| 00:07 | Okay. Mhm Yeah, I Hey folks, welcome, let me |
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| 00:41 | this up a little bit. Testing. Can you hear me? |
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| 00:47 | of low test casting, wow that's better. Okay, all |
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| 01:03 | let's see. So there shouldn't be new information to anybody. I hope |
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| 01:09 | , email. Uh So that's only in terms of being a little more |
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| 01:23 | . So instead of the usual uh questions, this one is about 25 |
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| 01:32 | , you'll have 45 minutes. Um basically cover stuff since last, I |
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| 01:40 | you can hear the 17th, 19th kind of only started a real |
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| 01:46 | So uh basically chapter 134 and summer , whatever we cover on five on |
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| 01:54 | . So Um so just know that have more time for this one |
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| 02:00 | So uh of course the smart, work. Um the only difference there |
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| 02:07 | you got something to do on but then two days later in Chapter |
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| 02:12 | , which is not long, it's short, but I did it that |
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| 02:16 | . So if you did have questions you at least have some time before |
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| 02:21 | exam. Okay, so that's why put the due date on Wednesday for |
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| 02:26 | . Um So again, exam is week from this friday, so I |
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| 02:35 | have over a week yet until So but we'll start before that it |
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| 02:42 | start viruses and this and this is one of those, I'll have the |
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| 02:52 | uh set up for you. So the flip classes we'll we'll do a |
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| 02:57 | of questions. Um too. that's not going to be on the |
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| 03:03 | example. Okay. But just to you know, So we've got plenty |
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| 03:09 | time, so likely um the the that ketchup day, we'll finish up |
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| 03:16 | last bit of chapter five. That likely not be a an hour and |
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| 03:22 | minutes class. Probably somewhere about 45 range. So anyway, so that's |
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| 03:27 | coming up. So uh let's uh a brief recap. Um So we've |
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| 03:36 | through, we haven't talked about this , but we'll go through that and |
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| 03:42 | this in this today and maybe uh two? Uh short by comparison. |
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| 03:53 | it only covers two topics. Uh films in those scores. Okay. |
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| 03:58 | , well, we may or may get to some of that today. |
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| 04:02 | see, but we've got plenty of . Um Alright, so we went |
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| 04:07 | kind of uh alright, here's what need for growth. Right? So |
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| 04:14 | kind of went down to the right? The C H O M |
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| 04:17 | S. Okay. And then of , depending on the nutritional, |
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| 04:25 | We need different forms of these Most of phosphate or phosphorous is a |
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| 04:34 | , sulfurous sulfate content typically and so . But the kind of the one |
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| 04:38 | the major dividing lines was a carbon C. 02 or something, a |
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| 04:42 | more complex. Like a glucose or carbohydrates, or fat or what |
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| 04:48 | Right? So you're distinction, So we looked at in terms of |
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| 04:54 | , electrons right to remember us hetero and uh more manual for our |
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| 05:01 | Right? We can eat carbohydrates and carbon from that. We can also |
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| 05:05 | energy from that. Right? And from that as we'll see when we |
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| 05:10 | into chapter two. Okay. So all depends on kind of the metabolic |
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| 05:15 | and then we put those components together we make a media, right, |
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| 05:20 | medium. And then uh we have types of growth media. We talked |
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| 05:24 | uh complex divine. Right? We'll about a couple others today. And |
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| 05:31 | and uh and ways to grow bacteria . So you can have can get |
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| 05:38 | you want, so to speak. . And then uh so well today |
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| 05:45 | get into uh growth dynamics. So growth, how fast can you |
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| 05:52 | Uh quantitative them. And uh we'll a couple of problems. You're allowed |
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| 05:58 | have a Hand held calculator. I care what type you just have to |
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| 06:04 | able to do multiplication, division log base 10 function. That's pretty |
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| 06:10 | . But there'll be a couple of . Maybe three at most have to |
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| 06:15 | with that. But we'll go through today. There's nothing complicated. But |
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| 06:22 | then it will end with this here of growth. So if we |
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| 06:33 | Yeah, don't memorize anything. Um the um you know, actually the |
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| 06:43 | you march your growth and you get get ages, right? They can |
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| 06:50 | in terms of length and the former not as we'll talk about it. |
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| 06:55 | uh that's the lag along station. read that face so I'm gonna talk |
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| 06:59 | some of the variations of growth. this is an area here that if |
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| 07:05 | a biotech major that's these are the of things you'll do actually we'll do |
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| 07:11 | of these things as well. And so anyway, we'll take a |
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| 07:17 | at that. So let's start with question. Okay. So it's kind |
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| 07:22 | relates to this kind of relate to medium. And this question here, |
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| 07:29 | . So you remember what is. . Um That's the key answering. |
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| 07:35 | just a yes or no question. . So look at that gross medium |
