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00:01 mm hmm. Oh but it's okay . Welcome. Alright. So I

00:46 a lot to get any kind of at this point in the semester?

00:54 we are on track, which I hard to believe not because of you

00:59 because I've never been on track. um so today we're gonna finish uh

01:05 quite finish up for but we're gonna uh at the end of sport part

01:10 next time chapter five is pretty um okay. So maybe like doesn't result

01:19 will go through mainly focusing on really focusing on control of microbial growth.

01:26 physical chemical methods of control is pretty working to focus on. So chapter

01:30 is not lengthy. So uh I'm only looking at a part of that

01:35 . So uh we'll do that on and then whatever we have left

01:41 we'll do Wednesday. So Wednesday is of those I called days. And

01:45 just built built into the, into of course schedule in case you

01:51 just to give some leeway if you know, get behind the have

01:56 to catch up basically. But we're much on track. And so if

02:01 have anything and just be a little of stuff to do on that

02:04 Um then we'll start no, yeah start then the following week we start

02:10 next unit. Okay, so you'll that material um begin to show up

02:19 week. And so have the unit two Which won't start until the

02:26 21st 21st is when that would Um Let's see. Okay. Uh

02:34 examples of the castle schedules open, know that the unit quiz. So

02:38 this, the unit quiz go different only in terms of a little bit

02:44 , just to be more comprehensive with unit quiz. So you know,

02:48 be questions over the areas in chapters and four, I'll uh five isn't

02:54 because we're not gonna get to it monday, but I'll um I'll post

03:00 questions on checker five on on so you can have those to look

03:05 as well. It won't won't count a quiz. It'll just be always

03:08 questions and then I'll have the Uh keyboard. So uh I'll uh

03:15 notice of that. I'm gonna send email tomorrow about stuff. Um What

03:21 ? Oh, there is one thing meant to pull up the blackboard.

03:27 , maybe I can do it and don't want to waste time with

03:30 But what I'll mention it in the tomorrow. So um so when you

03:37 to where you look at the the video lectures folder, right?

03:43 you'll see a link in there called link to a site called says video

03:50 . Video points site for lecture, ? All of this is, it's

03:55 available yet, but it will be . Uh It's the same identical lectures

04:00 they're used to seeing. Okay, just they're being uploaded to this site

04:05 well in addition to so nothing I wanted to say something extra. Um

04:10 video points does. It's uhh uhh and they they it's really just,

04:16 only difference is how you can navigate you can search student lectures, you

04:20 actually do a keyword search if you to look at a specific in this

04:25 lecture on a specific topic, you actually search that and we'll go to

04:29 . Okay, so so you had as an option? So I'm just

04:33 just making it available to you. You may find it very useful.

04:38 , so and then there's other features there as well, I'm not well

04:42 in all of it. So I there's even like a little help or

04:46 button if you want to look at the features, but it's just another

04:48 to present the material. Again, it's the same lectures, I'm uploading

04:54 one drive and just uploading it here well, but it gives you this

04:58 search options if you want to explore . So uh you will be able

05:02 see that tomorrow. Okay? Um don't I uploaded all the lecturers who

05:08 done so far to that site but think it takes a while for him

05:13 do that. Index is called So I think all but the last

05:20 are set but maybe by tomorrow that be they will be set. But

05:25 , it takes a little bit of to get the thing to the

05:27 but again, completely optional. look look at it for yourself.

05:32 again, I'll mention in an email . Okay, So let's go

05:41 Um Oh, I wanted to put in just for grins. Okay.

05:46 , um, the super Bowl is . Okay. Who will win the

05:50 bowl? This sunday? Any Okay. I'm not sure if I

05:55 Cincinnati. Right. I think that's . I had to tease in there

06:00 initially I thought none of them Right. There's two edges on

06:03 2. Um uh I remember when Bengals were in the Super Bowl last

06:18 . Tell you how old I 1984, I think. Mhm.

06:33 , mm hmm, mm hmm. prediction is um d is gonna be

06:42 majority answer. I think most people what the Super Bowl is, so

06:47 not gonna I'm not gonna pick Okay. And let's see if I'm

07:01 . Yeah. All right. I right. But you just picked that

07:05 I said that so split between the and rams. That's interesting.

07:11 Okay. I don't know. I to pick the rams, but I

07:17 the Bengals defense. Mm Hmm. see. Um Actually, I'm actually

07:22 the first time. Probably miss half the Super Bowl because I'm going out

07:24 my wife for valentine's day thing. , but I will record it.

07:33 anyway, let's get to some science guess. Okay. Um Alright,

07:41 kind of what we've been talking so we went through everything up to

07:48 dotted line last time, so we'll through the batch growth curve today.

07:54 stages uh bad batch, a little about bioreactor growth, those of you

08:01 biotech folks in here. I know of the curriculum is you do work

08:07 bioreactors. Um Do you do kind a mini uh I guess semester long

08:12 where you grow cells up in your and they're like um recumbent DNA plasmids

08:18 them. Many that you harvest the and something like that. I think

08:21 do things like that. Um So talk a little bit about fire,

08:25 growth um And then dynamics. So involves in growth calculations. Nothing super

08:31 . Okay. Um It's you it's it's logs, logs, the

08:37 tens and multiplication and division. Pretty . Okay, so we'll go through

08:42 F. Y. I. Um will be able to bring a handheld

08:48 of any type. Okay with you the cost of testing center, they'll

08:53 made aware that you're allowed this and that you won't be bothered about bring

08:59 calculator, so so that won't be problem. Um It's only like a

09:04 of problems, so it's not like a whole, you know, 12

09:07 or something, just a couple on . Um But we'll go through some

09:11 of that and then we'll talk we'll get through biofilm formation but we're

09:16 going to get through in those information that's why we have plenty of time

09:20 finish that up next week. Um , so let's uh any any questions

09:28 we go. Alright. My question not here today is just sitting right

09:34 . Okay. All right. Um , so let's take a look at

09:39 in case these are uh let's uh us over. And so while you're

09:46 at this, I just explained so growth, okay, why why that

09:51 ? So what it refers to? really a contrast between that and what

09:56 do in a bioreactor? Really. . A bioreactor, as you'll see

10:02 offers you really total control of your of your cells are growing so you

10:09 you can influence all the parameters, ph auction levels, etcetera.

