| 00:00 | mhm mm. Alright folks let's get . We've got we're gonna finish up |
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| 00:26 | six, finish up chapter six which the last unit that will be covered |
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| 00:34 | the exam don't have that much more do so like I'm gonna get out |
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| 00:38 | here early. Um So of course week remember exam one? Next friday |
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| 00:47 | will be multiple time slots there if haven't signed up. So the scheduler |
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| 00:53 | open. Um so of course look the exam one review sheet if you |
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| 00:59 | , if you're wondering what's gonna be the exam, that's what's on |
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| 01:02 | that's what's right in front of my when the test is made, so |
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| 01:05 | not on there. Not in the . Okay um Black bar, so |
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| 01:12 | quiz this week is not a typical know 567 question quiz. Okay um |
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| 01:23 | not it's about 2234 questions or Okay of course it's a little more |
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| 01:29 | to do it in as well so be you know just be aware of |
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| 01:34 | . So I think you have 45 to complete it. So account for |
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| 01:39 | little more time I guess trying to . So that of course is open |
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| 01:44 | through monday then um mastering. So have one assignment due on monday that's |
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| 01:51 | chapter six. So next Tuesday. we started unit to next Tuesday and |
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| 01:57 | material is available. Um So in seven is kind of an extension of |
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| 02:05 | in a way and six we're talking growth. Um seven, it's actually |
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| 02:11 | about how do we stop growth. it's about looking at the effects of |
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| 02:15 | , disinfectants, etc, that kind stuff. Okay, so um |
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| 02:21 | so with chapter six here we're looking growth, we talked about different types |
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| 02:28 | media. How you put these ingredients . Remember the C H O |
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| 02:31 | P. S. Right? We these in order to facilitate growth. |
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| 02:38 | ? The molecules ah biomolecules, carbohydrates, nucleic acid structure made of |
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| 02:49 | with these various atoms on them. , so we formulate these media with |
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| 02:55 | contain these things. Of course. . And so we know that |
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| 02:58 | different nutritional types require different combinations of things. Different forms, right? |
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| 03:04 | you're autotrophs we need co two if head or a trophy like us, |
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| 03:08 | need more complex organic materials. So account for those things, right? |
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| 03:13 | then of course things like temperature ph levels all have to be adjusted maybe |
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| 03:21 | to grow what you're trying to So today it's more of um looking |
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| 03:28 | kind of you might call growth how there's no calculations you'll have to |
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| 03:33 | but we're gonna look at kind of real basic things um basically how the |
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| 03:40 | , if you if you inoculate a growth medium of some type of |
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| 03:44 | you can track it from beginning to and it will show you some kind |
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| 03:50 | pattern. Ok, they all pretty kind of the same uh pattern but |
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| 03:55 | differ in the length of the different or how maybe how high it will |
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| 04:01 | in terms of how much cells you'll these kind of things. But the |
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| 04:04 | pattern, the kind of shape is same. So we'll go over that |
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| 04:09 | then I think we finished up with of what I guess a growth |
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| 04:14 | The biofilm formation, look a little of that and that will close out |
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| 04:20 | Six okay. So let's start with question. We had we had this |
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| 04:25 | not gonna be a clear question because already had it before. But it |
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| 04:28 | this question. I think in chapter , in the context of chapter one |
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| 04:33 | what we're talking about pure culture Remember I guess it was coke as |
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| 04:37 | was coming up with the germ had to isolate bacteria to develop techniques |
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| 04:43 | do this. So this kind one of the things we're talking about |
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| 04:46 | at the beginning is the the use growth medium types of growth medium and |
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| 04:53 | is their use for different things? . So this is kind of asking |
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| 04:56 | question really, what's the use the of a liquid media versus a um |
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| 05:06 | media, right, urine lab, obviously doing these things. And so |
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| 05:11 | not using flask, you're using a test tubes but nonetheless to contain liquid |
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| 05:16 | . And so if you have environmental sample or a patient sample |
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| 05:20 | You might want to grow up what's there and then see what you've |
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| 05:26 | So if you're doing a trying to a pure culture, in other |
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| 05:30 | um have a plate that will ultimately only one type of bacterium, one |
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| 05:37 | and one species only with nothing else it. Right? Your culture that |
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| 05:42 | media has to be a part of equation, right? Because yeah, |
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| 05:46 | may grow up in liquid, You'll do like maybe growth studies in |
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| 05:50 | . But if you want to if have a mixture of things in |
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| 05:54 | okay, you cannot hope to obtain pure culture because yeah, you can |
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| 06:00 | a sample in the microscope and oh yeah, there's like X, |
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| 06:03 | . Z. Whatever swimming around in . I can see there's like 10 |
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| 06:06 | 20 different things swimming in there. , that's fine. And well, |
