this is part one of two on chapter 3 Tools in microbiology this chapter is going to go through some of the techniques and tools that we're going to be using in lab so we're going to be reviewing these throughout the semester in lab in microbiology we have something called the six eyes and it first starts off in the middle and we actually have specimen collection that occurs first and specimen collection this can come from environmental samples it can be specimen's collection off of animals or people so like urine blood water um just surface swabs for example after you collect the specimen we're first going to go to the top right hand where it says inoculation so usually after you collect your specimen you inoculate some type of augur plate or some type of media or it can even be like a live um inoculation so in a bird embryo for example so we need to inoculate some type of media we then go down towards incubation so we're going to go clockwise around the circle so after you acculate your sample you have to incubate it so this allows the sample to grow over time and we'll be working with incubation quite a bit in lab after incubation you can isolate certain bacteria colonies and just focus on those species of bacteria most of the time in lab we're going to be working with peer cultures but every once in a while I may mix cultures together so you have two different bacteria and then you can actually isolate the bacteria from each other after isolation you can inspect your bacteria so this involves using microscopes we also are going to be using lots of different dyes to help us inspect the bacteria see what they look like and during that inspection you can gather information so information gathering is our fifth eye so you can get this information from your inspection part you can also do biochemical testing which will do the second half of the semester you can do drug sensitivity testing to figure out what drug works against your bacteria you can even do DNA analysis to identify that specific species of bacteria after you gather all that information a lot of times you actually want to identify what the bacteria is especially when we're looking at human or animals and we're helping to treat those humans and animals a really important part of microbiology is learning how to use the microscope and we're going to spend a whole Lab on using the microscope and understanding CU you're going to be using it pretty much every single time in lab so two really important things of a microscope so two key characteristics of reliable microscope are one we need a microscope that can magnify so we need to enlarge objects because bacteria are very very small so we need to make them bigger so we can actually see them and then as we magnify our object that we're looking at we also need to increase the resolving power or the ability to show the detail in the image we're looking at so this is showing magnification versus resolution so magnification you're just enlarging the image so a really good example of this if you think of like your digital camera you can take a picture you can zoom in you can magnify the image but a lot of times as you magnify an image it gets blurrier so that's where the resolution comes into play so the resolution is just your ability to distinguish between two individual points or to distinguish the detail so resolution this is um analogous to pixels on a camera so the more pixels you have the higher resolution the clearer the picture especially when you zoom in on the picture and magnify it so we need both of these things working at the same time in a really good microscope the magnification part happens in two phases so there's actually two different lenses the image passes through in order to be magnified so one type of lens is called the objective lens so so the objective lens we have four different objective lenses on our microscopes and they're set at different magnifications so objective lens you can actually change or rotate so this objective lens it forms something called a real image then after the image passes through the objective lens it goes through the second type of lens called the ocular lens so that real image is projected into the ocular lens these are the lenses you look through and all of our microscopes the ocular lens is 10x so it magnifies 10 times as the image passes through this ocular lens it creates something called a virtual image and this virtual image it's inverted so it's actually upside down and backwards to what is actually on the slide and we'll be working with this in lab when we use the microscopes so because magnification it happens in two phases to get your total magnification you take your objective lens power remember objective lens is the one that you can rotate or change and you take this times your ocular lens which again is usually 10 so objective lens times ocular lens that gives you the total magnification that you're viewing the image at you can also quantify the resolution or actually give it a number so your resolving power it just equals the wavelength of light in nanometers and this is usually given to you or you have to figure it out and then you divide this by the numerical aperture of the objective lens plus the numerical aperture of the ocular lens and your wavelengths can vary from anywhere from 400 to 750 NM the numerical aperture or the na it's written on the lenses it's anywhere from 01 to 1.25 and we'll again be working in lab on this resolution and trying to understand what it actually means so remember resolution is the clarity of the image or how well you can distinguish things when you're looking at them for one of the objective lenses on your microscope the biggest one it's 100x you're going to have to use oil in order to increase the resolution so I'll show you how to actually do this in lab how to use the oil because you need to use the oil objective lens in order to actually see the structure of bacteria in a lot of times to see the color