10:16 Uh in a batch growth mode you necessarily doing that. So think of

10:23 mode as you know, you make media in a cup in the flask

10:29 uh you inoculate right? You're gonna it with some cells. Okay.

10:34 then they will go through, they you a growth curve, something like

10:39 , not identical in terms of the necessarily the same inflection of the phases

10:46 things, but it's gonna give you those 1234 stages. Okay. Um

10:54 um Mhm interesting. The so what doing, the only manipulations you're doing

11:02 once you inoculate then at certain time after that you will take a sample

11:08 you will measure growth somehow. The common way to measure growth is with

11:14 absorbent students inspector for thomas. Or take a sample as a as a

11:19 culture grows initially. When you inoculate looks pretty much like water or clear

11:24 water and then as it grows it rick highly. It's called turbidity we

11:30 it. Okay. And so as turbidity increases you know it you can

11:37 light. Okay. And so you'll an increase in absorb into what we

11:40 optical density or O. D. uh increase as it grows and then

11:46 plateaus of course as you see there stage three and then um uh and

11:53 what you're doing is getting a growth what's called a batch of growth

11:56 Okay. Um and so it will , you can have the same species

12:02 grow e coli on various types of complex defined what have you uh the

12:11 media with different carbon torches maybe or you can manipulate. Okay. And

12:16 will give you like a different different different patterns in terms of the length

12:21 the phases and how sharp each phases so forth. So um so remember

12:29 why you're doing this for a Right? So you are typically do

12:35 growth curve um to see and then samples you know if you have a

12:42 for interest of an enzyme activity. cells may possess a protein they make

12:47 you want to analyze. Okay, um some other kind of activity And

12:53 and you take samples over time to Okay, where where along this growth

12:58 ? Am I getting maximal activity? it's typically it's going to be somewhere

13:03 this range or you will. And that's and so you may not

13:09 when it arrives at that point. ? So you didn't take samples to

13:13 . So that's why I do the curve because you know, oh at

13:15 time point here you can actually collect and then do my thing Or maybe

13:20 just want to see well how how cells right this level here. I'm

13:27 telling you how much souls you're getting . So maybe you want to see

13:33 how high that gets right whenever you to get the more cells than

13:38 Okay, so mhm So um the purposes. So you know, typically

13:46 for doing isolating proteins, isolating N. A. Doing an enzyme

13:52 , What have you? So there reasons for doing this. Maybe you

13:55 to find out what are the nutrients of mine. So what do I

13:59 to, what do they need to best? Okay, so you can

14:03 do growth curves to figure that out well. So um even um you

14:09 , and you're doing these things that this kind of a scale,

14:11 Called bench scales like this quality of ok. In industry you start there

14:20 kind of figure things out growth wise that level because it's easy right before

14:24 wrap up what we call um um scale up before we scale up to

14:34 hundreds of liters of quantum right? can have 500 liter tanks, 1000

14:40 and up. Right? So you out it's very expensive to do growth

14:45 at that scale. So you do bench scale and then scale it up

14:49 there. Okay um so uh so see uh let's see what we got

14:57 . Been rattling on here. so e none of the BDS parts

15:06 let's see change. The cell size during phases two and three. That's

15:11 . Exponential changes in cell numbers. two, true acclamation, phase one

15:17 . Um and yeah so none of of these were false are all true

15:21 . So um so yeah, so and four particular both exponential ones.

15:28 increase, one's exponential decrease. Okay so let's look at description of these

15:35 , right? So lag phase. it all begins again, you have

15:40 in a flask and you see this the inoculation is a seed, so

15:44 speak, um it contains a a of cells you would have grown

15:50 Okay initially and then take a small of that and then inoculate your

15:57 Okay, um now you always go a period called the lag phase.

16:03 . Which is here, in this from here to here. Okay,

16:08 can be lengthened or shortened depending on immediate types being grown between binoculars um

16:17 the batch medium. Okay, So this has grown in and be a

16:24 type That may be may or may be different from what you're putting into

16:29 that can influence your blackface length length the blackface. So, lag phase

16:34 general is really uh think of the of themselves in the United Kingdom,

16:38 . Maybe they were going for 12 24 hours somewhere in that range.

16:42 ? So now you're taking a small of those and you're putting them in

16:45 fresh medium. Alright, so now in this different new environment,

16:51 They'd likely have been in an environment they had been growing for some

16:55 maybe accumulating waste. Maybe some ph have occurred. And now they are

17:02 they're slightly oxygen deprived. Right? so now you put them in a

17:07 brand spanking new medium, Right? they're not going to take off growing

17:11 . Okay, I have to get to this around because that's that

17:15 Yes. Um maybe they need to express different metabolic pathways because of the

17:23 that's now given available to them. . And so they have to turn

17:27 certain genes and turn off certain genes proteins. So all that kind of

17:32 is going on in the lag phase then once they're set then boom,

17:38 can take off and take off And so so basically what I just

17:44 because some of the factors affecting lag , so inarticulate rage, right?

17:48 you're You know I claim is 12 or 14 hours old versus 48

17:53 that could make a big difference because might not be as many viable cells

17:57 after 48 hours as it was And so we're actually seeing it with

18:02 viable cells and it would be if were fresh culture. Um the amount

18:07 you inoculating 100 μm is you're likely is it one mil? Right,

18:12 that will make a difference. Um , the media composition. How different

18:16 the two types? Okay, um physical factors were just kind of the

18:21 environment that the cells were in previously you put them in something new,

18:25 know, the different, slightly different concentrations and things like that. All

18:30 collectively, all these factors can will the length of blackface. Okay.