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| 07:42 | history and of course is amino So could a history in grow on |
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| 07:50 | medium as is. Okay. All . Let's count down. Let me |
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| 08:28 | that again. Who answered? I you to see you are correct. |
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| 08:35 | , you're not correct. Alright there no C. Okay. Um Who |
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| 08:44 | a Hey police 61 of you. you can have you know your 61 |
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| 08:55 | you answered. Yes. Okay. um Why tenant girl This video? |
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| 09:08 | because that's right. So so number the history has to be supplied. |
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| 09:14 | that's that's number one. The definition . This is a this is one |
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| 09:22 | deficient in some sort of pathway can't in this case histamine or can't make |
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| 09:29 | or whatever whatever the word is before control it can't make that. So |
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| 09:33 | have to supply it's gonna grow. so knowing that then you look at |
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| 09:38 | words medium and uh what's what's supplying history and potentially it's gonna be these |
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| 09:47 | . Alright. Pepto beef extract, ? Complex nutrients. Let's think of |
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| 09:53 | tone and beef extract. It's a about that flesh and bone. What's |
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| 10:01 | to be in that? Everything amino , nucleotides, carbohydrates in different it's |
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| 10:08 | gonna be there. So um certainly will be among those amino acids in |
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| 10:16 | . Okay so yeah it will grow the complex meat. Now if you |
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| 10:24 | it on this medium. Yes, minus that. Okay. In other |
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| 10:33 | that would be a defined medium, , synthetic medium, minimal medium. |
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| 10:41 | if you you could use that if just added which you can buy and |
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| 10:47 | can buy history by itself and just that. You can do that. |
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| 10:54 | you were if you were really testing examining the properties of this and how |
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| 11:02 | could you know how much history would and that this and that that you |
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| 11:05 | want to do that a minimal medium defined with history alone. So how |
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| 11:11 | you control everything all the parameters? um But yeah so that's what we're |
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| 11:16 | with. Castro that's what you got think about. How do I supply |
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| 11:20 | nutrients. Okay so this question. so this isn't a quicker question. |
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| 11:28 | um just focus on the components of media. Just bringing us into the |
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| 11:35 | topic here. What would grow on medium Is something missing? It's something |
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| 11:45 | think of the thinking. Six Right? six elements. Alright. |
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| 11:53 | missing. Uh huh. Anybody else down mm hmm. Else it's not |
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| 12:13 | , correct nitrogen. Okay so what grow on this? Oh there's your |
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| 12:20 | . Right well couldn't grow on it the bacteria that are in this category |
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| 12:26 | here they can actually take into and that. We'll talk about this later |
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| 12:33 | the semester. And then that's that's nitrogen. Okay so this is the |
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| 12:39 | of medium that's agriculture. This is example example of regiment culture. Okay |
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| 12:46 | you formulate a growth medium with ingredients will allow for the growth of a |
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| 12:56 | type. Certain okay um now there's we'll talk about selectively during a second |
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| 13:05 | at me. It's a very subtle maybe between the two and all |
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| 13:12 | You might you might argue that any is selective, right? Neutrino? |
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| 13:19 | can be selective. It's not it's a second media and stand corrected. |
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| 13:24 | I'm just saying you might argue idiots you can't grow everything we grow everything |
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| 13:32 | that's able to be cultured. Certain can but not everything so well |
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| 13:38 | No not quite. Okay so selective is where you are purposefully add a |
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| 13:46 | component that you know will inhibit a particularly a certain class of bacteria. |
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| 13:53 | common is and chemical agents that inhibit positives and inhibit gram negative. So |
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| 13:59 | know and a and an antibiotic right any chemical that you know will inhibit |
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| 14:06 | growth of certain types. So these all selected media. Okay. Enrichment |
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| 14:12 | a little bit different to add a to the things you're trying to combine |
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| 14:19 | components of the type of what we're growth of the type you're looking |
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| 14:26 | Okay it would be right. You're adding something to inhibit chemical to inhibit |
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| 14:33 | . You're just producing a media that will allow for the growth of a |
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| 14:37 | select type of impact. Right? can adjust it right? But you're |
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| 14:43 | to enrich for those types right? you try to look for one of |
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| 14:48 | types. Okay one of these types . Okay use a nutrient auger. |
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| 14:58 | would get in a handful of soil ? Because these are from the |
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| 15:04 | You don't get all kinds of stuff out of plate and you may have |
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| 15:08 | them fixers but you'd never probably find . Right so let's throw it on |
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| 15:14 | medium instead. Okay and now only select few things are going to grow |
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| 15:22 | that You enrich for their presence if will. Okay so it's kind of |
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| 15:28 | little of a subtle distinction but that very 30 between the original culture and |
