| 00:00 | Alright so we're finishing up Chapter So uh so um it's it means |
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| 00:10 | blackboard quiz, it's just different only terms of the uh a little more |
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| 00:17 | . Okay so basically covering stuff that been talking about since the 19th of |
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| 00:23 | , I think we officially started discussing . So um so basically chapter 1234 |
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| 00:32 | of chapter five because we'll get into on thursday. Okay so there'll be |
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| 00:37 | of that on there too, So know more or less comprehensive. Uh |
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| 00:44 | because that's longer, it's gonna be 25 or so questions, you'll have |
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| 00:49 | 45 minutes to do it. So you know for planning purposes just know |
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| 00:56 | gonna take a little longer for some you, so plan for that. |
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| 01:01 | uh so that's coming up friday. um Smart work do, so Smart |
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| 01:07 | a little different. Chapter four is monday, so there's always stuff due |
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| 01:11 | monday, but then two days later the chapter five. So I I |
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| 01:15 | it that way so that you had about it, you could actually you |
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| 01:19 | actually have at least a couple of to ask a question before the exam |
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| 01:23 | up, right? So the exam on the 17th 17 18. So |
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| 01:30 | sign up on casa if you haven't And uh exam one is like this |
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| 01:36 | ordered like 35 questions plus remind us you have an hour. Okay so |
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| 01:46 | you got any questions or anything? . Alright okay so uh so I |
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| 01:56 | recap, so here's what we've gotten so far. We didn't cover uh |
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| 02:03 | yet. Okay we'll start with that but we'll get into dynamics of growth |
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| 02:13 | of growth. Uh concentrating on here's you need to grow of course focusing |
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| 02:19 | microbes. C. H. M. P. S. |
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| 02:22 | Um We provide those in different forms on the metabolic type. Let's say |
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| 02:31 | your chemo autotrophs better versus a chemo of growth. Right? Carbon requirements |
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| 02:37 | different C. 02 versus something more . Um And so then we put |
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| 02:43 | things together in the medium and a medium. And um and uh there's |
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| 02:50 | types of growth medium and complex right? And then uh then we'll |
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| 02:55 | a little bit today about different ways can use culture to kind of find |
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| 03:00 | you want. Um And then briefly forms of growth media and some other |
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| 03:08 | of growth media. And then we'll into the growth growth growth calculations which |
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| 03:14 | not complex but we'll go through a of examples. Uh I'll give you |
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| 03:18 | blow by blow. So as uh I haven't said this already, you're |
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| 03:23 | to bring a calculator. Right? this got to do is multiply divide |
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| 03:29 | and do a log function log base . In fact that's all that's required |
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| 03:35 | it's only two problems for sure. three but a couple of problems you'll |
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| 03:41 | the formula. You'll have the formula need. So you all you gotta |
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| 03:45 | is just punch numbers. Okay hopefully them in correctly. Right so we'll |
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| 03:51 | through all that. And what Uh Okay then we'll go through when |
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| 03:57 | inoculate a growth medium. What are stages of growth that goes through what |
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| 04:03 | at those stages? So that's kind pretty much uh Then that's you that's |
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| 04:07 | one. Part one compared to part of our floor is comparatively much more |
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| 04:13 | . Part two is already only 22 biofilms. And end of course and |
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| 04:17 | it. Okay we may get into think we'll probably get to biofilms at |
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| 04:21 | the start of that today and finish and then do chapter five or chapter |
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| 04:26 | is relatively short as well. It's control micro microbial growth. So looking |
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| 04:31 | some physical chemical um methods of Some of the terminology involved when we |
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| 04:39 | we talk about controlling growth like bacteria static these kind of firms. And |
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| 04:44 | uh and then what the other section is the response to oxygen. So |
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| 04:49 | have various responses to the presence of . Kill some some requiring some can |
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| 04:56 | with that. So we'll look at basically is gonna wrap up in this |
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| 05:02 | . So um let's look at this here. Okay so we've seen this |
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| 05:08 | before. Right. Hopefully we know kind of medium this is. All |
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| 05:13 | . The question is a little So what would just yes or |
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| 05:18 | Yes or no. What a bacterium to be a Histamine Octa trophy. |
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| 05:26 | able to grow on this medium. . Yay or nay. So obviously |
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| 05:33 | operative term to know there was just beans amino acid. Alright. And |
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| 05:41 | this type of medium is a defined , correct? Fine, is that |
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| 05:50 | ? The same one? Complex, beef extract make it complex. Um |
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| 05:59 | right. We'll put the timer You have to do too much thinking |
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| 06:02 | . It's you know yes or Okay, counting down from 10. |