18:37 now you can sometimes use this for advantage depending on you know, when

18:43 do this kind of stuff for a . You you don't necessarily want to

18:47 tied to the clock of the Right? You don't want to do

18:53 not necessarily do things on their Right? You don't want to wake

18:57 midnight I have to go in and whatever. Right? So maybe they

19:00 manipulate conditions and do it at reasonable . That's where you can intent intentionally

19:06 with conditions and maybe make a shorter longer lag phase so that you don't

19:10 to come in at weird hours. . Uh An easy way to do

19:14 is really what the with the size the nightclub just making it smaller than

19:18 can get a longer life or make a little bigger and then you so

19:22 can kind of manipulate some things to your schedule sometimes. Um Just that's

19:27 of those things you don't see in but if you are working on the

19:31 you might want to set them Eur hit okay um like so walk

19:38 . So once they're set they're going will go very quickly. And so

19:42 a lot of faces. The most active state. Lots of selves doing

19:49 um that appear like this. Let's go with the rod shaped cell.

19:57 lots of cells in this kind of of dividing. Right? God that's

20:03 . Try it again. Okay so of cells are kind of not much

20:09 . Okay but the point is you lots of cells in that where the

20:13 is formed and they split up because dividing very rapidly. So you see

20:16 of cells that are in that kind state in log phase because they're functioning

20:21 very rapidly. Lots of protein So we offer you often differentiate log

20:27 into mid log. Yeah in late . Okay and again middle august party

20:36 arranged or something like this. Late has arranged something like that.

20:41 Okay. So why that distinction? middle you know if you're interested in

20:47 in a and assessing measuring enzyme activity some sort in the cells uh they're

20:53 be most functional likely in mid log gonna be at they're not nutrient limousine

20:58 very fast. Very active. So it's mid log where you typically

21:03 want to do those measurements. Late log is maybe when you want

21:09 harvest cells. Okay if you want to be using a centrifuge to get

21:13 out to the store and save them or whatever. Um You could also

21:18 that mid log as well but um log is kind of becomes your near

21:23 tipping point. Okay So um the are at that point becoming nutrient

21:30 Okay And so they're gonna begin to down and then you get into a

21:34 phase. Okay Because at that point late log you get a lot of

21:38 there and you certainly don't have enough to sustain everybody to keep going growing

21:43 that rate. Okay to keep doing . Okay it's going to tip over

21:50 I? Um And so because it's batch growth, you're not doing any

21:54 relations to it. You're just letting go. All you're doing is measuring

21:59 the growth. Okay. Um You do those many collections as we'll talk

22:04 shortly. Okay. So I'll sell so cell size because the cells are

22:12 of in this state or in a it's a wholesome goodness. Okay.

22:19 have a I hope so here they kind of you'll see some information occurring

22:24 itself. So you're gonna have you're elongate if it's a rod shape.

22:28 course it's a caucus it'll get this , you know, browner. But

22:33 expand to a point where then they'll into two. So you're gonna see

22:36 lot of cells that kind of they're be the biggest size in in mid

22:41 log phase. Okay? Um as get to stationary phase. And of

22:47 that's right. And uh growth rate equalized because you know flat they're not

22:54 out of nutrients yet, There's still to feed folks. Okay. But

22:59 is limited. Okay. Um And kind of because of that kind of

23:03 stress responses are initiated uh due to limitation could be also due to oxygen

23:11 if it's an Arabic organism. Um so the the strategy now is let's

23:20 . All right, we're gonna go survival mode. So that means kind

23:23 let's reduce protein synthesis of non essential . Um Let's um let's get

23:34 It's a little bit smaller. That that's less material to keep up

23:37 Okay, so um and so all the the strategy of okay let's do

23:45 survival thing with hope that maybe more will appear okay uh You know,

23:52 as cells die right? The the the material from that cells that sells

23:58 now available as a food source as so they can actually live off of

24:02 for a period of time. Um of course eventually, you know I

24:07 here right here Is when completely out nutrients no way you can sustain anymore

24:14 at all and very rapidly goes off cliff. Okay, exponential decline.

24:19 so that's what we actually focus on Chapter five. Is this phase

24:25 Because we're trying to um control microbial and when we say that we're trying

24:31 control the pathogens obviously. So it's let's throw chemicals uh chemicals,

24:38 whatever the treatment is to make them as quickly as possible. So we

24:43 on how can we maximize that So that rate make them go really

24:49 , dying very quickly. Okay, that's what we focus on In Chapter

24:54 . So but the point is um phase and death phase can both be

25:03 change is exponential changes in cell numbers going up when going down. Okay

25:10 so this phenomenon of dormancy and persistence come to um ah significance, particularly

25:20 the context of pathogenic bacteria that can this that we call them per sisters

25:28 can um get around the effects of for example, um by not

25:36 Okay, so remember a lot of antibiotic targets um focus on components that

25:46 that are at their most active when are actively growing, right? Like

25:50 wall synthesis, right. Things. of targets are sell all sentences,

25:54 , example, ampicillin, etcetera. so those drugs are most active when

26:00 cells are actively dividing, Right? when they're actively dividing, okay,

26:06 they're when there's many cells along phase are kind of this sting,

26:13 This is that's this or this All . That septum that's cell wall

26:23 Right synthesis. Doing so so that's active. Of course the mark

26:30 Right. And that's when these kind antibiotics would be most effective.

26:36 And uh there are lots of targets the for the antibiotic in that

26:41 Right? It's not wrong then, really no targets. What's it gonna

26:45 ? There was no cell wall being . What's it gonna do?

26:49 so, so many other antibiotics that components of protein synthesis. Right?

