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00:01 | mm hmm. Oh but it's okay . Welcome. Alright. So I |
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00:46 | a lot to get any kind of at this point in the semester? |
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00:54 | we are on track, which I hard to believe not because of you |
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00:59 | because I've never been on track. um so today we're gonna finish uh |
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01:05 | quite finish up for but we're gonna uh at the end of sport part |
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01:10 | next time chapter five is pretty um okay. So maybe like doesn't result |
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01:19 | will go through mainly focusing on really focusing on control of microbial growth. |
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01:26 | physical chemical methods of control is pretty working to focus on. So chapter |
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01:30 | is not lengthy. So uh I'm only looking at a part of that |
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01:35 | . So uh we'll do that on and then whatever we have left |
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01:41 | we'll do Wednesday. So Wednesday is of those I called days. And |
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01:45 | just built built into the, into of course schedule in case you |
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01:51 | just to give some leeway if you know, get behind the have |
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01:56 | to catch up basically. But we're much on track. And so if |
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02:01 | have anything and just be a little of stuff to do on that |
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02:04 | Um then we'll start no, yeah start then the following week we start |
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02:10 | next unit. Okay, so you'll that material um begin to show up |
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02:19 | week. And so have the unit two Which won't start until the |
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02:26 | 21st 21st is when that would Um Let's see. Okay. Uh |
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02:34 | examples of the castle schedules open, know that the unit quiz. So |
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02:38 | this, the unit quiz go different only in terms of a little bit |
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02:44 | , just to be more comprehensive with unit quiz. So you know, |
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02:48 | be questions over the areas in chapters and four, I'll uh five isn't |
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02:54 | because we're not gonna get to it monday, but I'll um I'll post |
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03:00 | questions on checker five on on so you can have those to look |
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03:05 | as well. It won't won't count a quiz. It'll just be always |
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03:08 | questions and then I'll have the Uh keyboard. So uh I'll uh |
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03:15 | notice of that. I'm gonna send email tomorrow about stuff. Um What |
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03:21 | ? Oh, there is one thing meant to pull up the blackboard. |
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03:27 | , maybe I can do it and don't want to waste time with |
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03:30 | But what I'll mention it in the tomorrow. So um so when you |
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03:37 | to where you look at the the video lectures folder, right? |
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03:43 | you'll see a link in there called link to a site called says video |
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03:50 | . Video points site for lecture, ? All of this is, it's |
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03:55 | available yet, but it will be . Uh It's the same identical lectures |
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04:00 | they're used to seeing. Okay, just they're being uploaded to this site |
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04:05 | well in addition to so nothing I wanted to say something extra. Um |
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04:10 | video points does. It's uhh uhh and they they it's really just, |
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04:16 | only difference is how you can navigate you can search student lectures, you |
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04:20 | actually do a keyword search if you to look at a specific in this |
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04:25 | lecture on a specific topic, you actually search that and we'll go to |
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04:29 | . Okay, so so you had as an option? So I'm just |
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04:33 | just making it available to you. You may find it very useful. |
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04:38 | , so and then there's other features there as well, I'm not well |
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04:42 | in all of it. So I there's even like a little help or |
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04:46 | button if you want to look at the features, but it's just another |
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04:48 | to present the material. Again, it's the same lectures, I'm uploading |
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04:54 | one drive and just uploading it here well, but it gives you this |
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04:58 | search options if you want to explore . So uh you will be able |
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05:02 | see that tomorrow. Okay? Um don't I uploaded all the lecturers who |
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05:08 | done so far to that site but think it takes a while for him |
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05:13 | do that. Index is called So I think all but the last |
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05:20 | are set but maybe by tomorrow that be they will be set. But |
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05:25 | , it takes a little bit of to get the thing to the |
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05:27 | but again, completely optional. look look at it for yourself. |
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05:32 | again, I'll mention in an email . Okay, So let's go |
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05:41 | Um Oh, I wanted to put in just for grins. Okay. |
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05:46 | , um, the super Bowl is . Okay. Who will win the |
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05:50 | bowl? This sunday? Any Okay. I'm not sure if I |
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05:55 | Cincinnati. Right. I think that's . I had to tease in there |
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06:00 | initially I thought none of them Right. There's two edges on |
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06:03 | 2. Um uh I remember when Bengals were in the Super Bowl last |
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06:18 | . Tell you how old I 1984, I think. Mhm. |
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06:33 | , mm hmm, mm hmm. prediction is um d is gonna be |
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06:42 | majority answer. I think most people what the Super Bowl is, so |
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06:47 | not gonna I'm not gonna pick Okay. And let's see if I'm |
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07:01 | . Yeah. All right. I right. But you just picked that |
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07:05 | I said that so split between the and rams. That's interesting. |
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07:11 | Okay. I don't know. I to pick the rams, but I |
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07:17 | the Bengals defense. Mm Hmm. see. Um Actually, I'm actually |
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07:22 | the first time. Probably miss half the Super Bowl because I'm going out |
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07:24 | my wife for valentine's day thing. , but I will record it. |
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07:33 | anyway, let's get to some science guess. Okay. Um Alright, |
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07:41 | kind of what we've been talking so we went through everything up to |
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07:48 | dotted line last time, so we'll through the batch growth curve today. |
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07:54 | stages uh bad batch, a little about bioreactor growth, those of you |
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08:01 | biotech folks in here. I know of the curriculum is you do work |
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08:07 | bioreactors. Um Do you do kind a mini uh I guess semester long |
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08:12 | where you grow cells up in your and they're like um recumbent DNA plasmids |
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08:18 | them. Many that you harvest the and something like that. I think |
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08:21 | do things like that. Um So talk a little bit about fire, |
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08:25 | growth um And then dynamics. So involves in growth calculations. Nothing super |
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08:31 | . Okay. Um It's you it's it's logs, logs, the |
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08:37 | tens and multiplication and division. Pretty . Okay, so we'll go through |
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08:42 | F. Y. I. Um will be able to bring a handheld |
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08:48 | of any type. Okay with you the cost of testing center, they'll |
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08:53 | made aware that you're allowed this and that you won't be bothered about bring |
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08:59 | calculator, so so that won't be problem. Um It's only like a |
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09:04 | of problems, so it's not like a whole, you know, 12 |
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09:07 | or something, just a couple on . Um But we'll go through some |
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09:11 | of that and then we'll talk we'll get through biofilm formation but we're |
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09:16 | going to get through in those information that's why we have plenty of time |
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09:20 | finish that up next week. Um , so let's uh any any questions |
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09:28 | we go. Alright. My question not here today is just sitting right |
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09:34 | . Okay. All right. Um , so let's take a look at |
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09:39 | in case these are uh let's uh us over. And so while you're |
