we spend a lot of time looking underneath the microscope in microbiology and one of the things that I mentioned before in the recording for chapter one is that if you were in lab and you used to applaud your lab partner for doing such a great job focusing by the end of micro you are going to be applauding yourself because we spend quite a bit of time practicing using the microscope um especially under oil immersion um which not a lot of students have practiced with but definitely something that we're going to have to use in order to see our very tiny microbes so microscopy is basically making very small things visible to the human eye so we are going to be looking at very small bacteria if you look on a plate um that you're growing bacteria on you will be able to see a colony how however you're not going to be able to see individual cells in order to see individual cells we are going to have to make slides we're going to have to stain them and then we're going to have to look at them underneath the oil imersion lens if we're using a microscope most of the measurements that we use or at least talk about with our bacteria even eukaryotic cells is the micrometer range so just a little review on the metrics system if you remember there are 10 mm and 1 cm so if you think about how small a cenm is and then how small A millimeter is there is 1,000 micrometers in 1 millimeter so obviously that is very small we're not going to be able to see these things without a microscope so the two things that we talk about with microscopy so we talk about magnification versus resolution so magnification makes things bigger that's really why we're using the microscope you can make things bigger by using a magnifying glass in fact we use magnifying glasses in lab um but you're not going to be able to get the magnification that we need in order to see individual cells resolution helps us to see two separate objects so that they're not one big blur so that we can actually see them separately so in order to see that good resolution that means that we're going to need light passing up through those samples so that we can see two cells distinctly um instead of just seeing one blob so that's the difference between magnification and resolution our microscopes have pretty good resolution uh you could pick up a microscope on Amazon like a children's microscope and you'll be able to see things magnified but you're not going to see that Clarity a lot of times people think of resolution as being that Clarity really it's the difference between being able to see those objects as separate units um but it helps to clarify things so the difference between a good microscope and maybe a not so good microscope is that resolution it's also the difference between a cheaper microscope and a more expensive microscope so ours are thousands of dollars because they do have that really good resolution and we're going to be spending a lot of time talking about the use of the microscope care of the microscope so that we can get the most out of our microscope and also treat them with care so that we can continue to use them so this chart here kind of shows the relative sizes um I'm getting pretty blind now as I'm getting older but even I don't need a microscope to see my dog um but if you take a look at some of the smaller things like looking at um a eukaryotic cell like a human egg and then going down to looking at bacteria you'll see that there are different levels and we have our compound light microscope which is what we will actually be using so our compound light microscope that's that that yellow line that's going to allow us to see some good detail with our eukariotic cells and if you've taken anatomy and physiology no doubt you've spent quite a bit of time looking at eukaryotic cells underneath the microscope looking at neurons looking at muscle cells looking at blood cells and our compound light microscope does a pretty good job of magnifying those so we really don't have to go up to oil immersion of course we could if we wanted to see more detail but with our periodic cells those slides were pretty good at being able to see some detail under high power in order to see our bacteria uh we're going to be at about the five micrometer level we are definitely going to need our high power and our oil immersion lens and I'm going to talk a little bit more about oil immersion and why we have to use oil to clarify and get that resolution with our sample one of the things that we will be talking about this semester are viruses we cannot see viruses in lab you'll be probably happy to know we are not going to be working with viruses in in lab at all we will be talking about viruses in class but they are very small so we would actually have to use an electron microscope in order to visualize a virus so the thing about microscopes is that it all has to do with light and there's a lot of physics of light there's a lot of properties of light that really go into um magnifying and getting that good resolution of our samples um we're not going to get into a lot of the physics of light one thing though that we do talk about is resolving power and this tells us how well our lens can resolve so what this really means is how close to cells can be so that we can actually see them as being separate sprad so like I said before our microscopes have pretty good resolving power the thing about being able to see our samples is that because these are light microscopes we actually have to transmit the light through our sample so that means that when we're preparing our slides we have to do some practice with making sure that our smears are a good consistency so that we can get light through it if they are too clumped up and we tend to be a little heavy-handed when we first start making slides because we're afraid we're not going to get anything on our slide um then we're not going to be able to get the light through it so one of the things not only will we be practicing with using the microscope but we'll also practice with making our slides because getting that light to go through our microscope and through our our slide is going to be really important for us to to to see our sample so a little bit on light um so the two terms on this page that we talk mostly about with our microscopes and lab transmission so that means that light has to pass through the object so in order to