of it so basically what the oil does is it condenses the light or it captures the light better than just what air can do so it's going to increase the resolution it allows more light to get through so you get this clear image this image again is showing resolution to help you kind of understand this idea so at the top of the image we have bacteria cells that are considered to be non-resolvable so you can't see them as individual objects they're kind of smooshed together there's no space in between them on the bottom we have the same image but here our bacteria cells are very resolvable you can actually see space in between your bacteria cells so they're very very clear in lab going to be using a compound microscope and this is what it looks like and I'm not going to go through all of the parts cuz you're going to learn about them in lab but just to kind of familiarize yourself with the different parts um I can definitely show you so we have um the two objective lenses so you have the or the two different types of lenses you have the objective lens and we're going to have four of them including the oil lens so that's the first lens your image goes through and then the ocular lens those are way at the top those are what you look through and again our microscopes the ocular lenses are always 10x at least the ones that we have in lab so different compound microscopes or different Optical microscopes these all use light in order to see the object the microscopes we have in lab are considered to be bright field microscopes so these these are the most widely used microscopes the ones you're going to see in Labs pretty much everywhere so in your image when you look through these microscopes your specimen is going to be darker than your surrounding field so usually the surrounding field is going to be white or really bright and we use these bright field microscopes for live and preserve stained specimens and we'll get to different types of staining in a little bit other types of microscopes you can have a dark field this is basically just the opposite of a bright field so your specimen is really brightly illuminated and the background is black or really dark and these are used for live specimens or they can be used for unstained specimens another type of light microscope is called the face contrast and this uses subtle changes in light waves that are passing through the specimen and they turn it into different light intensities so these phase contrast microscopes are really good at looking at the inside of your cell and we have one microscope that does this phase contrast thing and then we also have fluorescent microscopes that you can see these ones you use dyes on your specimen these dyes admit visible light when we pass UV rays over them so they flues or they glow under UV light and this these are usually found in big research hospitals or research Labs they're really good at um diagnosing infections so we can actually see the bacteria they're going to be glowing or fluorescing the microscopes you've looked at so far give you a 2D image if you want to get a 3D image so you want to see the outside of the specimen or you want to see through the specimen you can use what's called a conf focal microscope so this uses a laser beam so more concentrated light and it scans the specimen and it can actually scan the specimen at different layers so we can have multiple depths depths or multiple planes so you can actually go from the outside of your specimen and you can go through it using these scanning con focal microscopes another type of micros scope besides the light mic microscopes are the electron microscopes so instead of using light to form an image electron microscopes they use beams of electrons to form the image so electrons are very very tiny if you remember from chapter 2 extremely tiny these electron waves are 100,000 times shorter than the waves of visible light so electron microscopes they allow us to magnify and to resolve to very very high numbers so electron microscopes they can magnify between 5,000 and a million times and our microscopes that we have in lab they only magnify up to a thousand times so you can see that you can magnify a lot more with these electron microscopes so light microscopes and electron microscopes they pretty much have the same idea behind them so you start off at the top light microscopes on the left they start with light electron microscopes start with an electron gun that creates this Electron Beam the lighter the electrons they first pass through a condenser lens so you have to condense down the light or condense down your electrons and then after that they go through the specimen that image of the specimen passes through the objective lens so you magnify it the first time and then your image will pass through your ocular lens so you magnify it the second time and you get your image at the bottom with light microscopes we can view these images just with our eyes with electron microscopes the image goes onto a viewing screen a lot of times nowadays it's just a computer screen that processes the image and basically that's because you can't look at electrons so you need to put it onto some type of viewing screen there are two types typ of electron microscope so the first type is a transmission electron microscope or it's abbreviated a temm transmission electron microscopes the electrons will go through the image they're transmitted through the specimen so electron microscopes or the transmission electron microscopes you get a 2D image it's really useful for seeing what's inside of a cell the other type of electron microscope is called a scanning electron microscope or abbreviated sem scanning electron microscopes they scan the outside of the cell so you get this really cool 3D image and a lot of times they'll add artificial color to these 3D images and they can be very very cool and interesting to look at and I'll try to incorporate some of these into lectures when we start