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09:46 | at this, I just explained so growth, okay, why why that |
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09:51 | ? So what it refers to? really a contrast between that and what |
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09:56 | do in a bioreactor? Really. . A bioreactor, as you'll see |
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10:02 | offers you really total control of your of your cells are growing so you |
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10:09 | you can influence all the parameters, ph auction levels, etcetera. |
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10:16 | Uh in a batch growth mode you necessarily doing that. So think of |
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10:23 | mode as you know, you make media in a cup in the flask |
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10:29 | uh you inoculate right? You're gonna it with some cells. Okay. |
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10:34 | then they will go through, they you a growth curve, something like |
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10:39 | , not identical in terms of the necessarily the same inflection of the phases |
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10:46 | things, but it's gonna give you those 1234 stages. Okay. Um |
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10:54 | um Mhm interesting. The so what doing, the only manipulations you're doing |
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11:02 | once you inoculate then at certain time after that you will take a sample |
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11:08 | you will measure growth somehow. The common way to measure growth is with |
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11:14 | absorbent students inspector for thomas. Or take a sample as a as a |
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11:19 | culture grows initially. When you inoculate looks pretty much like water or clear |
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11:24 | water and then as it grows it rick highly. It's called turbidity we |
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11:30 | it. Okay. And so as turbidity increases you know it you can |
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11:37 | light. Okay. And so you'll an increase in absorb into what we |
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11:40 | optical density or O. D. uh increase as it grows and then |
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11:46 | plateaus of course as you see there stage three and then um uh and |
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11:53 | what you're doing is getting a growth what's called a batch of growth |
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11:56 | Okay. Um and so it will , you can have the same species |
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12:02 | grow e coli on various types of complex defined what have you uh the |
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12:11 | media with different carbon torches maybe or you can manipulate. Okay. And |
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12:16 | will give you like a different different different patterns in terms of the length |
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12:21 | the phases and how sharp each phases so forth. So um so remember |
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12:29 | why you're doing this for a Right? So you are typically do |
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12:35 | growth curve um to see and then samples you know if you have a |
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12:42 | for interest of an enzyme activity. cells may possess a protein they make |
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12:47 | you want to analyze. Okay, um some other kind of activity And |
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12:53 | and you take samples over time to Okay, where where along this growth |
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12:58 | ? Am I getting maximal activity? it's typically it's going to be somewhere |
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13:03 | this range or you will. And that's and so you may not |
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13:09 | when it arrives at that point. ? So you didn't take samples to |
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13:13 | . So that's why I do the curve because you know, oh at |
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13:15 | time point here you can actually collect and then do my thing Or maybe |
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13:20 | just want to see well how how cells right this level here. I'm |
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13:27 | telling you how much souls you're getting . So maybe you want to see |
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13:33 | how high that gets right whenever you to get the more cells than |
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13:38 | Okay, so mhm So um the purposes. So you know, typically |
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13:46 | for doing isolating proteins, isolating N. A. Doing an enzyme |
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13:52 | , What have you? So there reasons for doing this. Maybe you |
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13:55 | to find out what are the nutrients of mine. So what do I |
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13:59 | to, what do they need to best? Okay, so you can |
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14:03 | do growth curves to figure that out well. So um even um you |
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14:09 | , and you're doing these things that this kind of a scale, |
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14:11 | Called bench scales like this quality of ok. In industry you start there |
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14:20 | kind of figure things out growth wise that level because it's easy right before |
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14:24 | wrap up what we call um um scale up before we scale up to |
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14:34 | hundreds of liters of quantum right? can have 500 liter tanks, 1000 |
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14:40 | and up. Right? So you out it's very expensive to do growth |
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14:45 | at that scale. So you do bench scale and then scale it up |
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14:49 | there. Okay um so uh so see uh let's see what we got |
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14:57 | . Been rattling on here. so e none of the BDS parts |
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15:06 | let's see change. The cell size during phases two and three. That's |
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15:11 | . Exponential changes in cell numbers. two, true acclamation, phase one |
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15:17 | . Um and yeah so none of of these were false are all true |
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15:21 | . So um so yeah, so and four particular both exponential ones. |
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15:28 | increase, one's exponential decrease. Okay so let's look at description of these |
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15:35 | , right? So lag phase. it all begins again, you have |
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15:40 | in a flask and you see this the inoculation is a seed, so |
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15:44 | speak, um it contains a a of cells you would have grown |
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15:50 | Okay initially and then take a small of that and then inoculate your |
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15:57 | Okay, um now you always go a period called the lag phase. |
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16:03 | . Which is here, in this from here to here. Okay, |
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16:08 | can be lengthened or shortened depending on immediate types being grown between binoculars um |
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16:17 | the batch medium. Okay, So this has grown in and be a |
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16:24 | type That may be may or may be different from what you're putting into |
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16:29 | that can influence your blackface length length the blackface. So, lag phase |
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16:34 | general is really uh think of the of themselves in the United Kingdom, |
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16:38 | . Maybe they were going for 12 24 hours somewhere in that range. |
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16:42 | ? So now you're taking a small of those and you're putting them in |
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16:45 | fresh medium. Alright, so now in this different new environment, |
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16:51 | They'd likely have been in an environment they had been growing for some |
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16:55 | maybe accumulating waste. Maybe some ph have occurred. And now they are |
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17:02 | they're slightly oxygen deprived. Right? so now you put them in a |
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17:07 | brand spanking new medium, Right? they're not going to take off growing |
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17:11 | . Okay, I have to get to this around because that's that |
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17:15 | Yes. Um maybe they need to express different metabolic pathways because of the |
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17:23 | that's now given available to them. . And so they have to turn |
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17:27 | certain genes and turn off certain genes proteins. So all that kind of |
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17:32 | is going on in the lag phase then once they're set then boom, |
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17:38 | can take off and take off And so so basically what I just |
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17:44 | because some of the factors affecting lag , so inarticulate rage, right? |
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17:48 | you're You know I claim is 12 or 14 hours old versus 48 |
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17:53 | that could make a big difference because might not be as many viable cells |
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17:57 | after 48 hours as it was And so we're actually seeing it with |
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18:02 | viable cells and it would be if were fresh culture. Um the amount |
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18:07 | you inoculating 100 μm is you're likely is it one mil? Right, |
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18:12 | that will make a difference. Um , the media composition. How different |
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18:16 | the two types? Okay, um physical factors were just kind of the |
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18:21 | environment that the cells were in previously you put them in something new, |
