| 00:02 | OK, folks, welcome to the part of chapter six where we're continuing |
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| 00:06 | discussion on microbial growth. So last we looked at um the physical and |
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| 00:12 | chemical requirements for growth uh as well growth media. And um and today |
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| 00:22 | gonna look at um growth dynamics a bit more about gross medium and end |
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| 00:30 | uh biofilm formation. OK. So always, there are the learning |
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| 00:38 | This is a checklist to, to through after you finish this module that |
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| 00:44 | uh um can, can answer OK. So uh continuing with the |
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| 00:48 | medium. So remember, we combine the chemical requirements for growth into a |
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| 00:55 | medium and then we of course incubate at proper conditions provide adequate levels of |
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| 01:03 | if required, et cetera. And we can also look at growth media |
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| 01:07 | terms of uh functional type we call . Um There's often what's called a |
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| 01:13 | purpose medium that will grow a number different types uh there, but there |
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| 01:18 | no one medium that will grow of course. But uh oftentimes we |
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| 01:22 | to what's called the heterotrophic medium. a general purpose medium is would be |
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| 01:26 | like a nutrient, a a nutrient that fits that description. Um generally |
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| 01:34 | type, a complex type of medium that um enriched medium. So there |
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| 01:40 | we kind of try to provide conditions to favor the growth of specific types |
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| 01:45 | others, particularly it comes in use uh environmental samples. Recall when a |
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| 01:51 | kind of did this to to be to uh discover the little tropes. |
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| 01:57 | so you provide conditions that will uh specific to a certain type you're looking |
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| 02:02 | and then that will tend to only tend to favor their growth over |
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| 02:06 | Ok. If you, especially using media, if you're trying to find |
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| 02:11 | types and samples that tend to be in number compared to their uh |
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| 02:16 | and you can oftentimes do this by using specific nutrients that you, |
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| 02:20 | know that favor, favors their Ok? Uh Anaerobic growth, of |
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| 02:25 | , you'll be dealing with uh anaerobes don't uh that cannot live in the |
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| 02:28 | of oxygen. There's, there's there are methods to do that. |
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| 02:32 | It can be kind of laborious, whatever means you use, you have |
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| 02:36 | keep oxygen out of it. So typically have to displace that by bubbling |
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| 02:41 | gas and it is very typical you um uh what are called aerobic chambers |
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| 02:45 | do your culture work. Uh The exclude oxygen. Uh So, um |
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| 02:51 | , it's certainly done and has been for a long time. But uh |
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| 02:55 | number of protocols are developed for But again, it involves uh it |
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| 02:59 | be a little laborious, having to , do the things you need to |
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| 03:03 | to keep it anaerobic um as So you can um different types of |
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| 03:10 | testers, different media formulations that will you to detect, for example, |
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| 03:15 | of certain types of sugars um to for different metabolic properties based on oftentimes |
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| 03:22 | reactions you see occurring in media uh well. You can just take samples |
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| 03:26 | , of cultures and, and test different types of enzyme activities, things |
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| 03:30 | that. So uh media is gonna that purpose as well. The the |
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| 03:33 | paced negative positive is an example of by chemical tests. They're is looking |
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| 03:38 | uh for the ability to, to fats, for example, and you |
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| 03:42 | see that by this organism producing a which degraded the fat in the media |
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| 03:48 | it has these light pace enzymes to that with. OK. Um The |
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| 03:54 | aero tolerance, the the fluid like looking at growth patterns. That's an |
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| 03:58 | type of medium that you're testing to what's the behavior uh to oxygen of |
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| 04:02 | microbe. OK. So a couple examples there then of course, we |
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| 04:06 | a differential medium and we'll talk about and out for this one. |
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| 04:12 | Um As well, you have, talked about forms immediate. So solid |
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| 04:17 | liquid. There's also a solid You see over here on the upper |
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| 04:21 | , called slants or auger slants can used as well. Typically, these |
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| 04:25 | used for um storing cultures because tubes take up small space in the |
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| 04:31 | uh which is typically where you'll store lot of your live cultures. And |
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| 04:35 | that's typically where slants come in to . You don't really use slants to |
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| 04:40 | single colonies as you can see from very thick yellow streak line there. |
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| 04:45 | but that's more for storage of cultures . Um So culture media types talked |
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| 04:52 | this last time and then the um purpose for each of those types uh |
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| 05:00 | the medium. So, selectively, uh uh there's additions to the medium |
