all right everybody we're going to get started on chapter 3 material uh chapter 3 is broken up into two powerpoints uh because it uh takes a a big change in uh the the material u partway through it so we're going to split it up into the first half being the topic on microscopy and then we'll follow up with a PowerPoint uh chapter 3B that'll cover the second half of the material relating to the cells um so on our topic on microscopy um the microscope is a very important tool when it comes to uh the study of microbiology as we said um microorganisms and microbes in general um that what defined them was that they were too small to be seen with the naked eye so we need the assistance of uh microscopes uh in order to allow for us to visualize uh these organisms and um uh the microbes that are even simpler than cells um and the microscope uh that you guys would be most familiar with would be the compound light microscope i'm sure you've seen uh or even worked with these before um they require a light source that is going to shine the light through the specimen that's on a glass slide we have um lenses that magnify the image and you look in through the ocular lens and you see uh the enlarged uh illuminated image of uh what is on the glass slide um so when it comes to this type of microscope we refer to this as being a bright field microscope because um the light source lights up the background of the image so um the uh area is illuminated and basically white in the background so bright field um the light is produced as we saw beneath the stage it passes through the glass slide and the specimen sitting on the slide and then the image is magnified by a combination of two lenses this is why this is called a compound light microscope because it compounds the magnification we have one source of magnification here called the objective lens and a second of magnification here the ocular lens so we compound the magnification we see that uh the ocular lens the one you're looking through has a constant magnification of 10x it magnifies the image 10 times uh larger than it actually is whereas the objective lens is the one that we can change so we see that by rotating the nose piece we can put in different size objective lenses and change the magnification the smallest of the objective lenses is 4x power and uh overall then the total magnification in this scenario would be 40 times magnified because the image is magnified four times by the objective lens and then that image that is four times bigger is then made 10 times bigger than that by the ocular lens so we have to multiply those numbers together 40x um so the um objective lenses are going to vary there is 4x then there's 10 x if that you're using 10 x 10 * 10 would be a total magnification of 100 there is 40x 40 * 10 is 400 times larger and then finally there is uh 100 uh x for an objective lens that times the 10 means that we are able to get a maximum magnification with a compound light microscope of 1,000 times so um so it's the objective lens that we change the ocular lens is 10x all the time now one of the things we need to be able to do with um this piece of equipment is focus the light on the specimen and that's what we use the condenser lens for so the condenser is right here underneath the stage and this is something we can move up and down to uh either spread the light out or focus it in on a smaller area um and this is going to allow for us to uh get the right amount of light um hitting the specimen so it's not um overly illuminated or on the flip side of it um too dark so the um condenser lens adjusts the quantity of light notice that this is not affecting the magnification and that's something we want to point out here is that the compound light microscope has a lot of working parts but none of them are going to impact magnification other than the objective lens and the ocular lens these magnify everything else just adjusts the image um to improve its clarity and illumination okay so you don't have to you know look at everything here and say "Oo these are all changing the magnification because this is a microscope that magnifies." Keep in mind again there's a lot more that we have to do to an image than just make it bigger and that relates to our next point here about resolution one of the features of a microscope is its resolving power or resolution this is the ability of a microscope to um be able to distinguish between two separate points in space um this is the minimum distance between two objects in which those objects can be seen as separate entities uh we see here uh the example of the minimum distance that these two dots can be seen as separate um so it's 02 micrometers in this case here we have um at this distance if they're 02 micrometers apart we can see that they are two separate dots however if they were 0.1 micrometer apart uh our typical brightfield microscope would not be able to separate those two points and so they would look like one continuous point resolution is best understood when you think about uh our cameras that we have gotten so used to using with our phones and everything um resolution is again that um that ability to get fine detail out of an image the ability to tell two points apart and so really resolution is about clarity um if you take a picture with an old phone's camera um it has poor resolution if you take that picture and you magnify it you make it just bigger and bigger and bigger the picture just gets blurry um so if you try to zoom in on something in the background um you can't even see what that something is because um even though you're making it bigger it's not clear where uh a camera with higher resolution will allow you to zoom in on that image in the background and and uh as it gets bigger you're still able to uh visualize it well and so the higher the resolution the better the clarity of the image once again resolution having nothing to do with