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18:25 | know, the different, slightly different concentrations and things like that. All |
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18:30 | collectively, all these factors can will the length of blackface. Okay. |
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18:37 | now you can sometimes use this for advantage depending on you know, when |
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18:43 | do this kind of stuff for a . You you don't necessarily want to |
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18:47 | tied to the clock of the Right? You don't want to do |
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18:53 | not necessarily do things on their Right? You don't want to wake |
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18:57 | midnight I have to go in and whatever. Right? So maybe they |
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19:00 | manipulate conditions and do it at reasonable . That's where you can intent intentionally |
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19:06 | with conditions and maybe make a shorter longer lag phase so that you don't |
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19:10 | to come in at weird hours. . Uh An easy way to do |
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19:14 | is really what the with the size the nightclub just making it smaller than |
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19:18 | can get a longer life or make a little bigger and then you so |
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19:22 | can kind of manipulate some things to your schedule sometimes. Um Just that's |
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19:27 | of those things you don't see in but if you are working on the |
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19:31 | you might want to set them Eur hit okay um like so walk |
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19:38 | . So once they're set they're going will go very quickly. And so |
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19:42 | a lot of faces. The most active state. Lots of selves doing |
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19:49 | um that appear like this. Let's go with the rod shaped cell. |
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19:57 | lots of cells in this kind of of dividing. Right? God that's |
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20:03 | . Try it again. Okay so of cells are kind of not much |
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20:09 | . Okay but the point is you lots of cells in that where the |
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20:13 | is formed and they split up because dividing very rapidly. So you see |
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20:16 | of cells that are in that kind state in log phase because they're functioning |
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20:21 | very rapidly. Lots of protein So we offer you often differentiate log |
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20:27 | into mid log. Yeah in late . Okay and again middle august party |
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20:36 | arranged or something like this. Late has arranged something like that. |
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20:41 | Okay. So why that distinction? middle you know if you're interested in |
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20:47 | in a and assessing measuring enzyme activity some sort in the cells uh they're |
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20:53 | be most functional likely in mid log gonna be at they're not nutrient limousine |
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20:58 | very fast. Very active. So it's mid log where you typically |
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21:03 | want to do those measurements. Late log is maybe when you want |
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21:09 | harvest cells. Okay if you want to be using a centrifuge to get |
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21:13 | out to the store and save them or whatever. Um You could also |
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21:18 | that mid log as well but um log is kind of becomes your near |
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21:23 | tipping point. Okay So um the are at that point becoming nutrient |
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21:30 | Okay And so they're gonna begin to down and then you get into a |
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21:34 | phase. Okay Because at that point late log you get a lot of |
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21:38 | there and you certainly don't have enough to sustain everybody to keep going growing |
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21:43 | that rate. Okay to keep doing . Okay it's going to tip over |
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21:50 | I? Um And so because it's batch growth, you're not doing any |
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21:54 | relations to it. You're just letting go. All you're doing is measuring |
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21:59 | the growth. Okay. Um You do those many collections as we'll talk |
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22:04 | shortly. Okay. So I'll sell so cell size because the cells are |
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22:12 | of in this state or in a it's a wholesome goodness. Okay. |
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22:19 | have a I hope so here they kind of you'll see some information occurring |
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22:24 | itself. So you're gonna have you're elongate if it's a rod shape. |
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22:28 | course it's a caucus it'll get this , you know, browner. But |
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22:33 | expand to a point where then they'll into two. So you're gonna see |
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22:36 | lot of cells that kind of they're be the biggest size in in mid |
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22:41 | log phase. Okay? Um as get to stationary phase. And of |
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22:47 | that's right. And uh growth rate equalized because you know flat they're not |
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22:54 | out of nutrients yet, There's still to feed folks. Okay. But |
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22:59 | is limited. Okay. Um And kind of because of that kind of |
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23:03 | stress responses are initiated uh due to limitation could be also due to oxygen |
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23:11 | if it's an Arabic organism. Um so the the strategy now is let's |
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23:20 | . All right, we're gonna go survival mode. So that means kind |
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23:23 | let's reduce protein synthesis of non essential . Um Let's um let's get |
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23:34 | It's a little bit smaller. That that's less material to keep up |
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23:37 | Okay, so um and so all the the strategy of okay let's do |
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23:45 | survival thing with hope that maybe more will appear okay uh You know, |
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23:52 | as cells die right? The the the material from that cells that sells |
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23:58 | now available as a food source as so they can actually live off of |
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24:02 | for a period of time. Um of course eventually, you know I |
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24:07 | here right here Is when completely out nutrients no way you can sustain anymore |
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24:14 | at all and very rapidly goes off cliff. Okay, exponential decline. |
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24:19 | so that's what we actually focus on Chapter five. Is this phase |
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24:25 | Because we're trying to um control microbial and when we say that we're trying |
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24:31 | control the pathogens obviously. So it's let's throw chemicals uh chemicals, |
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24:38 | whatever the treatment is to make them as quickly as possible. So we |
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24:43 | on how can we maximize that So that rate make them go really |
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24:49 | , dying very quickly. Okay, that's what we focus on In Chapter |
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24:54 | . So but the point is um phase and death phase can both be |
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25:03 | change is exponential changes in cell numbers going up when going down. Okay |
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25:10 | so this phenomenon of dormancy and persistence come to um ah significance, particularly |
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25:20 | the context of pathogenic bacteria that can this that we call them per sisters |
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25:28 | can um get around the effects of for example, um by not |
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25:36 | Okay, so remember a lot of antibiotic targets um focus on components that |
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25:46 | that are at their most active when are actively growing, right? Like |
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25:50 | wall synthesis, right. Things. of targets are sell all sentences, |
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25:54 | , example, ampicillin, etcetera. so those drugs are most active when |
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26:00 | cells are actively dividing, Right? when they're actively dividing, okay, |
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26:06 | they're when there's many cells along phase are kind of this sting, |
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26:13 | This is that's this or this All . That septum that's cell wall |
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26:23 | Right synthesis. Doing so so that's active. Of course the mark |
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26:30 | Right. And that's when these kind antibiotics would be most effective. |
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26:36 | And uh there are lots of targets the for the antibiotic in that |
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26:41 | Right? It's not wrong then, really no targets. What's it gonna |
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26:45 | ? There was no cell wall being . What's it gonna do? |
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26:49 | so, so many other antibiotics that components of protein synthesis. Right? |
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26:54 | gonna be more effective also when its growing. Right? That's when it's |
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26:58 | lots of proteins. Okay, so some some pathogens have adopted this |
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27:05 | of of arresting growth. Right? the presence of antibiotics. Okay, |