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| 15:32 | can not selected. Right. Any about that? So um and so |
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| 15:41 | talked about this before um Different types liquid and solid media, right? |
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| 15:47 | um there's utilities for both. You use liquid cultures that you're trying to |
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| 15:53 | large volumes of whatever microbe you're working because the amount of volume is |
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| 16:00 | Right? Um And that's before. like if you want to get some |
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| 16:06 | of molecule from yourself to grow them to get enough stuff, you can |
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| 16:10 | with whether it's D. N. . Or uh plate. You |
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| 16:15 | plates, let's call it a on plate. You would take something from |
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| 16:20 | and transfer to a plate. All to get visible colony. So think |
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| 16:27 | solid media gives you a way to . Um But this is a bowl |
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| 16:36 | of the micro it's kind of But you know what's what's calling you |
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| 16:42 | plate. You know that's that is representative of one type, right? |
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| 16:48 | a cure column, right? So can some of that material, you |
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| 16:54 | go back to liquid video or do , but you can now have to |
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| 16:58 | it in your culture. And so is essential for that. Getting pure |
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| 17:04 | . Look what could be a part the process. But ultimately you got |
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| 17:07 | put on a plate to see. gives you a visual representation of something |
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| 17:11 | can work with. You can look the microscope, right? But you |
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| 17:16 | sell a pair of tweezers. You need that plate for that. |
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| 17:21 | , so and the plate will tell Well, there are other things mixed |
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| 17:25 | there. Okay. So you can of weed things out. Alright. |
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| 17:30 | everything has kind of its own And so selected media and different uh |
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| 17:37 | , chemicals in there that will inhibit classes. A lot of these |
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| 17:41 | So, let me talk about this the context of selective and differential. |
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| 17:49 | . So both of these together. . Yes, there can be media |
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| 17:54 | medium that is just selective. But very often you combine two or |
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| 18:02 | selective differential. So differential medium gives a uh difference in terms of some |
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| 18:11 | of chemical property that the cell does doesn't have a lot of these selected |
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| 18:18 | media were developed for water quality analysis what I'm gonna tell you now, |
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| 18:29 | not gonna test you on but it to uh explain. It kind of |
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| 18:33 | in terms of what's going on So, the water quality uh one |
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| 18:38 | the things you're very concerned about is presence of by definition coliform is a |
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| 18:51 | a short program, gram negative lactose . That's what coliform is. All |
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| 18:58 | . So these are indicators of fecal of water. Find something like this |
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| 19:06 | your water. That's what you're looking a waterfall. Right? And e |
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| 19:11 | is Okay. As are some And so um of course you see |
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| 19:17 | in your water quality analysis. That's red flag obviously. So all these |
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| 19:22 | differential media can quantitatively qualitatively show this you. Okay. And this is |
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| 19:30 | type of such selective differential media. basically we're looking for a gram native |
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| 19:37 | this water quality. So you don't to deal with grand positive. So |
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| 19:41 | put a chemical here that will inhibit as a selective part, differential part |
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| 19:47 | let's put in lactose sugar to see you can ferment or not. |
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| 19:52 | And these visualizations of different colors on plate are typically due to ph differences |
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| 20:00 | ph indicators, alternative certain color, know, fermentation or acidic. So |
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| 20:06 | was fermenting. It will show either color or some other type of |
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| 20:11 | depending on the ph indicator. Uh an aunt from Atlanta's. You won't |
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| 20:15 | a color change. That's really a of what these are about. But |
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| 20:18 | metabolic property we're looking at. Um so with this particular medium this heck |
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| 20:31 | and you can differentiate coliform. So a coliform. They show up as |
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| 20:38 | . They may not be may not enough contrast there, but the yellow |
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| 20:42 | . So these are kind of the colorless opaque. And these are more |
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| 20:47 | the yellowish types here and yellowish ones the lactose fermenters. Okay. You |
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| 20:57 | there. So uh these two salmonella shigella most interest for museums? Both |
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| 21:10 | . Um they don't so they give these totals colonies. They go well |
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| 21:17 | can I differentiate? Um It's the of a black color. So you |
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| 21:22 | salmonella is only showing the black color that's due to production of H. |
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| 21:27 | S. There's like sulfur compounds that convert to H. Two. |
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| 21:35 | So you can differentiate between the So this is actually kind of |
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| 21:38 | You can differentiate, enters producers. so anyway the point is these color |
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| 21:48 | can tell you something about their metabolism ? Whether they have it or |
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| 21:53 | And so it gives you a really of a quick qualitative result. Um |
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| 21:59 | easy to interpret. Okay and the quality analysis you often have people that |