magnification um it's it's the ability to see something clearly that is being magnified our next thing to consider when using a microscope is the impact of refraction um refraction is the bending of light as it passes from one material into another material and that's going to happen when light is passing from this light source through air then hitting glass and then hitting air again and then hitting glass again uh the the light is passing through uh multiple objects of different uh material and so what will happen is there'll be some bending of the light as it passes from one to another so we see this here light comes into the glass slide and as it's uh coming in at this angle uh let's have it come in at this angle here we see it does not continue in a straight line but instead it bends as it goes into the air then it once again bends as it goes into the glass of the lens so this bending of light is something that can impact um our ability to clearly visualize a magnified image and we see the impact of the bending of light here when you see a straw in water once again the light that's passing through the water as it then passes through the air creates this shifted um image uh which again is is not an accurate image um due to the bending of the light so what we have to do when we um go to the higher and highest level of magnification is we have to take into account the bending of the light and uh we have to introduce um oil at our highest level of magnification that was the 100x objective lens uh which gives us a total magnification of 1,00x we have to introduce oil which has a similar refractive index to the glass of the slide and this will reduce the bending of the light and give us a a more accurate image we see what happens when we introduce oil on top of the glass the oil has a similar refractive index to the glass so we have glass oil glass we see that light passes straight on its path through the oil into the glass um so we have eliminated the bending and by eliminating the bending we've reduced the amount of light that is lost due to its bending and we've created an image that not only is better illuminated but also more accurately visualized so what did we just say out of this whole thing what we just said is when we go to the largest um objective lens we have to put a drop of oil on the glass slide and as we click this into place and that oil is going to fill the distance between the glass slide and the glass of the lens to give us the best visual uh of that specimen okay now we have been focusing so far on the compound light microscope because this is the most common uh microscope it's the easiest one to use um but there are many different microscopes that are available uh for us to use in research um so we're going to uh direct our attention then to um this um section in lecture content you come down to the lecture handouts and you have the microscope summary that is this document here and um what you want to get out of this document it's got a lot of information but let's reduce this down a bit for you two things you want to understand about each microscope the first is are you visualizing a living specimen okay so you want to know that are the are the cells that we're looking at alive or dead and the second thing you want to understand is just what makes this microscope different from the other ones okay so the first one the bright uh field microscope this is the one that we've just been talking about the compound uh brightfield microscope this is used to uh view specimens that are stained generally uh so more often than not we are going to be staining cells and what we're going to learn is that in order to see uh cells and uh use stains to visualize them we're going to have to uh heat fix the cells which means we're basically going to have to cook them onto the slide and in doing so we're going to kill them so anytime that we have to stain the cells we're basically killing them so the brightfield microscope is for the most part not being used to uh visualize uh living cells um and it's the most common type of microscope because it's it's easy to use and it's cheap um our next type of microscope the phase contrast um uses uh this refractive index that we mentioned the the bending of the light um to figure out um uh what objects are are interfering with the movement of the light and by detecting the bending of light it's able to uh provide an image of a specimen that is unstained uh so uh we're working with the bending of light as opposed to uh you know coloring the cells for us to see them and so since the cells are unstained the cells are still alive so living cells are v are visible with phase contrast uh and it uses the refractive index uh of the specimen to produce an image interference is another type of microscope that is going to be used for seeing living cells this is going to use um uh two light sources um and because it's going to use two light sources um it's going to allow for us to generate a 3D image uh so we're working with more information when we have two light sources and so we're able to u combine that information to create a 3D image the dark field microscope uh once again is not going to be using any staining so living organisms are visible and um the reason why this is dark field is because the light is not going to be shined from underneath the specimen where the background of the specimen becomes illuminated instead the light is shined toward the specimen um and then what we are capturing is the light that bounces off of the specimen so the light that does not hit the specimen is lost it's kind of like just going off into space so it's like looking at a night sky where um the the light source is the cell that you're looking at as opposed to in the background of the cells and so once again this does not require staining and therefore we have living cells that we're able to view we see um sometimes uh these uh