26:54 gonna be more effective also when its growing. Right? That's when it's

26:58 lots of proteins. Okay, so some some pathogens have adopted this

27:05 of of arresting growth. Right? the presence of antibiotics. Okay,

27:10 not exactly sure what the mechanism is inducing that, but we've seen that

27:16 so it can be not grow and viable. Okay, so maintaining that

27:22 membrane potential when we talked about we'll talk about that in the next

27:26 . But that's remember the proton pump talked about. Alright, so that

27:30 a membrane potential that can be used produce energy. Okay. And that

27:37 can Mhm allowed itself to sustain itself it's not growing. Okay. And

27:45 so because any antibiotics you take don't around forever because they will dissipate from

27:51 body. Right? And so when so when the organism's senses that then

27:57 they begin to grow again. So it's a um now that kind

28:03 mechanism is not really one that's I think it's one that's that's terrible.

28:13 . Maybe it is that it's not . But regardless, it certainly is

28:17 the individual or individuals with that particular type, they can they can um

28:25 be affected by antibiotics while antibiotics are . Okay, so so again,

28:30 what we call for sisters, which kind of a dormant state, but

28:35 do while the inbox around but then the grill when it goes away.

28:39 . Um Yeah, go ahead. I don't think so because that's I

28:48 they they are just resistant to So methicillin resistant staff lawyers, so

28:55 not saying they can't exhibit that but um but but I think there

29:00 particularly resistant to many antibiotics. Any other questions? Okay we'll talk

29:09 a little bit about antibiotics. I there is semester because of antibiotics can

29:13 in different ways. You can. often prescribed two antibiotics, not just

29:17 to you Because sometimes 200 biotics work together than they do alone. So

29:23 that as well. Um Like I we'll talk about some of that vaccines

29:28 talk about later as well. Um so let's look at this question.

29:34 is something I touched on just a minutes ago. Okay um let me

29:40 that up again. Okay so okay just go to the question here.

29:46 so a bacterial inoculate um has grown nutrient broth. Ok so nutrient broth

29:52 them. An eloquent of this. it's grown let's say overnight you're gonna

29:58 that to a batch growth medium. the bachelor growth medium is going to

30:01 M. M. Nine. What's M. Nine. You see the

30:05 there? Okay so you have nutrient and M. Nine formulations.

30:10 Fund inoculation of the batch medium. will the growth pattern most closely resemble

30:16 B. Or C. And so for for reference let's just say that

30:25 when the inoculate them has grown in broth and transferred to fresh nutrient

30:31 the growth pattern? It looks like does in a, Okay so it

30:37 from neutral breath nutrient broth looks like . Okay so what might you guess

30:44 when it goes into M. what would it look like? What

30:47 looks like A B. Or Yeah. Mm hmm. Bye.

31:28 . Okay, let me see. the timer on two. We just

31:35 down to a question of And the . nine might there be a

31:53 Yes. Okay. So let's see can see. Okay. So who

32:08 C. No. Happy. So why C. What?

32:18 One more time that so going into um So going into M.

32:35 Okay. It's gonna looking at sea means you're extending the like this.

32:42 ? And so you're sending the lag because the cell has so remember the

32:47 nutrients. Right. Mhm. So things gives provide the cells. So

32:53 one, when you see that I of this Pepto beef extract meat,

32:59 ? Meat. But also there's there's . Plant products like soy products are

33:03 fit in this category as well. So in doing providing complex nutrients,

33:10 providing. Not just providing C. . O. M. P.

33:14 in all different ways. Right? as carbohydrates as lipids as as um

33:22 acids um proteins, right? As as vitamins and amino acids. Write

33:28 these things because that's what's in Right. And then plant tissue.

33:34 uh so a lot of pre formed lot of preformed nutrients are present there

33:39 bacteria to grow up. Okay, an M9. Right. This is

33:44 minimal media a defined media. Okay in that medium It's going to basically

33:50 to make everything from scratch? There's gonna be a lot of when

33:53 when it goes from nutrient broth into . nine there's gonna be a lot

33:58 we have to turn these pathways on these off. A lot of turning

34:03 because now it's gonna have to you turn on the you know acid pathways

34:07 this and that to synthesize all it to grow and so that's time.

34:11 it's always always takes time to do things. So that's why you would

34:16 the lag phase for that reason because the time needed for the cells to

34:21 ready the things grow on this new or medium if you will. Okay

34:28 if you went from like I said it went from if you went from

34:31 . Nine and M. Two M medium but then they were already set

34:34 go as well. So you wouldn't wouldn't see a long life phase it

34:37 be relatively short because they already acclimated those minimal medium conditions. Right?

34:42 whenever you're going from the same medium another the same medium and batch it's

34:46 be you know, reasonable black faces you go from very different that it

34:52 it can be extended. Right? if you go from M. Nine

34:56 a rich medium like nutrient broth than can shorten? Right so again just

35:02 from the perspective of the cell what a cell have to do when it's

35:06 this medium now you have to do lot of stuff to get acclimated and

35:10 going or not so much. Many questions about that. Yeah.

35:16 it's big. Okay so yeah good . So these they have about the

35:27 black face. Right. But but different the amount of cells? All

35:35 so you're actually getting more cells The dashed line. Right so you've

35:41 gotten a read a quantity of cells be so maybe maybe maybe um you

35:50 be be could be just hypothetically. So you can make nutrient broth in

35:55 in that in what we call one . Right let's double it up and

36:00 it two X. Alright so then will give you more because you have

36:05 stuff to grow once you get more . So that that's that's really what's

36:08 on in B is the medium formulation you more cells. Okay that make

36:14 . All right. The same. . So does that mean?

36:26 Yeah I mean again it's it's I'm hypotheticals out there but yeah the point

36:31 yeah clearly in B. They could the same media they don't have to

36:35 but clearly B is giving it more source is my guest to give it

36:41 cells. Yeah. Yeah push it you can get from A to B

36:47 us to the next point in fed culture? Okay so that's how you

36:52 be growing like this in a. . Ah I'll live. Okay so

37:06 if I fed it like say here then I could get more sells right

37:12 give it more carbon. More carbon the thing that influences it the

37:16 And so get more carbon at that goes up. Okay I can just

37:21 my nutrient broth which has Pepitone and extract and add glucose to it.