getting into cells and looking at different types of organisms so in our light microscopes in lab we're going to be using light microscopes we don't have any electron microscopes on campus so for our light microscopes or Optical microscopes we have to prepare our specimen in order to actually see it so different types of preparation that we're going to be using a lab you can use a wet Mount so this is where you just take your bacteria mix it in with water and you can look at it when it's still wet another type is called a hanging drop mount a hanging drop mount Mount um we can attempt to do this in lab it's kind of hard to prepare but those wet mounts and hanging drop mounts they're used when you want to see bacteria alive and moving so we want to keep them alive the other type of Mount that we're going to be using the most of is called a fixed Mount or a smear or it's called heat fixed in your lab book so heat fixed Mount so this you start off with a wet Mount so you take bacteria you swirl it into a drop of water on your slide except now we're going to let your slide dry and then we're going to heat fix it so you're going to pass it over a flame two or three times and these fixed mounts or smears the bacteria get fixed and they actually get killed on the slide so when you look at them they shouldn't be moving if you did it the right way once you have your heat fixed Mount or your heat fixed slide that's when you can start doing the fun part which is the staining part so staining we use this to increase the contrast so we can actually see the bacteria cells are not just white um blobs that you can't really see very well so one type of staining we can have positive staining this is when your stain sticks to the surface of your microorganism so your surface of the microorganism they're usually negatively charged and then you add a basic or or it's a positively charged Dy so your negative cell attracts the positive Dy and so your positive D sticks to your actual cell so positive staining you're going to color your cell kind of the opposite of this is negative staining so this is when your microorganism has a negative stain or negative charge to it you add a negative or acidic d to it negative repels negative so you're actually staining the background and your cells are going to going to appear white when you look at this slide and we're going to be doing positive and negative staining in lab other ways to categorize staining so some stains are simple stains this is just where you use one dye or one stain to stain or give color to your bacteria cells and this is usually done to reveal the shape size and arrangement of your bacteria that you're looking at so I'm going to quickly run through the procedure of a simple stain so if we start at the top left hand side you're going to spread your culture in a thin film over the slide and usually you're going to spread it in water you're going to let that water dry in the air and then you're going to pass your slide through the flame to produce that heat fixed part of the slide once you have your slide heat fixed you're going to flood the slide with a stain so here we're using a pink stain you're going to let that stain sit on there for the amount it si in your labbook and then you're going to rinse the stain off with water and blot dry your slide then once your slide is dry you can look at it on your microscope and again we're going to have to be using your oil objective lens so I'm going to show you how to use the oil in lab different types of simple stains you can use are um we have Crystal Violet which is the image a on the right it's the purple image you have methylene blue which causes the bacteria cells to appear blue and then the third image the pinkish red that's a saffrin stain so we're going to be doing all three of these stains in lab to practice your simple staining one once you have your simple stains done and you feel comfortable with those the other type of stains we're going to do are called differential stains these stains are a little more complicated they have more steps to them so you usually have a primary stain and then in addition to your primary stain you can have a counter stain and this differential stain it helps you distinguish between different cell types or different parts of your cell and examples of a differential stain you can have a Gram stain which is shown on the left here and Gram stain we're going to do this multiple times it's one of the most important stains that you can learn in microbiology so in a gr stain you're going to have your primary stain which is Crystal Violet it's going to cause the bacteria cells to appear blue or purplish these blue or purple bacteria are considered to be gram positive the counter stain is going to be sa in which causes a pink or reddish to your gam negative bacteria another type of differential stain we're going to be using is an acid fastin these are good at um identifying acid fast bacteria so micro bacterium is an example of an acid fasting so on the acid fast stain you have a pinkish primary stain and then your counter stain is going to be methylene blue and it causes anything that's not acid fast to appear blue in color so I'll be doing these and again these have multiple steps to them so you're really going to have to pay attention takes a lot of practice to get a really good stain out of this the third type of stain is called a structural stain these stains they reveal certain cell parts that are not revealed by just normal staining methods or conventional methods so two examples of structural stains will do a capsule stain so some bacteria have a capsule around them which protects the bacteria cell from dehydration and the immune system so you can actually see the capsule another type of structural stain is the flag flag stain this is where you can see the flatula or the really long string like structures coming out of the bacteria and flula will get to this but they're used for cell movement