but eventually as we'll see it's going to be lost by the thin peptidoglycan layer of gram-negative bacteria so let's talk about how this actually works this is a flow chart and we'll actually see the steps of how you actually arrange to do a Gram stain okay so the first step actually is not technically part of the Gram stain we're actually gonna do something called heat fixation so we're gonna have usually a microscope slide and we're going to be we're gonna have a smear of the bacteria on there and we're actually going to do something called heat fixation and we'll watch a video of that in the demonstration video which will be posted and in heat fixation what we're actually doing is two things we're basically first of all immobilizing the bacteria but also heating them actually makes them more permeable to the dyes meaning we can actually get the dyes to go in and stick in these peptide in layers okay now now for the actual gram staining we're actually going to first use a dye like I mentioned called crystal violet crystal violet is violet it's purple okay when we add crystal violet dye it's actually initially going to get into both of these peptidoglycan layers in both gram positive and gram negative bacteria so we actually have it in both of them we're then gonna treat it with something called light stable iodine what light stable iodine does is it actually also goes into the peptidoglycan and it forms crystals with the crystal violet and basically helps them kind of stick in the peptidoglycan it crystallizes them within the peptidoglycan layers however we're then going to use something called a decolorizer which is usually a mixture of some kind of alcohols you can usually be f'n all and isopropyl alcohol in some kind of proportion okay the decolorizer is not able to remove the crystal violet dye from gram positive bacteria why is it not able to remove it from this peptidoglycan layer because it's a very thick peptidoglycan layer the crystal violet dye really cannot get out of this pepto glycan layer very effectively okay however in gram-negative bacteria where the peptidoglycan layer is much thinner the decolorizer has an easy time washing the crystal violet dye out of this and so after you use the decolorizer if you actually at this point looked at the bacteria gram-positive would still be purple they would retain the crystal violet dye however the gram negative would be you wouldn't be able to see them okay so in order to see gram-negative bacteria we actually do a counter stain with something called safranin and safranin stains anything pink that does not have crystal violet dye which in this case is only gram-negative bacteria so after we do safranin it has really no effect on gram positive bacteria because they re are staining purple or violet and gram-negative bacteria will end up staining pink so this is basically your flowchart for gram staining and what each type of bacterial positive or negative looks like at each stage you should understand this and hopefully the theory of why it looks like that and these are the results of gram staining also understand gram positive are designated the positive because they actually do absorb that crystal violet dye and these are the grand positive over here you can see that they're purple okay or violet in fact one thing that's important to note which will see much later in the semester is generally when we do any kind of test like this but if it's a Gram stain or we're gonna do biochemical tests later on in the semester we designate a positive sign or just call it a positive result whenever either the dye is absorbed or it possesses the enzyme or whatever whenever it has or does whatever we want it to do it's a positive result we wanted it to absorb crystal violet dye it did over here so these are gram positive bacteria if they do not do whatever we want if they don't absorb the dye or they don't have the enzyme whatever it's a negative result so because in gram negative bacteria they do not absorb the crystal violet died after decolorization their bare gram negative in fact because of the safranin they actually stain pinkish red which you can see over here on the left okay so hopefully this little theory behind gram staining make sense be sure to watch the demonstration videos where we'll look at both heat fixation and the actual process of doing these stages of the Gram stain thank you for watching