37:26 that will boost cell cells up. And so we call that fed batch

37:32 that you see there. Okay so is now where we're making manipulation.

37:36 batch growth you kind of just Let it go right to get a

37:40 curve. But now we're now we're uh the culture, we're adding

37:46 You could even go as far as could um You could put a ph

37:51 in your in your medium to see it gets acid or base. And

37:55 adjust right you want to keep it 6 to 8 for optimal growth.

38:00 it gets to lower to higher. can adjust it with neutralizes the acid

38:04 um at carbon to get more So you can kind of control things

38:08 now becomes a pain to do that the shake flask because you constantly have

38:12 monitor, you probably have to see how's it doing? Let me look

38:15 it. Is it getting acid You know, is this cell numbers

38:19 down or leveling off? So then have to do your manipulation. So

38:23 can be a pain in the Which is why we have the

38:26 which is all integrated computer control, program what you want to do.

38:31 you just but you don't have to all these manipulations by yourself. And

38:36 you look at it by reactor, itself is right. Is the tank

38:42 , which is been put into the ? Um you see all the pumps

38:47 here? Okay, so delivering um base uh could be delivering nutrients as

38:54 on a on a prescribed schedule. , So even if the fed batch

38:59 , you could feed again right here keep going up, feed again here

39:05 go up. So you keep doing even in shape fast will be become

39:10 in terms of volume. Uh and parameters start to limit you like

39:16 And so but with my reactor you control a lot of this stuff.

39:22 , So um so it fed I'll come back to Bayreuth for the

39:26 . Let's look at this question. If let's say for example, if

39:30 can add I don't know, 50 per leader glucose. Right? and

39:38 the typical medium may have like, we started out with 10,

39:44 10 g of glucose. And then then at at intervals here and here

39:52 maybe a third time we add like 10 or 15 g at a

39:57 Right? And then we get lots cells. Okay then you go then

40:01 think to yourself well I don't want keep adding at different times. Let

40:07 just do it all at once. , why not? At all The

40:11 at the beginning instead of doing it batch, right. Which you could

40:16 but you will find if you do the cells don't grow. Why is

40:25 ? And that strict? They typically don't grow if you add everything up

40:29 like that. Why why would that very simple chemical principle? You all

40:43 ? Any ideas don't meet. So wild at me. Well think of

40:54 you added 50 g per liter of inter medium at the beginning with your

40:58 stuff you need. Right, nitrogen source Blah Blah Blah. What's what's

41:04 to be different compared to that Or in the same medium with just

41:08 g per liter of glucose. What increased the total is concentrated the concentration

41:17 total. Mhm salute. So we a high salt use concentration. What's

41:23 going to affect? Hi platonic hype . The old osmosis. Right.

41:30 where it becomes inhibitory. So it's a high solid concentration. It was

41:34 hyper time. And that actually can the cells because they're fighting the water

41:39 and that kind of stuff. So go I don't like this, I'm

41:42 really gonna grow that well they may but not very well. I like

41:46 want them to or like you would them to. So for those for

41:50 reasons you don't add all this stuff front, you kind of do the

41:53 batch thing to keep them happy. The so if an aerobic bacterium and

42:02 in liquid culture is adequately fed So we do the fed batch thing

42:07 what can limit growth fairly quickly unless control this parameter. There's one word

42:14 the beginning here, that's the operative . Which one? Okay.

42:24 Yes option. Right. You gotta options limited fairly quickly. And so

42:31 there's no way to get around And so um one way is.

42:39 um has anybody grown cells in the flask anybody? Okay. I don't

42:49 the hands. So I'm missing Okay so um if you have an

42:54 aerobic bacterium and you can actually uh just like the one you see

43:00 Okay. Do you think you see difference if it were sitting static like

43:06 ? And just in the incubator or you do something else to a cancer

43:13 ? Get heroic time. So aerobic gets its uh Oh too from surrounding

43:24 . Right. What you need to up this analogy? Um How does

43:33 how is air mixed with very well some water gulfs and beaches waves

43:45 right? Air mixes into the Okay so if you take that shake

43:49 and do this. Yeah a lot growth. And just think it's ecstatic

43:55 now you're that's how air gets into system. You create turbulence to mix

44:00 air within water. Okay now you even go a step above that.

44:05 see this flask has these ridges in bottom of the flat, right,

44:11 getting a flat typical flat flash. things have what are called baffles,

44:16 little indentations, right? That creates more turbulence. You can actually compare

44:21 to a class like that with one flat bottom and you'll see a difference

44:26 the amount of turbulence being created more and mixed in. Um The other

44:30 you can do is jack up your on your shaker. So instead of

44:36 100 revolutions per minute do 200. then you'll see a difference as

44:41 faster growth. So obviously this is assuming you're working with Arabic organism.

44:47 ? So in the bioreactor then you control all these parameters. So you

44:51 um ph and temperature of course here can automatically add acid base. You

44:59 have set points For your p typically six or 8. Below six ad

45:07 above eight at acid. Um Then have oxygen concentration. So what's called

45:14 D. O. D. Pro oxygen that will measure the option

45:19 and um when and so again you it uh to get below experts and

45:28 then do this and to do this is to make this go faster.

45:34 is what we call um the right? Has these blades on

45:39 That twist turn? It's basically a mechanism. Okay so stir faster.

45:46 great more churches. Makes more air with the liquid so those can get

45:51 . And then the other thing is the amount of air coming in.

