| 00:00 | Yeah. Hey folks, welcome. of course schedule we were at, |
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| 00:24 | we're down there of course with the at the first time ever, I'm |
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| 00:29 | schedule, It only took me 16 to be able to do that. |
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| 00:36 | So uh next uh next lecture, not long um ketchup day will um |
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| 00:51 | , like they have some stuff that spill over into that day. Um |
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| 00:58 | uh you know, I don't anticipate they being on the length, but |
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| 01:04 | know, may have questions and So uh but any of those ketchup |
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| 01:09 | , you've seen the schedule, that's of just to give me some cushion |
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| 01:13 | I go over uh time on some the topics beforehand so that I can |
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| 01:17 | of use that day to catch up . So um uh then we'll start |
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| 01:24 | next unit uh to on that So a week from this thursday and |
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| 01:33 | see that, you know, material up um likely later this week. |
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| 01:41 | Okay, so the schedule is obviously um blackboard quiz. So blackboard quiz |
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| 01:53 | week, as you can see it's a little bit different only and |
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| 01:59 | con uh in the in terms of length, so it's a little more |
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| 02:04 | . So eun quizzes cover Uh the week's materials, so like this one |
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| 02:11 | cover basically chapters 1412, Alright, kind of meant to be like a |
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| 02:19 | I guess somewhat of that unit. there will be questions covering, you |
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| 02:26 | , not every single topic, but know, some of the big topics |
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| 02:29 | covered in three just four chapters. it'll be something like 25 something |
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| 02:40 | Uh 45 minutes or seven length. um a little bit longer. A |
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| 02:46 | bit more comprehensive than your typical weekly . Okay um uh Smart work. |
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| 02:52 | smart work. There wasn't nothing was last week but this week there will |
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| 02:56 | assignments uh 34 do on sunday then couple of days later chapter five. |
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| 03:04 | just kind of the give you some before the quick exam if you have |
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| 03:10 | questions about the homework stuff. So be aware of that. And obviously |
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| 03:16 | a reminder on thursday about this stuff again next week and so on. |
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| 03:23 | um All right. No they were up chapter four uh before we go |
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| 03:32 | kind of administrative questions? My Okay. Alright so let's go to |
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| 03:40 | is really review again the bacterial growth . Okay so we use the term |
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| 03:47 | culture. Okay so uh in the that would be um like an early |
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| 03:57 | right? It could be any any . But typically and uh labs around |
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| 04:02 | is gonna be maybe 100 mils of . Right? So you you prepare |
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| 04:07 | growth medium uh liquid from a liquid medium here liquid growth medium in a |
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| 04:13 | vessel, autoclave it and then you oops inoculated. Okay. Inoculated |
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| 04:23 | What you're gonna do is the only you're doing after that. Once you |
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| 04:28 | inoculated. Inoculated means the seed if will to provide some cells that are |
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| 04:33 | be like they're gonna grow. And the so following inoculation, all |
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| 04:41 | doing after that is taking samples at time intervals in measure and growth and |
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| 04:47 | can measure growth in different ways. most common way is using a spectrum |
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| 04:51 | tom occur which I'm sure you've always multiple labs uh measuring turbidity, |
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| 04:59 | So the culture of growth which it like clear as water but then it |
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| 05:04 | very cloudy and as it grows more more cells appearing and it was just |
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| 05:09 | turbidity turbidity color. Okay. And can and that will absorb light so |
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| 05:16 | see an increase in absorb ints over as growth increases. Okay uh ultimately |
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| 05:23 | should do are are in addition to can take samples and dilute them |
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| 05:29 | It's actually what we call viable cell . Okay measurement has uh called |
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| 05:35 | F. U. S. Colony units per mil. Okay. Um |
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| 05:41 | way uh the absorbent measurements um of increase over time as the as the |
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| 05:48 | grows, the bible count gives you of a quantitative value of actual cell |
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| 05:54 | but that will D. O. . Is the is the parameter for |
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| 05:59 | optical so you can equate O. . To a actual cell number if |
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| 06:06 | wanted to. Okay. Um Now of how you measure you're gonna see |
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| 06:12 | curve like this. Okay, now it will, parts of the curveball |
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| 06:17 | so you have relied in the beginning phase. Okay, and so that |
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| 06:25 | uh today knocking them itself, What's the source you're using to inoculate |