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| 22:03 | working there don't have. So you something that will be easily interpreted. |
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| 22:10 | , so this is just another So blood auger is one that's used |
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| 22:16 | often in uh diagnostic purposes uh throat depending what you get. You can |
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| 22:25 | can indicate strep throat uh the bacterium causes it. Uh And so reactions |
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| 22:31 | blood are are used for that So especially with the group called |
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| 22:40 | Okay, this encompasses and we'll talk this later this semester but streptococcus has |
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| 22:46 | number of pathogens um strep throat. called fever, flesh eating disease? |
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| 22:57 | cavities Alright, tooth cavities uh gastrointestinal and so a lot of them. |
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| 23:05 | they historically that you can kind of the types on how they behave on |
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| 23:10 | augur right? Some some groups some group a strep licenses these red blood |
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| 23:18 | so it gets a clear zone, zone around them. Others kept different |
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| 23:23 | . So you kind of differentiating that . So anyway, differential selective |
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| 23:28 | Okay. Just on the tool you use you know depending on kind of |
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| 23:33 | you're doing in this case, water analysis or diagnosis of something can |
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| 23:38 | Okay. Um All right. Any about that in the next 15 20 |
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| 23:47 | on quantity of bacterial growth? Okay we'll go through kind of step by |
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| 23:56 | . Do a couple of problems and I said it's not that complicated but |
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| 24:01 | get through it. So this is kind of intro intro not a clicker |
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| 24:05 | . Um so it's just a It's kind of it's not gonna shock |
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| 24:11 | . I don't think that just kind to show you, you know double |
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| 24:16 | time and how that can be a that can indicate a lot of |
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| 24:22 | Okay and so what we have here a bacterial two types of bacteria, |
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| 24:30 | has a four hour doubling time and 1 15. Right so doubling time |
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| 24:42 | the same thing as generation time. , bacteria that binary vision one generation |
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| 24:49 | be one cell divide into two. . More practical standpoint. We use |
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| 24:55 | We use generation time uh population to right when we're measuring growth. Okay |
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| 25:04 | it's it's uh typically use optical We measure the cloudiness of liquid culture |
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| 25:13 | as a parameter of their growth. optical O. D. Units refers |
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| 25:18 | optical density. So it has an of 00.5 versus a 12 is much |
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| 25:27 | . 1.5 indicates absorbed into light as grow as a culture of. So |
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| 25:35 | you do is it's prone to .5 that time there was a generation |
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| 25:42 | Okay. Time for it to So um so in on both sides |
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| 25:48 | we have so so the equation we and this is the real basic equation |
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| 25:54 | this thing here um Alright here my is not working. Okay. So |
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| 26:03 | one here uh so in this population . So what's the population of eight |
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| 26:10 | zero versus sometime in the future T Okay. And is generation |
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| 26:18 | Okay Um so at 20 hours, many cells do we have, what |
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| 26:25 | looking for on each side? How many generations are there in 20 |
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| 26:30 | ? OK And so it's a generation or doubling time? On the left |
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| 26:37 | one generation every four hours and 40 ? We've got five generations. And |
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| 26:44 | on this side. Oh I'm we keep going. So now we're |
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| 26:48 | gonna plug in the values. So this is in is is that's |
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| 27:00 | Not working? That's in. Okay we just plug it in and zero |
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| 27:06 | 10 cells right from up here. and uh multiplied by two to the |
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| 27:13 | right to the ends we get 320 . But that was for anyway on |
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| 27:21 | inside It's one generation. Every point every 15 minutes, 15 minutes, |
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| 27:29 | generation every 15 minutes or .25 Right? 5 28 generations. And |
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| 27:36 | think you already know how much we're to differ here numbers. Right? |
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| 27:41 | that's two years of being an 10 to 25th, 7, 10 |
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| 27:47 | 25th versus versace versace, jesus, on. Um this versus that huge |
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| 28:00 | . Right? So just that what not seem like a big 10 times |
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| 28:05 | huge in terms of how many cells get. E coli has something in |
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| 28:08 | order of about 15, 20 minutes optimal conditions. Okay now um the |
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| 28:16 | how fast can attend something in nature produce that many cells in a short |
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| 28:24 | . Not very often, but there be influx of nutrients somehow that can |
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| 28:29 | this to happen. We'll look at of those things when we talk about |
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| 28:33 | ecology because there can be an influx nutrients that can cause of birth but |
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| 28:39 | days are finite and generally there's very strict competition and nutrients are typically limiting |
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| 28:46 | you don't get this kind of explosion growth but it can't happen here and |
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| 28:50 | . So um as we look at , we're going to kind of get |
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| 28:56 | somewhat from this, this basic Right? And then solve producer producing |
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| 29:03 | we're in is more easily figure it . Okay, Because once we can |