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| 05:06 | will uh inhibit certain types. Uh an example of that here. So |
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| 05:13 | media is one that produces color, typical kind of uh reactions that produce |
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| 05:19 | uh that can tell you something about metabolism. OK. So uh there |
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| 05:24 | be media that can be only there can be a media that can |
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| 05:27 | only differential, there can be a that can be both types. |
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| 05:31 | So the Heins and a you see is is both, both of |
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| 05:36 | it's both selective and differential. So it has chemicals that prevent growth of |
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| 05:41 | positive bacteria. And so it favors growth of gram negatives. That's how |
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| 05:46 | selects it's differential because among gram negatives grow on it, you can detect |
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| 05:51 | they ferment lactose or not or produce sulfide, which can be a metabolic |
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| 05:56 | product. And so the hydrogen sulfide up as a black color, uh |
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| 06:03 | fermentation. So shows up as kind yellowish colored colonies. Ok. So |
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| 06:08 | actually differentiate two different features here, fermentation plus or minus uh H two |
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| 06:15 | formation. So again, it's both and differential blood art is a type |
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| 06:20 | only differential because there's no agents in in agents in there that will inhibit |
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| 06:25 | microbial types. But um for, other types that have certain types of |
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| 06:31 | enzymes called hemolysin homos, break down blood cells. Uh there can be |
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| 06:36 | degrees of that. So in you see a, a microbe that |
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| 06:40 | cleared the area where it's growing because lied to red blood cells. Uh |
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| 06:45 | a type of hemolysis, we call beta hemolysis. The middle one in |
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| 06:49 | B is one exhibiting what's called alpha . So it's like a partial um |
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| 06:57 | . So it kind of basically it the hemoglobin in the red blood |
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| 07:01 | creating this kind of greenish color with little bit of lysis. So, |
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| 07:04 | we call a partial hemolysis and then is one where there's none. So |
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| 07:09 | A is actually used oftentimes to cultivate . Uh strep throat organism is one |
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| 07:16 | would show an a type of complete hemolysis, oftentimes it can be |
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| 07:20 | and you see the clearing like you in a and you do microscopy and |
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| 07:27 | gram positive cox chains. That's pretty presumptive for uh strep throat bacterium. |
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| 07:34 | So the blood art was used a in clinical type of microbiology. |
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| 07:40 | It's a little bit about dynamics of . So uh we all we know |
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| 07:44 | of course, bacteria divided by binary , right? And so when we |
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| 07:48 | about uh generation time, OK. Generation time is really the, you |
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| 07:54 | of it two ways it can be time for a soap to divide in |
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| 07:57 | as you see there. Uh But it can be the time for a |
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| 08:01 | to double both those, both those you the generation time. OK? |
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| 08:06 | in growth, you're looking for really , what's the rate at which these |
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| 08:11 | are forming? OK. So you an idea there in a green box |
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| 08:15 | , of um really how fast the cells can grow or I can go |
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| 08:20 | one to a million uh in 6 8 hours for E coli, for |
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| 08:25 | , under optimal conditions, uh we look at the population size represented by |
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| 08:30 | equation where in its number of cells at uh at some 0.0 times zero |
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| 08:36 | the start and in t at, some point in the future, and |
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| 08:42 | can, if you calculate the number generations and we can go to, |
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| 08:45 | the two to that power times number you started with in zero, we |
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| 08:50 | you the number of cells at any time. OK. Of course, |
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| 08:53 | have, you have to figure out many generations have passed. OK. |
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| 08:57 | um uh the, the rapid rate growth uh certainly can, certainly happens |
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| 09:05 | a laboratory because we control conditions and , and we use pure cultures. |
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| 09:10 | uh but here in the lab, can grow, um we'll grow at |
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| 09:15 | we call unlimited or what's called exponential . OK. Kind of the growth |
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| 09:20 | see in that green box there uh to 2 to to 4 to |
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| 09:25 | et cetera, right? That kind uh exponential logarithmic if you will progression |
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| 09:30 | typically logarithmic exponential growth are kind of interchangeably in microbiology to express this really |
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| 09:36 | rate of growth. OK. And base 10 comes in because whenever you're |
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| 09:41 | , you're looking at numbers which are by a big range, right? |
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| 09:46 | talking about one digit up to 10 the sixth range or beyond, we |
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| 09:51 | to compress the scale. So we um make it more manageable. |
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| 09:55 | you see the launch of the base think of the PH scale. It's |
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| 09:58 | same thing. It's also a long 10 scale. Uh because to reflect |