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| 06:30 | medium can be uh obviously the solar , right, Come from a |
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| 06:35 | Okay. It will come from another culture and you're just uh you're not |
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| 06:40 | a sample that into batch growth Okay. And so the lag phase |
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| 06:48 | is, so imagine those cells in Okay, how long have they been |
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| 06:54 | in there. What kind of medium they growing in that or not? |
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| 06:59 | . Um at what point did you cells out and then put them into |
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| 07:03 | next batch? Okay, so Right, so all these things factor |
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| 07:08 | the length of this lag things, . For the souls coming in relatively |
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| 07:13 | , is it old culture and not a lot of viable cells, Is |
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| 07:17 | , is the medium were growing in curriculum very different from what they're going |
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| 07:21 | ? Okay, what is it? identical. Right. Um what's the |
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| 07:27 | , what was there? A sample of binoculars? Was it one million |
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| 07:31 | $10? Okay, so these are factor into the length of this period |
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| 07:39 | . Okay. And so the, know, what's the micro environment of |
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| 07:44 | ? You know, it's an item been growing for 24 hours, maybe |
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| 07:47 | like a ph changes that occurred in now it's going into fresh medium |
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| 07:52 | you know, a more optimal ph it's gotta be just right, so |
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| 07:57 | uh acclimates the surroundings, that's what that involves, right? So I'm |
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| 08:02 | use the surroundings right now, I'm fresh medium in the ph is, |
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| 08:07 | know, more more suitable, I to kind of get ready to |
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| 08:10 | Gene expression may be going on Okay. Turning genes on turning genes |
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| 08:17 | , certainly if it's two different media , it may have to um turn |
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| 08:22 | certain pathways, turn off certain pathways all takes time. Right? So |
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| 08:27 | that's all represented in that lag so that faces flat because that's what |
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| 08:31 | doing, is it's getting ready to , okay, not growing yet. |
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| 08:36 | so once um all that is set then, boom, it goes and |
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| 08:43 | grows like crazy of course, or under optimal conditions. Alright, this |
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| 08:48 | exponential corrupt law of faith, so and then lost. Okay, and |
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| 08:56 | of course that's going to be the growing a portion of the batch |
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| 09:03 | And so we sometimes even um differentiate up log phase into talking about mid |
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| 09:13 | . Okay, And late log. . And so in mid log is |
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| 09:21 | kind of in this range here. , of course, it's going to |
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| 09:26 | most the most active cells. lots of cells dividing rapidly. Um |
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| 09:35 | in, this is often broken down log, it is often times when |
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| 09:40 | might um take if you're if you're in some kind of activity that they're |
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| 09:46 | right and enzymatic activity you might get log somewhere where you want to take |
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| 09:51 | samples to see what's the what's really maximal activity here and it's gonna generally |
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| 09:57 | in that mid log phase because the are very healthy and growing and most |
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| 10:02 | . Um And so their size changes occur and the whole uh in the |
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| 10:09 | stages of growth of the back So um initially in this blog log |
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| 10:18 | growth lots of cells dividing. So get slightly bigger and then then |
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| 10:25 | And so so tend to be a bit bigger on average in log things |
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| 10:31 | that changes the stationary phase. They actually get a little smaller than the |
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| 10:36 | phase. Okay so um the so a late log. Okay so late |
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| 10:44 | now is it becomes almost a tipping where it is running out of |
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| 10:50 | Okay the growth rate begins to slow in late log um you know nutrients |
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| 10:58 | to sustain that level of cell density that point. So then We get |
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| 11:04 | go into um stationary phase. So basically death rate is equal to |
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| 11:09 | three flat flat and this is where becomes a thing of the survival mode |
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| 11:20 | in. Okay and so what's the gonna do in the state. Um |
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| 11:28 | if it becomes a little bit smaller size, that's less cytoplasmic material to |
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| 11:34 | up with it. In fact, a way to conserve energy. Um |
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| 11:41 | responses may occur, proteins may become . And so we don't want to |
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| 11:48 | of just slow down growth. you know, express so many |
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| 11:52 | Kind of slow down proteins. It's remember that takes energy. Okay. |