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| 29:09 | that number for a lot of different as well. See Okay, so |
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| 29:16 | so as mentioned you can look at that's certainly producing one generation. They're |
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| 29:23 | but again we use typically the time a population that double as our generation |
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| 29:28 | unlimited growth. You may have. recalled from ecology you talked about um |
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| 29:37 | in the context of microbes necessarily but all fits um remember recall the J |
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| 29:44 | curve. Right? So if you in our context here with time, |
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| 29:52 | axis number of cells on this A J shaped curve gives you that |
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| 30:01 | rapid growth. Of course that doesn't on forever as we all know. |
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| 30:06 | , it flattens out, right, talk about this in the next section |
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| 30:11 | on growth curves. Right. But exponential growth can be on occasion where |
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| 30:16 | does happen, but again it's a thing. And so with the way |
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| 30:22 | course microbes grow Exponential growth and exponential growth growth are kind of used |
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| 30:31 | Okay, it just means a very growth 4-8-16 and so forth. This |
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| 30:37 | of progression here. Okay. And um and so we're dealing with a |
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| 30:45 | that can change and very big, range of numbers from small to very |
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| 30:52 | , right? Like a ph scale also a water based and scale. |
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| 30:57 | ? So you try to kind of compress so you can make it more |
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| 31:02 | . And when you plot data in way there is a typical growth curve |
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| 31:08 | would uh get here. So here's density. So again using measurement of |
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| 31:16 | cloudy liquid getting is bacteria growing um taking the log values of that log |
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| 31:25 | gives you a this would be the of the very rapid growth that would |
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| 31:32 | . Okay. And so um so very easy to measure. Okay this |
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| 31:38 | respected thermometer and liquid culture of bacteria just take samples and you measure any |
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| 31:46 | . So um so let's uh look uh converting this equation here here into |
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| 31:58 | that is more manageable as we'll see . Okay. So we're gonna do |
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| 32:03 | we're gonna solve this equation for little Number of generations. Um so what |
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| 32:10 | do is we start to start going a lot of the base 10. |
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| 32:14 | . And we we remember logs Multiplying dividing of logs is a little |
|
| 32:22 | . Okay. And so this expression can be simplified you see in the |
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| 32:33 | . Right? So this will simplify that so long as the base 10 |
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| 32:40 | the end is the same as Times log base 10 of two. |
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| 32:45 | that number equals 20.301. Right? that equals that. Okay so you |
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| 32:56 | so think of that as the as power of growth. Right? This |
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| 33:01 | growth. Okay? And so then can just solve for n. And |
|
| 33:06 | get that. Okay, so uh very often what we like to do |
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| 33:14 | a time element to that. so measuring number of generations over time |
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| 33:19 | can do that. We can get it's called a growth rate comes about |
|
| 33:24 | . Notice just adding time to right? Right here. T. |
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| 33:31 | T. Okay, so every species kind of growth rate constant, |
|
| 33:37 | Uh brought you grow it there will a growth rate you'll get every time |
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| 33:45 | grow it you can go the same your bras, you'll always get that |
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| 33:49 | growth rate constant. Okay, For any species. Okay. And |
|
| 33:55 | the other thing to value here is generation time rights. Okay, this |
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| 34:02 | per time. Right? So remember this, right? This this right |
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| 34:12 | , is that which is generations. ? And so we're just putting a |
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| 34:18 | element to it as well, generations time. So the inverse of that |
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| 34:22 | the generation time. Okay, so gonna use and these will be again |
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| 34:29 | to you. So we're gonna use We're gonna use this one. |
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| 34:38 | And this one for our problems. And it's typical that generation times in |
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| 34:48 | but you may not see it in for something that's really slow. |
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| 34:53 | pretty much it's usually minutes. so we're gonna look at this |
|
| 34:57 | I'm not gonna ask you to derive equation on a on a test, |
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| 35:04 | be given this formula. Um So won't need to memorize that. Okay |
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| 35:12 | as we do the problems, you'll how this all works. Okay so |
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| 35:16 | first one here, let's try it your own first. Yeah you do |
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| 35:23 | not sure about it. It's That's why we're gonna go through |
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| 35:26 | Um step by step. So the houstonians, cougar Insys has a generation |
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| 35:40 | . 40 minutes. Okay. Starting five cells in log phase. How |
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| 35:46 | minutes to produce? About 10,000 Okay so the way I'm gonna show |
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| 36:25 | you're gonna think it's probably kind of but I just kind of set it |
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| 36:29 | up. Right? So you kind know logically how we go through each |
|
| 36:35 | . Okay. Okay. It's just camp down. Yeah if you're not |
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| 37:05 | , just Give it your best Okay. Alright I'm counting down from |
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| 37:18 | . You anything. Alright so let's through it. So step one like |
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| 37:30 | said I keep it kind of basic set everything up here so you can |
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| 37:34 | the logic. So uh so we're zero N. T. Perhaps. |
|
| 37:39 | so going for 5 to 10,000, long is it gonna take? Right |