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| 06:11 | you've established that. But there's something can do with that unless unless you've |
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| 06:15 | to the future and come up with super micro tweezers and you can actually |
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| 06:18 | out individual cells. Obviously that's You can't do that. So what |
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| 06:22 | you do? You try to put on plate because now you get a |
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| 06:26 | representation something, a physical entity that can deal with work with. |
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| 06:33 | You can take that. Here's a of what was in here now in |
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| 06:39 | form and you can isolate these different into pure culture. Right? So |
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| 06:46 | so solid media is always gonna have be a part of that somewhere if |
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| 06:50 | what your goal is. Okay, obviously we're trying to figure out growth |
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| 06:56 | of organisms. X. Right? here is X. Right here and |
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| 07:03 | a bunch of other stuff. Um you need to get this one away |
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| 07:09 | everything else and in pure culture. now you can start with that plate |
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| 07:15 | pure culture and do what you do you want to do. Knowing with |
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| 07:19 | knowledge that any kind of data you're now from that pure culture, you |
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| 07:24 | , applies to that organism and you're dealing with a mixed culture anymore. |
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| 07:29 | . So um so again this kind goes hand in hand with we just |
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| 07:33 | just literally just talked about liquid versus media. The utility of liquid media |
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| 07:40 | if you want um growth studies because very common to get your growth |
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| 07:47 | which you'll see here in a Okay So number of cells and |
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| 07:54 | Okay. And you'll see an S curve. And so you want there's |
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| 08:00 | uses to get something like that. . As we'll talk about shortly but |
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| 08:06 | get these values right? These are values that you get and you plot |
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| 08:13 | . Okay. And very common. absorb ints is how you do |
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| 08:18 | You would take for example that flask test tube popping in the spectrum odometer |
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| 08:26 | absorb light. The more dense cloudy sample is, the more light's |
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| 08:33 | Um The less cloudy less light. your values will increase as they grow |
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| 08:38 | you'll get something that looks as I like this. That's as shape, |
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| 08:42 | ? So that's what it is for . Um You don't really use solid |
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| 08:46 | to get a growth curve. You really do that. But um the |
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| 08:50 | use of course, is to get of cells right if you're growing um |
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| 08:55 | know, there's lots of examples in book of how um using DNA for |
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| 09:02 | different things like sequencing D. A. Seeing what kind of jeans |
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| 09:06 | there that begins with growing it in and then you get lots of cells |
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| 09:10 | way and you harvest them and manipulate . Okay, so it's kind of |
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| 09:15 | 22 uses for liquid media. So and of course over here you can |
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| 09:22 | at growth characteristics on a plate. . They all have different shapes, |
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| 09:26 | is and you can use that as way to identify. So um point |
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| 09:30 | there's different uses for these things. so um and so again we talk |
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| 09:35 | functional type using the media to for testing different functions perhaps. So something |
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| 09:42 | an assay right yourselves in that test are producing some kind of enzyme and |
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| 09:51 | want to measure how active it right? So you would grow it |
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| 09:54 | samples under a specific type of growth that would test for that particular |
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| 10:02 | Okay it could be something like So light pace is ace if you |
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| 10:09 | see a c. At the end world that typically means an enzyme |
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| 10:12 | You're looking for enzyme activity that will down fats lipids or fats. Okay |
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| 10:18 | this can show you that. So is a a plate I think this |
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| 10:22 | corn oil. Okay with a blue in it. And if the bacteria |
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| 10:27 | on it as you see here is um swab of growth then there's a |
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| 10:33 | around the growth that halo represents enzyme been released. Breaking down the corn |
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| 10:39 | . Right? The fat And you it as a halo around there. |
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| 10:42 | activity. So it's like paste Right? Control of being like paste |
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| 10:47 | there. You don't see that. so that's just a simple kind of |
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| 10:51 | test and a visual you can see the plate. Um We've already talked |
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| 10:56 | this one. This was the aero . Right? So you can use |
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| 10:59 | as an about chemical test or anaerobic . Uh yet special conditions for |
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| 11:06 | Right. Keep you have to keep out um General purpose. So this |
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| 11:12 | this is where nutrient you've probably all about that. You've heard about that |
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| 11:16 | lab. So nutrient aga is a hetero trophic medium. You just use |
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| 11:22 | It grows most of your basic types bacteria like E. Coli and salmonella |
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| 11:28 | lots of lots of bacteria that are and they are quite happy on that |
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| 11:32 | . But there's lots of things that grow on it. But it's it's |
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| 11:37 | used for that kind of purpose heavy in the lab uh enrichment. So |
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| 11:42 | where you can manipulate the components in media to um favor the growth of |
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| 11:49 | certain type of microbe. Maybe it's that grows in soil and it has |