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| 11:56 | it really becomes a a survival mode the idea of being okay, I'm |
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| 12:02 | gonna kind of hang on as best can until uh you know, maybe |
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| 12:07 | internet will come clomping down and I'll growing again. Okay. But in |
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| 12:12 | growth, right? Aside from taking samples to measure growth, you're |
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| 12:17 | doing anything. You're not adding anything doing anything like that. You're just |
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| 12:22 | Before four phases of growth. So, um and so there's a |
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| 12:28 | of someone of the same stuff I really touch on his. Why why |
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| 12:33 | you didn't care about gross? Because you know, if you're in |
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| 12:40 | mm hmm. Really in lots of fields, you use gross cells for |
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| 12:45 | purposes. Um biology grow cells to proteins to to isolate D. |
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| 12:52 | A. For various purposes. Um um in order to get those |
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| 12:58 | you need quantities of that stuff. . And so you can grow cells |
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| 13:03 | liquid culture to get a lot of that then get a lot of your |
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| 13:07 | whatever it may be of interest. . And so um and uh and |
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| 13:14 | maybe take samples and maybe making an activity in the in those cells. |
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| 13:19 | you want to take samples at different . You want to know what's the |
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| 13:22 | time to do that one of the active. So they grow curve is |
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| 13:29 | is the first basic thing you do then analyze along the different time points |
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| 13:35 | it's doing Okay. And so maybe on different media and tests at work |
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| 13:40 | point, it gets to here and . So these are all things you |
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| 13:44 | . Okay. Maybe figuring out what it likes best. These are all |
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| 13:49 | you do usually this kind of Okay. Um and that can |
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| 13:57 | you know, if you're in an lab you're basically working with. It |
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| 14:02 | a material, right? You go an industrial lab where you're growing those |
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| 14:08 | for commercial purposes. Um a spoonful stuff doesn't work. You need buckets |
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| 14:16 | , hundreds of thousands of gallons. , so you grow, grow these |
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| 14:20 | , you're doing the stages, you out small scale, maybe go from |
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| 14:26 | leaders to 10,000 m, 200,000, do it in a chain like that |
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| 14:33 | ? To get uh lots of right? So for commercial purposes, |
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| 14:38 | what you do. If you biotech in the earth that's what you'll be |
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| 14:45 | growing up to those kind of Okay, so but it all starts |
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| 14:49 | the system at the small scale and scaling up. Okay um rambled too |
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| 14:55 | on that subject. So stationary So according to something you're talking about |
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| 15:03 | so explore relationships. We're talking about second. That's certainly something that would |
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| 15:08 | in stationary phase. Okay. In sense that stress occurring, it was |
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| 15:16 | of forming endospore, it's very likely to begin doing this. Uh Certainly |
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| 15:23 | um That present forming indoor sports certainly in the stress conditions. Okay. |
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| 15:31 | Alright. The death phase so Important remember especially in Chapter five, Were |
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| 15:39 | mostly discussed. How do you how you kill bacteria? Okay and so |
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| 15:44 | really focused on track five on this of the curl. Okay how fast |
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| 15:53 | make it die? You know we we add chemicals here okay or whatever |
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| 15:59 | treatment is, how fast will it ? And so that phase much like |
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| 16:05 | phase. That phase is also log . Okay it can decrease exponentially just |
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| 16:11 | log phase can increase actually. Okay that's really what one tries to do |
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| 16:16 | different types of disinfectant treatments and what you is too get you know very |
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| 16:23 | water ready. Okay so point being that death? And so certainly there |
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| 16:31 | there are still some nutrients available to phase. Just very limiting and becoming |
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| 16:36 | less so as we go on. my death phase beginning you've run out |
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| 16:44 | there are no nutrients. Okay And quickly Death occurs exponentially. Okay. |
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| 16:52 | and by that time you know in growth where you haven't really done anything |
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| 16:57 | than just taking samples um You know ph changes maybe have become very acidic |
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| 17:02 | basic depending on the media. By point waste materials are accumulating so not |
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| 17:09 | good situation. Okay. Um now one phenomenon dormancy persistence. This is |
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| 17:17 | we've seen in uh certain types that generate a resistance to antibiotics. Okay |
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| 17:27 | cells may um in different responses some types will um not responding by kind |