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| 06:31 | , Let's see. Is it Okay. Um Of those 93 who's |
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| 06:43 | 93 that answered? Yes. Why it? Yes. Right. |
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| 07:02 | Well you got a right um It yes. Okay. So all those |
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| 07:10 | which is the one that can supply history? Probably yeah, beef |
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| 07:18 | Right. So it's yes. So the is deficient in this case deficient |
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| 07:24 | being able to make history. Okay , this is supply. Okay, |
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| 07:30 | if you had a complete a complete uh just defined medium. Right? |
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| 07:36 | only only having these as your Right? Just those that are boxed |
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| 07:45 | . That's the middle meeting that won't the beach. Right? You're gonna |
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| 07:49 | to add history into it to make grow. Right. So these two |
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| 07:56 | are literally when you see these two or you see soy extract or soy |
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| 08:03 | . Right. Think about a plant plant. Just in your media. |
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| 08:10 | ? Beef extract kept own think of cow is in your media okay, |
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| 08:16 | muscle, right? What's in All kinds of stuff? All kinds |
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| 08:20 | preformed nutrients, certainly all the amino are gonna be in there ready to |
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| 08:25 | . Right. So yeah it's so you're studying these kinds of oxygen tropes |
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| 08:34 | of different types you have to make you're supplying nutrients, you can supply |
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| 08:41 | in different ways. You could just could use just these. Okay and |
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| 08:51 | these make that user defined media but and you can buy off the shelf |
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| 08:57 | anxious to dean to it. You do that. So any questions about |
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| 09:04 | . So alright, what would This introduces this isn't a clear |
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| 09:11 | Um So this introduces another subject. what would grow on this medium? |
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| 09:20 | don't focus on the amounts. Just on the ingredients. Okay um Is |
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| 09:30 | missing? Remember your your six Yeah six left. Remember your six |
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| 09:44 | . Something missing there. There's a , here's a hint what's missing |
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| 10:01 | nitrogen. I'm old. I can't in a a so uh so it |
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| 10:11 | grow on this thing. These types bacteria into fixing bacteria. Um They |
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| 10:20 | into from the atmosphere and bring it the environment whether it's aquatic or terrestrial |
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| 10:26 | , that's what they do. We essential. That's an essential function for |
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| 10:30 | because doing that ultimately allows you to these things. Which plants allergy other |
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| 10:43 | approach need that stuff to grow, ? We'll talk about natural cycle |
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| 10:47 | But um so this is an example so in our context right now this |
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| 10:53 | an example of enrichment culture. so you are purposefully manipulating the nutrients |
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| 11:01 | your growth video so you can savor growth of a certain type. |
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| 11:07 | now there's a nuance are subtle distinction this medium and selectively. You can |
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| 11:22 | think of any growth medium as being because not everything grows on some things |
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| 11:28 | grow up on neutral dog, But a selective meeting. Okay. |
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| 11:35 | one where you are actively adding agents chemical too. That will prevent something |
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| 11:43 | growing. Okay, that's what it's . You are actually getting a page |
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| 11:49 | you know when you hit it. type that type would happen. |
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| 11:55 | Enrichment culture is we're not adding something , you know, chemical inhibit growth |
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| 12:01 | something. We're just modifying nutrients to the growth of what we want to |
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| 12:06 | this medium. Or nitrogen fixing That's how you find it. You |
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| 12:12 | you could change something else to find types, right? Because these are |
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| 12:16 | found in soil. If you're in oil and just put it on like |
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| 12:23 | auger, you're gonna get so many bro right? Yeah, maybe a |
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| 12:28 | fixer grows in there. It's gonna so far outnumbered by these other |
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| 12:32 | Okay, so you favor the growth giving something that they were like all |
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| 12:39 | , take it or not. That's limit lots of stuff. Okay, |
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| 12:43 | just want to fixing bacteria so it enriches for those types. Any question |
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| 12:51 | that? Okay, so um So this is fastidious bacteria. So |
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| 13:02 | anybody know just out of the Forget the context of bacteria. Just |
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| 13:07 | word in the dictionary, anybody Fastidious? Are you fastidious? Are |
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| 13:13 | fastidious? Big and fastidious meaning high . Are you high maintenance? Okay |
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| 13:29 | uh we all of us are fastidious certain ways. It means we're very |
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| 13:37 | exactly right. You're fastidious. You're one who said salad restaurant notice I |
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| 13:44 | . Do you have I don't want cheese on it. Do you have |
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| 13:47 | dressing made in the south of You can put on it, you |
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| 13:50 | carrots out of radishes. Takes matters . Put that right. That's a |
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| 13:55 | person. Very exactly. You Okay. That's all right. So |