|
| 37:44 | what we're asking. Okay so we this right generation time is minutes per |
|
| 37:50 | so if you can figure out right basically asking actually uh Yeah, |
|
| 37:58 | we can figure out in, So we can figure out this that's |
|
| 38:05 | end value, Right? Number That's in. Alright. So that's |
|
| 38:08 | this right here. Okay. So know N. T. And |
|
| 38:12 | Zero right here and there. So plug it in. Right? So |
|
| 38:18 | we'll do is we know that. right. 40 minutes per generation is |
|
| 38:21 | we're given. Um If you can a number of generations, right? |
|
| 38:27 | figure this out. Um Then we use that to multiply by our generation |
|
| 38:36 | . Not give us minutes because generations cancel out. Okay? So here's |
|
| 38:41 | we do it, right? So then plug in R. N. |
|
| 38:46 | . Uh N. Zero values five 10,000. Do the math. And |
|
| 38:51 | get 11 generations. Okay. Then we take that multiplied by generation |
|
| 38:59 | is canceled, right? We have minutes, which is 60 minutes in |
|
| 39:05 | hour, 60 times seven is 4 at seven hours. A little over |
|
| 39:09 | hours back to right this one lower . Okay. Um That's okay. |
|
| 39:22 | if you just if you set it like I like I did then I |
|
| 39:27 | it makes sense logically. Okay. wise. Okay. Um And uh |
|
| 39:34 | questions. It's not clicking yet. mean I also know that um because |
|
| 39:43 | says here uh There's like there's like practice problems. Either two of |
|
| 39:50 | There's three new ones in there, there is a sheet in there that |
|
| 39:54 | you the blow by blow like I did it here. It works it |
|
| 39:57 | out. So you're having it's not quick and look through that, |
|
| 40:03 | If you have questions let me Okay um Okay so here's another way |
|
| 40:09 | ask a question. Okay this is for a generation time. Okay. |
|
| 40:15 | actually calculated generation time, so that's generation time is, right? So |
|
| 40:22 | if we have not heard cells for hours That produces over three million |
|
| 40:30 | what will be the generation time? So let's see if we can do |
|
| 40:34 | one timer's on. Okay let's count 54. Okay uh see what we |
|
| 42:19 | here. So we set it Okay parameters 900 minutes. So if |
|
| 42:31 | ever M. T. And Zero. So we figure out a |
|
| 42:35 | of generations, right? And we how much time has elapsed that will |
|
| 42:40 | calculate generation time, right? So . Is almost 12 generations And our |
|
| 42:49 | spans 900 minutes. So divided by and you get 76 minutes. |
|
| 42:55 | So anyway you may might need may may not need to do the set |
|
| 43:00 | kind of a thing, but I if you do that you're less likely |
|
| 43:03 | make mistakes. So but it's up you. Okay but like I said |
|
| 43:07 | more practice problems if you want to through those questions. Uh let me |
|
| 43:13 | . Okay everything questions now. Alright so um of course what do |
|
| 43:23 | do with this stuff? Well if have for example uh you threw at |
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| 43:30 | time when you're growing the bacteria on certain growth medium and you want to |
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| 43:33 | can I improve that by including some the nutrients? This will give you |
|
| 43:38 | quantitative answer of that. Uh Maybe testing some kind of antimicrobial and you |
|
| 43:45 | to your culture of control, it's added. Look at generation time. |
|
| 43:50 | there a difference there? So you there's of course practical applications to |
|
| 43:54 | Okay um The okay so speaking of let's go into um growth curve. |
|
| 44:02 | here's a question. Okay so there's parts of this growth curve of course |
|
| 44:09 | different things happen. Each grows Okay so you're looking at that um |
|
| 44:18 | me just explain what batch grows a growth curve is. Okay does anybody |
|
| 44:23 | a water bottle on or other water ? So let's say this is so |
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| 44:40 | rose just simply you not claim Okay and then the only time I'm |
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| 44:47 | do anything with this after that point to take samples and measure growth. |
|
| 44:53 | that's what I'm gonna do and then gonna follow it all the way through |
|
| 44:58 | it dies. Okay I'm gonna see kind of growth curve I get and |
|
| 45:04 | all I'm doing. So that's basically one batch right? And we're just |
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| 45:08 | the cells from birth to death if want to think about it that |
|
| 45:12 | that's what batch growth way there are of that. Uh So uh let's |
|
| 45:25 | what we have here. Okay and like I said we can do manipulations |
|
| 45:34 | just that basic batch growth to get sells. Right? Um And so |
|
| 45:41 | kind of things you know on a scale if you especially biotech majors working |
|
| 45:49 | industry and you're gonna commercialize something maybe micro or some kind of enzyme or |
|
| 45:55 | protein of interest um that you it's practical to work. I mean not |
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| 46:03 | just to just have tea spoons and . You need buckets and barrels and |
|
| 46:08 | right? Which means you have to these cultures of the high density to |
|
| 46:12 | in the product right? If you're commercialize and make money on it. |
|
| 46:16 | So there's ways to do that as see. Okay so correct answer here |
|
| 46:30 | yes is e. Okay all these true. There's no false ones. |
|
| 46:40 | So you do get cell size changes time. Okay? Um We'll explain |
|
| 46:48 | exponential changes. So certainly there's exponential here. There can also be exponential |
|
| 46:54 | here. There is in death right? So when you're given plentiful |
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| 46:59 | you can grow like crazy. Okay when you're when nutrients are completely gone |
|
| 47:07 | equally die like crazy going this way it's really this part of the curve |
|
| 47:15 | we focus on in chapter five? looking at control of microbial growth? |
|
| 47:18 | you want to add stuff and kill whether physical or chemical means to make |
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| 47:25 | die as fast as possible. Um So remember d is about rapid |
|
| 47:31 | , right? When you have rapid is typically when penicillin has better action |
|
| 47:37 | . So it's gonna be in phase . Uh So let's look at the |
|
| 47:43 | of these. Okay so uh so batch growth curve. And um so |
|
| 47:52 | face. Okay which is right here part here now whether um whether how |
|
| 48:06 | this is Right that slag. So begins once you inoculate inoculate needs to |
|
| 48:13 | , it's like planting seeds and adding . Plant growth. Right? You |
|
| 48:16 | cells to a liquid medium. Not . Okay that's what they call it |
|
| 48:23 | . There's a lag before it picks . Okay so that lack many things |
|
| 48:30 | . Is the lag like this? it shorter? Is it maybe gross |
|
| 48:37 | here instead or is it long? . The length of that, like |
|
| 48:43 | of things factoring. Okay so picture bacterium. So what's the nature of |
|
| 48:52 | because that came from something? It be from a plate culture. It |
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| 48:57 | be from a liquid culture. How was that inoculation growing in liquid culture |
|
| 49:02 | three days? Or the large cells ? Okay. How much did you |
|
| 49:10 | it you had when Miller had 10 did you are the media media in |
|
| 49:17 | one the same. Right. Are , are they completely different? |
|
| 49:21 | Uh so all these factors. So think of kind of the micro |
|
| 49:27 | of the cells. Right. So in an in ocular and that gross |
|
| 49:31 | , whatever it is. And now plop them into something super fresh. |
|
| 49:35 | . Well that's gonna be slight subtle in temperature, ph salinity concentrations before |
|
| 49:44 | to it. So all these factor while the lag place. Okay, |
|
| 49:51 | if you're going from a medium medium its very different. Right? So |
|
| 49:56 | a medium that's very rich. lots of nutrients that can just take |