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27:10 | not exactly sure what the mechanism is inducing that, but we've seen that |
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27:16 | so it can be not grow and viable. Okay, so maintaining that |
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27:22 | membrane potential when we talked about we'll talk about that in the next |
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27:26 | . But that's remember the proton pump talked about. Alright, so that |
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27:30 | a membrane potential that can be used produce energy. Okay. And that |
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27:37 | can Mhm allowed itself to sustain itself it's not growing. Okay. And |
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27:45 | so because any antibiotics you take don't around forever because they will dissipate from |
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27:51 | body. Right? And so when so when the organism's senses that then |
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27:57 | they begin to grow again. So it's a um now that kind |
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28:03 | mechanism is not really one that's I think it's one that's that's terrible. |
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28:13 | . Maybe it is that it's not . But regardless, it certainly is |
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28:17 | the individual or individuals with that particular type, they can they can um |
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28:25 | be affected by antibiotics while antibiotics are . Okay, so so again, |
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28:30 | what we call for sisters, which kind of a dormant state, but |
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28:35 | do while the inbox around but then the grill when it goes away. |
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28:39 | . Um Yeah, go ahead. I don't think so because that's I |
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28:48 | they they are just resistant to So methicillin resistant staff lawyers, so |
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28:55 | not saying they can't exhibit that but um but but I think there |
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29:00 | particularly resistant to many antibiotics. Any other questions? Okay we'll talk |
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29:09 | a little bit about antibiotics. I there is semester because of antibiotics can |
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29:13 | in different ways. You can. often prescribed two antibiotics, not just |
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29:17 | to you Because sometimes 200 biotics work together than they do alone. So |
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29:23 | that as well. Um Like I we'll talk about some of that vaccines |
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29:28 | talk about later as well. Um so let's look at this question. |
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29:34 | is something I touched on just a minutes ago. Okay um let me |
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29:40 | that up again. Okay so okay just go to the question here. |
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29:46 | so a bacterial inoculate um has grown nutrient broth. Ok so nutrient broth |
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29:52 | them. An eloquent of this. it's grown let's say overnight you're gonna |
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29:58 | that to a batch growth medium. the bachelor growth medium is going to |
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30:01 | M. M. Nine. What's M. Nine. You see the |
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30:05 | there? Okay so you have nutrient and M. Nine formulations. |
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30:10 | Fund inoculation of the batch medium. will the growth pattern most closely resemble |
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30:16 | B. Or C. And so for for reference let's just say that |
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30:25 | when the inoculate them has grown in broth and transferred to fresh nutrient |
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30:31 | the growth pattern? It looks like does in a, Okay so it |
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30:37 | from neutral breath nutrient broth looks like . Okay so what might you guess |
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30:44 | when it goes into M. what would it look like? What |
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30:47 | looks like A B. Or Yeah. Mm hmm. Bye. |
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31:28 | . Okay, let me see. the timer on two. We just |
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31:35 | down to a question of And the . nine might there be a |
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31:53 | Yes. Okay. So let's see can see. Okay. So who |
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32:08 | C. No. Happy. So why C. What? |
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32:18 | One more time that so going into um So going into M. |
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32:35 | Okay. It's gonna looking at sea means you're extending the like this. |
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32:42 | ? And so you're sending the lag because the cell has so remember the |
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32:47 | nutrients. Right. Mhm. So things gives provide the cells. So |
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32:53 | one, when you see that I of this Pepto beef extract meat, |
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32:59 | ? Meat. But also there's there's . Plant products like soy products are |
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33:03 | fit in this category as well. So in doing providing complex nutrients, |
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33:10 | providing. Not just providing C. . O. M. P. |
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33:14 | in all different ways. Right? as carbohydrates as lipids as as um |
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33:22 | acids um proteins, right? As as vitamins and amino acids. Write |
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33:28 | these things because that's what's in Right. And then plant tissue. |
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33:34 | uh so a lot of pre formed lot of preformed nutrients are present there |
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33:39 | bacteria to grow up. Okay, an M9. Right. This is |
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33:44 | minimal media a defined media. Okay in that medium It's going to basically |
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33:50 | to make everything from scratch? There's gonna be a lot of when |
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33:53 | when it goes from nutrient broth into . nine there's gonna be a lot |
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33:58 | we have to turn these pathways on these off. A lot of turning |
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34:03 | because now it's gonna have to you turn on the you know acid pathways |
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34:07 | this and that to synthesize all it to grow and so that's time. |
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34:11 | it's always always takes time to do things. So that's why you would |
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34:16 | the lag phase for that reason because the time needed for the cells to |
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34:21 | ready the things grow on this new or medium if you will. Okay |
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34:28 | if you went from like I said it went from if you went from |
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34:31 | . Nine and M. Two M medium but then they were already set |
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34:34 | go as well. So you wouldn't wouldn't see a long life phase it |
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34:37 | be relatively short because they already acclimated those minimal medium conditions. Right? |
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34:42 | whenever you're going from the same medium another the same medium and batch it's |
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34:46 | be you know, reasonable black faces you go from very different that it |
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34:52 | it can be extended. Right? if you go from M. Nine |
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34:56 | a rich medium like nutrient broth than can shorten? Right so again just |
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35:02 | from the perspective of the cell what a cell have to do when it's |
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35:06 | this medium now you have to do lot of stuff to get acclimated and |
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35:10 | going or not so much. Many questions about that. Yeah. |
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35:16 | it's big. Okay so yeah good . So these they have about the |
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35:27 | black face. Right. But but different the amount of cells? All |
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35:35 | so you're actually getting more cells The dashed line. Right so you've |
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35:41 | gotten a read a quantity of cells be so maybe maybe maybe um you |
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35:50 | be be could be just hypothetically. So you can make nutrient broth in |
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35:55 | in that in what we call one . Right let's double it up and |
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36:00 | it two X. Alright so then will give you more because you have |
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36:05 | stuff to grow once you get more . So that that's that's really what's |
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36:08 | on in B is the medium formulation you more cells. Okay that make |
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36:14 | . All right. The same. . So does that mean? |
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36:26 | Yeah I mean again it's it's I'm hypotheticals out there but yeah the point |
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36:31 | yeah clearly in B. They could the same media they don't have to |
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36:35 | but clearly B is giving it more source is my guest to give it |
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36:41 | cells. Yeah. Yeah push it you can get from A to B |
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36:47 | us to the next point in fed culture? Okay so that's how you |
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36:52 | be growing like this in a. . Ah I'll live. Okay so |
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37:06 | if I fed it like say here then I could get more sells right |
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37:12 | give it more carbon. More carbon the thing that influences it the |