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| 14:08 | this means context here is you know dealing with a fastidious microbe when you |
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| 14:14 | a list a mile long of you have a Okay, so you |
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| 14:20 | see the growth meeting here. You we the basic stuff right that we |
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| 14:25 | familiar with here. Right? But all of this amino acids nucleotides, |
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| 14:32 | . This is very very uh a bacteria. I've never had a list |
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| 14:39 | long. Okay so it's a lot requirements. Typically they have a lot |
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| 14:44 | deficiencies and you have to add a of things for them to grow. |
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| 14:47 | the definition of um. Alright Culture . We talked about this before. |
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| 14:56 | liquid versus solid medium. There's uses both. Okay um you know the |
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| 15:03 | medium gives you a a manipulable version the microbe that you can do stuff |
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| 15:14 | . That's kind of a I just that that definition it's but you put |
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| 15:18 | on you can take out of a and put on a plate right like |
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| 15:24 | . And get now you can see the different types of you can then |
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| 15:28 | a column and transfer it to a . Work with it in pure |
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| 15:33 | Right? You can then put this into liquid media and growing up. |
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| 15:39 | ? So each has the right place culture um liquids if you want to |
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| 15:47 | large volumes of cells up and you to harvest them and you can use |
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| 15:50 | for different purposes. So each has use um selected media just mentioned. |
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| 15:56 | So they contain specific components that will the growth of certain types. Very |
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| 16:03 | is to add chemicals that inhibit growth of gram positive types. Some that |
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| 16:08 | inhibit growth of gram negative types. there's chemicals that can be kind of |
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| 16:13 | much specific for those groups. Okay the and that's the nature of a |
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| 16:19 | medium. So very all but a number of these selected were developed for |
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| 16:27 | uh uh wastewater treatment for the purpose providing clean drinking water water quality |
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| 16:36 | That's what I'm trying to say were for water quality analysis. Right? |
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| 16:41 | you want your drinking water of course it's tested to be free of water |
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| 16:46 | don't need to know this, but just gonna let you know of holo |
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| 16:51 | . Okay, performs in water are of people contamination. You don't want |
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| 16:58 | take that. Right? And so a lot of the media the selected |
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| 17:03 | were developed for to be able to these quantitatively. Right and two first |
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| 17:10 | by definition of gram negative um lactose . Okay and so the media was |
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| 17:20 | media were formulated to kind of identify and there's like a lot of different |
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| 17:25 | that that are used here. And one of them you see here is |
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| 17:30 | tones and Terek Auger. And so can actually differentiate three properties on |
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| 17:35 | It's selective for um gram negative. there's asians and pentagram positive bacteria. |
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| 17:42 | and so um the lactose fermentation Is seen as a color change. |
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| 17:49 | let me just bring this up so can have selective media that is completely |
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| 17:56 | to favor one of one type or . You get a chemical 20 it |
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| 18:03 | you combine that with um you combine with um a differential medium. Okay |
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| 18:11 | a differential medium is one that gives a color reaction. Some other visual |
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| 18:21 | reaction that occurs. Okay so over for example, it's not easy to |
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| 18:32 | . Probably. But here you have a yellowish colony. Okay. And |
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| 18:40 | more opaque colony like that. that's difference between and non okay, |
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| 18:47 | colony electrodes fermenter colorless, Right? lactose remember. Right. I said |
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| 18:54 | right. Yellow colony black toaster. so um and then there's agents in |
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| 19:00 | 200 g positive gram negative bacterial girl this. They ferment lactose you say |
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| 19:05 | that's a cool thing. Like any . Okay black color. This actually |
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| 19:12 | for um H. Two S. . Okay, you put things like |
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| 19:19 | in the medium and the material can that. It gives off HTE two |
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| 19:23 | . And it precipitates as a black . Right? So actually you can |
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| 19:27 | differentiate, right? That's the differentiate between black plus fermentation, non |
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| 19:32 | verse and production. Okay and this specific for these two types here. |
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| 19:42 | I'm another. Okay so both gram , both nonblack tremendous. So you |
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| 19:49 | differentiate them based on production and so . One does one does. |
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| 19:52 | So just an example of that's what media does for you. So if |
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| 19:58 | combine it selected narrow down but can and then making differential you can see |
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| 20:04 | metabolic properties in the cells. And for wastewater treatment or for water quality |
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| 20:11 | . Right? Like to have something can easily be used by perhaps people |
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| 20:17 | don't have the microbiology background but they easily identify. Hope this one is |