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| 11:52 | particular property and you kind of want get it away from these other types |
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| 11:57 | soil. So you have a formulation for it and it enriches for those |
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| 12:03 | over the others. So really just manipulating the growth conditions. Okay now |
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| 12:09 | that's kind of on the fringes of . So when you look at enriched |
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| 12:15 | can often um confused. Okay I difference for me between these two because |
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| 12:23 | both are kind of in the enrichment you want to enhance the growth of |
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| 12:28 | certain type by giving it the nutrients once. Right in selected medium. |
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| 12:35 | are trying to just get some types of the way and look for one |
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| 12:40 | . Right? But typically this involves adding an inhibitory agents. Okay you |
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| 12:48 | do that in enrichment culture inhibitory chemicals . Okay so you're actively adding a |
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| 12:59 | antibiotic you can add certain chemicals will growth of gram negatives others inhibit growth |
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| 13:05 | gram positive. Right? So you're adding something that will inhibit select against |
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| 13:10 | from growing enrichment is not really that just you're using your C. |
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| 13:15 | O. M. P. Right? And changing those up to |
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| 13:18 | of to fit one particular type of for um in selecting media you're adding |
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| 13:25 | chemical that says you're gonna you guys grow because you can't survive with this |
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| 13:29 | in there. So that's that's the between those two types. Okay um |
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| 13:35 | differential medium I think I have a on that. Let's just look |
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| 13:41 | Yeah selected media. So here's selective . So again they have specific chemical |
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| 13:47 | in it meant to to stop the of certain types certain types and very |
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| 13:55 | . Um Many of these media were for really wastewater analysis. Okay so |
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| 14:04 | wastewater of course you've got lots of in wastewater. Okay so water quality |
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| 14:10 | you're trying to look for types that indicators of fecal contamination of water. |
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| 14:18 | ? You don't want to have those your drinking water obviously. So a |
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| 14:23 | of this media is kind of developed that. So you're types that are |
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| 14:27 | one indicator of fecal contamination is anybody very common part of everybody knows this |
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| 14:35 | begins with e coli thank you. E coli so that's an indicator |
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| 14:41 | It's by definition gram negative um ferments . Okay and so you look for |
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| 14:48 | of waste water that are that? and so how do you find it |
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| 14:52 | waste water when there's all the other in there. That's where you select |
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| 14:55 | media. So you add agents that inhibit grand positive. You don't want |
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| 15:00 | in there. So you can add things like a don't even know |
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| 15:04 | But one of the chemicals in these called bile salts. Okay. And |
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| 15:09 | actually inhibits gram positives. So you you're going to favor the growth of |
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| 15:13 | negatives which e coli is one of . So that's what the selected media |
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| 15:17 | do. Now this selective media is this heck tones and terek. So |
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| 15:24 | also doubles as being differential. So media Is one that you're looking |
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| 15:32 | You're adding things into the medium to the appearance of a certain metabolic |
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| 15:40 | And it gives you a visual if positive or negative we just saw the |
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| 15:46 | light paced activity is one of Okay. So that could be differential |
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| 15:51 | seeing if bacteria has the enzyme or . Right. So it's a visual |
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| 15:56 | you a visual whether it's a halo around the growth or here again features |
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| 16:04 | all around the growth. So this actually blood on them. Okay. |
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| 16:09 | this is used for uh streptococcus strains causes things like pneumonia. Some types |
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| 16:18 | strep throat um uh scarlet fever. other types cause like uh cavities and |
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| 16:27 | on your teeth. So it's a to each each of the different types |
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| 16:31 | streptococcus have a reaction on blood. there's three types A. B. |
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| 16:37 | C. I'm not gonna worry about specific reactions now at the point now |
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| 16:41 | it's a differential media that will produce reaction to whether it can lice blood |
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| 16:49 | or not in this particular medium. ? A complete clearing zone around the |
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| 16:54 | be forms like a greenish color that's of of reacting with the blood cells |
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| 17:01 | blood cells have hemoglobin and it kind reacts and forms of green color. |
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| 17:05 | call this partial hydraulics. Sis again worry about it. We'll talk about |
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| 17:09 | later. Just this is just in context of what differential media is and |
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| 17:13 | is one that doesn't do it at . So you can group streptococcus side |
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| 17:17 | are medically important uh This way. so just a differential test. Um |
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| 17:23 | you can combine, you can combine and differential together. That's that's what's |
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| 17:29 | here. Okay so this is both . It has chemicals to inhibit gram |
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| 17:36 | . So your favorite growth of gram And this is one that selects |
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| 17:42 | I'm sorry that differentiates between. So see black colonies and it's not really |
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| 17:48 | clear here. The black is obviously but it's kind of yellowish colonies. |
