this is part two of two on chapter three tools of microbiology in the first part of this chapter we looked at the different types of microscopes properties and microscopes and then we also looked a little bit at different types of staining this part of the chapter is going to really focus on other parts of those six eyes of microbiology so just to remind you here are our six eyes of microbiology we're not going to go into a lot of detail on each of these you can actually find them in your book to help you finish with your homework packet so we kind of focused on the inspection part and information gathering a little bit and identification in the previous part so now we're going to be looking at isolation techniques and inoculation techniques as well so looking at how to isolate colonies and how to actually inaugurate inoculate or use different types of auger to grow your bacteria or specimen that you collected so start off with isolation techniques so isolation a lot of times when you get a sample you have lots of different bacterias species mixed into that sample so you're going to take that sample you're going to spread it out on a plate or put it in media for example and then put it on a plate each of these different bacteria species they will get onto the plate and then they'll divide and grow so they'll go through binary fission and then you'll end up with something called a colony so on our plate we have white colonies and yellow colonies and you can tell colonies apart they're going to have different colors different sizes different shapes different textures and we'll look at colony or the macroscopic part of colonies in one of our labs so because they look different these two colonies look different from each other we can assume that they're different species of bacteria so some isolation techniques you can use something called a streak plate technique or a four quadrant technique and we're going to be using this in lab so you get to practice this but the street plate technique you take a sample you put it in one corner of your plate and then from that one corner you spread it out into the second quadrant the third quadrant and the fourth quadrant and you're not adding any more bacteria to your plate you're just spreading it out or diluting down your sample as you go around from your first quadrant to your fourth quadrant another type is called a pore plate technique so this is where you put your sample into a liquid or a broth and then you're going to dilute that broth down and put those samples on your plate so you have a sample that has lots of colonies lots of bacteria if you dilute it down you end up with fewer colonies on your plate and then hopefully you can see the two different types of bacteria in your sample a third type is called the spread plate technique this is where you take a very tiny amount of your sample and you spread it around using a hockey stick so you spread it around on the plate and then hopefully it's dilute enough where you get two different colonies you can tell them apart each of these three isolation techniques if you look on the far right hand side you can see that each of these techniques you end up with individual red colonies and individual yellow colonies and then from that you can pick up one of those colonies and that's called the isolation you can grow that specific bacteria species from then on so after isolation you can also do inspection so if you have a single species growing on your plate or on your slant or in your auger that's called a pure culture so all of the colonies look the same but if there's multiple species then you can have a mixed culture and mixed cultures usually we want to isolate out the different species of bacteria other times you can have something called contaminants this is something that we don't want at all and it's usually some type of mold or it can spread out or just appears in one place on your plate not everywhere on the plate so if we look at the images down here so if we start on the left so image a on the left we have yellow colonies and kind of a whitish cream colony as well so this is a mixed culture for image a image b we have orange colonies but then we have kind of this whitish blob that whitish blob is called a contaminant so your culture is mostly pure but it is contaminated and then image c these are agar slants they're not just flat plates they're slants in a tube we have three tubes that each have different type of bacteria you can tell by different color different growth pattern these are all pure cultures i don't see any mixed cultures or contaminants in these identification of a microbe so once you have your pure culture you've isolated it you want to identify what that bacteria is so we usually use this when you want to identify bacteria species that causing a animal or human to become sick so we can identify microorganisms based on their cell and colony morphology so you can look at the colony write down what it looks like if it has any color to it you can take a sample of your colony you can stain it so you can use simple staining you can use gram staining so some of those differential stains we talked about another way to identify microbe is to sequence the dna you can also do biochemical testing so looking at how that species of bacteria reacts to different environments or to different chemicals that we're going to be testing and biochemical testing we're going to do a big project the second half of the semester you can also do immunological testing and we're not going to do that in labs so i'm not going to talk about that and you can read about it in your book finally i want to talk about media so in order to