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| 20:21 | fermented. It's not it's a cold . Okay, so on this side |
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| 20:26 | a strictly differential media blood dog. the little halos you see here the |
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| 20:34 | areas, that's where they've basically lISZt red blood cells and growth. So |
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| 20:40 | that do that. This is typically um streptococcus species, we'll talk about |
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| 20:47 | later. Um But uh in this a bunch of different types of strep |
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| 20:55 | , scarlet fever, rheumatic fever, etcetera. Right, so different members |
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| 21:03 | that group will get different reactions of up. Right, you can kind |
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| 21:07 | identify with groups based on do they this complete what we call complete Hamal |
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| 21:13 | or something else? Right, so , differential differentiator between this and that |
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| 21:22 | and like the Bose. Okay, in lab will be doing this in |
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| 21:29 | weeks lab six. Okay. Um questions? Yeah. Right uh |
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| 21:43 | So if you have strep throat it's you'll take that swab and throw her |
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| 21:48 | . So if they see this kind reaction that's indicative of streptococcus which is |
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| 21:54 | the strep throat and they administer the antibiotics. So but things are getting |
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| 21:59 | diagnosis. Yeah. Yeah. Mhm. Oh I'm sorry. |
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| 22:10 | you're right. Okay, no No, I need everybody to see |
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| 22:13 | so that's good. Yeah, All right. Um Okay, switching |
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| 22:20 | to growth now. Okay so we're go through this doesn't have to question |
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| 22:24 | . So um I'm just gonna introduce terms. We're gonna talk up here |
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| 22:30 | 15 minutes, 20 minutes about Quantitative bacterial growth. This is just a |
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| 22:36 | up. I'm sure you know this not gonna be a jaw dropping thing |
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| 22:40 | you. But anyway it's a way kind of introduce some terms. |
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| 22:44 | so we have a bacterial population starting 10 sets. Right? This is |
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| 22:51 | just illustrating how the difference in doubling which is the time for a population |
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| 22:59 | cells to double. Okay. And how do you think? four |
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| 23:06 | W just 15 minutes time. It was like that big a difference |
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| 23:10 | terms of results that it actually Okay, so 10 cells every four |
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| 23:18 | ? What's the population sign? 20 ? What if it doubles every 15 |
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| 23:22 | ? So two different types. One a 15 minute time when it's a |
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| 23:27 | hour. Okay. Same parameters for . Right. 10 cells starting 10 |
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| 23:32 | . 20 very basic equation. Uh zero N. Is population size, |
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| 23:41 | ? Population size at some point in zero. And then sometime later right |
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| 23:46 | time this parameter here relates to the kind of exponential growth. Right? |
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| 23:56 | is a number of generations. Okay what this is. And so The |
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| 24:02 | you know, exponential growth is like . So just two to the N |
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| 24:09 | . Okay so how many generations in hours? Well yeah before are doubling |
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| 24:18 | ? Uh you have five generations? equates to just plug that value |
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| 24:30 | They're the end value to the Right? So I just thought it |
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| 24:34 | a place here then over here with 15 minute time that's one generation every |
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| 24:42 | out like .25 and 15 minutes. uh about 20 hours we get 80 |
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| 24:49 | generations versus five. Right well 27% to 25th. Right? That's a |
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| 24:58 | obviously. Okay so definitely time obviously a you know a significant difference in |
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| 25:08 | . Okay between these two um Now you achieve this growth in nature? |
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| 25:18 | I'm not gonna get 10-25 cells. doubt it. Okay but there can |
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| 25:23 | spurts where you have rapid growth but in nature of course everything's competing right |
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| 25:31 | nutrients and space and resources. So there's not any comment that you'll get |
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| 25:39 | growth. Right? But that it happen on occasion. Okay so what |
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| 25:44 | gonna look at is um how we up with these numbers. Okay we |
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| 25:50 | to start with cells we get a time. Um And so we look |
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| 25:55 | a couple different equations. So let's of look at it here. Okay |
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| 26:01 | of course we're talking about in a of course you can think of a |
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| 26:07 | in different terms right? It can be that one cell doubling, forming |
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| 26:12 | selves. That's a generation. But from a more practical standpoint we |
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| 26:18 | refer to as time for a population double. So we can take a |
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| 26:22 | of a culture and they go okay long does it take for that number |
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| 26:27 | double? Right. And that gives generation time. Okay. Very common |
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| 26:32 | used in measuring microbial growth. Okay as mentioned you can have exponential |
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| 26:39 | Which is this uncontrolled growth. And um intro bio you went through |
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| 26:46 | ecology and you learned the J. growth? Right? You can have |
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| 26:53 | . Right? But this is time in our case we're right number of |