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| 17:52 | there's more colorless colonies. That's the of ones that are hard to distinguish |
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| 17:56 | least from this image here. But this is like the yellowish that's like |
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| 18:01 | uh more colorless colonies and kind. But so two things being differentiated. |
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| 18:09 | can it ferment lactose. Can it ferment lactose? Right? And so |
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| 18:14 | you can plus then you have a colony. If you can't it's more |
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| 18:18 | color less less yellow or more And H. Two s ability to |
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| 18:25 | sulfur. Okay and rather reduce And you produce this which is a |
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| 18:31 | precipitate. Okay and so we basically three types. You see one that |
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| 18:37 | um use utilize sulfur. One that utilize lactose and one that can do |
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| 18:44 | . You can differentiate these three Okay and so you don't need to |
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| 18:50 | this either. But the salmonella and are two types that will you'll separate |
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| 18:54 | on this medium. Uh They're both negatives. They both are lactose |
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| 19:01 | And but salmonella is able to use sulfur so it's a way to distinguish |
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| 19:06 | those two. Anyway so and salmonella like e coli are gram negative enteric |
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| 19:12 | . So in land and land bill with these things as part of the |
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| 19:17 | projects like E coli, salmonella klebsiella blah blah. They're all like gram |
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| 19:24 | fitness group. Okay so selective differential . Any questions about that you'll work |
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| 19:32 | in lab, you work with, work with these next week in the |
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| 19:36 | and A. T. A. one of them. And but progress |
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| 19:38 | there too. Um Okay growth so so one way to kind of visualize |
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| 19:48 | course is one of the terms we in describing growth or growth measurements is |
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| 19:55 | we call generation time. Okay and on it's on this slide coming up |
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| 20:01 | time is different ways to think about . So uh that what you see |
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| 20:06 | is literally the production of one generation ? In a bacterial cell of |
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| 20:12 | dividing to make two, that's one . Okay so in measuring bacterial growth |
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| 20:18 | look at um how quickly can it generations? Okay and how can |
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| 20:26 | from a growth standpoint, how can enhance in terms of growth features and |
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| 20:32 | components to maximize that? Okay, often a consideration. So um so |
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| 20:39 | measure using the term generation time. so how many generations have passed and |
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| 20:44 | . Amount of time. So you want to put a time element to |
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| 20:48 | a rate and see how fast it's doing this. Okay. And so |
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| 20:54 | also when we talk about the growth , well here is one kind of |
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| 20:58 | curve. And so what we call growth, okay in ecology they call |
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| 21:04 | a. J. Shaped growth which why this looks like a. |
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| 21:08 | Right there. And so it's very growth occurring very quickly. What we |
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| 21:13 | exponential or logarithmic growth is very fast . That 1 to 2 to 4 |
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| 21:19 | 8 to 16. Very fast. ? And so you know under optimal |
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| 21:26 | like an E. Coli can Double every uh 15, 20 minutes. |
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| 21:32 | very rapid. So just to put in perspective, think how fast it |
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| 21:36 | to produce, generate generations of Right? So 20 generations takes maybe |
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| 21:43 | hours in bacteria. Um I can coli for humans it takes 400 years |
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| 21:50 | produce 20 generations give or take. ? So it's a puts it into |
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| 21:55 | . Okay so they can grow very under optimal conditions. So here's a |
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| 22:00 | time. So you can look at as length of time for a cell |
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| 22:03 | divide into two or more practical? the length of time for a population |
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| 22:09 | double. That's kind of what you when you're actually measuring unit measurements, |
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| 22:13 | can't really measure how long you're not not looking at one cell dividing the |
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| 22:17 | , You're looking at a population that's growing. So you look at how |
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| 22:21 | it takes to double the population. . And so a number of variables |
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| 22:28 | of course influence um if you're growing coli on nutrient broth. Ok. |
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| 22:36 | basic formulation or are you growing it a defined medium? Okay, so |
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| 22:43 | sources defined. Right, so which would you think it would grow faster |
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| 22:48 | ? Would it be faster on rich or on defined medium? Yeah because |
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| 22:54 | has a bunch of preformed stuff made it. Right. So things like |
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| 22:58 | are probably in their things like amino . So it doesn't have to make |
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| 23:02 | stuff like it wouldn't define medium. whenever you have to stop and make |
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| 23:07 | that's going to stretch out your time you can grow as fast um if |
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| 23:11 | got a lot of stuff made for that equates to faster growth. |
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| 23:16 | so there's that there's a temperature, , all the kind of parameters. |
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| 23:20 | talked about well influence, you know , how long, how fast they'll |
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| 23:26 | . Okay. Um and so just great idea here is this is a |
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| 23:32 | question. Again, you're not going do any calculations, but I just |
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| 23:35 | to show you uh one of so in the end and of course |