see our cells we're going to have to transmit light through our sample that's why practicing making our slides um is going to be really important so that we get that good light transmission so that we can see between those cells so that we can make out the different shapes and Arrangements that we want to see underneath the microscope the other term that we use is refraction so as that light bends so as it's going through all of the different media of the microscope and through the slide and through our sample it starts to bend so that light is then refracted so what happens as we start to get up to higher and higher magnification with our oil immersion lens because of that refraction because of that bend of light we actually have to correct for that so that it's clearer and so we have that good resolution so in order to do that to correct that bend what we do is we add immersion oil now this to me was always counterintuitive because I know if I've ever gotten like something oily on my eye I couldn't see it was very blurry so I always found that to be counterintuitive this actually helps to correct for that bend and so it does the opposite of what you would expect it actually helps with that resolution and it helps to clarify it I know it seems really strange to think about adding oil to make something clearer um but when we get into lab you will actually see that if you don't add enough oil when you're under oil immersion it is going to make a difference um and it's not going to be clear and sometimes just adding a little more oil um is going to help to to clarify it and make sure that you get that good resolution so if you're a visual person like I am this is actually a light source that is um that is interacting with an object um and you can see those different pictures with those different terms you can see B is transmission so that's our light that's going through our sample and then D you can see our refraction so as it as it hits It's actually creating that angle or that bend we're going to have to add that oil to correct for that bend so this next part I'll talk a little bit about some different types of U microscopy that there are we are going to be using a compound light microscope um if you've use these microscopes in a general biology or anatomy and physiology they are very similar even if they're a little different from what you've used um the principles are basically the same they all have their little idiosyncrasies and nuances that you have to get used to um but they're going to be using light we're going to be using bright field so when we stain our cells they are going to have a contrast and they're going to be on a light background that's mostly what we would call a bright field so if you take a look at these two pictures here these are actually cheek cells cheek cells to me almost look like a fried egg with that yolk that's in the middle if you took a general biology course one of the things we often do is to stain our cheek cells with methylene blue so they look blue underneath the microscope you could also choose to use a dark field this has a condenser and a condenser is a part of the microscope that helps to focus the light through the sample this type of microscope has a condenser that deflects light so it adds a little different contrast so you can see how these pictures look a little different where one looks a little more three-dimensional and the other one looks a little more two-dimensional um our bacteria are not going to be jumping out at us looking three-dimensional they will look two-dimensional and rather flat but we will be able to make out shape Arrangement and depending on the stains that we're doing we'll be able to see different colors phase contrast microscopy this is used for samples especially life samples that can't be dyed or stained so if we want to see specimens in their natural environment we might choose to use a phase contrast microscope so if we wanted to look at Pawn swimmy things underneath the microscope of course we want to see them moving so we're not going to kill them we're not going to stain them so that would allow us to see them in their natural environment so we can see these um almost a three-dimensional look because of a special condenser that allows different indices of refraction so it just allows us to get a little better picture especially if we want to see living moving organisms our bacteria are not going to be living they are going to be dead we are going to kill them as we make our slides and I'll talk a little bit about that coming up another type of microscope um uses UV light and this is fluorescence microscopy um so that things kind of glow underneath the microscope so it uses different wavelengths and they can often use this to um diagnose things if they're looking for particular microbes um we do not have a fluorescence microscopy uh microscope rather um when I worked in pharmaceutical we did have uh fluorescence microscopes um and we looked at uh different crystallography um they use them in different labs to look at microbes um but we unfortunately do not have access to one that we are going to be able to look at another one that we're not going to have access to these are electron microscopes these are very powerful microscopes and these use beams of electrons rather than light and there are two types there's transmission electron microscopes that they can magnify up to 500,000 times so they can actually see inside of microbes and specimens have to be embedded in a resin a plastic and then they're cut into very very thin sections using this machine this this microtone that basically allows to cut like super super thin slices um I had a friend that I was in graduate school with and with her thesis uh we did not have a transmission electron microscope but she did need it for her thesis so she actually sent out her samples so she embedded her samples in the resin and then cut them and then sent them out to a lab these are huge machines this is not one of those things that they have sitting in a lab that you're like hey I think I want to use the electron microscope today you can walk up and you can start focusing they have dedicated Personnel you have to be trained on it um and um universities would would have that my university did not um the one University I did go to did have an electron microscope um another type is scanning