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| 27:01 | . Right? So you can have J. Shape exponential. This doesn't |
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| 27:07 | forever. Right eventually this well it out at some point so we'll go |
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| 27:16 | that in a little bit. But point is is not it's a it's |
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| 27:20 | finite thing. Exponential growth. Okay while that's occurring you especially with bacteria |
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| 27:27 | gonna have lots of cells being produced quickly. Okay um And so we |
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| 27:33 | started equation second go right for but publishing sizes another generation which is end |
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| 27:43 | haven't seen the example we know you that numbers can increase very quickly. |
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| 27:49 | uh so I'm sure you also know log base 10. Use that in |
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| 27:57 | When when numbers or rapidly increasing immense . Right So I think of the |
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| 28:06 | scale. Right? That's a log base 10 scale. Okay. Um |
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| 28:12 | so kind of you can kind of that scale to make it more more |
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| 28:16 | by using log base 10. And that just shows your typical growth |
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| 28:22 | that you where you would use log 10 and this represents that kind of |
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| 28:28 | growth. Okay? So so let's at so this equation here has limited |
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| 28:40 | it has some use. Okay. generally when you begin talking about really |
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| 28:45 | numbers, um making other kinds of and estimates you'd like to more easily |
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| 28:53 | in. Okay. And so what gonna do in the next slide, |
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| 28:58 | not gonna I'm just gonna show I'm not gonna expect you to derive |
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| 29:02 | on an exam the equation. But the bottom line is we're |
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| 29:06 | We're gonna take that end value and able to calculate that more easily. |
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| 29:12 | . And that's what we're doing Right? So this this is all |
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| 29:17 | using logs, right? Remember when find the logs just a little bit |
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| 29:22 | differences there. Okay. So all gonna do is we want to be |
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| 29:26 | to set this up so we can and equals our equation and then be |
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| 29:32 | to figure out him rather easily. ? That's the end line. End |
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| 29:36 | . So we go through log base . We can see that here. |
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| 29:40 | ? And then um again we're using equation and that's what we're modifying. |
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| 29:46 | . And so we ultimately get in step right to so we can really |
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| 29:53 | can uh we arranged that because again logs it'll look like this form. |
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| 30:02 | log base 10 to the end is same as N. Times log base |
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| 30:06 | of two. That's all we're Okay. And so that in itself |
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| 30:12 | term place 2.3 oh one. So so basic thing of that to |
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| 30:20 | enter that's that represents that exponential Right? So we're gonna put that |
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| 30:25 | that that is what the 10.301 is here. Okay so now we can |
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| 30:32 | solve for n. Okay that's what get here in the in the |
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| 30:39 | Okay. So that's and this is question of be honest, you need |
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| 30:43 | memorize this necessarily. Okay so um now with that in hand we can |
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| 30:52 | have population, no population sizes at times and then come up with a |
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| 30:57 | time rather easily. Okay and then that in different ways. We'll do |
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| 31:03 | couple of examples like that. So other thing here is this this is |
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| 31:10 | to use but often times we also to put a time parliament because we're |
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| 31:14 | in, you know, a growth constant. Right? So a growth |
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| 31:20 | constant. So every bacterium for every will have a under optimal conditions, |
|
| 31:29 | ? We'll have a growth rate depending how you grow, right? You |
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| 31:33 | grow the coli on nutrient auger nutrient and measure growth. It'll have a |
|
| 31:40 | rate then you do the really cool on a minimal media will have a |
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| 31:46 | rate likely much less. Right? um there is even on those media |
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| 31:55 | can there will be a an intrinsic rate. Such that no matter how |
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| 32:00 | nutrients you add, it will only at that particular growth rate. |
|
| 32:05 | this is just the inherent growth for species. Okay. You can't make |
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| 32:10 | grow faster than normally would. Okay , so that's what the K. |
|
| 32:16 | that's the growth rate constant. So we have a time element now |
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| 32:21 | that gives us the growth rate Okay. And so k. So |
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| 32:27 | is generations over time. Right? if we invert that, that's basically |
|
| 32:35 | generation time is. Right? It's for generation. That's the generation time |
|
| 32:40 | . Okay, so we will use much when you use this and this |
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| 32:51 | our equations. Okay. In a of problems around with. So let's |
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| 32:59 | at That. Here is number Okay, so The bacterium, Houstonians |
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| 33:09 | , not a real bacteria. At the generation time of 40 |
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| 33:15 | Start with five cells in log how many minutes Does it take to |
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| 33:21 | ? About 10,000 cells assume all the are viable. That's pretty much always |
|
| 33:28 | assumption. Okay. So there is equation. And let's see what we |
|
| 33:36 | here. So if you get an in in minutes, you may need |
|
| 33:46 | look at that in other time parameters fix and if you're not sure it's |
|