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| 17:35 | slowing their growth down. Okay, . The growth. Okay but remain |
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| 17:41 | . Okay. So you cannot be but still be alive. All |
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| 17:45 | And they do so by maintaining a potential. No we haven't talked about |
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| 17:52 | . We'll talk about that unit But the proton pump that talks about |
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| 17:57 | , that creates a difference in the potential. Okay. That is a |
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| 18:01 | to provide energy. Okay, so you can kind of maintain that a |
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| 18:06 | bit of that membrane potential, absolute pump generates some https. It can |
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| 18:12 | so and by doing that to remain . Right, So it's not really |
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| 18:18 | demand for energy but nonetheless has to some activity. And that's enough to |
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| 18:23 | it. Right. And we've seen phenomenon and that can present some types |
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| 18:30 | bacteria can induce this state. And remember with antibiotics many of these um |
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| 18:38 | best on fast growing populations. Because that's when the targets for antibiotics |
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| 18:44 | most plentiful I think of cell wall . Right? One is cell wall |
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| 18:50 | is going to be occurring like like . Right? During log phase. |
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| 18:54 | ? So that's when an antibiotic that trigger things like um cell wall |
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| 19:00 | components of protein synthesis. Right? are the processes that can be very |
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| 19:05 | when soldiers growing. Um But if slows down then those antibiotics that target |
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| 19:12 | components don't aren't as effective. And so if a cell can he |
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| 19:21 | to antibiotics then say okay I'm gonna my growth and you won't be able |
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| 19:26 | work with me as well. So a way to kind of. And |
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| 19:29 | the antibiotic um you don't when you taking the exit your system. And |
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| 19:38 | then that's the opportunity for the per and then I'm gonna start growing |
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| 19:42 | Right? Cause disease. So they be a problem. Um Now that |
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| 19:51 | Sanoma is about. Okay. Um questions about that? Yeah, mm |
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| 20:00 | . Mhm. For some it's not for certain pathogens. We've seen that |
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| 20:07 | that they can induce this dormancy. hmm. Yeah. In stationary |
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| 20:20 | Yes. Because it's gonna be a situation and if it can perform in |
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| 20:24 | portals that would be the time to it. Okay before it gets the |
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| 20:28 | thing volunteer. Sure. There are types of antibiotics that can they can |
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| 20:39 | on it. Um It may be like a Yeah, There are two |
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| 20:46 | of antibiotics that work as as I the analogy of like sticking with strong |
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| 20:51 | commemorate stuff leaks out. So those of antibiotics don't dependent on growth. |
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| 20:57 | those are the electric types that you use those types. So um but |
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| 21:01 | may not it may be something you to figure out by trial and |
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| 21:05 | Initially that was any other. if you limited volume but still absolute |
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| 21:22 | of. Okay. Oh, so you have like a one male |
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| 21:30 | Um So okay, what's the It's able to get enough. |
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| 21:43 | you still you still get a You still would get broken. It's |
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| 21:48 | it would be a growth curve that represent a high selling but you will |
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| 21:54 | get a Yeah, but it would that's not something that's done. Very |
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| 22:06 | sampling life kind of stuff. But minimum life I've seen for like these |
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| 22:12 | of like in 50 mils, 50 100 in that range. Um |
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| 22:22 | Yeah. What is this I'm going get at the same time. I |
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| 22:34 | I don't think so. So I'm sorry. Ok, I |
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| 22:39 | Um The the thing with I don't . I don't think that is because |
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| 22:46 | antibiotics work well together this is what's a synergistic effect. Um Well, |
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| 22:52 | actually talk about that. Uh There a good example is one that's um |
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| 22:59 | gosh escapes me out. But yeah have they'll have a better effect together |
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| 23:06 | separate or by themselves but it's not dormancy but right if I forget mine |
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| 23:14 | we do talk about that and uh think I want to talk about it |
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| 23:17 | time. So do you mind about ? Yeah so neither of penicillin or |
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| 23:31 | because it's likely of those types that do that. Those who may maybe |
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| 23:38 | . Right? Because those rely on rapid growth rates typically they work |
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| 23:42 | Yeah. Okay so here's the Um So a bacterial inoculate has grown |
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| 23:52 | nutrient broth? Okay And Allah Quantas to a batch growth medium. So |