microscopes uh can can have specific functions relating to particular organisms and uh we see the causitive organism of syphilis trapema paladum um is actually better visualized um as a a spyroet cell type um using this this dark field microscope next we have the fluoresence uh microscope and this one is going to be unique because this one actually allows for us to identify an organism one of the things that we're going to learn about throughout microbiology is that um bacterial cells um don't have many physical differences between them most of them are going to be small circles or small rods so you will never look at a bacterial cell and go that bacterial cell is E.coli i know it's E.coli i would bet my life on it you cannot identify a bacterial cell based on how it looks so none of these microscopes none of these methods of imaging gave us an identification of the organism we simply were looking at the cells um in their which uh are in a very simple form but with fluorescents this um uses um antibodies that have a fluorescent u marker attached to them and the antibodies hopefully you guys remember this from um A&P antibodies are proteins that u stick to antigens molecules on um cells and so the antibodies are very specific to the antigen that they stick to so let's imagine we have um three cells that look the same okay we used any of the other microscopes and we see these three cells we have E.coli uh let's do uh Salmonella and uh let's do Lebella three entirely different organisms they all look the same but we introduce an antibbody that uh is an antibbody for E.coli so the antibbody for ecoli uh we are going to uh add to this slide that has all three of these cells it's not just an antibbody said it has a fluorescent protein attached to it so it's got this molecule that glows green and we mix this antibbody with these cells and what happens is that antibbody is going to stick only to E.coli and as we said it's going to glow so the cell that glows we can identify as E.coli so over here what we can conclude is this um has to be E.coli e coli and everything else is not E.coli right that's what we've done with this um this tool if the antibbody sticks to the cell and it's an antibbody that sticks to an antigen on E.coli then we've identified it as E.coli otherwise the cells that the antibbody does not stick to uh we don't know what they are they are um but we know for sure that they are not uh ecoli okay and so um next uh we are going to oops hop back into here and look at the um conffocal uh microscope so in con focal we're using a laser beam that is going to uh gather information from multiple levels um you can kind of think of it like um like the meat slicer that's taking you know a a sausage and cutting through it into thin slices um it's gathering information with each of those slices and then putting that information stacking it back together to reconstruct the whole sausage basically in this case we're getting information from each layer and then we're um g stacking that information um together to create a three-dimensional image and this is unique then because this allows for us to view uh multiple layers of cells uh so um this will be important for um organisms that grow as as um communities of cells we'll talk about things like slime layers which are going to be uh groups of cells that are all built together in um in an extracellular slimy material and uh we can use a uh a tool like the con focal microscope as a way to see the internal structure of this three-dimensional specimen um so what made this unique is this is allowing for us to look at something that is um many layers deep where all the other ones so far we've had to lay them out as a single layer on a surface in order to view them okay now the next group of microscopes the electron microscopes and the atomic force microscopy these ones are unique compared to everything else that we've learned so far because these all have far better resolution okay so what this means is that um these are the best at being able to zoom in and see really really fine details that you would never be able to see with the other microscopes that we've described the first type of electron microscope is a scanning electron microscope this one covers cells in metal therefore the cells are going to be dead here so we're not dealing with living cells and um the way that this is going to work is we're going to fire electrons at these metal covered cells um so here's our slide and here are the cells on the surface of the slide and we're going to cover them in metal so the whole surface gets covered in metal and so this is our metal layer oops hit the wrong button there okay and then what we do is we fire electrons at it and we get information about the surface as we fire electrons at the surface uh so we're going to fire electrons at the surface and what we'll see is that um this cell right here is higher up than the glass surface and so what we're going to get information from is this electron makes contact with this cell and then bounces back giving us information sooner than this electron so this electron is going to bounce back and give us information this electron is going to bounce back a little bit later and give us information that um that this surface is deeper than this surface so what it does is it gives us a um a great view of the surface of this metal covered slide slide okay um so what that does for us is it gives us a a very detailed three-dimensional image of the surface of the cells transmission electron microscopes go even further in that we are going to thin slice or section the cells so this one's pretty crazy going back to the idea of a meat slicer cutting up a sausage so you can look inside of it in this case the cells are prepared and then sliced and as they're sliced we're then able to um look at the internal structures of the cells so this one is very unique because it gives us the the