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37:16 | And so get more carbon at that goes up. Okay I can just |
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37:21 | my nutrient broth which has Pepitone and extract and add glucose to it. |
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37:26 | that will boost cell cells up. And so we call that fed batch |
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37:32 | that you see there. Okay so is now where we're making manipulation. |
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37:36 | batch growth you kind of just Let it go right to get a |
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37:40 | curve. But now we're now we're uh the culture, we're adding |
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37:46 | You could even go as far as could um You could put a ph |
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37:51 | in your in your medium to see it gets acid or base. And |
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37:55 | adjust right you want to keep it 6 to 8 for optimal growth. |
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38:00 | it gets to lower to higher. can adjust it with neutralizes the acid |
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38:04 | um at carbon to get more So you can kind of control things |
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38:08 | now becomes a pain to do that the shake flask because you constantly have |
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38:12 | monitor, you probably have to see how's it doing? Let me look |
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38:15 | it. Is it getting acid You know, is this cell numbers |
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38:19 | down or leveling off? So then have to do your manipulation. So |
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38:23 | can be a pain in the Which is why we have the |
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38:26 | which is all integrated computer control, program what you want to do. |
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38:31 | you just but you don't have to all these manipulations by yourself. And |
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38:36 | you look at it by reactor, itself is right. Is the tank |
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38:42 | , which is been put into the ? Um you see all the pumps |
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38:47 | here? Okay, so delivering um base uh could be delivering nutrients as |
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38:54 | on a on a prescribed schedule. , So even if the fed batch |
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38:59 | , you could feed again right here keep going up, feed again here |
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39:05 | go up. So you keep doing even in shape fast will be become |
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39:10 | in terms of volume. Uh and parameters start to limit you like |
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39:16 | And so but with my reactor you control a lot of this stuff. |
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39:22 | , So um so it fed I'll come back to Bayreuth for the |
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39:26 | . Let's look at this question. If let's say for example, if |
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39:30 | can add I don't know, 50 per leader glucose. Right? and |
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39:38 | the typical medium may have like, we started out with 10, |
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39:44 | 10 g of glucose. And then then at at intervals here and here |
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39:52 | maybe a third time we add like 10 or 15 g at a |
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39:57 | Right? And then we get lots cells. Okay then you go then |
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40:01 | think to yourself well I don't want keep adding at different times. Let |
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40:07 | just do it all at once. , why not? At all The |
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40:11 | at the beginning instead of doing it batch, right. Which you could |
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40:16 | but you will find if you do the cells don't grow. Why is |
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40:25 | ? And that strict? They typically don't grow if you add everything up |
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40:29 | like that. Why why would that very simple chemical principle? You all |
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40:43 | ? Any ideas don't meet. So wild at me. Well think of |
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40:54 | you added 50 g per liter of inter medium at the beginning with your |
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40:58 | stuff you need. Right, nitrogen source Blah Blah Blah. What's what's |
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41:04 | to be different compared to that Or in the same medium with just |
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41:08 | g per liter of glucose. What increased the total is concentrated the concentration |
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41:17 | total. Mhm salute. So we a high salt use concentration. What's |
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41:23 | going to affect? Hi platonic hype . The old osmosis. Right. |
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41:30 | where it becomes inhibitory. So it's a high solid concentration. It was |
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41:34 | hyper time. And that actually can the cells because they're fighting the water |
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41:39 | and that kind of stuff. So go I don't like this, I'm |
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41:42 | really gonna grow that well they may but not very well. I like |
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41:46 | want them to or like you would them to. So for those for |
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41:50 | reasons you don't add all this stuff front, you kind of do the |
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41:53 | batch thing to keep them happy. The so if an aerobic bacterium and |
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42:02 | in liquid culture is adequately fed So we do the fed batch thing |
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42:07 | what can limit growth fairly quickly unless control this parameter. There's one word |
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42:14 | the beginning here, that's the operative . Which one? Okay. |
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42:24 | Yes option. Right. You gotta options limited fairly quickly. And so |
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42:31 | there's no way to get around And so um one way is. |
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42:39 | um has anybody grown cells in the flask anybody? Okay. I don't |
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42:49 | the hands. So I'm missing Okay so um if you have an |
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42:54 | aerobic bacterium and you can actually uh just like the one you see |
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43:00 | Okay. Do you think you see difference if it were sitting static like |
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43:06 | ? And just in the incubator or you do something else to a cancer |
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43:13 | ? Get heroic time. So aerobic gets its uh Oh too from surrounding |
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43:24 | . Right. What you need to up this analogy? Um How does |
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43:33 | how is air mixed with very well some water gulfs and beaches waves |
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43:45 | right? Air mixes into the Okay so if you take that shake |
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43:49 | and do this. Yeah a lot growth. And just think it's ecstatic |
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43:55 | now you're that's how air gets into system. You create turbulence to mix |
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44:00 | air within water. Okay now you even go a step above that. |
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44:05 | see this flask has these ridges in bottom of the flat, right, |
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44:11 | getting a flat typical flat flash. things have what are called baffles, |
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44:16 | little indentations, right? That creates more turbulence. You can actually compare |
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44:21 | to a class like that with one flat bottom and you'll see a difference |
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44:26 | the amount of turbulence being created more and mixed in. Um The other |
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44:30 | you can do is jack up your on your shaker. So instead of |
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44:36 | 100 revolutions per minute do 200. then you'll see a difference as |
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44:41 | faster growth. So obviously this is assuming you're working with Arabic organism. |
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44:47 | ? So in the bioreactor then you control all these parameters. So you |
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44:51 | um ph and temperature of course here can automatically add acid base. You |
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44:59 | have set points For your p typically six or 8. Below six ad |
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45:07 | above eight at acid. Um Then have oxygen concentration. So what's called |
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45:14 | D. O. D. Pro oxygen that will measure the option |
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45:19 | and um when and so again you it uh to get below experts and |
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45:28 | then do this and to do this is to make this go faster. |
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45:34 | is what we call um the right? Has these blades on |
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45:39 | That twist turn? It's basically a mechanism. Okay so stir faster. |
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45:46 | great more churches. Makes more air with the liquid so those can get |
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45:51 | . And then the other thing is the amount of air coming in. |
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45:55 | so a spar Jer is really just device that just has little tiny |
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46:01 | tiny holes in it. Okay and you're forcing the air through these tiny |
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46:06 | and that works better than just kind bubbling it in through a you know |
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46:11 | of the tubing or something because tiny mix better with water than large |
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46:17 | And so it dissolves more quickly in water. If they're tiny bubbles and |
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46:21 | does that so increasing the RPMs, more turbulence, mixing and mixing small |