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| 23:58 | the M9, that's the sea. you see both formulations down there um |
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| 24:04 | of defined medium, minimal medium following of the batch medium. But what |
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| 24:12 | the growth pattern most closely resembled? so nutrient broth inoculate into M. |
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| 24:18 | . Okay so here's the choices. the other piece of information is when |
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| 24:25 | night is growing nutrient broth and transferred fresh nutrient broth it looks like |
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| 24:33 | Okay um So what would it look if neutral broth in Oculus going into |
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| 24:40 | . Nine? You look like B. Or seek. So when |
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| 24:49 | think about that you just mentioned um medium. So minimal medium is also |
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| 24:59 | sight of the fact that you, way I think of it is if |
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| 25:03 | give you a calculator and periodic chart can figure out all the old National |
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| 25:06 | Adams in there, right. It contain this complex meetings. Um But |
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| 25:13 | say about your willingness. Um It's have to synthesize all of all of |
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| 25:19 | constituents. Okay. To synthesize it's me go ask. It's vitamins. |
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| 25:29 | and that can um produce some different . Okay I want that one this |
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| 25:43 | . Okay. Timers on. Yeah. Okay. Alright. Here |
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| 26:19 | go. And it's 5-5 seconds. positive too. Alright hold on. |
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| 26:28 | we go. 654. Mhm. . Okay. All right. Um |
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| 26:39 | who answered C. Y. It's longer like why is that? |
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| 26:48 | Where did it come from? Like . Okay. Yeah that's correct. |
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| 26:58 | there's less. Um So what What the nutrient broth provide that? |
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| 27:04 | nine does not anybody. I mean provides C. H. O. |
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| 27:11 | . P. S. Is the in which they provide some of the |
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| 27:14 | . H. O. M. . S. Which is because for |
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| 27:19 | um They had, so any minimum . They just said the soldiers have |
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| 27:25 | make basically make all their stuff from , so to speak. Okay. |
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| 27:30 | not preformed material for them. There an accomplice in a complex nutrients like |
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| 27:38 | you'll have preformed vitamins. Preformed amino and the like and so what that |
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| 27:46 | is going from a what color rich rich broth or a complex medium. |
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| 27:55 | where you have all these preformed Okay um and now you're locked into |
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| 28:01 | middle media where you can hear it scratch that's going to put on the |
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| 28:06 | in terms of now you gotta not that turn on different pathways right to |
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| 28:12 | the size of the glass and so and so forth. That that's a |
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| 28:16 | more time to acclimate now that those . So life life stays is gonna |
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| 28:23 | . Okay Because that it sells an down in 10 9. I don't |
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| 28:27 | I'm not getting you know preformed amino . Preformed vitamin is gonna make it |
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| 28:31 | myself and that's that's time turn expressed make proteins blah blah blah. Okay |
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| 28:39 | that equates to a longer lag Okay no problem. Yes. Thank |
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| 28:47 | . But if you went the other around from a memorable and not getting |
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| 28:51 | media into Richmond and generally lacks, should decrease compared to if we're going |
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| 28:58 | a minimum immediate. Okay so it's about from the standpoint of the cell |
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| 29:05 | how much work do I gotta do get going? Bye. So um |
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| 29:15 | why because my boss set up my job. Think like that back |
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| 29:20 | Okay. Sounds weird but put yourself the bacteria shoes and in this |
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| 29:25 | Okay. How long is it gonna me to get going? Yeah so |
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| 29:31 | many questions. Yeah. Okay so well I'll answer that in the next |
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| 29:39 | . Okay. Um b equates to . Okay so let's say you have |
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| 29:49 | just talk about right, you're not anything to your medium other than taking |
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| 29:54 | eye clinic and taking samples to measure . That's it. Right now. |
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| 29:58 | could be more rigorous. You could more attentive to the to the flask |
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| 30:06 | you wanted to um If you want get more sells out of it you |
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| 30:13 | add stuff to it. It's gonna the thing you want to add. |
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| 30:18 | we had if we had this growing M9, what would you want to |
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| 30:22 | to get more sells out of it quick. Any idea If you were |
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| 30:32 | on M9 And you are growing in M9 medium then you plateau mode. |
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| 30:39 | now you want to get you I want more cells out of this |
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| 30:43 | . What would I add on that of ingredients to get that effect |