electron microscopy this allows magnification of 50,000 times so it's not as strong as transmission but allows you to see sort of the topography so you can see like 3D images outside of organisms they have to be coated with heavy metal something like gold in order to actually see them so here are a couple of pictures of transmission versus scanning so you can see with the scanning electron um micrographs you can see where you they look three-dimensional where they've got those shapes um you can see looking at the pollen at the picture above you can see um lots of lots of different um textures that are happening on the outside and then the transmission is where you're seeing that crosssection where you can see right through it the bottom picture is a bacteria so that's showing you the outside and then kind of looking through it with that crosssection so there have been lots of advancements in microscopy scanning tunneling microscope this uses a very thin wire to move over a specimen it kind of gives you a topography where you can kind of see Peaks and valleys Atomic Force by microscopy this allows 3D imaging of structures so they can use this to visualize DNA um scanning tunneling microscopy they might use um maybe to look at different receptors on cells Atomic Force microscopy they might use to look at DNA RNA or even bonds between um atoms uh which is amazing so it's really come a long way from from certainly um Robert Hook and um and Tom Von Luen hook um if you remember them from the first chapter when we talked about um a little history of microbiology so in order to see things underneath the microscope we're going to have to prepare samples so if you have taken a general biology course at some point in your academic career you might have looked at maybe your cheek cells underneath the microscope or pond water that would be a wet Mount slide so we would add a liquid or a little bit of pond water we'd put a cover slip on it and then we could actually visualize it underneath the microscope um that's going to allow us to see moving living things um it never gets old for me when we look at pond water and in BIO 109 um and this whole new world opens up and um everybody can see these these living swimming things underneath the microscope um that they had no idea were actually in that that pond water specimen that they took we're going to be making smears we are not going to be looking at Living bacteria we are going to fix our bacteria onto the slide so like I said earlier we're going to be spending a lot of time practicing making our smears so we will go through that protocol in lab um so first we will adhere the bacteria to the slide then we're going to heat fix it so we will dry it on a heat block and then we'll pass it through a flame and that's going to allow us to heat fix our organism this does three things first off it kills the organism now the organisms that we are working with are fairly lowlevel biohazard but if you are working in a lab um or if you have no idea what unknown microbe you are staining um you want to make sure that you kill it because you don't want it to cause any harm to anybody in the lab so a it's going to kill the organism then when we heat fix it it's going to allow the organism to stick to the slide because we're going to be doing some staining and some washing and we don't want to wash it away in our waist bin because then we're not going to be able to see anything the other thing that the heat does is that it helps to open up the cell wall of the bacteria um in the upcoming chapters we're going to be talking a little bit more about the external and internal structures of the bacteria but bacteria have a special cell wall and in order to open that cell wall up so that they can actually get that dye so that dye can penetrate and that specimen can hold on to that dye if we Heat fix it zap it with heat it's going to open it up so heat fixing is going to do those three things kills the organism allows it to stick to the slide so we're not washing it away and opens up that cell wall so it can be stained more easily like I said we're going to have a lot of practice with that there are different types of stains that we can use in lab we mostly use cat iic stains cationic stains if you remember learning about cat I cation have a positive charge so I often think about a cat um with positive eyes I usually draw picture of a cat with positive eyes cats have paws so some people say oh it's positively charged because it has Paws um but they are positively charged and they actually work better than anionic stains anionic stains are acidic they also have a negative charge the way that I remember that is an an ion a negative ion the reason that cat iic stains work better is that that cell wall that I was just talking about with bacteria is slightly negative so as you know Opposites Attract so that positive D is actually going to stick to that cell wall so if you have done some simple staining in the past um You probably used methylene blue so that all of your cells are blue uh we also use crystal Violet which is a purple dye malachite green is another Dy that we'll be using which is green and safranin or saffrin however you prefer to say it is a red dye so those are the dieses that we will be using we do not use any anionic dyes um and it's because those cionic dyes are going to stick better to that bacterial cell wall we're going to be starting off with our staining with a simple stain and a simple stain uses just one type of dye so if you've stained cheek cells before that was a simple stain so you added methylene blue and then you looked at your cheek cells underneath the microscope that's a simple stain what is that going to allow us to do it's going to give a little bit of contrast so that we can see the shape of the cell and what the cell looks like with our bacteria and we're going to be getting into the different names of shapes and different names of arrangements the shape has to do with whether or not it's a circle or whether or not it's a rod shape Arrangement has to do with if those cells are stuck together so a simple stain is going to allow us to see the the shape as well as the arrangement so we could use any of those cationic dyes we could use methylene blue we could also use crystal Violet but it's only going to show us that one color if we're using methylene blue all