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| 23:40 | population size. Biggin. Biggin population uh little end is number generations. |
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| 23:46 | this is so if you want to out how many cells you had at |
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| 23:49 | time point, you would use the you started with times two to the |
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| 23:56 | of generations. Okay, so this here kind of represents that That doubling |
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| 24:02 | . Okay, the to the And so for example, if we |
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| 24:07 | 10 started with 10 cells. and we went five generations. |
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| 24:13 | so we have that equates to 32 . Okay, times 10. So |
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| 24:20 | started with 10 times two to the , we get 320 cells in in |
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| 24:26 | time frame. And so that's another , you can also put time on |
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| 24:29 | as well to see how fast this occurring in. Okay, so really |
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| 24:33 | some kind of basic equations. But see um numbers wise, right, |
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| 24:38 | of cells. 12 1,000,020 generations that bacterias can occur as I said, |
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| 24:46 | will be 10 to 12 hours very . Right? And and typically when |
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| 24:51 | dealing with a wide range of numbers this, you use a lot of |
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| 24:57 | base 10 to kind of compress the . Okay. The same thing you |
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| 25:01 | with ph ph is also a lot the base 10 scale. Um so |
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| 25:08 | and so here's one way you would log base 10 values. Okay. |
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| 25:14 | you can use uh absorbency measurements, put in spectral Radiometer and can use |
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| 25:20 | number and take the log to the 10. But it will give you |
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| 25:24 | a where it's in the call log growth, you'll see a straight line |
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| 25:29 | . Okay. And that can give an idea of of the growth rate |
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| 25:35 | getting this line. Okay. And if you didn't use log base |
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| 25:39 | this kind of shows you that because numbers are so big, right? |
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| 25:44 | be kind of deceiving looking at You might think, oh that's not |
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| 25:47 | growth there. Right. But certainly is Right. We're going we're going |
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| 25:51 | 30 0 to 32 to 32,000. ? So if you put it in |
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| 25:55 | base 10 it becomes really apparent. know that it's Yeah, it's growing |
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| 26:00 | fast. Okay so again there's that shaped growth. Okay But we put |
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| 26:07 | a linear form using log base 10 really see that. Okay so um |
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| 26:15 | a common thing you do when you're with values that are so can be |
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| 26:18 | wide right? In terms of Um Now so this is again just |
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| 26:25 | of give you a A bit of in terms of how really how powerful |
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| 26:31 | bacterial growth thing is. Right so with 10 cells and bacterial bacterial type |
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| 26:37 | every four hours. Okay. Will the population size after 20 hours. |
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| 26:42 | happens if it doubles every 15 Right so on this side 20 hours |
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| 26:49 | have um five generations. Okay um then plug in the values. Right |
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| 26:59 | from the previous example 320 cells. if we do the same parameters |
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| 27:05 | Uh Start with ourselves in 20 How many generations? In 20 hours |
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| 27:11 | ? Right but doubling time is now shorter. four hours down to 15 |
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| 27:17 | . Okay. And you get to 80 times 10 and that's that's a |
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| 27:24 | difference. Obviously 10 of the 25th . That's like holy cow. Alright |
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| 27:30 | that 23 20 that's just taking the time down from 4 to 15 |
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| 27:35 | Okay So um so you know think a uh a cell that's infecting |
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| 27:45 | An infectious agent. And how gross very quickly bacterial infection can very quickly |
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| 27:52 | serious? Of course. Right? you start out maybe with have a |
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| 27:56 | and a few cells are in there very quickly they can grow and can |
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| 28:00 | be a problem uh maybe causing systemic so that bacterial growth can be a |
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| 28:08 | thing. Hence uh you know finding to stop growth of infectious agents. |
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| 28:14 | . Um many questions. Okay So here's the questions here is a clicker |
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| 28:24 | . So this will take us into this is all about. So let's |
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| 28:29 | How do we do here? So batch growth curve is shown which the |
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| 28:34 | among eight D. Is false. what you're looking at is so the |
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| 28:38 | batch growth. So let's see um . So batch growth is you would |
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| 28:47 | for what you're gonna measure growth in you're gonna grow it in. Make |
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| 28:52 | medium uh told up to about here inoculate okay. And then likely put |
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| 29:00 | in an incubator, shaking it. ? And then every at periodic intervals |
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| 29:07 | would take samples out and you somehow growth, right? You can do |
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| 29:11 | in different ways but that's what you're . And so you're basically following doing |
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| 29:16 | . Getting a pattern like you see and then you're just following all the |
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| 29:20 | to the end. Okay. And . Just taking samples out to measure |
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| 29:26 | . That's all you're doing right? that's that's what we call batch |
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| 29:30 | Okay? There's variations of that you do but when you see batch |
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| 29:35 | that's what it is. Right. set amount of media, you take |
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| 29:39 | to measure growth and that's it. . You're not doing anything else manipulated |
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| 29:43 | any other way. Right? You're trying to get this kind of a |