45:55 so a spar Jer is really just device that just has little tiny

46:01 tiny holes in it. Okay and you're forcing the air through these tiny

46:06 and that works better than just kind bubbling it in through a you know

46:11 of the tubing or something because tiny mix better with water than large

46:17 And so it dissolves more quickly in water. If they're tiny bubbles and

46:21 does that so increasing the RPMs, more turbulence, mixing and mixing small

46:29 . That's what the sparkly gives. . See a lot of oxygen better

46:32 uptake by the cells that way So in all bioreactors are equipped like

46:39 . Okay so we can control all things and you see here will be

46:43 in nutrients where you add your inoculation the system, you can also on

46:49 timed program at nutrients to it. in that system you're the cells are

46:55 happy because everything is controlled right, everything optimal ph optimum nutrients, optimal

47:02 . Okay. And they grow like . Okay. Um so the one

47:07 that that is critical in the system this the cooling? Because we didn't

47:16 would grow so fast, they would themselves because of all the heat being

47:20 , right? Thermodynamics, right? lots of heat and so hot that

47:26 it worked, I didn't experience But somebody mentioned to me that the

47:33 system on their vessel uh was non and then they came back and it

47:40 like they couldn't even touch the reactor so hot because the cells have grown

47:43 much. So uh so water is important. And so um so

47:49 you know, biological control all these get lots of cell yield, which

47:53 what you need to do for a process. If you're in an industrial

47:59 , you're growing lots of cells for it's to make a lot of,

48:04 lot of it is to for enzyme . There's enzymes, a lot of

48:09 commercial products, laundry detergents and And these bacteria, it's beautiful to

48:15 grow to make these proteins and harvest . So enzymes, enzymes in

48:21 big business and using bacteria to produce them at large scales is what they

48:29 any questions about. Okay, so you are in biotech major, you

48:35 would have your hands somewhere in this because they're also I'm not going to

48:40 about it now. But aside from vessel then you can go elsewhere with

48:46 material with what we call uh you what that's called biotech majors, blank

48:55 , downstream processing and that involves a of other stuff, engineering stuff and

49:02 . Where you may be taking themselves have a process to break open the

49:06 , get the protein or whatever it . So that's that's the whole thing

49:10 itself. Okay. Um Alright, let's talk a little bit about dynamics

49:16 growth. So um obviously having just at the growth curve, we can

49:23 that. So numbers can increase Rather . Right? 1, 2 million

49:30 generations. You collect and do that 12, 10 to 12 hours or

49:35 . Okay. Where it takes humans do about 400 years to do

49:39 Right? 20 generations. So um when you're talking about um increases of

49:46 numbers like this. Okay. Just a ph scale hydrogen ion concentrations from

49:52 to large numbers. You use log base 10 to kind of compress that

49:56 . Make it more manageable when you're to quantitative these things. So another

50:02 generation time is what we use to to growth of a bacteria. You

50:09 any any life really. Um And you can look at the different ways

50:15 time. Basic way is what you here right time for oneself dividing the

50:20 is obviously a generation. Um But the more practical value is what we

50:26 doubling, doubling time. Okay. you you and you can measure that

50:31 any point in the growth curve. go, here's my cell numbers at

50:35 time. How long did it take double? Okay. And that too

50:39 generation time. Okay. And so calculations will look at and so you

50:46 before I mentioned that. So you here the equation basic equation right.

50:53 if you know the number of generations have passed the end number here.

50:59 . And you know how many cells started out with? Right? You

51:02 out with one cell. Okay. zero. Um and generation number generations

51:10 let's say five. Right? Use value. So one cell times to

51:16 fifth. Well that's 32 cells. ? Um but we often don't want

51:22 put in. It gets more complicated that. So we we we take

51:27 equation really? And we rearrange it that we can solve for n

51:33 So we can go and we set up to be in equals.

51:37 So we can easily calculate the number generations uh in a culture, you

51:43 , beginning an end, right? then we can figure out generation time

51:48 we can do some more of these of growth problems. So the next

51:55 here and you don't need to know it's derived and all of a sudden

51:59 that complicated. Right? So we're to take this equation here right?

52:05 just rearrange it so we can solve the number of generations, right?

52:12 we're gonna use log base 10. . And so we're just gonna multiply

52:18 By blocking the base 10. So gonna take this equation alright and we'll

52:22 it through stepwise like this right? long as the base 10 all the

52:26 through um log to the base 10 and T. Equals log base 10

52:32 zero times log base 10 2 to right. So to the answers up

52:38 power of two that grows right 1 2 to 4 to 8. So

52:43 . So um so then that log the base to to the ends because

52:48 the way law work. You can it like that. Okay Or in

52:54 that equals in times long to the 10 of two. Right? Then

52:58 value Actually equals .301. Alright so we're just going to um rearrange to

53:08 it equal to the small and and number of generations. So we just

53:12 this right? Basically we divide through .301 right? And then this expression

53:20 is the same as running it like . Okay. Again it's just that's

53:24 logs work. Okay log base 10 so long. Today is 10 times

53:30 minus log base 10 zero is the as log to this tinge of

53:36 T. Over N. Zero. so that's our equation that we use

53:40 do our uh problems. Okay so time we can as long as we

53:46 N. T. N N zero can figure out the number of generations

53:49 do things with that number. And so one of the things of

53:53 that's important is the time element. . So what's called a growth rate

53:59 ? So bacterial you know every species a growth rate constant. Okay um

54:07 will be an optimal right on the conditions there will be a growth rate

54:11 . That's but that's the max value that species under optimal conditions. Of

54:16 that can change depending on how you the cells. Okay. Um but

54:22 it is is just taking this Same equation here. I'm just putting

54:26 time element in and you can see right there. T for time.