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46:29 | . That's what the sparkly gives. . See a lot of oxygen better |
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46:32 | uptake by the cells that way So in all bioreactors are equipped like |
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46:39 | . Okay so we can control all things and you see here will be |
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46:43 | in nutrients where you add your inoculation the system, you can also on |
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46:49 | timed program at nutrients to it. in that system you're the cells are |
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46:55 | happy because everything is controlled right, everything optimal ph optimum nutrients, optimal |
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47:02 | . Okay. And they grow like . Okay. Um so the one |
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47:07 | that that is critical in the system this the cooling? Because we didn't |
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47:16 | would grow so fast, they would themselves because of all the heat being |
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47:20 | , right? Thermodynamics, right? lots of heat and so hot that |
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47:26 | it worked, I didn't experience But somebody mentioned to me that the |
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47:33 | system on their vessel uh was non and then they came back and it |
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47:40 | like they couldn't even touch the reactor so hot because the cells have grown |
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47:43 | much. So uh so water is important. And so um so |
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47:49 | you know, biological control all these get lots of cell yield, which |
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47:53 | what you need to do for a process. If you're in an industrial |
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47:59 | , you're growing lots of cells for it's to make a lot of, |
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48:04 | lot of it is to for enzyme . There's enzymes, a lot of |
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48:09 | commercial products, laundry detergents and And these bacteria, it's beautiful to |
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48:15 | grow to make these proteins and harvest . So enzymes, enzymes in |
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48:21 | big business and using bacteria to produce them at large scales is what they |
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48:29 | any questions about. Okay, so you are in biotech major, you |
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48:35 | would have your hands somewhere in this because they're also I'm not going to |
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48:40 | about it now. But aside from vessel then you can go elsewhere with |
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48:46 | material with what we call uh you what that's called biotech majors, blank |
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48:55 | , downstream processing and that involves a of other stuff, engineering stuff and |
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49:02 | . Where you may be taking themselves have a process to break open the |
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49:06 | , get the protein or whatever it . So that's that's the whole thing |
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49:10 | itself. Okay. Um Alright, let's talk a little bit about dynamics |
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49:16 | growth. So um obviously having just at the growth curve, we can |
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49:23 | that. So numbers can increase Rather . Right? 1, 2 million |
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49:30 | generations. You collect and do that 12, 10 to 12 hours or |
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49:35 | . Okay. Where it takes humans do about 400 years to do |
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49:39 | Right? 20 generations. So um when you're talking about um increases of |
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49:46 | numbers like this. Okay. Just a ph scale hydrogen ion concentrations from |
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49:52 | to large numbers. You use log base 10 to kind of compress that |
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49:56 | . Make it more manageable when you're to quantitative these things. So another |
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50:02 | generation time is what we use to to growth of a bacteria. You |
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50:09 | any any life really. Um And you can look at the different ways |
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50:15 | time. Basic way is what you here right time for oneself dividing the |
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50:20 | is obviously a generation. Um But the more practical value is what we |
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50:26 | doubling, doubling time. Okay. you you and you can measure that |
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50:31 | any point in the growth curve. go, here's my cell numbers at |
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50:35 | time. How long did it take double? Okay. And that too |
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50:39 | generation time. Okay. And so calculations will look at and so you |
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50:46 | before I mentioned that. So you here the equation basic equation right. |
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50:53 | if you know the number of generations have passed the end number here. |
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50:59 | . And you know how many cells started out with? Right? You |
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51:02 | out with one cell. Okay. zero. Um and generation number generations |
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51:10 | let's say five. Right? Use value. So one cell times to |
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51:16 | fifth. Well that's 32 cells. ? Um but we often don't want |
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51:22 | put in. It gets more complicated that. So we we we take |
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51:27 | equation really? And we rearrange it that we can solve for n |
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51:33 | So we can go and we set up to be in equals. |
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51:37 | So we can easily calculate the number generations uh in a culture, you |
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51:43 | , beginning an end, right? then we can figure out generation time |
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51:48 | we can do some more of these of growth problems. So the next |
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51:55 | here and you don't need to know it's derived and all of a sudden |
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51:59 | that complicated. Right? So we're to take this equation here right? |
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52:05 | just rearrange it so we can solve the number of generations, right? |
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52:12 | we're gonna use log base 10. . And so we're just gonna multiply |
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52:18 | By blocking the base 10. So gonna take this equation alright and we'll |
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52:22 | it through stepwise like this right? long as the base 10 all the |
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52:26 | through um log to the base 10 and T. Equals log base 10 |
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52:32 | zero times log base 10 2 to right. So to the answers up |
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52:38 | power of two that grows right 1 2 to 4 to 8. So |
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52:43 | . So um so then that log the base to to the ends because |
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52:48 | the way law work. You can it like that. Okay Or in |
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52:54 | that equals in times long to the 10 of two. Right? Then |
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|
52:58 | value Actually equals .301. Alright so we're just going to um rearrange to |
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|
53:08 | it equal to the small and and number of generations. So we just |
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53:12 | this right? Basically we divide through .301 right? And then this expression |
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53:20 | is the same as running it like . Okay. Again it's just that's |
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53:24 | logs work. Okay log base 10 so long. Today is 10 times |
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53:30 | minus log base 10 zero is the as log to this tinge of |
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53:36 | T. Over N. Zero. so that's our equation that we use |
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53:40 | do our uh problems. Okay so time we can as long as we |
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53:46 | N. T. N N zero can figure out the number of generations |
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53:49 | do things with that number. And so one of the things of |
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|
53:53 | that's important is the time element. . So what's called a growth rate |
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|
53:59 | ? So bacterial you know every species a growth rate constant. Okay um |
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54:07 | will be an optimal right on the conditions there will be a growth rate |
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54:11 | . That's but that's the max value that species under optimal conditions. Of |
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|
54:16 | that can change depending on how you the cells. Okay. Um but |
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54:22 | it is is just taking this Same equation here. I'm just putting |
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54:26 | time element in and you can see right there. T for time. |
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54:31 | . And so Kay, what they the growth rate constant N over |
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|
54:37 | Um is the inverse of that. , so remember and there's generations |
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54:47 | And tea time that the inverse is time. Okay. Generation time is |
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|
54:54 | time per generation. Right? Typically minutes because bacteria grows so fast Could |
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55:00 | estimate minutes? 50 hours as Okay and so uh so that equation |
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|
55:09 | can look at a couple of different of problems. Okay. On blackboard |
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55:15 | um and where the electron notes are as a document called bacterial growth |