| 34:03 | . Take a stab. Mhm. , time around, but I'll pause |
|
| 34:21 | couple of times. Let's count down positive vibe. Okay, start up |
|
| 34:57 | and here we go. Okay. , let's go through the process. |
|
| 35:09 | . All right, so here's our . So is that we're trying to |
|
| 35:15 | ? Right and zero equals five. how many generations to get to |
|
| 35:22 | Okay or how many minutes? So we know that generation time is |
|
| 35:30 | per generation. Okay. So we're That value 40 minutes per generation. |
|
| 35:39 | , So calculate number of generations produced going from 5-10,000. That's what we're |
|
| 35:43 | to do. Right? So I this may be super basic, |
|
| 35:47 | But all I'm doing here is just up what's going on. Okay. |
|
| 35:52 | uh so catholic emerge generations produced and from 5 to 10,000. Use this |
|
| 35:58 | and multiply by generation time to yield like this. Okay, so we're |
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| 36:03 | do let's figure this out. Figure value out and then plugging it. |
|
| 36:10 | . So uh do that are. is 10,000 R n 05 We get |
|
| 36:19 | which is 11 generations. Okay. just multiplied by 40 440 minutes, |
|
| 36:27 | is right here is seven hours. , 440 minutes. Uh 60 minutes |
|
| 36:43 | hour, right? Seven hours is 20 minutes. Okay so seven hours |
|
| 36:54 | 20 minutes. A little over seven . Yeah. Alright so back to |
|
| 37:03 | again. Okay. Any questions about ? And there's there's um these these |
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| 37:11 | plus three more three others are on borders that she called bacterial growth |
|
| 37:17 | If and it's one side has the . The other page has it all |
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| 37:23 | out. Okay? So if you help look at that of course let |
|
| 37:28 | know let's have another problem. So little bit different. Uh So we're |
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| 37:35 | here for the to calculate the generation . So Kathleen generation time of 900 |
|
| 37:44 | cells growing 15 hours produced three million cells. Okay? Um No I |
|
| 37:56 | I had that written everybody. Alright So there's your two things for |
|
| 38:36 | . The old two heads are better one operate and put the timer |
|
| 39:21 | Yeah. All right, counting Let's see. Okay. Through |
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| 39:34 | E. No it's not. It's you know, majority wins. Okay |
|
| 39:44 | let's look at the breakdown here. so um Alright so 15 hours, |
|
| 39:54 | minutes obviously. Okay so let's go . R. N. Zero and |
|
| 40:00 | . T. Wright. And there's formula. So I'm trying to do |
|
| 40:07 | if we need to figure out uh trying to figure out this is what |
|
| 40:11 | trying to figure out. We of need to have that value. |
|
| 40:17 | And so that's what we're doing. so our generation is almost 12. |
|
| 40:24 | . And through the math there and get 76 minutes. Okay. So |
|
| 40:32 | problems you'll see aren't gonna be any complicated than this. Okay. And |
|
| 40:37 | , there's the practice problems you can through and they're all worked out, |
|
| 40:41 | ? So go through those. Um course you have if you have questions |
|
| 40:47 | me know. Okay. But Um Alright. Um Really should. |
|
| 41:02 | another question. So this relates to growth phases. So uh so batch |
|
| 41:09 | , right? They call us a growth curve. What that means is |
|
| 41:16 | uh also here um water bottle. so this uh meeting All right, |
|
| 41:30 | gonna throw it in our flask. . We would inoculate inoculate, |
|
| 41:37 | Just incubate let it go. Then take samples and quantitative number of cells |
|
| 41:42 | getting over time that will plot it . Uh Once we're the only time |
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| 41:49 | doing anything with this class, once inoculated is only take samples at periodic |
|
| 41:56 | . That's it. Alright. We're gonna go its course right? That's |
|
| 42:01 | growth we're having we have a batch growth with this monitoring and that's |
|
| 42:05 | That's that's what that means. Uh you can do variations of that that |
|
| 42:13 | talk about but that's what match growth . Okay so um and from practical |
|
| 42:28 | , I'd say in my experience uh quantitative doing this kind of calculations we |
|
| 42:37 | went through. It's good to know , Right? But my interest is |
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| 42:44 | in terms of what the growth curve like, how to influence that. |
|
| 42:51 | so you will cross the map. . That's all right. So um |
|
| 43:05 | changes in south science. That is . Okay. Um Hold on that's |
|
| 43:15 | . Um Come on. My pen not working right? That's true dang |
|
| 43:21 | . Hold on. Okay. Check. Exponential changes occur in both |
|
| 43:31 | and four. That's true. Yes. penicillin susceptible to most likely |
|
| 43:40 | to penicillin. Remember? That's fast typically. Okay so um none of |
|
| 43:50 | is false. Okay so they're all . All right, so um growth |
|
| 44:02 | curve match growth curve. Um So inactivated growth liquid growth medium and monitor |
|
| 44:10 | . You're gonna see some type of . Now they can differ in terms |
|
| 44:14 | lag phase. Okay. Maybe there longer lag phase. Maybe it's the |
|
| 44:23 | height of the curve doesn't get as . Okay. Uh Maybe the inflection |
|
| 44:32 | more like that. Okay. Not steep. Right, so all those |
|
| 44:36 | are possible depending on the organism, growth medium, the conditions etcetera. |
|
| 44:43 | . Regardless of the lengths and you're always gonna have these four stages |
|
| 44:51 | batch growth. Okay. And so lag phase. Right so you inoculate |
|
| 44:59 | the medium, right? Planting seeds the soil, water landing the |
|
| 45:05 | Okay so depending on the source. so there was a that's coming from |
|
| 45:17 | typically it's coming from a liquid growth that you're putting into? A fresh |
|
| 45:21 | medium. It could be from a . Yeah. Um The uh the |
|
| 45:29 | of that in Oculus is that has inoculate been growing for a week before |
|
| 45:35 | transferred It? Is the medium is in a way different from what you're |
|
| 45:42 | into? Okay. How much are transferring transferring point? Oh one mills |
|
| 45:49 | your batch growth or one meal? that makes a difference. Okay. |
|
| 45:56 | What about you know, think of the the I guess micro conditions. |
|
| 46:01 | ? So you're in a in Oculus and it's grown to the point and |
|
| 46:07 | you're putting in the fresh meat. ? So now it's in these new |
|
| 46:11 | , right? Likely differences in albeit slight. Okay, um differences |
|
| 46:19 | osmolarity. Right, So you concentrations be different ph may be slight |
|
| 46:24 | Okay um a different growth meeting right , all that are those different, |
|
| 46:32 | those differences. I mean the cell not going to start growing like crazy |
|
| 46:38 | you put it in there that has adjust their surroundings. Okay. Um |
|
| 46:44 | may have to be turning on genes turn on certain pathways because of the |
|
| 46:47 | . It now has to eat or has to be has to make a |
|
| 46:51 | of stuff to be able to So all that translates into time, |
|
| 46:56 | ? In this lag phase and so length of this lag phase, whether |
|
| 47:01 | this long that short for longer, on those factors, depends on all |
|
| 47:08 | factors. Okay. Another type of it is. Take a look. |
|
| 47:13 | , so you can you can influence by transferring the cells when there if |
|
| 47:23 | haven't to a point where they haven't so long, right? Where they're |
|
| 47:26 | growing actively and transfer them to fresh . Even even the best case |
|
| 47:32 | you're still gonna have a bit of life. Right? Maybe not so |
|
| 47:35 | . But there'll be some Yes, acclimation phase here. Okay. But |
