Notes

Growth media

We'Re still good on the screen is that correct. You can still awesome fantastic all right. Let me know basically I'm mute yourself and tell me if something is going wrong. It'S gon na be fine, all right, sterile collection, as we mentioned, you don't want to inoculate to contaminate your medium of your sample by any unknown microorganisms that can reside your non sterile instrument, god forbid so medium. We discussed the concept of medium. Anything that supports microbial growth - and you will see many examples as we go through different systems of classification for the medium. So first, a classification approach is based on the physical state, so liquid medium liquid temperature can contain a variety of nutritional. Let'S say mixtures derived from soy like trip DK soya gar derived from yeast yeast, extract their media based on milk. It can contain blood for certain reasons that we will discuss, but basically the main idea it can allow. It allows you to first of all accumulate large amounts of microorganisms. save draft on instagram can easily cultivate liters or hundreds of liters of liquid medium that contains bacteria, for instance, or viruses, and produce an enormous amount of microorganisms. It can serve as the indicator medium. So, for instance, in this image in the bottom, you can see on inoculated control on the left once you inoculate this liquid medium that contains litmus indicator Decatur that changes its color. I don't remember if it's litmus or phenyl of telling the point is this medium is red because it contains pH indicator when you inoculate it with the fermenting bacteria. The result of fermentation in this case is generation of lactic acid lactic, acid decreases pH and decreased pH is followed by the change. The indicator color, so you see progressively it goes orange and then yellow so liquid medium is really convenient way to detect bacterial growth. Based on different indicator, approaches on the other end of the spectrum is the solid medium. You can see Anna gar, I believe it's a Tellarite agar, but I'm not sure hundred percent. You can see this agar plate, so it has a solid surface which allows you to characterize colonies from the standpoint of the appearance. When you grow bacteria, you can say on this agar my bacteria grow as the round sharp-edged convex colonies. I don't know white-collar whatever you know motile non-motile glossy, so you can do a lot of characterization a lot of descriptive analysis of the appearance of your microorganisms for the convenience we use agar, which then you know actually it can be found in various. What'S the proper word like baking products, I guess like jell-o's, it's basically a derivative, it's derived from algae, it's a powder. You can buy it in a grocery store if you mix it with water or a buffer, and you heat it up. It'S gon na. It'S called melt, not really melt, well kind of melt, and then, when you pour it into the petri dish like this, it will solidify. This is why we often call the agar medium liquefiable, solid medium. You can liquefy it another. What'S that is that a comparator'real another material that can be used for bacterial cultivation is gelatin? If you decide to do a home-based microbiology and do gelatin petri dishes, beware, some bacteria can eat gelatin. So, in the end, you may end up with no medium and no bacteria in your plate between the liquid and the solid medium. There is a semi-solid medium without a solid surface I mentioned yesterday. It is like a clot, so here in this image, you can see four so-called slant tubes. They called slant because well the surface is slanted now. If you will hypothetically take this tube turn it upside down and live it like that for a few days, the medium will slowly slowly drip out of that tube. Very excruciatingly Li slow so really like nobody doesn't so what are main uses for semi solid medium? Obviously, it's not good for accumulating large amounts of microbes. How you're going to filter them out how you're going to separate them from the medium and actually, neither is agar. Agar is not good for accumulation, it's good for characterization in isolation, okay, so if we can't accumulate large amounts of microbes in the semi solid medium, what do you use it then, for first of all, you can determine the motility of microorganisms. So how would you do that? Imagine that this is the tube with semi solid medium inside what you would do. You would take a wire sample, your bacteria and inoculate your medium in the vertical motion in the center. Essentially, you create a trace of bacteria in the very center of your medium. If your bacteria cannot move, you will observe growth exactly where you inoculated. However, if your bacteria can move, then you will see the spread of bacterial growth to the site. I hope it makes sense. Another useful application of semi-solid medium is to identify different bacterial species based on the growth appearance, and this is one of my favorite examples really so these four tubes all represent so-called triple sugar, iron, agar or iron slant. So what does that mean? Triple sugar part refers to the presence of three different kinds of sugars, carbohydrates, glucose, lactose, and, if I remember it correctly, Manos, but that's not so much so important. The third one is not important, so glucose and lactose, first and foremost, now iron refers to the presence of soluble iron salts. Three valent iron, I'm not gon na. Ask the valence. Believe me don't worry about it. So here's the point on the right. You can see. Uh nanaka lated medium, that's pretty clear! So, what's on the left on the left, you can see slant inoculated with the bacteria that can ferment glucose ferment lactose and on top of that Ron, aerobic respiration producing co2 that is essentially will be equal. I you know intestinal bacteria so and so forth right this one appears slightly different and you can see the change in color right change in color, it's yellow because of the fermentation this empty space at the bottom of the tube, that's co2. So that's very characteristic appearance. Next one, this one yellow ish, only glucose for matter, no lactose, fermentation, okay or very weak, lactose fermentation and no co2 production, so that is different. That will be either Serratia, marcescens or enterobacter. Arrow Jenna's also two microorganisms that can be found in a human GI tract and finally, this one that if ur, is totally black so why it is black. It turns out that some microorganisms, namely produced mirabilis and Salmonella typhi, I believed named salmonella, sounds unfortunately very familiar. Both these generous microorganisms can generate hydrogen sulfide hydrogen sulfide binds to three valent iron, and that leads to the formation of iron sulfide, which is a non soluble B black. Basically, there is a video if you curious, there is a video in the in the lab playlist with the lab. I believe it's lab number 10 called entry tube. I mean it's not G or anything. You can just watch it and in this video I discuss one of the tubes enter tubes that have this very characteristic: hydrogen sulfide