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55:20 | It has one that has the ones you're about to see in addition to |
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55:25 | couple more um the back of the or the bottom of the sheet has |
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55:30 | the answers all worked out. So just take a look at |
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55:34 | There'll be a there'll be a question the blackboard quiz. That's a problem |
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55:38 | this as well. Okay again I think it's anything that complicated. But |
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55:42 | let's go through a couple of Okay so start here give it your |
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55:47 | shot. Okay if you're not sure it, that's okay because we're going |
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55:51 | work and work through it. And that is not a real bacterial |
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56:02 | by the way. So we've got bacterium that has a generation time of |
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56:11 | minutes. Mhm. Starting with five in log phase. How many minutes |
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56:18 | it take to produce? 10,000 Okay. So 5-10,000. So those |
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56:23 | two year values right here in zero . T. Okay. You know |
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56:28 | generation time. Right? That's um per generation. Yeah. And these |
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56:44 | will be part of the part of question as well on the test. |
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56:48 | you don't have to memorize the equations . But I wouldn't know what you |
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56:52 | what obviously what the variables meaning the but you'll have the equation there. |
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57:41 | . Mhm. So the to me key key but the way I do |
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57:51 | is always set up set the thing first and you know have my variables |
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57:55 | what their equal to and then kind logically think through it again if you're |
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58:01 | 100% sure that's okay. Just for sake just punch something in. |
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58:09 | Yes. Okay two more seconds. here we go. Two 210. |
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58:26 | so uh the majority says be alright see. Alright so let's go through |
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58:34 | here. So um that's our Alright so we're going from 5 to |
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58:40 | cells so we want to figure out many generations is it to get from |
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58:45 | to there? Okay. Our generation Is this minister generation. So we |
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58:53 | given that 40 minutes right? And so if we calculate the number of |
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59:00 | end which we can figure out by equation. Uh Going from 5 to |
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59:06 | cells. Use the value multiply by time to yield minutes. So these |
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59:12 | of course cancel out. Right? we just need to figure out |
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59:16 | Okay and so uh n. 0 10,000. Do I have my |
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59:23 | ? So long as the base 10 that? Is that Divided by |
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59:28 | So it's about 11 generations. Okay so it didn't just multiply that by |
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59:33 | generation time gives you 440 minutes which think is about seven hours. What |
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59:44 | choices were? Take a little over hours. So D Okay. |
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59:53 | Yeah Um 60 times seven is 420 . Yeah it's over seven hours. |
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60:01 | just look like logically makes sense. well um let's look at another |
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60:11 | Okay so calculate the generation time. this time we're going to catholic generation |
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60:17 | sort of being given to you. if the 900 bacterial cells growing 15 |
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60:23 | produced and then lower three million Okay so there's your equations so you |
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60:30 | your N. T. And Zero here. Alright you have a |
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60:33 | frame so just a matter of plugging the into this over here. |
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60:49 | Do that again. Okay and so do these kinds of calculations. You |
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60:57 | maybe grow if you're interested in the of maybe disinfectant or antiseptic or |
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61:04 | Maybe you you do growth curve with it and then without it and then |
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61:10 | the numbers you know to get the . Let's see if there really is |
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61:12 | difference or not one example but certainly you just use these values these values |
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61:18 | lots of things. Right so figure a generation time on when medium. |
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61:24 | would it be in a different medium so forth. So any reasons to |
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61:28 | this timer on. Yeah. Okay I'm gonna go ahead and um |
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62:34 | time I'm gonna let it go Okay here we go. Three Okay |
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62:54 | B that is that is correct So let's just go through not that |
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63:00 | surprised. Um let's look at the . Okay, so 15 hours, |
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63:08 | minutes. There's 900 minutes. Then go to uh are NT and zero |
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63:15 | then solved in and then plug that To get our generation time administer divided |
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63:24 | 11, almost 12 generations 76 Okay. So okay. These are |
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63:30 | kind of things. You'll see a , look at the problems on blackboard |
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63:35 | . Certainly let me know. Um but every everybody okay with |
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63:41 | Okay. Alright. So let's switch then at least talk about. So |
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63:48 | look at this question real quick. so the next couple of phenomenon we're |
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63:53 | about um are as many processes as see going through the semester that are |
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64:05 | or driven by the presence or absence nutrients. Okay. That lack of |
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64:13 | a bunch of literacy can cause different to happen in certain bacterial types. |
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64:19 | these next two phenomenon we're talking about certainly fit that category. Okay. |
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64:25 | so this is so for this this is a nutrient driven phenomenon initiated |
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64:33 | a black or depletion of nutrients. . Mhm mm hmm. Yeah. |
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65:12 | . Okay. Alright. Here we to one. Mm hmm. All |
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65:19 | . So it was it is in sport formation. Okay, so bio |
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65:27 | information is actually the opposite of that opposite. Right? It's all about |
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65:33 | abundant nutrients. It wouldn't be lag because lag phase. There are plenty |
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65:40 | . The soldiers haven't gotten activated yet begin using them. Ok. And |
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65:44 | log phase is all about lots of and lots of growth. Okay, |
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65:49 | um let's look at biofilms examples. , we'll do that question next |
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65:58 | So biofilms uh have um their role important biofilms. Right. And become |
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66:07 | become a problem um In the last , 20 years. These are staff |
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66:16 | example is on your skin or mucous anyway and they can be responsible for |
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66:21 | called hospital acquired infections. So you to the hospital for let's say you |
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66:28 | a get your knee scoped or some procedure or whatever. But while there |
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66:33 | get a bacterial infection of some Okay. And very common that they're |
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66:38 | very common. But certainly you these accounts for maybe 10% of these |
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66:43 | acquired infections is a medical device, Catheter, a hip replacement, knee |
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66:50 | , heart valves replacement um that these handled properly. Maybe they the the |
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67:00 | with healthcare workers hands or something like . You don't handle it properly. |
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67:04 | breathing tube. Another example these will surfaces. So biofilms are all about |
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67:08 | surface and uh material that formed their specific types that do this and |
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67:17 | have specific features that enable them to this. Okay. But it's all |
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67:21 | a surface growth on the surface, growth on the surface. And so |
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67:27 | use of a catheter when it comes of the packaging and you put it |
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67:30 | a person that can lead to um for information. And the thing about |
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67:35 | biofilm is they're not easy to get of. Okay, because I'm making |
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67:39 | very big large, massive um you have uh types that are on the |
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67:45 | of the biofilm, does it on interior of the biofilm that are harder |
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67:49 | get at. So antibiotic therapy You take not just a week or 10 |
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67:54 | , but months to get over these of infections or if the infection can |