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| 29:47 | , whatever it may be. And of course there's lots of uses |
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| 29:51 | that. Um Very often if you studying a particular microbe, this is |
|
| 29:59 | of the first things you do because may want to use that microbe to |
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| 30:04 | experiments with or test in time activities or whatever. And you might like |
|
| 30:11 | um do those studies a time point it's going to be most functional, |
|
| 30:18 | active. Right? So if you know what the growth curve looked |
|
| 30:21 | then when it got to each you know you may you may have |
|
| 30:26 | this culture at at one and one not going to be very active. |
|
| 30:31 | ? Rather you want you want to to get somewhere or two is at |
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| 30:36 | then, you know, you wouldn't that where that would be unless you |
|
| 30:40 | it and kind of growth curve. that's that's one use and so but |
|
| 30:44 | you can compare growth curves, you have the same media and maybe you |
|
| 30:48 | to test a some kind of chemical a antimicrobial agent. How well does |
|
| 30:53 | work? Well you would grow it on the medium then you add agents |
|
| 30:58 | see how it affects growth and so like that. Um But the other |
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| 31:04 | is of course um if you are to see how can optimize growth, |
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| 31:09 | fast can I get to get up of cells? Um The and so |
|
| 31:16 | different uses for this. And so so for this question so changes in |
|
| 31:21 | size occur during phases two and That is actually true. Okay we'll |
|
| 31:27 | into that. So there are subtle changes sometimes not so subtle but there |
|
| 31:32 | definite changes in size as you go the growth curve and there's reasons for |
|
| 31:37 | B is also true. Okay so are exponential changes obviously here. Okay |
|
| 31:46 | as well here in four. And so it's really in in this |
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| 31:53 | That that's that's what Chapter seven is controlling microbial growth because you're trying to |
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| 31:59 | different agents and see how fast can make that that death become very |
|
| 32:07 | Right? Very rapid. Um So because it is exponentially going down not |
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| 32:13 | of course. Okay. Acclamation phase does fit with 11 is what we |
|
| 32:19 | lag phase. Okay. Um So would have been inoculated right here. |
|
| 32:28 | means kind of to seed it. take your un inoculated sterile media and |
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| 32:34 | add cells. That's the inoculation. and that's what the kick starts the |
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| 32:39 | thing but you always when you do you always have some it's always gonna |
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| 32:44 | flat but how long it stays flat dependent on various factors. But it |
|
| 32:49 | does that. Um most likely to forming in those sports in those spores |
|
| 32:55 | a kind of a stress response. ? So it's going to begin to |
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| 32:59 | under stress right in here? For . Right. So it's likely where |
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| 33:06 | going to be forming spores called stationary . It's not completely limited for nutrients |
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| 33:14 | it's getting there. So that's gonna the signal in time. And say |
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| 33:18 | you can form in the sports let's it now. And then by the |
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| 33:22 | we get to, by the time get to hear we'll be finished and |
|
| 33:27 | all in those sports. Right? if you wait too long you get |
|
| 33:30 | four and there's absolutely no no food because it does takes energy to make |
|
| 33:35 | endo sports. You have to have to be able to do that with |
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| 33:37 | you wait too long, not gonna . Okay, so all of these |
|
| 33:44 | true none none of these are Okay, so I went through a |
|
| 33:48 | bit of these points but let's talk them again, add a little bit |
|
| 33:52 | to it in here. Okay, again this is the batch growth |
|
| 33:58 | And so every time you grow bacterium , a really anything protozoan and |
|
| 34:07 | Any kind of microbe or any kind living organism. You're going to get |
|
| 34:12 | kind of a curve. Okay um it's gonna look like this. What |
|
| 34:18 | what will vary will be the length a lag phase. Okay. The |
|
| 34:25 | steep the exponential growth is and maybe like that or like that. What |
|
| 34:32 | you? Um The length of stationary may differ how high it gets, |
|
| 34:39 | . The number of cells you attain vary. Um And of course the |
|
| 34:45 | phase, maybe it's like that. it's like that, it's like |
|
| 34:48 | So the basic pattern is the It's just you can have variations in |
|
| 34:53 | of these stages based on what the is, what you're doing to |
|
| 34:58 | what you're growing on. Okay. So the uh so in blackface. |
|
| 35:05 | starting here. Okay, lag face there. Um Okay, so cells |
|
| 35:13 | the cells are coming from or what are, they're obviously coming from either |
|
| 35:18 | plate that you're just transferring to liquid they came from another liquid you're growing |
|
| 35:24 | . Right? So whatever the state that, the cells on that part |
|
| 35:29 | will affect what happens when they go the new stuff. Okay, so |
|
| 35:33 | have to think about. Okay, yourself down to the level of the |
|
| 35:36 | , what's going on? Well, could be slight ph changes, slight |
|
| 35:41 | in 02. Maybe uh slight solute differences between the two uh enough to |
|
| 35:48 | of maybe a slight temperature difference enough kind of, you know, put |
|
| 35:54 | on hold for a little bit right it gets adjusted. Okay, After |
|
| 35:58 | . Excuse me, after that then okay. How long are they on |
|
| 36:02 | plate before you transferred? How long they growing in that liquid before you |
|
| 36:05 | ? Which is a very old. there's hardly any cells that are left |
|
| 36:09 | in there. Right? That that have an influence. So if you |
|
| 36:11 | start this, if your inoculation is one cell or is it 1000 cells |
|
| 36:18 | gonna make a difference. 2000 cells to grow faster. Um the difference |
|
| 36:24 | the media types, Right, are growing it on minimal media and now |
|
| 36:29 | gonna transfer to rich media or vice that will influence okay, if you're |
|
| 36:35 | rich media and you're going to the , well the brakes are gonna be |
|
| 36:39 | on because now the cells going, I don't have all the pre made |
|
| 36:44 | I did before, I'm gonna make and when it still has to make |
|
| 36:49 | , that means turn genes on translate, blah, blah, |
|
| 36:53 | blah. Right, that takes So that's, that's going to stretch |
|
| 36:57 | blackface. Okay, but you can it around if you go from a |
|
| 37:02 | to a rich that can turn it because now it's in a situation where |
|
| 37:06 | doesn't have to make a bunch of in the last phase can be |
|
| 37:10 | Okay, so, so a lot things go into into, but it |
|
| 37:14 | an acclamation phase for sure, Turning different genes etcetera, that kind of |
|
| 37:19 | . Maybe making stuff until but when gets going it does go pretty |
|
| 37:26 | So, and then we're in log . So as mentioned, you may |
|
| 37:31 | the term at some point. They about, you hear mid log |
|
| 37:39 | Okay, that's typically the most The cells are at their biggest, |
|
| 37:44 | you have cell cell size changes. they tend to be a little bigger |
|
| 37:49 | middle because remember they're dividing them to a rod shaped cells, right? |
|
| 37:55 | it's a singular form. And then they're actively growing, you'll see lots |
|
| 37:58 | forms like this. So in the the life of a cell, |
|
| 38:04 | it will begin to get a little bigger. So it elongates, it |
|