|
| 47:40 | it gets going, it grows Okay. Typically. Okay, so |
|
| 47:46 | phase is gonna be the most active and it's not surprisingly where let's |
|
| 47:51 | do you often hear the term, may hear the term mid log |
|
| 47:55 | right? Or late mid log phase typically somewhere in here. Right. |
|
| 48:04 | It too. This is called mid phase. Okay. That area that |
|
| 48:12 | um are you the most active? if you're interested in some kind of |
|
| 48:19 | activity, it has that that's likely point where it's going to be most |
|
| 48:23 | . Where you'll take samples and measure activity. Okay. Or maybe it's |
|
| 48:28 | point where you'll you'll harvest the cells that's what I have the most maximum |
|
| 48:34 | of activity in the in the So it just depends, not always |
|
| 48:38 | mid log but it kind of depends what you're doing, but that's that's |
|
| 48:42 | reason to make a growth for one to see if you are interested in |
|
| 48:46 | that's making or the activity of an or what have you, is that |
|
| 48:51 | measure it across the whole time You see when it is most active |
|
| 48:56 | that's, that tells you that's when need to be there to to test |
|
| 49:01 | . Okay, so early on, kind of figure these things out, |
|
| 49:07 | ? If it's especially biotech people, ? If you have a culture and |
|
| 49:13 | new and it makes some kind of commercially commercial product you want to make |
|
| 49:19 | product you want to commercialize. You figure out these basics in the |
|
| 49:23 | what's the growth look like? What's grow best on how can I maximize |
|
| 49:28 | a short lag phase and a high density very quickly. Right, where |
|
| 49:35 | the activity of my product most What? These are all things you |
|
| 49:40 | know ahead of time. Okay, definitely these are the things you do |
|
| 49:45 | you're on the bench top, You don't want to figure this stuff |
|
| 49:48 | when you're trying to grow it in gallon tank, right? Because that's |
|
| 49:55 | and money wasted. We don't know going on ahead. So a lot |
|
| 49:59 | this kind of basic work is Small scale, of course, usually |
|
| 50:03 | up. Okay, so um but , so cell size. Right. |
|
| 50:08 | cell size will change, you for some of the more dramatic than |
|
| 50:13 | but size will change very for most as they're growing. Okay so long |
|
| 50:19 | remember you're gonna have a lot of I'm just gonna use a rod shaped |
|
| 50:23 | . You may have a lot that in this dividing state. And so |
|
| 50:33 | they're actively growing and dividing right and they will elongate and then they'll split |
|
| 50:39 | they'll be in there probably the largest certainly mid log phase. Okay. |
|
| 50:45 | compared to when you look at them stationary phase for example. Okay so |
|
| 50:51 | signs will be bigger in their in log phase. Okay um as we |
|
| 50:56 | to like around here late log Okay those things would rapidly begin to |
|
| 51:05 | down because now you have a population here right that is in late log |
|
| 51:12 | that you can't sustain that rate forever you're not gonna have enough resources to |
|
| 51:19 | everybody to be able to grow Right so you're gonna rapidly level off |
|
| 51:24 | that's what stationary phase were stationary phase and um that will of course obviously |
|
| 51:35 | we have balanced growth which means they themselves dying is balanced by the rate |
|
| 51:41 | that are better increasing. Right so death and cell growth or equal. |
|
| 51:48 | hence a flat curve. Okay and of course now we're not completely out |
|
| 51:54 | nutrients yet. Okay so it becomes survival mode for the for the for |
|
| 52:00 | micro okay and what are things that survival? Well if you become smaller |
|
| 52:08 | less material to keep up with um obviously slowing down significantly um stress can |
|
| 52:16 | . Okay so uh so you produce that help you kind of stress um |
|
| 52:25 | happens in stresses proteins begin to break ? And so you kind of have |
|
| 52:30 | system that kind of maybe help some those along the functioning. Um So |
|
| 52:34 | really about maintaining survival. This kind just getting a little smaller, eliminating |
|
| 52:42 | metabolic processes. Okay and kind of hanging out hoping that new principal |
|
| 52:50 | Okay um you know they can sputter because cells are gonna be dying, |
|
| 52:59 | ? Cells die that actually can be for them for others. Right so |
|
| 53:03 | can kind of subsist on that for while until of course that goes |
|
| 53:07 | Right and then at that point now death so there's no nutrients left to |
|
| 53:13 | anything, right? And that's why fast as you grew, maybe not |
|
| 53:19 | fast as you grew this way but do die rapidly. Right? So |
|
| 53:25 | that's why that can be a lot as well that you can also go |
|
| 53:29 | quickly. So uh and that's what really this part. This is what |
|
| 53:38 | focus on in chapter five. Control my program. How do we kill |
|
| 53:44 | things and throw them fast? Right um but obviously here we we've run |
|
| 53:50 | of everything can start dying off Okay. And so um okay so |
|
| 53:57 | phenomenon of dormancy persistence. I mentioned a little bit in the context of |
|
| 54:03 | resistance. So there are cells that of be a little smaller and they |
|
| 54:09 | of just um are able to survive an industrial period that way. |
|
| 54:15 | so we'll talk about this in um chapter are Sorry, unit two coming |
|
| 54:25 | . Let me try this again And this membrane potential to provide |
|
| 54:32 | Okay, we mentioned that earlier in context of chapter three, right cell |
|
| 54:38 | . The proton. All right, a form of energy. And so |
|
| 54:44 | able to sustain that in that non state to provide some energy. |
|
| 54:51 | Um and that they can remain in state for quite a while. So |
|
| 54:56 | them to when they're in a stationary to really drag that out. |
|
| 55:03 | not forever, but for a pretty period. And so that can you |
|
| 55:08 | if they find themselves environment now nutrients coming in and that may enable their |
|
| 55:12 | . Okay. Um The of course uh we'll talk about that next time |
|
| 55:22 | forming in the sport. And the is also something that you might form |
|
| 55:26 | it's capable it would do so in phase. Right, Those spores form |
|
| 55:31 | that's the way for it to kind be a dormant state for a long |
|
| 55:35 | . And those spores that can be been found that are like 250 million |
|
| 55:40 | old. So that's that's uh potentially mode if the cell is capable of |
|
| 55:45 | that. Okay so um. Is are where are there any |
|
| 56:00 | Alright so let's look at this question relates to growth curves in a different |
|
| 56:06 | . Okay. A bacterial inoculate um grown in nutrient broth. And you |
|
| 56:12 | the formula not what is then transferred a batch growth medium of M9. |
|
| 56:19 | we're going from we're going from this that. Okay flying inoculation of the |
|
| 56:27 | medium, what will the growth pattern closely resemble A. B. Or |
|
| 56:33 | . Okay so um the night has on nutrient broth then transfer to freshen |
|
| 56:40 | broth. Growth pattern is like Okay so just one little hit |
|
| 56:48 | But now we're doing this of course putting it in that. So it's |
|
| 56:53 | if you put it in M. , will it look again like a |
|
| 56:57 | like B? What will it look ? C. So A. |
|
| 57:03 | Or C. I predict 90% will this correct, 95%,, 99%. |
|
| 57:37 | let's uh Okay calm down. Right um Who entered C. |
|
| 58:16 | C. That's that's it. This what you need to look at is |
|
| 58:33 | face. Right? But from rich middle meeting right in this we got |
|
| 58:41 | of preformed stuff, amino acids and does not make it I can grow |
|
| 58:48 | quickly compared to growth in this. , so nutrient broth and you plop |
|
| 58:55 | into minimal medium. It's also whoa, put the brakes on, |