ability to visualize what's inside of cells so uh when we look at images of like a virus inside of a bacterial cell the way that we're able to visualize something that small that is also inside of a cell is with the transmission electron microscope in both cases the electron microscopes involves processing the cells and therefore the cells are not alive okay finally we have the atomic force microscopy um this one is uh more comparable uh as an imaging source to something like an MRI does not require um uh the cells to be um prepared for being visualized and so it gives us a very detailed image of the surface of the cells with greater resolution than the electron microscopes and what uh is great about it once again is that it does not require that the cells are prepared to be viewed uh so u this is going to give us the opportunity to visualize uh living cells at a high resolution uh without um you know without having to of course kill the cells and the preparation that we do with electron microscopes okay so with that handout hopefully um you guys um understand once again which microscopes are visualizing um living cells versus dead cells and what is um a unique characteristic what makes each one special compared to the others all right so speaking of getting a good visual here um yeah not sure what kind of microscope we need in order to see that question there okay so uh we'll move on to explain more information about um the slide preparation that we have to do with our compound light microscope that bright field microscope so the problem with a brightfield microscope is we are shining light through the bacterial cells um and the bacterial cells we see they're so small they are nearly transparent so if we were to just take bacterial cells put them on a slide and shine light through them we really wouldn't get a good visual of them because again they're just so small and fine that uh they wouldn't look any different than the background additionally if we have them in uh a water source uh the bacterial cells would be able to move in the water and so it would be difficult for us to uh zoom in and look at one while it's also uh freely moving so what we do in order to um uh visualize cells using a brightfield microscope is we immobilize the cells through a process known as heat fixation and the way that heat fixation works is you put cells um into a drop of water on the slide and then you evaporate the water off by passing the slide over a flame so I want you guys to think about this for a second compare this to let's say a a cooking pan with water and pieces of chicken in it if you cook the water off what happens to the chicken on that pan i hope you guys all know that that chicken would stick to the pan right so this is the thing the reason why we have to oil up pans and everything is because uh when you get a surface really hot and the water evaporates away the uh cells stick to that hot surface that's exactly what we're doing here with bacterial cells we evaporate the water and we leave the cells glued to the slide that does two things for us one it allows for the us to keep the cells still so we can look at them we said the downside of this is we've also killed the cells but at least we can see them now but the other thing this allows for us to do is stain the cells um because what we're going to do is add a stain to the surface and then we're going to wash it off if the cells are not stuck then when we wash the stain off we would wash the cells off with them so we need to heat fix the cells we need to stick them to that surface so that we can hold them still and stain them okay so now we're going to look at the different staining methods the first staining method is what's called a negative stain um two ways of looking at this one a negative stain the image you get is like the photo negative um which is where the everything's backwards uh so the things that would normally um you know dark or light and vice versa so we see an image where it it looks like the the photo negative um but the other thing to associate with negative staining is that this is an acidic dye that has a negative charge the surface of cells o overall have a generally a positive char uh I'm sorry have a negative charge to them as well um so the negative stain that is negatively charged when it's combined with a cell that has a negative charge to its surface um ends up being repelled by the surface of the cells uh so negative charges repel negative charges what happens here is the acidic negative stain does not stick to the cells and so the cells are left unstained so with negative staining the things you want to understand it's an acidic negative stain it does not stick to the cells it leaves the cells clear with a background that is stained okay that's one way of looking at cells another way of looking at cells is simple staining which is where we use a single stain that is we see here basic or positively charged and because the stain is positively charged it will be attracted to the surface of the cell that has a net negative charge to it and so what we see then is the stain sticks to the cells when we wash the slide after adding the stain the stain washes off of the glass but sticks to the cells so the cells are colored and the background is clear the reason why this is called simple staining is because all cells will pick up the same color so if I had a a slide that had ecoli and salmonella and strapus and stafloccus if I had all these different bacteria on the slide they would all come out looking this same color simple staining everything comes out looking uh the same color the next thing we want to consider the opposite of simple staining is differential staining differential staining allows for us to get different outcomes based upon the characteristics of the cells that we are staining so we can use differential stains to distinguish bacteria from one another our first