of the cells no matter what type of cell stain is going to be blue if we use crystal Violet no matter what um cell we are using they're all going to look purple a differential stain is going to give us additional information so if we were to find an unknown for example an unknown bacteria a differential stain is probably going to be better for us to use because it's going to give us more information so it's it's going to be a series of stains so it's not just one stain it's going to be series usually there could be two or three there might be a couple of different steps in between and it's also going to allow us to see differences so differential differences kind of begins with the the same part of the word so these are a little more involved but they are going to give us more in so some differential stains that we're going to be using this semester the Gram stain we'll talk a lot about the Gram stain and what it means If an organism is gram positive or gram negative we will do an acid fast stain that's going to allow us to see an acid fast bacteria from a non- acid fast bacteria again we're going to talk a little bit more about that and those different types of bacteria and Spore stains a Spore stain is going to allow us to see if the organism makes spores and a Spore is not a reproductive thing like you might think of with some plants this is actually a protective structure so if there is a bacteria that is very resistant and we're curious to find out if it does go into that Spore State that's the type of stain that we would do so those differential stains are going to be able to show us the shape and Arrangement but then give us additional information so that we can tell differ is between different types of cells and maybe between um different parts of one cell itself so this is actually the gramain procedure that you are seeing in this picture and I'm showing you this not because I want you to memorize this in fact you are never going to have to memorize the gramain procedure because in lab we have these little cards with the directions I call them recipe cards they never leave the sink they are always there but this is just to show you the different dyes that we use and the different techniques so there's a primary stain which is Crystal Violet then there is a Morant treatment that's actually iodine what that Morant does is it allows uh a complex to form with a crystal Violet so that it adheres to certain types of cells a decolorizing step that uses alcohol to wash the color away and then a counter stain that gram negative bacteria will actually take up so this allows us to see the shape and Arrangement but then it allows us to see differences so if you look at this picture you'll notice that on the one side it says gr positive they are purple cells underneath the microscope where gram negative organisms would be red so that's going to allow us to see the difference between a gram positive and gram negative organism we're going to talk a lot more about the Graham stain coming up this is a picture of a Graham stain so this is a differential stain we can see the differences so here we've got two organisms one is gram positive one is gram negative it allows us to see the shape and Arrangement so you can see that that pink one is a rod shape that's what we would call a basili where that purple one is a round shape that's what we would call a coxy and the arrangement how those cells are stuck together are a little different as well so this is a very good Gram stain because it allows us to see those differences so we can tell that we have a gram negative and gram positive organism in this slide so some other types of stains a negative stain this allows us to see if an organism has a capsule and a capsule is a really thick glyco Kix like this polysaccharide layer that's on the outside of the cell and that allows the organism to be sticky if it can stick to cells that means that it can be hazardous to sell so that's not a good thing at least for us anyways capsule also helps to protect them uh which is a good thing for the bacteria we really won't be working with any organisms with capsules so we won't be doing any negative stains uh but if you were to see a negative stain underneath the microscope you could see where there's almost like a layer on the outside of the cell if you can see from that picture fella stains what's a flagella flagellum is a tail and probably um about half of bacteria will contain some sort of arrangement of these Tails these Vellum very similar to a eukariotic uh flagellum but it's just the makeup is a little different and the way that they move it is a little different so in order to see the fella we would actually have to coat it so here in that picture you can see it almost looks furry almost like it's hairy in that picture um we are going to be working with bacteria with fella but we are not going to be doing um one of those stains one stain that we will be doing is a Spore stain and endospores are protective structures and this is when a cell can go into a dormant state so it's just DNA and a protein coat just kind of waiting better times organisms that can form endospores are very resistant they're tough to kill so it's difficult to normal disinfectant techniques don't always work medicines don't always work as well um when the cell is just kind of doing its thing um undergoing metabolism going about its day that's what we call the vegetative cell so a Spore stain is going to allow us to see when those bacteria have gone into the dormant State and now they're in that endospore form or when they're in that vegetative state they haven't quite gotten the memo yet that um conditions are deteriorating and so they're just kind of going about their business so that little picture that you see um you can see differences there's one that looks a little like oval and bluish and one that looks a little longer and red so that allows us to see the differences between the vegetative cells and those endospores um again because we're going to be doing the stain we're going to be spending a lot of time talking about that practicing it as well as talking about some examples of some organisms um that I know that you're going to recognize that um are actually spor formers so that is a little bit on microscopy and uh like I said we're going to be spending a lot of time in lab using the microscope