54:31 . And so Kay, what they the growth rate constant N over

54:37 Um is the inverse of that. , so remember and there's generations

54:47 And tea time that the inverse is time. Okay. Generation time is

54:54 time per generation. Right? Typically minutes because bacteria grows so fast Could

55:00 estimate minutes? 50 hours as Okay and so uh so that equation

55:09 can look at a couple of different of problems. Okay. On blackboard

55:15 um and where the electron notes are as a document called bacterial growth

55:20 It has one that has the ones you're about to see in addition to

55:25 couple more um the back of the or the bottom of the sheet has

55:30 the answers all worked out. So just take a look at

55:34 There'll be a there'll be a question the blackboard quiz. That's a problem

55:38 this as well. Okay again I think it's anything that complicated. But

55:42 let's go through a couple of Okay so start here give it your

55:47 shot. Okay if you're not sure it, that's okay because we're going

55:51 work and work through it. And that is not a real bacterial

56:02 by the way. So we've got bacterium that has a generation time of

56:11 minutes. Mhm. Starting with five in log phase. How many minutes

56:18 it take to produce? 10,000 Okay. So 5-10,000. So those

56:23 two year values right here in zero . T. Okay. You know

56:28 generation time. Right? That's um per generation. Yeah. And these

56:44 will be part of the part of question as well on the test.

56:48 you don't have to memorize the equations . But I wouldn't know what you

56:52 what obviously what the variables meaning the but you'll have the equation there.

57:41 . Mhm. So the to me key key but the way I do

57:51 is always set up set the thing first and you know have my variables

57:55 what their equal to and then kind logically think through it again if you're

58:01 100% sure that's okay. Just for sake just punch something in.

58:09 Yes. Okay two more seconds. here we go. Two 210.

58:26 so uh the majority says be alright see. Alright so let's go through

58:34 here. So um that's our Alright so we're going from 5 to

58:40 cells so we want to figure out many generations is it to get from

58:45 to there? Okay. Our generation Is this minister generation. So we

58:53 given that 40 minutes right? And so if we calculate the number of

59:00 end which we can figure out by equation. Uh Going from 5 to

59:06 cells. Use the value multiply by time to yield minutes. So these

59:12 of course cancel out. Right? we just need to figure out

59:16 Okay and so uh n. 0 10,000. Do I have my

59:23 ? So long as the base 10 that? Is that Divided by

59:28 So it's about 11 generations. Okay so it didn't just multiply that by

59:33 generation time gives you 440 minutes which think is about seven hours. What

59:44 choices were? Take a little over hours. So D Okay.

59:53 Yeah Um 60 times seven is 420 . Yeah it's over seven hours.

60:01 just look like logically makes sense. well um let's look at another

60:11 Okay so calculate the generation time. this time we're going to catholic generation

60:17 sort of being given to you. if the 900 bacterial cells growing 15

60:23 produced and then lower three million Okay so there's your equations so you

60:30 your N. T. And Zero here. Alright you have a

60:33 frame so just a matter of plugging the into this over here.

60:49 Do that again. Okay and so do these kinds of calculations. You

60:57 maybe grow if you're interested in the of maybe disinfectant or antiseptic or

61:04 Maybe you you do growth curve with it and then without it and then

61:10 the numbers you know to get the . Let's see if there really is

61:12 difference or not one example but certainly you just use these values these values

61:18 lots of things. Right so figure a generation time on when medium.

61:24 would it be in a different medium so forth. So any reasons to

61:28 this timer on. Yeah. Okay I'm gonna go ahead and um

62:34 time I'm gonna let it go Okay here we go. Three Okay

62:54 B that is that is correct So let's just go through not that

63:00 surprised. Um let's look at the . Okay, so 15 hours,

63:08 minutes. There's 900 minutes. Then go to uh are NT and zero

63:15 then solved in and then plug that To get our generation time administer divided

63:24 11, almost 12 generations 76 Okay. So okay. These are

63:30 kind of things. You'll see a , look at the problems on blackboard

63:35 . Certainly let me know. Um but every everybody okay with

63:41 Okay. Alright. So let's switch then at least talk about. So

63:48 look at this question real quick. so the next couple of phenomenon we're

63:53 about um are as many processes as see going through the semester that are

64:05 or driven by the presence or absence nutrients. Okay. That lack of

64:13 a bunch of literacy can cause different to happen in certain bacterial types.

64:19 these next two phenomenon we're talking about certainly fit that category. Okay.

64:25 so this is so for this this is a nutrient driven phenomenon initiated

64:33 a black or depletion of nutrients. . Mhm mm hmm. Yeah.

65:12 . Okay. Alright. Here we to one. Mm hmm. All

65:19 . So it was it is in sport formation. Okay, so bio

65:27 information is actually the opposite of that opposite. Right? It's all about

65:33 abundant nutrients. It wouldn't be lag because lag phase. There are plenty

65:40 . The soldiers haven't gotten activated yet begin using them. Ok. And

65:44 log phase is all about lots of and lots of growth. Okay,

65:49 um let's look at biofilms examples. , we'll do that question next

65:58 So biofilms uh have um their role important biofilms. Right. And become

66:07 become a problem um In the last , 20 years. These are staff

66:16 example is on your skin or mucous anyway and they can be responsible for

66:21 called hospital acquired infections. So you to the hospital for let's say you

66:28 a get your knee scoped or some procedure or whatever. But while there

66:33 get a bacterial infection of some Okay. And very common that they're

66:38 very common. But certainly you these accounts for maybe 10% of these

66:43 acquired infections is a medical device, Catheter, a hip replacement, knee

66:50 , heart valves replacement um that these handled properly. Maybe they the the

67:00 with healthcare workers hands or something like . You don't handle it properly.

67:04 breathing tube. Another example these will surfaces. So biofilms are all about

67:08 surface and uh material that formed their specific types that do this and

67:17 have specific features that enable them to this. Okay. But it's all

67:21 a surface growth on the surface, growth on the surface. And so

67:27 use of a catheter when it comes of the packaging and you put it

67:30 a person that can lead to um for information. And the thing about

67:35 biofilm is they're not easy to get of. Okay, because I'm making

67:39 very big large, massive um you have uh types that are on the

67:45 of the biofilm, does it on interior of the biofilm that are harder

67:49 get at. So antibiotic therapy You take not just a week or 10

67:54 , but months to get over these of infections or if the infection can

68:01 , can spread from just where it's there and go elsewhere in the

68:05 which of course is a dangerous So uh so these can be quite

68:11 . Okay, uh so again about surface. So pipes, sewer pipes

68:16 other types of pipes or a surface you often see biofilms form.