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| 10:02 | big increases in in hydrogen ions that occur over the span of a PH |
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| 10:07 | , for example, OK. So lot of base 10 is typically used |
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| 10:10 | , to when you're dealing with numbers vary by a very large amounts. |
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| 10:14 | ? To kind of help compress And uh we can see that |
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| 10:19 | OK. So it's just, I do this in just to show you |
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| 10:22 | of, you know, if if you plot a growth using a |
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| 10:26 | based 10 on just regular graph which is what you would see um |
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| 10:32 | kind of here on this curve OK. Uh rather you plot it |
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| 10:38 | , not using log to base but you use kind of the numbers |
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| 10:41 | see there that it doesn't really reflect rate of growth going on. But |
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| 10:45 | you, if you convert to log base 10 number of cells, |
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| 10:52 | as you see here, then you this linear function. And so that |
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| 10:58 | really the fast rate of growth that's , right? Which you really wouldn't |
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| 11:02 | from this curve by not using log base 10 because the numbers are so |
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| 11:06 | and spread out, right? But we compress the log to base |
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| 11:09 | then you can really see the rate growth and we can use that information |
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| 11:14 | calculate how fast it's growing. And do comparisons like maybe you want to |
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| 11:18 | an antimicrobial agent and and how it's growth and you could actually get the |
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| 11:23 | numbers and see, OK. Is uh is growth going down something like |
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| 11:30 | or like this? Ok. Is affecting growth? So those are kind |
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| 11:34 | things you can do? Ok. the point is that this rate of |
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| 11:38 | can be very fast under optimal Ok. If you provide bacterial species |
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| 11:45 | a in a shape, fast growth with everything it needs, they can |
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| 11:49 | at these very fast rates. Uh not. So the case in |
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| 11:53 | because of course, they're competing with , they are oftentimes nutrients are |
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| 11:58 | So they there can be a limited in nature, but it occurs in |
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| 12:03 | here and there where there happens to an influx of nutrients, let's |
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| 12:06 | OK. But typically it's not the um in nature. OK? But |
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| 12:11 | it is in lab because you can everything. And just to give you |
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| 12:15 | idea of the power of the doubling , right, the generation time. |
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| 12:20 | this is what this example is meant show. So if we have uh |
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| 12:23 | start with 10 cells and a bacterial doubles every four hours, what will |
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| 12:28 | the population size after 20 hours? if the doubling time is 15 |
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| 12:33 | So we're, we're doing the same , 10 cells. How many do |
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| 12:36 | have it for 20 hours with two doubling times? OK. So here's |
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| 12:42 | we set up the problem. And so this is basically what we're |
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| 12:48 | on both sides here, right? with different generation times. So before |
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| 12:52 | doubling time that we are that for value represents one generation every four |
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| 12:59 | That's what doubling time refers to generation , right? Is how many uh |
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| 13:03 | the number of the for two One generation? And how many hours |
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| 13:08 | minutes? OK. So we're asking 20 hour timeframe, what that would |
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| 13:13 | , that would equal five, And so that's the number, this |
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| 13:17 | the number of generations that have passed that time frame. With that doubling |
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| 13:22 | , we just plug in numbers right? NT equals N zero. |
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| 13:26 | started with 10, this is N N two to the fifth, which |
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| 13:29 | what we calculate this with 320 cells 20 hours with that doubling time starting |
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| 13:35 | 10 cells. So we go from to 3 20 in 20 hours with |
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| 13:40 | four hour doubling time with a 15 doubling time. So that's one generation |
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| 13:44 | quarter hour, right? Or 15 gives us 80 hours cancels out. |
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| 13:50 | to two to the 80th, so can see that significantly four hour versus |
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| 13:56 | minutes doesn't seem like a lot but in the end, right? The |
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| 13:59 | of cells you're getting 10 to the cells in that 20 hours compared to |
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| 14:04 | that for our doping time. So you an idea of really how fast |
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| 14:09 | optimal conditions bacteria can grow. So we look at what's called batch |
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| 14:14 | so here we are looking at um in batch growth, what you're basically |
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| 14:23 | is taking a a receptacle that will your growth medium and then you |
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| 14:33 | OK. So we begin to batch by basically not turning a growth medium |
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| 14:38 | then basically just monitoring the rest of way. And we don't do any |
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| 14:41 | manipulations to the culture other than taking and and getting a growth curve, |
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| 14:46 | is what we call a batch growth . OK. So again, with |