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68:01 | , can spread from just where it's there and go elsewhere in the |
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68:05 | which of course is a dangerous So uh so these can be quite |
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68:11 | . Okay, uh so again about surface. So pipes, sewer pipes |
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68:16 | other types of pipes or a surface you often see biofilms form. |
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68:21 | Um the other thing is whether it's caterpillar or something similar to that or |
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68:28 | pipe like that, there's typically a of nutrients, right? A pipe |
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68:32 | typically has fluid going through it, will be have organic material and typically |
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68:37 | that can sustain the cells in a catheter. Of course the body fluids |
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68:44 | have nutrients in them that can sustain organism. So it requires that you |
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68:49 | it requires because it represents massive growth so obviously if they have supply of |
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68:55 | that are feeding this thing and enabling to develop. Okay, so here |
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69:01 | to show up very well but starting and going clockwise that if you can |
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69:08 | it kind of a dot in the , there is what we call a |
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69:11 | colony. So it's it's not visible the naked eye. But it will |
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69:17 | by phone will start that way with few cells but then begin to collect |
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69:22 | then proceed to grow and you know two dimensions initially on the surface. |
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69:27 | then here you see it's going in dimensions and coming up off of the |
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69:32 | like like a mountain almost okay what call um bio stone towers. And |
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69:38 | that represents obviously a lot of cell and like I said before when it |
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69:44 | to be this massive, you'll have on the periphery out here and sells |
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69:48 | the interior and there can be two types of environments there. If we're |
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69:53 | about a medically important type. Well you may have types on the outside |
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69:58 | are susceptible to antibiotics or maybe those the inside aren't because they can't penetrate |
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70:03 | they may become resistant types of So it's a uh so the the |
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70:10 | are buying from our surface because we're the surface lots of growth supply of |
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70:17 | so it's not nutrient deprivation but nutrient that can sustain this thing. And |
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70:23 | here are a few more pictures um the process of that information you might |
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70:31 | well it was just a random association cells that come together and they just |
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70:37 | growing And it's far from that. a jean included process occurs in |
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70:43 | Uh And a species specific, not bacterium can do this many can, |
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70:48 | it's a it's a species specific And so yes, because of the |
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70:54 | can get massive other things can of come into contact with it in the |
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70:58 | . And you know, you might they're part of it but there was |
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71:01 | because they get stuck to it Have you? Okay. But the |
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71:05 | of initiation, attachment, maturation maintenance dissolution. Okay, So there can |
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71:12 | typically are two types of cells the ones that are motile. We |
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71:17 | plant tonic cells. There are Those that are lose their flagellum once |
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71:22 | on the surface and become what they stickers stick to the surface. |
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71:26 | So because of that phenomenal surface being , february pili are important features. |
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71:34 | biofilm formers have those structures. And so um the the I'm just |
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71:42 | to show you the picture here. the process is all orchestrated by the |
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71:47 | chemicals, chemical signals. Right? so the cells the species is not |
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71:54 | to form a biofilm on the surface there is not kind of nutrients because |
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72:00 | going to eventually form billions and billions cells and you have to have a |
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72:04 | supply of nutrients to sustain that. the initial process is going to be |
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72:09 | to kind of figure out, is this a surface environment that is |
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72:14 | to even forming a biofilm? And this quorum sensing thing phenomenon is all |
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72:22 | cell density. Right? So if is, so you have these swimmer |
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72:27 | , right? Plank tonic cells that out a favorable surface. So if |
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72:34 | land on a surface um if more more cells are get going there, |
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72:43 | it may be a fair favorable Okay, Because they're able to there's |
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72:48 | they're they're able to divide and And so if that's the case then |
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72:53 | reach a threshold level, Right? they're producing these chemicals today signals. |
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72:59 | if there's enough cells there, presumably are in themselves because it's a favorable |
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73:05 | . Right? And so if that's case, they will reach the threshold |
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73:09 | of those chemical signals and then that induce than the initiate the biofilm |
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73:16 | Okay, If you don't reach a level, then presumably it's not a |
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73:22 | environment, then they won't go through process. Okay. And then these |
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73:27 | cells will go elsewhere. Okay, because it's a commitment one way or |
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73:31 | other either form it or not. ? Because if you're going to commit |
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73:34 | it, then that means you're going be investing a lot of, you |
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73:38 | , gene expression and making things you to make a biofilm. Okay. |
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73:43 | it's not a trivial thing. And one of the first things is this |
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73:47 | policy Sacco information that's kind of the holding everything together. Okay, so |
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73:52 | the sugar carbohydrate rich. You can have proteins in there as well. |
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73:57 | . But it's it's a gene encoded material. Okay. That's only induced |
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74:04 | those chemical signals get the threshold that sensing phenomenal color. Okay, there |
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74:10 | be motility here. So they lose flagellum okay, when they're on the |
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74:15 | and now live from forming. But so that's where the came to me |
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74:20 | attachment. And so the pill I can have that twitching motility. So |
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74:26 | may have that twitching motility on the through those Pilates. Uh then you |
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74:32 | you have what are called micro They're kind of growing on the surface |
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74:36 | then that two dimensional growth becomes three . And so the biofilm towers, |
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74:43 | call it right now you're talking about significant number of cells growing. Multiplying |
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74:51 | within this matrix of this extra polish . So that's again that's kind of |
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74:55 | glue holding everything together because you can so dense the cells on the interior |
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75:02 | can be nutrient deprived. So you to see holds form. Okay, |
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75:07 | that material can flow through. And feed everybody out. Okay so |
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75:13 | have to what you don't see here what you have to kind of imagine |
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75:16 | envision is through through out here, nutrients. Okay, so whether you're |
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75:24 | a whether it's in a pipe or , right? You're having a flow |
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75:29 | organic material that's what's sustaining me. Okay, so they're stuck to the |
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75:34 | with this material flowing through nutrients. . And of course if that uh |
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75:42 | if the nutrient flow reduces or goes then you know you have to sustain |
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75:47 | and then dissolution occurs. These revert to plant tonic forms to swim and |
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75:55 | another favorable area. Okay? But biofilms can sustain themselves quite a period |
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76:01 | time if there is a so going um so obviously so the main things |
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76:09 | are surface features like are important for um and quorum sensing. Okay, |
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76:18 | we'll review a little bit of this next time and then in those floors |
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76:22 | the Chapter five next week. See you on |
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