68:21 Um the other thing is whether it's caterpillar or something similar to that or

68:28 pipe like that, there's typically a of nutrients, right? A pipe

68:32 typically has fluid going through it, will be have organic material and typically

68:37 that can sustain the cells in a catheter. Of course the body fluids

68:44 have nutrients in them that can sustain organism. So it requires that you

68:49 it requires because it represents massive growth so obviously if they have supply of

68:55 that are feeding this thing and enabling to develop. Okay, so here

69:01 to show up very well but starting and going clockwise that if you can

69:08 it kind of a dot in the , there is what we call a

69:11 colony. So it's it's not visible the naked eye. But it will

69:17 by phone will start that way with few cells but then begin to collect

69:22 then proceed to grow and you know two dimensions initially on the surface.

69:27 then here you see it's going in dimensions and coming up off of the

69:32 like like a mountain almost okay what call um bio stone towers. And

69:38 that represents obviously a lot of cell and like I said before when it

69:44 to be this massive, you'll have on the periphery out here and sells

69:48 the interior and there can be two types of environments there. If we're

69:53 about a medically important type. Well you may have types on the outside

69:58 are susceptible to antibiotics or maybe those the inside aren't because they can't penetrate

70:03 they may become resistant types of So it's a uh so the the

70:10 are buying from our surface because we're the surface lots of growth supply of

70:17 so it's not nutrient deprivation but nutrient that can sustain this thing. And

70:23 here are a few more pictures um the process of that information you might

70:31 well it was just a random association cells that come together and they just

70:37 growing And it's far from that. a jean included process occurs in

70:43 Uh And a species specific, not bacterium can do this many can,

70:48 it's a it's a species specific And so yes, because of the

70:54 can get massive other things can of come into contact with it in the

70:58 . And you know, you might they're part of it but there was

71:01 because they get stuck to it Have you? Okay. But the

71:05 of initiation, attachment, maturation maintenance dissolution. Okay, So there can

71:12 typically are two types of cells the ones that are motile. We

71:17 plant tonic cells. There are Those that are lose their flagellum once

71:22 on the surface and become what they stickers stick to the surface.

71:26 So because of that phenomenal surface being , february pili are important features.

71:34 biofilm formers have those structures. And so um the the I'm just

71:42 to show you the picture here. the process is all orchestrated by the

71:47 chemicals, chemical signals. Right? so the cells the species is not

71:54 to form a biofilm on the surface there is not kind of nutrients because

72:00 going to eventually form billions and billions cells and you have to have a

72:04 supply of nutrients to sustain that. the initial process is going to be

72:09 to kind of figure out, is this a surface environment that is

72:14 to even forming a biofilm? And this quorum sensing thing phenomenon is all

72:22 cell density. Right? So if is, so you have these swimmer

72:27 , right? Plank tonic cells that out a favorable surface. So if

72:34 land on a surface um if more more cells are get going there,

72:43 it may be a fair favorable Okay, Because they're able to there's

72:48 they're they're able to divide and And so if that's the case then

72:53 reach a threshold level, Right? they're producing these chemicals today signals.

72:59 if there's enough cells there, presumably are in themselves because it's a favorable

73:05 . Right? And so if that's case, they will reach the threshold

73:09 of those chemical signals and then that induce than the initiate the biofilm

73:16 Okay, If you don't reach a level, then presumably it's not a

73:22 environment, then they won't go through process. Okay. And then these

73:27 cells will go elsewhere. Okay, because it's a commitment one way or

73:31 other either form it or not. ? Because if you're going to commit

73:34 it, then that means you're going be investing a lot of, you

73:38 , gene expression and making things you to make a biofilm. Okay.

73:43 it's not a trivial thing. And one of the first things is this

73:47 policy Sacco information that's kind of the holding everything together. Okay, so

73:52 the sugar carbohydrate rich. You can have proteins in there as well.

73:57 . But it's it's a gene encoded material. Okay. That's only induced

74:04 those chemical signals get the threshold that sensing phenomenal color. Okay, there

74:10 be motility here. So they lose flagellum okay, when they're on the

74:15 and now live from forming. But so that's where the came to me

74:20 attachment. And so the pill I can have that twitching motility. So

74:26 may have that twitching motility on the through those Pilates. Uh then you

74:32 you have what are called micro They're kind of growing on the surface

74:36 then that two dimensional growth becomes three . And so the biofilm towers,

74:43 call it right now you're talking about significant number of cells growing. Multiplying

74:51 within this matrix of this extra polish . So that's again that's kind of

74:55 glue holding everything together because you can so dense the cells on the interior

75:02 can be nutrient deprived. So you to see holds form. Okay,

75:07 that material can flow through. And feed everybody out. Okay so

75:13 have to what you don't see here what you have to kind of imagine

75:16 envision is through through out here, nutrients. Okay, so whether you're

75:24 a whether it's in a pipe or , right? You're having a flow

75:29 organic material that's what's sustaining me. Okay, so they're stuck to the

75:34 with this material flowing through nutrients. . And of course if that uh

75:42 if the nutrient flow reduces or goes then you know you have to sustain

75:47 and then dissolution occurs. These revert to plant tonic forms to swim and

75:55 another favorable area. Okay? But biofilms can sustain themselves quite a period

76:01 time if there is a so going um so obviously so the main things

76:09 are surface features like are important for um and quorum sensing. Okay,

76:18 we'll review a little bit of this next time and then in those floors

76:22 the Chapter five next week. See you on

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