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| 14:50 | inoculum inoculation of the medium, we um be in what's called lag |
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| 14:59 | OK. So that's the rest stark think of this as an acclamation |
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| 15:05 | Uh the cells are gonna use their , so to speak, there may |
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| 15:08 | slight temperature changes uh that have occurred where they came from. So |
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| 15:12 | you're not getting as coming from some , whether it's a plate or another |
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| 15:15 | culture, they're coming from there into new medium and there can be changes |
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| 15:20 | may occur. Maybe it's a completely growth medium with different nutrients. Uh |
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| 15:24 | may mean it may have to um different genes in order to be able |
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| 15:29 | utilize new nutrients, uh may have turn genes off. So uh collectively |
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| 15:34 | all amounts to there being a period adjustment, adjustment and, and reading |
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| 15:41 | before uh vigorous growth can begin. all that's what we call is occurring |
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| 15:46 | the lag phase. OK. So no growth. The soldiers just have |
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| 15:50 | entered this new medium and they're getting and doing what they need to do |
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| 15:54 | adjust and then growth will begin. when it does, it can happen |
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| 15:59 | rapidly under optimal conditions. Ok. of things can affect lac face. |
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| 16:03 | can be, you know how, many cells are you adding to |
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| 16:07 | Are you adding to this new Uh is the incony coming from a |
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| 16:12 | that's very old. Maybe there's not lot of live cells there or maybe |
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| 16:16 | , maybe it's a very fresh medium it's the same medium you're inoculating |
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| 16:20 | So um maybe the media comp compositions very different or maybe they're the |
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| 16:26 | All right. So all these can will determine how long or short the |
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| 16:31 | phase is and it, and it vary. OK. So once active |
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| 16:36 | has begun, then we're in lock . And of course, this is |
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| 16:40 | be its most about the active Uh fastest growth rate you see, |
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| 16:44 | cell size even increases a bit because , you're gonna see lots of cells |
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| 16:48 | are in states of dividing. So of cells will be looking uh if |
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| 16:54 | a rod shaped cell will be a getting, then they may have a |
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| 16:57 | here that will form, that will the cell into two. So lots |
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| 17:01 | cells kind of uh many cells are of kind of in this state of |
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| 17:06 | bigger than dividing. And so that's you see this in long phase. |
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| 17:11 | the uh um and it's really as if you're measuring some kind of uh |
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| 17:19 | with the cell, some kind of , that's when you would do it |
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| 17:21 | a log phase because you know, gonna be at, at its highest |
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| 17:24 | typically will be in, in mid , mid log phase approximately. |
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| 17:31 | Now, as we get down to late log phase, which is probably |
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| 17:35 | be somewhere around here, excuse So as you get the late lock |
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| 17:53 | , excuse me, kind of around phase, uh then we're gonna begin |
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| 18:03 | become limited for nutrients. Ok? of course, at that point, |
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| 18:07 | have such a high cell density, not gonna have enough nutrients to feed |
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| 18:10 | . So of course, the growth is going to begin to slow down |
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| 18:13 | we get the stationary phase. Um of course, the growth rate and |
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| 18:18 | rate are pretty much equal. So makes us a flat uh plateaus. |
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| 18:23 | phase is characterized by a plateau And then of course, it becomes |
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| 18:27 | matter of survival now. So, are becoming nutrient limited. Uh And |
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| 18:32 | it becomes a matter of they're And so it becomes a matter of |
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| 18:36 | doing things that will enable their survival if you know, nutrient, maybe |
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| 18:43 | are added or something favors their Of course, it won't in batch |
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| 18:47 | , they will eventually die. but they try to prolong death as |
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| 18:51 | as they can. And, and of the things they do is to |
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| 18:53 | a little bit smaller, smaller not less uh cell mass to keep |
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| 18:57 | with. Um they'll begin to uh off a lot of functions and only |
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| 19:03 | functions that are necessary. So a of uh gene expression is turned |
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| 19:09 | Um So again, it's all in effort to kind of conserve energy. |
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| 19:14 | uh waiting it out, prolonging survival the hope that maybe things will get |
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| 19:21 | . OK. But eventually, of , in match growth, it doesn't |
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| 19:24 | they'll, they'll run out of food . And then of course, cells |
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| 19:29 | to die and just like growth can logarithmic. Uh There can be an |
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| 19:35 | algorithmic decline in death phase and that can happen. So you can see |
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| 19:40 | in log face growth very rapid. too will death be very rapid as |
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| 19:47 | ? And that's what we focus on chapter seven is, is, is |