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| 30:50 | Right well, M nine has So just add more glucose. |
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| 30:54 | add uh two more grams of glucose cells will do that. So that's |
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| 31:00 | we call fed batch growth feed it it. So add more partner. |
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| 31:06 | um and you know in terms of are you gonna become limiting for? |
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| 31:15 | because you can do this multiple Keep feeding, feeding at sea it |
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| 31:20 | that keeps going up. Okay. limitations. Especially if you're just doing |
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| 31:25 | , shake flask. Okay, um you wanted ph issues that will stop |
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| 31:32 | cells from growing. Um but it lie to um to to in that |
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| 31:40 | to crank up, get more, add more carbon. And only do |
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| 31:44 | just have a sterile, You have sterile um 20% glucose solution, |
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| 31:51 | And just add, you know, few millimeters of that and it takes |
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| 31:56 | . Um That's what we call fred , right? Basically, feeding it |
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| 32:01 | feeding the match. Okay. if you want to be super |
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| 32:05 | you can actually account for ph changes the class. You could have a |
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| 32:10 | indicator that will turn color yellowish sugar to an acid register, gets too |
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| 32:16 | . And you can never anyone there , you know, acid or base |
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| 32:18 | neutralize it until the, until the color goes more to read or read |
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| 32:22 | yellow in that range of 6 to . And that was so you could |
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| 32:28 | done that and put directly, like this crap. Okay, And so |
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| 32:32 | looking at it right, you can all that stuff, right? And |
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| 32:38 | will work. You'll get a lot selling out of that shape class. |
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| 32:43 | uh again, it actually becomes limited one of the things that's why you |
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| 32:48 | buy reactor. So you see a Bayraktar controls everything controls the pe |
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| 32:54 | . You see these things over our pumps, okay, that are |
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| 33:00 | , it's all integrated. All automatically ? Let's say, Okay, add |
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| 33:05 | base when it gets the ph six that's when it gets to. |
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| 33:12 | just automatically. Um There's also another parameter. This control, they can |
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| 33:17 | feed automatically. Okay. And that's you do growth curves initially in the |
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| 33:22 | class to kind of say, so when this is when it runs |
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| 33:25 | of with this much carbon upfront, is when it kind of begins to |
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| 33:29 | it all up and flat. Now go, okay. At that time |
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| 33:33 | , I need to add more carbon you can program all this stuff into |
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| 33:36 | reactor. Alright, we'll actually get of cells. Okay. Now the |
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| 33:43 | is so back to the red So for this for anything food carbon |
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| 33:52 | , you know, every eight hours 10 hours or whatever? Why? |
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| 33:57 | do that trouble? Why don't you add it all up front? |
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| 34:01 | I'm gonna add it all in the . I don't have to come up |
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| 34:03 | get out in the middle of the and add stuff. Why why couldn't |
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| 34:07 | do that? Because you actually can't that? It won't work. |
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| 34:15 | No, it's more of a fake um Think more in terms of |
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| 34:22 | Mhm. Chemistry taking chemistry terms, ? But why why couldn't you add |
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| 34:32 | my why can't I put all my upfront. So that medium had two |
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| 34:36 | per year. Why can't I put g sugar, glucose right off the |
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| 34:40 | . It won't brother. Why is set that ph which one? What |
|
| 34:52 | you say? Yes, concentrations too to too much site right now. |
|
| 34:59 | mostly won't won't go 20% glucose. put that in right at the |
|
| 35:05 | Um Yeah it's gonna be an issue to high solute concentrations as most of |
|
| 35:10 | issues. Right? So they actually grow. Okay that's what I can |
|
| 35:14 | it piecemeal. Okay. Um if bacteria growing in liquid culture is adequately |
|
| 35:22 | throughout. Okay. So you add carbon when you when you when you |
|
| 35:27 | what can limit growth fairly quickly. you control this physical parameters. There's |
|
| 35:33 | keyword sense. That's the clue. auction become limited very quickly. So |
|
| 35:43 | a shake flasks. How could you can actually kind of stave off that |
|
| 35:54 | of oxygen for a bit. And would you do that? She had |
|
| 36:01 | or flask? Yes, that's Okay. What if uh So in |
|
| 36:12 | flask didn't become limited for monster pretty . But what can you do to |
|
| 36:19 | that to kind of increase until you limited for that? What could you |
|
| 36:27 | ? What not? He said I like that. No that's one |
|
| 36:36 | But these are way is putting the . You always broke when we were |
|
| 36:42 | bugs uh bugs bacteria in shake flasks always have in the shaker. You |
|
| 36:48 | crank up the RPMs and that will more, that's how you get air |
|
| 36:52 | there and creates turbulence. Okay. We have pictures. Yeah. So |
|
| 36:58 | you look at the shake flask what's that? You see that you |
|
| 37:03 | you're used to the typical urban Meyer you see in chemistry lab which has |
|
| 37:07 | flat bottom, right? You see this has these affordable, affordable ridges |
|
| 37:14 | . That's what we call baffles, shaped flags. Okay, so when |