|
| 50:01 | and I have to make anything and it's going to a million the meeting |
|
| 50:08 | are gonna be put on because I'm be able to start with because it |
|
| 50:10 | to turn on different metabolic pathways to able to grow in this meeting. |
|
| 50:14 | . And all that takes time. . So again, what factors into |
|
| 50:21 | ? How long a lag faces are things growth media type? Physical |
|
| 50:27 | there's probably some slight differences in 02 media. Okay, the age of |
|
| 50:33 | size etc. So all these things in to that lag face. And |
|
| 50:38 | um but once it is out of it goes right, so once it |
|
| 50:44 | acclimated then it takes off. And that can be quite rapid depending |
|
| 50:50 | the growth medium and these other Okay. So of course log represents |
|
| 50:59 | most active metabolically speaking. And so not uncommon that uh in we refer |
|
| 51:07 | what's called mid log phase. Call that mid log if it's producing |
|
| 51:16 | of interest enzyme or whatever that you will like the sample during that time |
|
| 51:21 | figure out to get a good measure the activity. Okay. Now it |
|
| 51:27 | vary depending on what the product is the microbe. Um that's why you |
|
| 51:34 | in the early stages of you are to commercial process a biological process. |
|
| 51:40 | figure out all these parameters called bench which is like small flats and small |
|
| 51:47 | . Right? You can take a of measurements very easily. Right? |
|
| 51:52 | simple experiments very easily with smaller maps stuff. But the ending wrap it |
|
| 51:59 | scaling up. Okay. And so the bench scale you figure out, |
|
| 52:04 | , what is the basic growth curve this meeting? What do I see |
|
| 52:09 | take samples all along the way to , Okay, what is the activity |
|
| 52:13 | my product at different points? Where I measure it at? Where is |
|
| 52:17 | optimal? These are all things you out when you know at this |
|
| 52:22 | Okay, then it becomes Okay, here's the baseline. Now, how |
|
| 52:28 | I enhance things. Right. That's that's now where you manipulate growth |
|
| 52:33 | Right? You have different things To enhance the product formation. |
|
| 52:39 | And so that's a whole other So ultimately you get to a stage |
|
| 52:45 | this is the one that works and scale that up. Okay and scallop |
|
| 52:50 | be anything from 500 Uh 20 gallons two 1000 leaders to um 10,000 or |
|
| 53:05 | seen 100,000 liter tanks. So it depends. Okay but it's all these |
|
| 53:11 | things we're talking about that you do the early stages. Okay so log |
|
| 53:16 | aside from fast growth and lots of , his cell size changes. So |
|
| 53:23 | actually growing lots of cells and that of any kind of this uh if |
|
| 53:27 | drawing out a rod shaped cell and of this kind of state of |
|
| 53:32 | right? Not to sell her in form right? As they split and |
|
| 53:38 | two cells. Right? So the of the cell itself, right? |
|
| 53:43 | get they can elongate and then they big enough and they split and form |
|
| 53:49 | selves. Right? So their biggest log phase. Okay whether a rod |
|
| 53:54 | a caucus, they're gonna be at biggest size and likely in this mid |
|
| 53:59 | phase. Okay. Um So but course you're gonna get to a point |
|
| 54:07 | , sorry up here, Lake Right, and so now that tipping |
|
| 54:16 | where you get so many cells we have enough nutrients to sustain everybody. |
|
| 54:20 | ? So the road is gonna rapidly and death growth rate. Death rate |
|
| 54:25 | much equal. And I'm getting the phase. Okay so uh so now |
|
| 54:30 | kind of becomes survival mode. Okay we're not out of nutrients yet, |
|
| 54:38 | we're certainly limits getting more limited. now these kind of like a stress |
|
| 54:43 | . So as it as the cells kind of limited nutrients, they tend |
|
| 54:49 | then shut off or minimize metabolic pathways don't need. Okay especially those that |
|
| 54:58 | energy. Okay, so like protein is limited to only only critical type |
|
| 55:06 | or four critical type of functions. So size, it's small, |
|
| 55:11 | Um smaller cells just to keep up the right, better able to survive |
|
| 55:17 | being a big one. Big cell it was in the log face. |
|
| 55:22 | And so you know of course as has become limited, you know, |
|
| 55:27 | more cells are dying, right? Dead cells are actually food sources |
|
| 55:33 | right? The celeb diet, they their full of carbohydrates and protein. |
|
| 55:38 | that could be used as a food . So the cells can live off |
|
| 55:40 | for a while. But ultimately when get to hear right, then you're |
|
| 55:47 | . Zero, right? Nothing to . Right? And that's why death |
|
| 55:52 | exponentially right? There's there's nothing no brooks and curves, they're gonna start |
|
| 55:57 | dying very rapidly. Okay, So but there are we mentioned I mentioned |
|
| 56:05 | these types that are per sisters or types. Uh They can prolong they |
|
| 56:16 | stationary phase. I'm not gonna say forever but for a good while they |
|
| 56:23 | rely on a member they can maintain member of potential. So we can |
|
| 56:29 | about the proton pump. So they maintain kind of something like that to |
|
| 56:35 | them for a while. Not enough really gross but remain viable at this |
|
| 56:41 | about remaining viable. Not so much . Okay so but that can sustain |
|
| 56:47 | for a while. Another example of will talk about next time it's forming |
|
| 56:53 | industry. And those sports are kind normal forms of itself. Okay so |
|
| 56:58 | that can be an option depending on cell type. Okay so some things |
|
| 57:02 | can happen when so it's getting these limited states. Some can form a |
|
| 57:08 | some control what's called a cyst which kind of spore like. So there |
|
| 57:12 | be some forms that can allow them survive for a period of time in |
|
| 57:17 | kind of stress states. Okay now not not all not all bacterial or |
|
| 57:23 | types can do this but there are that can. Okay um Okay so |
|
| 57:31 | any questions? So let's look at question here. Okay yeah so this |
|
| 57:44 | let me just get this here. so this one I kind of mentioned |
|
| 57:51 | already but let's do this anyway so bacterial inoculate um Has grown nutrient broth |
|
| 57:58 | sample of his column is transferred to . That's basically. So we're going |
|
| 58:05 | here to hear. Okay so this our kill him this is our batch |
|
| 58:16 | medium. Okay so we're not playing with the culture growing nutrient broth. |
|
| 58:24 | so we have these patterns A. . And C. So note right |
|
| 58:32 | the in Oculus um is grown in broth. Right? It looks like |
|
| 58:37 | . Right so we go from that that we get that. Okay just |
|
| 58:46 | a comparison. So again not like put it in M9. What's it |
|
| 58:57 | look like? Is it gonna look a as well look like be like |
|
| 59:02 | okay time around. Okay. Yeah predicted that. Yeah focus on |
|
| 60:10 | Right. It's all about changing the phase. Right? So um they're |
|
| 60:17 | to a middle meat. So now got a nutrient broth it can get |
|
| 60:24 | nutrients from these components. Give me acid etcetera. You're going to go |
|
| 60:30 | M. Nine. It's gonna it's only got the basics here right? |
|
| 60:34 | so it's gonna have to make these track so to speak. And so |
|
| 60:40 | gonna take time and turning on genes cetera and that means more time. |
|
| 60:46 | lag face. Uh So alright so from that one so let's think about |
|
| 60:52 | in terms of a different example here say that this is N. |
|
| 61:01 | And this is N. B. broth. Okay both those RNB. |
|
| 61:07 | And so the cells the same cell say just say it's equal equal. |
|
| 61:12 | hear an equal here. So number . So the curve, you know |
|
| 61:21 | inflection and whatnot. Right? All kind of looks the same. More |
|
| 61:27 | less. Okay. But of course number of cells. Right. So |
|
| 61:30 | have here and then up here. , so there's a difference there and |
|
| 61:40 | yield. Right? Both grown and but B has more cells. |
|
| 61:45 | So they're both grown on this on nutrient broth. But yet he |
|
| 61:53 | more cells. So that that's that's to what we call batch back |