|
| 59:01 | , We're not we don't have any preformed nutrients that tone and actually give |
|
| 59:08 | right now, we've got to start everything for ourselves. We have to |
|
| 59:13 | amino acids, we have to make and that right if we're gonna grow |
|
| 59:17 | him not right? And that means on different pathways, turning off their |
|
| 59:22 | . Uh That means time translates into longer lag phase. Okay, what |
|
| 59:28 | that when you go from nutrient N. B. Two NB. |
|
| 59:35 | looks like that there's there's um there's short life is one from the same |
|
| 59:43 | same. You don't have to make adjustments and then kind of just adjusting |
|
| 59:46 | the temporary changes that may be temp and then get going around. |
|
| 59:53 | But you're drastically changing the meeting from complex rich medium to a defining. |
|
| 60:00 | definitely gonna we're gonna see that translators into a longer life phase and |
|
| 60:06 | a lot of stuff pathways etcetera. um Okay, so of course the |
|
| 60:14 | there is C. Okay, now take a look at A and |
|
| 60:17 | Real quick. So um this they the same lag relatively the same. |
|
| 60:27 | this part of the curve is very . Okay, but you notice, |
|
| 60:33 | assume A and B. The same that's going on the same medium in |
|
| 60:38 | A. And B. But you the difference there and sell yield |
|
| 60:46 | The number of cells cells is bigger B. Than A. So if |
|
| 60:51 | have let's say it's a cola growing A. And B. And |
|
| 60:55 | Book medium but it's more cells on . You can do that by doing |
|
| 61:04 | . This is the fed batch. ? So if you're growing it in |
|
| 61:10 | batch growth where you inoculated just take to measure growth and that's it. |
|
| 61:15 | get A. I like you see . Okay But if you then at |
|
| 61:21 | point ad nutrients add nutrients you can it. What's the obvious nutrient you're |
|
| 61:31 | add to see that effect begins with seat ends in Farber. Right? |
|
| 61:40 | had carbon that will enable more That right? Of course here we're |
|
| 61:50 | about the head of A pro. and if you give it more more |
|
| 61:56 | to it, more cells right, more carbon to provide an opportunity to |
|
| 62:05 | more carbon based life forms like Okay um the same thing but that's |
|
| 62:15 | . Right? I just sit and eating candy bars all day. I |
|
| 62:22 | multiply into 2-4 that hey but I get right I'll go write write more |
|
| 62:30 | . Okay so um and fifth batch an industrial scale stuff. That's what |
|
| 62:38 | do is this okay. And so that's the only way you're gonna get |
|
| 62:45 | high remember in uh commercial setting or you're taking stuff for commercial products you |
|
| 62:53 | deal in uh tablespoons of stuff. gotta deal with buckets, barrels. |
|
| 62:59 | ? So you're gonna need lots of and lots of cells. Okay. |
|
| 63:03 | that's where you got to keep it it, feed it. Okay? |
|
| 63:08 | so you can get and so not feeding but controlling all the parameters. |
|
| 63:15 | ? So what different practice is focused nutrients but th there's oxygen in this |
|
| 63:23 | . Um And then defeating. And so this uh this is a |
|
| 63:30 | controlled operation over here. You see the right this bioreactor that's probably like |
|
| 63:38 | five liters. Um You see all stuff on top. Right? All |
|
| 63:44 | wires and this and that up Right. So um and then of |
|
| 63:50 | these are pumps on the right. . It was all computer control program |
|
| 63:56 | ph between six and eight right acid base as needed. Um Set the |
|
| 64:04 | called A. D. O. ? Deal with dissolved oxygen. |
|
| 64:09 | So we can control the amount of going into the system, right? |
|
| 64:15 | call that D. O. And can measure we can measure and and |
|
| 64:22 | everything temperature controlled as well. One of the things that happens fast |
|
| 64:28 | growth is a lot of heat produced of heat. And if you don't |
|
| 64:33 | control it, that glass will get hot as a stove top. |
|
| 64:37 | Of course the temperature kills them as . So you have to one jacket |
|
| 64:42 | control attempt. Okay so so you all those things you baby it so |
|
| 64:48 | speak by maintaining ph 02 temperature and go grow like nuts. Okay? |
|
| 64:56 | they're in pure culture not competing with and they can just feed them what |
|
| 65:00 | want as much as they want. grow. Okay? So but of |
|
| 65:04 | you can do this and you have add things in at certain times. |
|
| 65:08 | ? Why not? Double owner once to worry about doing all this control |
|
| 65:14 | . Why can't you add it all once? It actually doesn't grow if |
|
| 65:18 | do that. Yeah. Your said wanted to let's say part that can |
|
| 65:44 | you a so you know you get oh just put all in the |
|
| 66:01 | Very close. So 100 g of you need at the beginning what you |
|
| 66:08 | live. What's that sells matrix. all these things around it burn those |
|
| 66:19 | And immediately 100 past years. Um where the take ordinary. So |
|
| 66:34 | becomes an osmotic issue. Right? so high platonic right? He has |
|
| 66:41 | lose water. So that's that's what you try to add all the digits |
|
| 66:45 | front to give it maximum growth then to its osmolarity issues. So to |
|
| 66:52 | tonic to the solids out there compared what's in the cell and then inhibit |
|
| 66:56 | . Okay? That's why you can't why you have to uh batch feed |
|
| 67:02 | in increments. Okay. But it it won't grow otherwise. But you |
|
| 67:06 | do it this way then it works . And so so you may have |
|
| 67:13 | end, you know you feed it multiple times. This will keep going |
|
| 67:20 | . Alright. There was a Okay? But you can get a |
|
| 67:25 | of cells. Okay? And so anaerobic bacteria growing in liquid culture adequately |
|
| 67:30 | you out. So what would and wrote it come in the it's you |
|
| 67:53 | take an option constantly, right? you begin to run you run |
|
| 67:58 | right? Began to breathe heavier. ? Running out of oxygen. You |
|
| 68:02 | to get more. You can stuff oxygen tank but that's not practical. |
|
| 68:07 | you just stop and rest. So bacteria growing like crazy. They have |
|
| 68:14 | so you have to feed oxygen to . Okay? And so um and |
|
| 68:20 | have you have this you have all parameters in here. You have a |
|
| 68:28 | oxygen program measure oxygen that was E start mixing it faster yet more |
|
| 68:37 | air into it, right? And it up real well, lots of |
|
| 68:41 | off the road. Beat up beat to it. Right? So if |
|
| 68:45 | biotech you'll work with these things. ? Uh That's it, folks go |
|
| 68:50 | lab or wherever you're headed to Yeah. Yeah. Oh yeah let's |
|
| 69:57 | uh That's right. So over here would be ah there we go. |
|
| 70:11 | it would let's say we went the way then it would go. Yeah |
|
| 70:16 | maybe short. So what is the we have to make a bunch of |
|
| 70:30 | ? It's going to have to make from scratch. What I was thinking |
|
| 70:36 | essential because I mean if you're going other way because remember what's in the |
|
| 70:53 | extract, fully formed, fully formed kinds of molecules they don't have to |
|
| 71:00 | in this media. These are all of the different elements. These these |
|
| 71:09 | exactly exactly. Yes. Which is probably on thursday. Yeah sure |
|
| 71:31 | Oh yeah I got you. Let's what why are bacteria about it |
|
| 71:45 | are they? Yeah. See Uh Because they multiply by dividing. |
|
| 71:58 | like I was thinking like they had because I've heard when it's like a |
|
| 72:06 | magic, something bad in biology and like because he can't tell the |
|
| 72:13 | Um I think I was like yeah so popular. Popular. Uh |
|
| 72:24 | Because they're uh they're fun guys. course. Yeah. Yeah. |
|
| 72:29 | Yeah. Yeah. Yeah. No shortage of those jokes. I'm |
|
| 72:38 | I've counted how many professor? Yeah, |
|