| 38:09 | a rod shaped, it was, clock will get bigger as well, |
|
| 38:13 | it'll kind of elongate somewhat and then a signal for it then, |
|
| 38:17 | let's just divide. Okay, so odd shaped they go kind of like |
|
| 38:21 | and then they become too and so volume will increase during log phase for |
|
| 38:28 | reason. Okay, and mid long again, it's kind of their most |
|
| 38:32 | , right? So if you're interested finding in in analyzing themselves saying, |
|
| 38:37 | , this is the point where they're have their biggest enzyme activity for whatever |
|
| 38:41 | studying then, that's probably where I to take samples at or harvest the |
|
| 38:46 | harvest means to basically take what's grown here and there's different ways to concentrate |
|
| 38:53 | down to a really thick mass of . You can centrifuge it. Alright |
|
| 38:57 | will get the cells out of solution splatter against the bottom of the tube |
|
| 39:03 | ? Making it thick, what we paste. Okay because you pour off |
|
| 39:08 | liquid enough with a thick then sell right? And that's what you can |
|
| 39:13 | and isolate D. N. Or whatever. Okay so um but |
|
| 39:18 | mid log is kind of the most um the most functional state you might |
|
| 39:23 | . But then um then as we to here this is let me erase |
|
| 39:28 | of this stuff here. So as get to about this point, okay |
|
| 39:34 | we're kind of at the tipping point into stationary phase. And right before |
|
| 39:40 | of course you've amassed a lot of at this point here. Okay and |
|
| 39:46 | you and of course you're rapidly running of nutrients, you can't sustain that |
|
| 39:49 | population anymore and that level of growth in batch culture. Right so they're |
|
| 39:56 | to begin to slow down very Okay they're not out of nutrients |
|
| 40:01 | Right so in in stationary phase basically growth rate equals death rate kind of |
|
| 40:10 | , right? So they're still hanging . So now you get another cell |
|
| 40:14 | size change. Okay so you have think of the situation they're in so |
|
| 40:20 | stationary phase limited for nutrients which is be a stress condition. Okay and |
|
| 40:26 | we'll assume they can't make an indoor right? So now what are they |
|
| 40:30 | do? Try to survive? Well strategy is to get smaller so they |
|
| 40:35 | get slightly smaller because now you have material to keep up with. Um |
|
| 40:43 | and so it's it's a survival Okay? And so they kind of |
|
| 40:48 | that they kind of then shut down essential functions. Okay so again the |
|
| 40:54 | is let's conserve energy until we can out of this mess, so to |
|
| 41:00 | . Okay of course if you're if in batch culture nothing's gonna help, |
|
| 41:04 | gonna it's going to die because you're gonna do anything to help them. |
|
| 41:07 | ? Um So they can even um of course there's dead cells around so |
|
| 41:12 | can kind of feed off the dead for a bit. Alright but eventually |
|
| 41:16 | gonna run out of food okay? you're gonna be in death face. |
|
| 41:20 | so very quickly with no nutrients it's wonder that that's exponential as well, |
|
| 41:27 | nothing to sustain anybody so they're gonna okay now and so uh so those |
|
| 41:35 | your four stages. Okay? Now just so contrast to batch crow so |
|
| 41:42 | at this right here, okay is curve we just looked at, so |
|
| 41:49 | this time point or so we can stuff. Okay. And if you |
|
| 41:57 | that you can get a response like . So you see now we've raised |
|
| 42:02 | growth amount of cells that we've achieved sell more cell growth. We can |
|
| 42:09 | more cells. That's what you wanna . You can just feed them uh |
|
| 42:15 | log or mid log phase and then continue to grow right up upwards before |
|
| 42:21 | go into stationary phase. So you them another boost basically. Okay and |
|
| 42:26 | uh so the thing you do so would you add what's gonna give it |
|
| 42:32 | biggest boost? Right so if you're it's growing glucose just give it more |
|
| 42:39 | . Okay add more shovel more glucose midway through growth and then it'll keep |
|
| 42:43 | okay. The um so if we let's say the batch growth medium has |
|
| 42:55 | translator glucose. Okay now we add 20 here. Okay so you might |
|
| 43:06 | well that seems kind of inefficient. not add all of it up |
|
| 43:12 | Why not add 40 g per liter at the beginning. 40 right |
|
| 43:18 | Okay um so the when you do , what you see is this So |
|
| 43:28 | you're not in the media with 40 per liter it would just be like |
|
| 43:34 | wouldn't do much. Okay and there's reason for that which is why you're |
|
| 43:41 | to do this you have to do in stages feeding stages. Does anybody |
|
| 43:44 | why you see this response? You 40 g from your glucose at t |
|
| 43:54 | zero anyway based yeah tom exactly. has to do with the osmolarity so |
|
| 44:04 | much of glucose in the beginning it be too much. So you're gonna |
|
| 44:07 | that effect. Cells are gonna be from growing. So you do it |
|
| 44:12 | feeding stages because it's just too much terms of solute concentrations. So um |
|
| 44:19 | and you can feed more than once you know? So um so the |
|
| 44:26 | so you know those I know my classes, the one that has the |
|
| 44:30 | makers in it, that's the kind stuff you do. Uh biotech is |
|
| 44:36 | of the departments will be responsible for lots of cells, be they bacterial |
|
| 44:42 | you know other cell types. And often about getting lots of stuff and |
|
| 44:47 | that's the kind of things you do get there. Okay. Um Another |
|
| 44:53 | you do uh is used basically a computer controlled uh bioreactor. Okay because |
|
| 45:05 | this you're basically controlling all the physical . You're controlling ph you're controlling oxygen |
|
| 45:11 | , you're controlling um feeds into the . Okay? And in something like |
|
| 45:19 | which is typically a shake flask, don't have that capability. There's some |
|
| 45:23 | you can do but you don't have control like this a computer controlled system |
|
| 45:28 | you can set parameters like add base it gets up to down to this |
|
| 45:34 | add acid begins to this ph um more auction. If we get to |
|
| 45:38 | level so all those things are programmed controlled. And under that situation you |
|
| 45:44 | gross that growth that you can hope achieve in something like this. And |
|
| 45:50 | these vessels can be. I've seen vessels be as big as 100,000 gallons |
|
| 45:57 | stories high blow your mind. And uh but that's what you gotta |
|
| 46:03 | for industrial scale you know because that's teaspoon stuff. That's like oil barrels |
|
| 46:08 | stuff you need. Okay. And you've got to grow this way. |
|
| 46:11 | all the parameters and get lots of that way. Okay. Um The |
|
| 46:18 | thing you do production of vaccines You have to have lots of of |
|
| 46:22 | if you're using the parts of the as antigens to make a vaccine, |
|
| 46:25 | do the same kind of things to lots of growth. Okay. Um |
|
| 46:31 | questions about that. Okay so growth the name of the game. Um |
|
| 46:39 | so the last segment. Okay, and the ability to attach. Let |
|
| 46:46 | open that our absolute requirements for I don't think that's gonna be lag |
|
| 47:01 | in that one. Okay, counting . You're not sure the shot. |
|
| 47:41 | . 21. Yes of course biofilm . I've been shocked if you don't |
|
| 47:49 | that one. Um And so then next part is uh let me push |
|
| 47:58 | up. So for this reason bow forming bacteria must have what they may |
|
| 48:09 | more than one of these but they have this. Okay. Okay. |
|
| 48:58 | . Any stragglers. 1 2nd. . Yes. Right. That's for |
|
| 49:07 | . Okay so surface um the to the biofilm you also need to have |
|
| 49:16 | steady supply of nutrients. Right? one thing about biofilm formation as you |
|
| 49:21 | see from some of these is it a lot of growth. Okay so |
|
| 49:30 | has to be some kind of a continuous supply of nutrients feeding the |
|
| 49:34 | Okay. Which is via very often found in a pipe for example which |
|
| 49:41 | water and then likely organic material flowing continuously. Maybe at different rates but |
|
| 49:47 | it's it's typically always there a mat top of this probably algae mat on |
|
| 49:54 | of pond water in the pond or . Your teeth. They've kind of |
|
| 50:00 | this with purple dye. It kind showed the the biofilm itself is not |