differential staining method is Graham staining we see our two possibilities with Graham staining are that the cells will end up this dark blue purple color or the cells will end up this light red pink color differential there are two different possible outcomes okay the Grahamstaining helps us learn more about the organism and we'll see in the second part of this chapter why um Grahamstaining uh ends up with two different outcomes we'll see that gram positive cells the dark purple ones will have a different cell wall than gram negative cells the pink ones and that's going to be the reason why they stain differently okay so with Graham staining uh this is uh first of all the um most widely used procedure uh with identifying bacteria because um it is a characteristic that is special for each bacterium so uh we will learn this all throughout the semester every time we learn about a bacterium we'll start off by saying whether it is gram positive or gram negative gram positive means that when we do the staining procedure to them they come out dark purple gram negative means that when we do this procedure to them they come out uh red pink okay the way that this procedure works is that um we have two different cells here we have a gram positive cell and a gram negative cell we have heat fixed them so they're stuck to the slide and then we add a stain called crystal violet crystal violet makes the cells this dark blue color we wash off the crystal violet and add iodine when we add iodine it combines with the crystal violet it gives us this purple color overall and so right now if we stop right here both gram positive and gram negative cells are dark purple but what is critical to this process is this decoloring step when we decolorize we add a solution that removes the crystal violet and if we do this right we will remove the crystal violet from just the gram negative cells but the gram positive cells will still be dark purple so if we time it right this is the outcome of decolorizing and then finally we see um the last thing that we do is add saffronin saffronin is that red pink color and when we add red pink to purple it doesn't change its color it's still purple when we add red pink to clear it comes out that red pink color so if we do this procedure correctly gram positive cells come out purple gram negative cells come out pink what we'll learn about in the lab is that this decolorizing step is is essential you have to do this right if you decolorize for too long then you take the purple out of the grandpositive and then that means that the grandpositive cell would end up decolorized and so the grandpositive cell would end up looking pink if you decolorize for too short of a period of time then you don't take the purple out of the gram negative cell and so the gram negative cell would end up looking purple at the end so it it's all about timing this just right in order to get the outcome that we see here where gram positive cells come out purple and gram negative cells come out in pink red okay the differential there are different outcomes here acid fast staining is another differential staining procedure what we see here is that acid fast staining is used to detect the uh presence of a uh lipid layer that is around the outside of um bacterial cells and these bacterial cells are specifically uh referred to as acid fast cells with an example um genus of acid fast cells called mcoacterium so these cells um are different from the gram positive and gram negative cells that we just learned about because they have this extra kind of waxy coat and so we can't stain those cells with this Graham staining procedure if we did the Grahamstaining procedure to them they would come out clear because the the wax would be repellent so we need to introduce a different kind of stain in order to uh visualize these cells and what we see here is the outcome of the acid fast staining procedure where the acid fast cells are the ones that pick up that specific dye that is used um under a high heat condition uh in order to um penetrate the waxy coat and stain the cell uh we'll learn more about that in the lab um but for now an acidifast stain um is used to uh detect cells that are considered to be acidfast finally we have a whole bunch of other special stains that are used to detect other features of cells which we will learn about in the rest of chapter 3 so in the rest of chapter 3 we'll learn about structures called capsules which are around the outside of the cell they're an expanded protective coat around the cell and we have stains that we can use to negatively stain for capsules where we see the capsule is unstained um and and it shows us just how large the capsule is around the cell we have endospore staining we learned about endospores in the first chapter they were that uh dormant cell type um we'll learn about how we do endospore staining in the lab and the idea here is that we once again need a special stain to penetrate the cell um the cell's outer protective coat um and so we can't use a conventional simple stain to show them bleella stains show the whip-like tail that projects from cells uh fluorescent dyes we talked about um how we can use fluorescent stain uh procedures including antibodies uh in order to um uh specifically identify certain organisms um and that was the process of amunofllororesence in which the antibbody tagged specific cells um so if we look at this here um take for example this cell that is yellow with a whole background of red cells it's that yellow cell that we can say this one is different from the other cells because it uh was tagged with a different antibbody so amunofllororesence um was the microscopy method and the staining method combined that we use to identify specific organisms based upon um whether or not um specific antibodies stick to them okay and so with that I hope you guys have a good understanding of the staining process