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| 19:51 | negative slope of death phase? How we make that fast? And, |
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| 19:54 | , and very, you know, negative slope. That's what we're interested |
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| 19:58 | in, in when we're controlling growth , and adding antimicrobial disinfectants is is |
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| 20:03 | really increase death. OK? Because can, it does, it can |
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| 20:08 | does decrease at a rate that's logarithmic exponential like log phase was time um |
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| 20:17 | one kind of uh thing you can . All right is with the batch |
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| 20:22 | , you can actually uh feed nutrients you wish to get more cells. |
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| 20:27 | if you're at, so if you're a point like you see here, |
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| 20:31 | ? And batch growth, right? we can add nutrients here, |
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| 20:39 | And that will prolong the growth. so we can actually get beyond what |
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| 20:44 | would if we just were stated batch . So fed batch growth allows you |
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| 20:48 | add nutrients to, to the growing so that they can achieve a higher |
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| 20:53 | density if that's what you're looking OK. And that's, that's, |
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| 20:58 | is actually kind of common to do you, if that's what you're |
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| 21:01 | if you're trying to achieve a higher yield for various reasons, it's what |
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| 21:05 | call fed bats growth and the, the, the nutrient you basically |
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| 21:10 | it's gonna be carbon, carbon is be the, the nutrient has the |
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| 21:14 | effect in terms of determining cell final yield. So if it's a |
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| 21:19 | you could add glucose to it. example, uh if you know the |
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| 21:23 | can use glucose, you'd add glucose glucose at that point of say maybe |
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| 21:27 | mid log phase, that glucose and it will, you use that |
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| 21:30 | and grow beyond what it would if , if it didn't have the |
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| 21:34 | OK? Um As well, you even control growth even more right, |
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| 21:40 | can uh have a microprocessor that uh you to grow the organisms. Uh |
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| 21:46 | an integrated system using a bio reactor has uh automated pumping. So you |
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| 21:52 | pump acid and base to maintain ph you can add more 02 to, |
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| 21:59 | supply the needs for an aerobic organism as it grows, uh you can |
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| 22:04 | faster to, to add more air it. So, and this is |
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| 22:08 | automated. So you can have, can attain very high yields basically because |
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| 22:12 | controlling all the all the growth parameters um making the cells very happy, |
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| 22:18 | , which enables them to grow to high cell densities. Uh those that |
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| 22:22 | work in industry in uh biotech. example, these are the kind of |
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| 22:26 | that you might do if you were uh working in, in um in |
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| 22:33 | field. OK. So we'll close session with biofilm formation. OK. |
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| 22:40 | biofilm formation. Uh uh So there's number of phenomenon that microbes that are |
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| 22:47 | to microbes that relate to, to nutrient conditions. OK. We talked |
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| 22:54 | uh couple modules ago about endospore That's one of the triggers for that |
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| 23:00 | actually lack of nutrients, right? can trigger that. Uh This phenomenon |
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| 23:05 | one that's actually triggered by plentiful OK. The other thing that's essential |
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| 23:11 | biofilm formation is um its formation is surface. So biofilms are all about |
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| 23:19 | . OK? That's, that's where initiate and that's where they grow from |
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| 23:23 | surf a surface right now, surface be your teeth if it's plaque, |
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| 23:26 | example, uh it could be uh a rock in a stream where mats |
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| 23:32 | standing water, even a form of biofilm. Uh This being pre nursing |
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| 23:40 | and many of you being in health eventually uh biofilms are important there because |
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| 23:45 | um different types of uh uh devices in um health care such as |
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| 23:55 | um uh heart valves, uh knee , hip replacements, um breathing |
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| 24:06 | All these are devices that are, are packaged sterilely, but if not |
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| 24:10 | properly can lead to infections. And of these are biofilm producers, these |
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| 24:17 | that will form on the surface and from a medical standpoint, these |
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| 24:21 | these can be quite, quite problematic in many cases dangerous and hard to |
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| 24:27 | . Ok. So we'll look at that here in the process, you |
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| 24:33 | , right. Again, it's all adherence. Soria is typically very |
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| 24:37 | Fimbria coli are important in this process that's what enables adherence to a |
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| 24:42 | And so if we look at kind the process here, right. So |
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| 24:45 | , it certainly it's characterized by a , it's characterized by having lots of |
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| 24:49 | there and that triggers the accumulation of and lots of bacteria to form these |
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| 24:56 | mats of growth that you see OK. And um so basically the |