grow bacteria in the lab you need to provide those bacteria with nutrients so media can be classified according to three properties so it's classified based on the physical state so you can have a liquid which is called a broth you can have semi-solid this is what we're going to be working with the most so augur it's kind of a gelatin like substance you can also have solid media which we're not going to be using in lab most of ours are semi-solid we'll also be using a few broth or liquid medias you can classify media based on chemical composition so we have synthetic media it's chemically defined or we can have complex media and then last you can classify your media based on what actually does what the function of it is so we have general purpose media this is nutrient agar or tryptic soy agar it grows pretty much anything we can have enriched agar where we add something to it selective auger differential anaerobic auger or media where we grow it in areas with no oxygen transport media if you're taking a sample and sending it to a lab essay media enumeration media so we have lots of different media that can be used for lots of different functions depending on what you need to do so we're using mostly semi-solid media in the form of agar so this is a commonly used solidifying agent it's kind of like gelatin so agar is solid at room temperature which is convenient it liquefies at boiling and above so you can heat it up you can liquefy it you can pour it into a plate you can pour it into a tube and cause it to solidify to slant and it re-solidifies at about 42 degrees celsius so as it cools it solidifies so the agar it does not provide any nutrients it just provides a framework where we can hold moisture and hold nutrients so the auger itself is not actually used by the microorganism you have to add some type of food or nutrients to the auger so commonly used media i mentioned nutrient agar we also have nutrient broth so nutrient broth is just a liquid media contains beef extract and peptone so it has protein in it the cells use that protein to grow nutrient agar it's solid media or semi-solid media so it has beef extract it has the peptone and then it has agar as well for the chemical composition of the media you can have synthetic media so this contains pure organic and inorganic compounds and an exact chemical formula so synthetic media we know exactly what is in this media you can also have complex or non-synthetic media where it contains at least one ingredient that's not chemically definable so you can have synthetic or non-synthetic media we have general purpose media this is like the nutrient broth or nutrient agar i just looked at this grows a broad range of microbes and it's usually non-synthetic and it grows pretty much anything another type of media is enriched so these contain complex organic substances such as blood blood serum hemoglobin or special growth factors that are required by fastidious or picky microorganisms so enriched media you have to add something more to it to get certain microbes to actually grow so examples of enriched media we can have something called blood auger which we're going to be using in lab so blood agar has blood in it it's sheep blood in with the auger and some bacteria are going to break down the blood cells and that's called hemolysis so if a bacteria species can break down blood cells it usually can cause some type of disease so we need to watch out for these microorganisms that can cause hemolysis or destruction of red blood cells other species of bacteria do not destroy red blood cells and that's shown in the image on the right hand side so these bacteria cells they produce colonies they can grow but they don't have a clearing around the colonies that shows hemolysis some media are selective and other types of media are differential so selective media they grow one type of bacteria and not another type so it's selective on what it can actually grow on this agar plate differential media this causes different species to grow but they have different reactions to the media so usually they turn different colors so selective media we use this we want to grow one type of bacteria we want to inhibit other types differential media is when we want to be able to identify different bacteria species on the same media some media can be both selective and differential at the same time the one that we're going to be using in lab is called mcconkey auger mcconkey auger is selective because it grows gram-negative bacteria but it does not grow gram-positive so it grows one type of bacteria inhibits another type it's also differential so those gram-negative bacteria they can either grow white or pink so if they grow to be white that means that that species cannot ferment lactose it can't break down lactose if it grows to be pink it means that gram-negative bacteria can ferment lactose so it's lactose positive other types of media you can have reducing media so reducing media contains a substance that absorbs oxygen or slows down the movement of oxygen so these reducing medium specifically a theo media t-h-i-o media it's used for growing anaerobic bacteria so bacteria that do not want to be an oxygen or they need a reduced amount of oxygen and then another type of media is called carbohydrate fermentation media so these different types of media they have sugars that can be fermented once it's fermented it's converted into an acid and that changes the ph and then your tube can actually change color so i'm not going to talk about these a lot you can again read about them in your book to learn more about them we're going to use these in the second half of lab when we get to our unknown project