|
| 37:20 | is shaking those battles creates even more . Okay, so you can actually |
|
| 37:26 | shake flats like that with the shake with a flat bottom at the same |
|
| 37:31 | and you'll get more growth in the flat because you create more turbulence. |
|
| 37:35 | mixing of air in there. Of it is. So you're growing aerobic |
|
| 37:41 | that they're gonna like that. Um Yeah the the the other thing |
|
| 37:47 | with bioreactor is I mean I'm giving more information I need to know but |
|
| 37:52 | can learn this stuff in the Okay? So for those of you |
|
| 37:55 | go on to do this kind of um you'll know so with bioreactors again |
|
| 38:02 | controlling everything. Okay. And you , auction limitation is one of the |
|
| 38:07 | things. And so you see that impeller is the name for this uh |
|
| 38:15 | all the way through. And you these blades on it, right? |
|
| 38:18 | this is going to be rapidly And you see that there is an |
|
| 38:23 | probe. Okay, That will measure level of dissolved oxygen in your |
|
| 38:27 | Right? And so that's it. it can be controlled so that can |
|
| 38:32 | controlled to increase the speed of the to get more music. Right? |
|
| 38:38 | also have what's called a sparkler down and it's however, is forced into |
|
| 38:45 | system. Um And as part of tiny little holes in it. |
|
| 38:50 | So, it creates small bubbles. bubbles dissolved better than big bubbles. |
|
| 38:57 | right, create small air bubbles. the option can mix more quickly into |
|
| 39:03 | liquid rather than for big bubbles. , So, uh that But this |
|
| 39:09 | can can you see here is um , to ask base control nutrient |
|
| 39:21 | Um Anti forms Another thing. Uh need lots of the aeration and some |
|
| 39:27 | the end product they got lots of well. Um The other thing here |
|
| 39:35 | this right here cooling jacket. Um If you didn't have that, |
|
| 39:42 | also would bring your capacity here I guess re controlling everything, |
|
| 39:50 | This is gonna be super happy. ? At ph control? They've got |
|
| 39:56 | control. They get they get more they need it and they're gonna be |
|
| 40:00 | so happy. Right? And you that happens in the levels of soldier |
|
| 40:05 | . Okay. But you didn't have from the water um jacket around |
|
| 40:11 | right? Thermodynamics, Right? Those are growing so fast and so much |
|
| 40:17 | incredible. Okay? And so that need that cold water jacket to keep |
|
| 40:22 | , keep them happy there because the of heat they're producing his roof and |
|
| 40:29 | so you have to have that water to keep continually cooling it down, |
|
| 40:33 | that back. So uh but yeah you get uh you know and this |
|
| 40:40 | how these things operate from one liter up to 100,000 m size. |
|
| 40:47 | Of course bigger scale and the limiting really on the size of how bigger |
|
| 40:53 | it can be. Um Is really the motor. Okay. And the |
|
| 41:00 | to drive the same. Okay. turned out to be the thing that |
|
| 41:06 | you in terms of economics and it boils down to money. Alright. |
|
| 41:10 | gotta you can't you gotta make sure high chromium isn't costing more than what |
|
| 41:14 | is to actually run the unit. uh I can attest to that have |
|
| 41:18 | laid out five times in the industry the company so not because of |
|
| 41:23 | I don't think. But um anyway any questions? Okay. Yeah. |
|
| 41:40 | . Okay. So what what stage you know what the bacteria in and |
|
| 41:43 | to add stuff? Great question. you're always gonna monitor, you always |
|
| 41:47 | take samples even if you have something a bazillion gallons, you're still gonna |
|
| 41:52 | samples but that's because you will want know that um the you know when |
|
| 41:59 | do this for a long time, can kind of see you can look |
|
| 42:03 | you can see the parameters of the or the ph is really kicking in |
|
| 42:09 | my my acid basically is really kicking now when my my D. |
|
| 42:13 | Is causing the thing that really go can give you a clue that yeah |
|
| 42:16 | guys are doing pretty well but you take samples and put them in perspective |
|
| 42:22 | and you get a growth curve for . Yeah all the time. So |
|
| 42:26 | curve isn't just for for this Okay. It's for every scale that |
|
| 42:32 | were doing to monitor what's going on . Yeah Russian is it difficult? |
|
| 42:44 | can be it um That's why you're careful. That's why you know the |
|
| 42:49 | you're allowed and accepted technique is really . And uh obviously you sterilize everything |
|
| 42:55 | everything that goes into the tank will you know sterile equipment. So it's |
|
| 43:00 | not impossible. It's not an impossible you just have to be careful |
|
| 43:04 | And so it's uh you know I didn't work but from we called fermenters |
|
| 43:12 | it's part of the right term. I didn't want protects that big. |
|
| 43:15 | worked with maybe up to 1000 leaders I wasn't that common to have contamination |
|
| 43:23 | . So if you're careful about It's certainly absolutely doable. It could |
|
| 43:26 | more of a problem with tissue culture culture versus bacterial cells tissue coach tend |
|
| 43:35 | be more finicky more. They More rich types of medium? More |
|
| 43:39 | types of medium can invite more chances contamination because lots of stuff can grow |
|
| 43:43 | it versus the minimum media. So gonna be some more issues with tissue |
|
| 43:48 | than bacterial cultures. But again, is this is an all day all |
|
| 43:52 | time. So you just have to careful what are you doing in addition |
|
| 43:56 | that? Yeah, bottom. Um All right. So let's look |
|
| 44:03 | little bit about growth. And so terms of dynamics of growth more. |
|
| 44:08 | this kind of growth calculations. So we're well aware of having to |
|
| 44:14 | at the batch growth curve that is exponentially. And when they say is |
|
| 44:20 | . Really the terms of use See there's a lot of rhythmic |
|
| 44:23 | exponential growth, right? They they used to anonymously to indicate obviously really |
|
| 44:29 | growth. Okay. And so a way to to measure cell numbers. |
|
| 44:36 | the this equation here. Okay. what are we what are what are |
|
| 44:41 | starting with? Zero? What's our of cells that we start with? |
|
| 44:46 | where do we get to at some in the future? Okay. We |
|
| 44:50 | calculate that by basically multiplying what we with by this to the end and |
|
| 44:56 | the number of generations. Okay. that value? Okay. So um |
|
| 45:03 | you start out with one cell. . And In uh five generations. |
|
| 45:12 | to through the fifth times two to fifth we go five generations. What |
|
| 45:18 | we? How many cells do we ? Well, we have 32 |
|
| 45:22 | Right. So um now it's not so easy to calculate. And just |
|
| 45:29 | by itself, you know, as get more and more numbers um higher |
|
| 45:34 | . So we tend to use um can we convert that equation so we |
|
| 45:40 | solve for n different generations will show that in a second. So um |
|
| 45:45 | other thing is when you're talking about increases in numbers, like we're going |
|
| 45:50 | one in this example one to a . And for Ireland e coli can |
|
| 45:56 | like eight hours, six hours, hours under optimal conditions that um that |