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| 25:06 | form biofilms can actually have two what are called planktonic cells and those |
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| 25:10 | are called um uh often called swimmers and stickers, but they |
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| 25:18 | they adhere to the surface. platonic cells are kind of your motile |
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| 25:23 | that uh will seek a favorable And then once they do, they |
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| 25:28 | of lose their flagellum. And now all about the Riri allowing them to |
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| 25:32 | to a surface and they may collectively together to form micro colonies that then |
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| 25:38 | on to form uh larger colonies as one of the initial phases of this |
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| 25:45 | to form this very thick sugar, called exo polysaccharide layer that, that |
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| 25:52 | all of the members of the OK. So they kind of all |
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| 25:55 | this the of this material is triggered there's enough cells present on the |
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| 26:01 | And then this begins the the formation the biofilm and, and enables. |
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| 26:07 | we now have initially it's growth in two dimensions on the surface. It |
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| 26:11 | extends beyond that to three dimensional growth forming these biofilm towers that you see |
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| 26:17 | . OK. And again, this gonna be able to only be able |
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| 26:20 | be sustained as long as there's a of nutrients coming through. So it's |
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| 26:24 | common to see these in, in things like uh in pipes. Uh |
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| 26:28 | example, sewer pipes, et cetera there's basically a steady flow of nutrients |
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| 26:33 | by the platform formation on your teeth you're constantly, you know, you |
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| 26:36 | saliva and nutrients in there that can the biofilm. Um And so as |
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| 26:42 | as you have a supply of nutrients that can sustain the biofilm. Um |
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| 26:48 | of course, as you get into dimensional growth, you know, to |
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| 26:52 | that all the members of the population be fed, actually, holes can |
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| 26:56 | created so that nutrients can flow in out and around these towers to, |
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| 27:00 | be able to feed everybody. Uh Eventually though it, it can |
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| 27:05 | that the biofilm disappears because simply there's enough nutrients to feed everybody and then |
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| 27:10 | begin to kind of dissolve and then cells revert back to their motile form |
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| 27:16 | the plantal planktonic types. And then will go seek out uh a more |
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| 27:21 | environment and start another biofilm. So that's how these things uh form |
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| 27:28 | and disappear. But um you for a bio that's, that's uh |
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| 27:34 | and and is is sustained these of , can looking at the BioFoam |
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| 27:39 | you can see there can be several here and that's what makes uh that's |
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| 27:44 | makes um uh let's let's look at real quick here. So we look |
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| 27:50 | the uh uh catheters uh insertion of cat or some other kind of device |
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| 27:56 | provide the surface for that, And so a biro can form a |
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| 28:01 | thick layer and that thick layer will can actually retard the diffusion of antibiotics |
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| 28:08 | them. And so that's why it's hard to get uh biofilm infecting types |
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| 28:13 | bacteria uh because of their resistance to things. And so uh antibiotics have |
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| 28:19 | hard time penetrating. And oftentimes there be differences in, in, |
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| 28:24 | in the cells themselves, types that on the periphery of the biofilm versus |
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| 28:29 | that are more internal and you can different resistances uh evolve. And so |
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| 28:34 | infections with these biofilm types are, very, very um problematic. Um |
|
| 28:41 | And, and it's certainly something if , if you're gonna, if you're |
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| 28:45 | end up in health care, you're gonna be likely see, see these |
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| 28:49 | some of your patients. And um it requires um again, this is |
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| 28:55 | treatment with antibiotics that takes not uh week or 10 days, but more |
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| 29:00 | uh several weeks and even into months , to take care of this. |
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| 29:06 | , um so certainly many of these , are medically important types. |
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| 29:11 | So uh that closes out then the on growth. And um uh so |
|
| 29:20 | covered here in this section uh uh little bit more about growth and |
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| 29:24 | such a differential medium uh growth including like growth generation time, uh |
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| 29:31 | phases of growth. So lag log death phase. So you should be |
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| 29:35 | with those and of course, ending bio films. OK. So next |
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| 29:41 | , we will go into chapter seven we're looking at control of, of |
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| 29:44 | microbial growth. So we'll touch on of these uh topics of um the |
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| 29:51 | and chemical growth factors. Uh But in, in, in controlling |
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| 29:55 | it's about um really focusing on the on the death phase and, and |
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| 30:01 | and reducing microbial numbers and to do at fast rates. And so we'll |
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| 30:06 | at the, uh what's involved in . Ok. All right. Till |
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| 30:10 | time. Thank you folks. |
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