black appearance. It'S like you see that you immediately say it's either produce or salmonella one out of two. No doubt nothing else can do that. Okay, so that's that's convenient so next topic of conversation, our classification of media, by composition and by purpose so composition, complex sorry, anyway, it's good complex media, peptone yeast extract milk, beef and heart infusion, for instance. Yes, there is such a thing, so bovine heart, not beef, a brain and heart infusion. Sorry, so bovine brain and bovine heart are dried out and then can be dissolved in water to produce medium. Obviously, when you take card and brain and dissolve it in water that that's gon na provide a lot of nutrients, so complex media are great to cultivate a variety of microbes from the sources. You don't know what what's gon na grow there, but something will actually. I plan on inoculating some of that gar plates for environmental samples to see you know what I can isolate from my own house. However, you can't truly control the nutritional composition, for instance, different batches of yeast extract or different batches of brain heart infusion. They may differ slightly because well, yeast, the different cows are different, so that may affect the outcome of your experiment. To avoid this, to control for the variable of the nutritional composition, scientists use synthetic medium. I mentioned it yesterday you take buffer with a known. Well, we take water, you start with water, it's salts, yeah, carbohydrates, amino acids, various chemical components which you can exactly measure the amount of which you can exactly measure. So at every given moment. You know what is in your medium and then you can add, remove alter. You know, change the amounts and therefore you can see how medium composition affects the growth of microorganisms. It'S basically, the main purpose now, based on the purpose media, can be divided into general purpose. Media ts a triptych: a soil, gar, a whole slew of media grow. Okay, enriched this one's. This media can support the growth of fastidious microbes. I mentioned new Syria meningitidis in Syria gonorrhea yesterday plague. Your senior best is an example of a fastidious microbe. They may require various growth factors or essential nutrients, so, for instance, for me, Syria, blood or chocolate agar both of them contain blood in either denatured or Nnamdi, natured form, meaning that they contain a lot of ayran. Our selective media blocks growth of certain microorganisms, for instance mannitol salt agar, contains a lot of salt. Salt is not really conducive to bacterial growth, except if bacteria are what's called hallow filling and interestingly enough. Staphylococci bacteria that we discussed yesterday are indeed hollow philic. So you can use that mannitol salt agar to selectively culture staphylococci, while inhibiting the growth of other microorganisms. Another good example is EMB agar, which contains two guys Erin and methylene blue, and these dyes inhibit the growth of gram. Positive bacteria. Now, unlike selective medium, that selects for a certain kind of microorganisms differential media allows you to distinguish between different microbial species distinguished by their appearance, so, for instance, on the blood agar, which contains intact, basically sheep red blood cells. You can see the difference between various microorganisms. In terms of their ability to destroy red blood cells, some of them will completely obliterate any traces of red blood cells, and we call such microorganisms beta hemolytic. Some of them can partially destroy red blood cells and they will be alpha. Gamma lytic and some bacteria will not do anything I mean they will grow red blood cells will still remain intact. We call them a gamma gamma lytic macconkey agar, for instance, allows you to distinguish bacteria that can or cannot brain fart. I'M sorry can or cannot. Ferment lactose, so many of them grow some of them ferment, some of them don't now the question sort of that may have. You may have now about selective and differential media. Can they be both? The answer is sure. For instance, macconkey agar very strongly inhibits gram. Positive bacteria allowing the growth of gram negatives: is it selective? Yes, it also allows you to differentiate between lactose fermenters and non for matters is a differential, absolutely it's both selective and differential, so the bacterial cultures that you obtain your medium, whether it's liquid or solid or semi-solid, can be pure or zenok single species. We will try to produce an acidic culture during a small part of the lab number four, which is the streak flight. We can be mixed, that's what I expect to see on our and wash sample, I'm kind of hesitant to check on it. I really want to see growth, but I know hopefully it's not incubator and they can be contaminated, undesirable microbes. Unfortunately, it's a damnation of the microbiological lab to avoid contamination. It'S really hard now. Another question that we need to answer is the cultivation of viruses. How how the heck do we do that viruses, as I mentioned, are obligate intracellular parasites so for a very long time they were cultivated in animals? I mean animals consists of cells right, you can inoculate animals and viruses will drown the cells, but that's a pretty darn expensive method to grow viruses because animals are expensive. So, in the late 40s early 50s Italian scientists worked in england, renato dulbecco, developed a revolutionary method that is called a cell culture. So what it is exactly the right on the slide. You can see a flop, a set of flasks, let's sit on the side and inside of the flask, you can see a red liquid, so I can tell you right away that on the kind of on the bottom of this flask each flask here here on these Sides of the flasks there will be cells growing in a single layer, so they grow on the plastic and this red liquid is nutritional medium that supports cellular growth. But the point is, you can add virus to these cells? Wait! No, it did. Oh, can you see that now yeah? Sorry, it was at the very bottom sorry yeah. So this this is the virus thing. Okay, so at the you can add viruses, okay and viruses will infect those cells and the cells will grow. It'S basically: okay, not cell. Sorry, viruses will cultivate, will grow in those cells and you can collect them because viruses will leave the cell you know so, but the point is you really need cells to replicate viruses? This is why developing a cell culture modal is so so important and so crucial for virus cultivation. You can just you, can just take virus, throw it in the medium and then same huh, yeah, that'll work. Actually, one of the one of the interesting questions that we discussed when you hypothetically, when you tap the tsa agar to see what's on your hands, you can detect a bacteria. You can detect fungi fungible grown tsa as well, but viruses you cannot because there are no cells that viruses can grow in. Does that make sense to you? Okay, before we kind of cut the recording off any questions? Okay,

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