|
| 62:02 | Okay, So you have batch growth then you can feed that batch, |
|
| 62:07 | can add stuff, right? And growth. Okay, so uh exactly |
|
| 62:15 | we're doing. So we can get a point. So maybe like right |
|
| 62:20 | , right here, we would there at that point we'd add at what |
|
| 62:33 | gonna be the biggest influence in So you another worker glucose would be |
|
| 62:41 | . Right? So added carbon. ? And you can take a trip |
|
| 62:46 | even though it doesn't have broth, can add just add carbon. Doesn't |
|
| 62:51 | . But for me it matters that has to be heated but um he |
|
| 62:56 | lots of things. So and add glucose to your broth that will increase |
|
| 63:02 | . Okay, and uh that's what do. And these kind of biotech |
|
| 63:11 | settings, right? When you're trying get lots of cell yield the way |
|
| 63:16 | get maximum yield is to control everything temperature and ph control oxygen levels control |
|
| 63:25 | source, right? If you do that they'll grow insane. Okay. |
|
| 63:32 | high yields. Okay. You just to figure out what it is that |
|
| 63:36 | like and need makes them happy. . So what you see here on |
|
| 63:40 | left, okay, is a computer controlled, computer controlled setup by |
|
| 63:48 | . Okay. Where you control all parameters? Right over here, our |
|
| 63:54 | again, all programmed for maintaining ph of them are being nutrients. So |
|
| 64:01 | source, likely timed intervals um and all the stuff up here uh tubing |
|
| 64:10 | whatnot coming out the top of the . These are different other controls. |
|
| 64:16 | ? So if you grow uh let's we can do this batch growth. |
|
| 64:28 | ? And we get um that and get a lot of growth. The |
|
| 64:35 | batch thing, right? And we we do it multiple times. Do |
|
| 64:41 | maybe two or three more times. ? And we get up to say |
|
| 64:45 | get up to up to here Let's say we get I don't know |
|
| 64:50 | make up a number. We get 100 od 100 0. D. |
|
| 64:56 | . Optical density, super thick Right? And it takes us let's |
|
| 65:02 | let's say it takes us I don't um you have to add make up |
|
| 65:08 | crazy number 1000 g of glucose and get that high density super high. |
|
| 65:20 | have to do it over three or feedings or more. Okay. But |
|
| 65:25 | get that high. Now the question why Why can't we all be at |
|
| 65:30 | beginning? We have to have it a pain in the butt come in |
|
| 65:33 | add stuff every three or four hours front. You have to worry about |
|
| 65:41 | you do that. It turns out they don't grow. Why was |
|
| 65:46 | The property is something we talked about you three. What might be the |
|
| 65:55 | that they wouldn't uh all this stuff . 1000 g of glucose poured in |
|
| 66:02 | the beginning plus the other stuff. actually high amount of what in that |
|
| 66:12 | ? Yes, maybe uh osmosis So be super hyper tonic. If |
|
| 66:22 | do that. Okay. And that or inhibit growth. So I just |
|
| 66:27 | water. It's fighting water coming out the cell into the surrounding. So |
|
| 66:32 | focused on that and not focused on . Right? So that's why you |
|
| 66:37 | do that. Okay. You have do it, get the meter it |
|
| 66:42 | right over time. Uh If the bank chairman culture is medically fit. |
|
| 66:48 | if you do all this stuff right reactor very quickly become oxygen. |
|
| 66:55 | So heroes in this case and that's what you're growing is ending its |
|
| 67:00 | cell densities and error because that's going give you the most cell density. |
|
| 67:05 | so you control that as well. we can control all these things. |
|
| 67:09 | ? So here's an example of a and a different control. Right? |
|
| 67:14 | we have nutrients oxygen. There's a that measures oxygen concentration, ph and |
|
| 67:21 | probe. So I'm measuring all these . Okay, acid base edition. |
|
| 67:28 | ? Um So um so you measure things, right? And particularly |
|
| 67:34 | you wouldn't think temperature but if you have temperature control, so the water |
|
| 67:40 | , cold water helps to maintain If you didn't have that e coli |
|
| 67:45 | growing up to like 100 ods, glass would be super hot. In |
|
| 67:51 | , itself would die. It gets hot. Remember bio energetic giving out |
|
| 67:56 | as as a part of the And so it can get super hot |
|
| 68:00 | quick when they're growing like crazy. you have to water water control the |
|
| 68:05 | . Um And then of course uh ph is relatively simple. Use ad |
|
| 68:11 | bases needed uh at nutrients um as . And typically these tie together nutrient |
|
| 68:21 | and D. O. Can kind tell you resolve options to kind of |
|
| 68:24 | you when you add more nutrients But then you need to um uh |
|
| 68:30 | auction you can add air, And so it comes in and then |
|
| 68:37 | thing here called the Spar Jer this so that's sort of the area isn't |
|
| 68:43 | bubbled in, right? It's forced through a Spar Jer. Right? |
|
| 68:48 | that does is creates tiny bubbles. ? Gasses makes better into a quick |
|
| 68:55 | if they're tiny bubbles, big So spark. There's always a part |
|
| 69:01 | the process to make it gas more in the liquid. Right? So |
|
| 69:05 | greatly increases absorption. And then the these things here are the whole thing |
|
| 69:14 | called an impeller blades and the terms right, rotate, creating turbulence, |
|
| 69:21 | ? To help mix those gas bubbles the medium as well as other stuff |
|
| 69:26 | . So so typically when D. . Dissolved the auction goes down, |
|
| 69:31 | increase it a combination of things to the turbulence and get mixing, you |
|
| 69:39 | compressed air into the system to get in there. So a combination of |
|
| 69:43 | those things will help you get enough itself will grow very quickly. |
|
| 69:50 | high cell density. Now, if don't have all the sophisticated equipment, |
|
| 69:56 | . And I've been there you can a lot of stuff with just this |
|
| 70:02 | surprisingly okay with just that okay, can control ph now you're doing all |
|
| 70:11 | this. You're doing manually, It's not computer controlled. It's it's |
|
| 70:16 | controlled. Okay. That can be pain in the butt, okay because |
|
| 70:20 | have to constantly monitor it. You to go in at all hours of |
|
| 70:24 | night and whatnot. But nonetheless you have a ph indicator so it turns |
|
| 70:30 | when it's acid ready when it's So you just add the original and |
|
| 70:35 | the red color range. Pop right if it's um uh the the |
|
| 70:41 | . O. Part of the Right we don't have a D. |
|
| 70:43 | . Probe in here but so we can't monitor that. But what we |
|
| 70:49 | do is maximize the amount of air into it. Okay so you notice |
|
| 70:54 | have these little indentations here in the bottom. Right? And so that |
|
| 71:02 | helps create more turbulence, more Okay surprisingly it makes a difference if |
|
| 71:07 | look at a just a flat bottom glass where there's one like this there's |
|
| 71:11 | significant difference in terms of how Okay that plus you can these things |
|
| 71:16 | always in a incubator course and help temperature so you can do that and |
|
| 71:24 | you shake them. It's a platform shakes so you can increase rpm and |
|
| 71:34 | can increase rpm. Have these little they call those baffles baffled flask. |
|
| 71:41 | that that combination can help you get of errands. So you're never gonna |
|
| 71:46 | the equivalent of what you get in thing but I was shocked myself how |
|
| 71:52 | growth you can get with just controlling that way and my manual addition and |
|
| 71:57 | these other things. So again I'm gonna get what you get in a |
|
| 72:01 | . Plus the bioreactors. All computer . Right? But nonetheless you know |
|
| 72:06 | can you can do some things depending the resources you've got. Okay um |
|
| 72:14 | a lot of the stuff I've just talking about this for you, biotech |
|
| 72:17 | . I'm not gonna give you an questions on what esparza is. |
|
| 72:22 | but hopefully you can apply this knowledge . Okay? Um Any questions? |
|
| 72:32 | . So I'm gonna keep. Perfect. Alright, so almost quarter |
|
| 72:38 | , so thanks folks, we'll see on better |
|