|
| 50:04 | but the dye they used to enhance I guess it was. But yeah |
|
| 50:08 | sticky feeling you feel in your teeth the morning. That's typically biofilm. |
|
| 50:13 | Obviously you think of the food that's there that they can eat, you |
|
| 50:16 | ? Not surprising. Um But in of from healthcare standpoint uh definitely different |
|
| 50:24 | of medical devices. Um Things like you put in the body like a |
|
| 50:32 | replacement, the hip replacement heart valve Things you insert like a catheter, |
|
| 50:39 | breathing tube. Um These kind of are all potentially or surfaces that can |
|
| 50:47 | inhabited by a biofilm. Okay and biofilms that are medically important are very |
|
| 50:55 | and problematic treat because of the nature the biofilm of antibiotics being able to |
|
| 51:01 | in there and penetrate. Okay so uh in fact I think I I |
|
| 51:09 | have it on me. I I was walking to my other class the |
|
| 51:12 | day, I actually took a picture two biofilms on campus. One is |
|
| 51:16 | looks very much like this is a over there by SnR SnR one that's |
|
| 51:21 | as a wooden board in front of . So it's dripping down and the |
|
| 51:24 | is just full of this slimy It was also on the on you |
|
| 51:29 | look on the windows and many of buildings um STL building windows. There |
|
| 51:33 | like black grimy biofilms on those as , so they're fairly prevalent if you |
|
| 51:39 | around. Um And so looking in , okay so here is the actual |
|
| 51:46 | . So how this happens is really because these things all come in sterile |
|
| 51:51 | . So if you mishandle them, them out and inserting them, why |
|
| 51:55 | wouldn't wear gloves while doing this. seemed crazy. But you know your |
|
| 51:58 | have staff on them or you touch nose because membranes of staff and you |
|
| 52:03 | potentially be transferring that to the You insert into a person and now |
|
| 52:08 | introduced a potential pathogen, like a biofilm. Okay. And um you |
|
| 52:14 | see the number of cells are growing and these can break off or elsewhere |
|
| 52:19 | the body um and and cause more illness. So this is definitely a |
|
| 52:25 | uh that you will definitely see in hospital setting. Hopefully not because of |
|
| 52:31 | , but uh you very likely will these things. Um Now, so |
|
| 52:38 | of biofilm formation. Okay, so not as you might think a it's |
|
| 52:46 | random coming together of cells and they this kind of sticky glue that all |
|
| 52:52 | out together. Right? It's it a orchestrated process. Number of genes |
|
| 52:59 | involved. There's chemical signaling going on kind of orchestrate this thing. It |
|
| 53:04 | a species specific phenomenon. So not bacterial types can do this. |
|
| 53:09 | But many types can And of course like we mentioned earlier, what they |
|
| 53:15 | have the capability of or having there specific to attach to a surface. |
|
| 53:21 | . And that's that's an important, important characteristic of biofilm form. Now |
|
| 53:27 | also have in these types that do . You have types they're called. |
|
| 53:33 | actually call these swimmer's. Okay. of a fancy name for this is |
|
| 53:42 | tonic cells that you see there. . This term basically means their motor |
|
| 53:49 | and they can come around. So the swimmers are kind of the |
|
| 53:53 | that are think of them as the looking for a place to land that |
|
| 53:58 | be a nutrient rich place. so then because biofilms are all about |
|
| 54:03 | surface they're gonna have to be able attach. Okay. So what happens |
|
| 54:08 | swimmers stick? So swimmers become stickers they lose the flagellum? Okay And |
|
| 54:22 | attach the surface. Okay so swimmers . Right? So swimmers swim looking |
|
| 54:28 | a good spot and land lots of . Okay? And then they lose |
|
| 54:34 | and they plant themselves. Okay? so initially they form what are called |
|
| 54:40 | colonies. So very tiny colonies of few cells um Now whether you go |
|
| 54:48 | further from this point okay is dependent chemical signals. Okay so the chemical |
|
| 54:59 | . Okay let me put this down stickers. The chemical signals are put |
|
| 55:08 | . And so if you have enough that accumulate right here, okay, |
|
| 55:16 | cells accumulate. You get enough of chemical signals. So the chemical signals |
|
| 55:21 | to reach a threshold. Okay? if you get there so it's like |
|
| 55:25 | uh you know your parliamentary procedure right an organization you have to have enough |
|
| 55:30 | there to constitute a quorum that you then vote. Right? So you |
|
| 55:34 | to get enough bacteria there on the because then you'll have enough signal produced |
|
| 55:41 | you reach the level at which the they can trigger the biofilm formation. |
|
| 55:46 | ? And so it will then progress there's enough cells there. Okay so |
|
| 55:51 | really dependent on having a lot of around you to start the biofilm. |
|
| 55:55 | logic the logic is if you've got more and more cells are sitting down |
|
| 56:02 | and giving up chemical signals, it be that there are lots of nutrients |
|
| 56:06 | for them to eat. Right? then more common more calm because it's |
|
| 56:10 | favorable environment in terms of nutrition. ? That's kind of one of the |
|
| 56:14 | things as well. Is there food us? Right. And so um |
|
| 56:20 | presumably you get enough cells reach You form the biofilm because it's favorable |
|
| 56:26 | do so because why else would the b more and more be accumulating if |
|
| 56:31 | weren't an indicator of good a good site, so to speak. Right |
|
| 56:36 | then that will kick in or the produce the Xo policy Saccharine, this |
|
| 56:42 | this is the the extra sailor This is the glue that binds everything |
|
| 56:48 | . Okay so again that's all. is a gene controlled process. |
|
| 56:55 | So there'll be genes will be turned to make the this sugary material again |
|
| 57:02 | of think about the glue. Okay then so you have growth really kind |
|
| 57:07 | initially on the surface in two Right then very quickly it goes in |
|
| 57:14 | dimensions. Right this way and so called biofilm. Towers right goes |
|
| 57:21 | not just this way on the surface then up. Okay. And so |
|
| 57:27 | can see that from the top little here right now, it's growing three |
|
| 57:33 | . And then of course you have flowing in around right through here and |
|
| 57:40 | may get holds form. Okay? then that will be that will more |
|
| 57:45 | get nutrients to those who are kind stuck in the middle, right? |
|
| 57:48 | dense. So everybody gets happy and nutrients okay. And but then you |
|
| 57:54 | it's it's likely or certainly likely that there's just not enough food anymore to |
|
| 58:00 | everybody. Okay? And so then kind of begins to dissolve and so |
|
| 58:05 | will come off and now you see cells have their um fragile and |
|
| 58:13 | So now we go back to being again. Right? So we're stickers |
|
| 58:17 | 234 and now we're going back to a swimmer again because they want to |
|
| 58:22 | find another another more favorable spot to up the biofilm again. Okay. |
|
| 58:28 | you know if there is like the the pipe, you know if you're |
|
| 58:33 | steady flow of nutrients, I mean bio foam can last a long |
|
| 58:36 | Okay. So it's just a matter having the nutrients to sustain everybody. |
|
| 58:42 | . But those that can't that don't enough and they can get their tail |
|
| 58:48 | and start swimming. Okay? And another spot. Okay? Um So |
|
| 58:56 | any questions about that information. And so it becomes a problem in |
|
| 59:01 | of a medical medically important type of , getting antibiotics that can diffuse into |
|
| 59:08 | and then even have an effect. . So that's why these biofilm infections |
|
| 59:14 | like weeks and months to really get be able to control them. |
|
| 59:19 | And you may have differences in the types. Maybe more resistant types are |
|
| 59:24 | the in the interior, less resistant the outside. So you can develop |
|
| 59:28 | kind of a resistance as well as result of this. So they're a |
|
| 59:32 | . Um Alright so that closes out or check for six. So that's |
|
| 59:41 | for today uh test next week. if you got questions stopped by officers |
|
| 59:53 | . Yeah. Um Never officially met |
|