|
| 46:04 | wide. So typically put it in log based tents to kind of compress |
|
| 46:08 | scale much like ph right, ph vary Hydrogen Ion concentration can vary from |
|
| 46:15 | range. So we put that also the log base 10. So when |
|
| 46:18 | have Values that can occur in a range rather quickly, we kind of |
|
| 46:24 | that using a lot of base Um So generation time. So that's |
|
| 46:32 | primary views all the time in growth growth. So here you see one |
|
| 46:37 | occurring generation time obviously is. How does it take to get there? |
|
| 46:42 | . There's different ways that that's you know, one basic way as |
|
| 46:45 | . The time it takes for one to make two. Right. Well |
|
| 46:48 | don't really measure it that way in real world. Typically measure what's the |
|
| 46:54 | for the population to double. So measure it at a certain point. |
|
| 47:00 | then when when the I wanted to for that population to double from that |
|
| 47:04 | . And that's also considered generation time well. Okay so um so doubling |
|
| 47:10 | , generation time. I mean the thing. Okay And so um the |
|
| 47:17 | of the generation time. So it you know it can you can have |
|
| 47:23 | generations time when you grow up on media. So you have the same |
|
| 47:27 | species growing on different media. It certainly have can certainly have different generation |
|
| 47:32 | . Especially if it's mineral media complex . That can certainly change it. |
|
| 47:37 | . Um So just to kind of mentioned um thinking that equation and then |
|
| 47:45 | that to basically solve for n to that an easy thing to do. |
|
| 47:50 | basically multiply through. And I put in there. You don't need to |
|
| 47:54 | how how the things derived. But it is it's not complicated at |
|
| 47:58 | . So what we're doing is basically through by log base 10. |
|
| 48:03 | And then um that's what we see . Okay just taking that equation and |
|
| 48:10 | through by log base 10. Okay we get to hear so log to |
|
| 48:16 | base 10 times to the end equals . Right? N times log base |
|
| 48:22 | . And to just how do you with logs? Okay and then this |
|
| 48:29 | equals that. Block to the base of two is .301. Right so |
|
| 48:34 | think that as that power of two right? That we see in this |
|
| 48:38 | growth. Um So then after this it's just solve for M. Alright |
|
| 48:45 | we divide through by .301 right? this can be expressed in that |
|
| 48:52 | So against how how logs work. ? So log base 10 of N |
|
| 48:58 | minus log base 10 of 10 It's the same as saying log base |
|
| 49:03 | of N. T. O grande . Check so so that equation Okay |
|
| 49:10 | what is what we use typically for solving problems? Okay and so we |
|
| 49:17 | express this as well. Time is of interest. Okay so generations. |
|
| 49:24 | gives us number generation. But what the time? Right. And so |
|
| 49:28 | we call the growth rate constant Is basically just that equation with time |
|
| 49:34 | now as part of the equation right . Okay so in over t. |
|
| 49:42 | and that equates to generations over time generation time is the inverse. Is |
|
| 49:49 | ? Okay? So g just time generations. Typically since bacteria grows so |
|
| 49:56 | it's typically in minutes it can be hour but it's very common to see |
|
| 50:02 | . See that term in minutes. and so we'll go through a couple |
|
| 50:08 | problems there's more practice problems with, them work out. I mean, |
|
| 50:13 | the exams, exams really full of things, It was like maybe two |
|
| 50:17 | on the exam. Um uh There also be one on the blackboard |
|
| 50:23 | a couple on the blackboard quiz. But I think if we go through |
|
| 50:27 | um with the examples that are on um uh it's all it's just a |
|
| 50:34 | of setting it up and what are solving for? So let's let's look |
|
| 50:38 | just a couple of problems here. . Uh and this is not a |
|
| 50:43 | bacterial species in case you're wondering. . Uh so this bacterium, houstonians |
|
| 50:51 | has a generation time of 40 Starting with five cells in log |
|
| 50:58 | How many minutes does it take to ? About 10,000 cells. Assume all |
|
| 51:03 | remain viable. Okay. Um So just think about it. So |
|
| 51:10 | these kinds of problems you will have you'll you'll you'll be allowed to have |
|
| 51:15 | calculator, don't care what kind in in casa. They'll be aware of |
|
| 51:21 | that you will be allowed to have calculator. Just don't bring a cellphone |
|
| 51:25 | kind of handheld capitalist one. And I said, there's only a couple |
|
| 51:30 | problems uh on the test that the will be there for. You don't |
|
| 51:36 | to memorize the equation. Thank Oh, sorry about that. |
|
| 51:52 | So if you're not, if you're sure about the answer here. It's |
|
| 51:56 | . Don't Well through it. I I want you to think, you |
|
| 52:01 | it worked out it's gonna be pretty . Mm hmm. Okay, get |
|
| 52:37 | timer going. Mhm, mm Uh Hold on. Yeah, it's |
|
| 52:47 | question now. No. Okay. any kind of calculators? Fun. |
|
| 52:58 | not a cellphone calculator. Okay. . Here we go. You're not |
|
| 53:10 | . Just answer anything. Okay, little bit around the map. Let's |
|
| 53:15 | what we come up with here. right, so the way I do |
|
| 53:18 | things is um Alright. So what trying to do is what R |
|
| 53:24 | Zero N. T. Values, ? So we're starting with five Going |
|
| 53:29 | 10,000. The question mark is how does it take to get there? |
|
| 53:34 | , Generation time is Minister generation and given that right? And so we |
|
| 53:42 | Catholic number of generations produced and going 5-10,000 cells and multiply this value by |
|
| 53:49 | time to yield minutes. Okay, we figure out the number of generations |
|
| 53:55 | we should be able to plug in figure out the answer. Okay, |
|
| 54:02 | there's our equation plug in N. . And N. Zero. |
|
| 54:06 | 10,000 cells to five and then 5000 five, sorry? Um and then |
|
| 54:13 | get that over 50.301 The 11 And so it should be 40 times |
|
| 54:22 | , minutes, which is a little seven hours. Yeah, I think |
|
| 54:28 | was it was D.A. over seven . Yeah. All right so again |
|
| 54:39 | kind of set up the yeah you the values you know plug them |
|
| 54:46 | Okay. Um let's look at another , calculate the generation time If 900 |
|
| 55:00 | cells growing 15 hours produced three million cells. Okay. So you know |
|
| 55:15 | . T. And N. You know the time element 15 |
|
| 55:23 | Yeah. So again it becomes down figuring out a number of generations and |
|
| 55:28 | it in. Oh I'm sorry. me open the pool again. Okay |
|
| 56:08 | hmm. Yeah. Okay timer's going hmm. Okay and the strategy, |
|
| 57:27 | not sure. Just punch something Okay. 210. Sure. Alright |
|
| 57:43 | again dot process. Okay. There's 15 hours, 19 minutes. So |
|
| 57:53 | . Zero N. T. We those plug him in. Sure. |
|
| 58:01 | that many generations. Okay so that generations to go from 900 to 3 |
|
| 58:05 | million Then uh this generation time Right? So our time is that |
|
| 58:13 | occurs over 900 minutes. Okay. is 76 b. Try um A |
|
| 58:25 | about this one of the previous So look ah I had those material |
|
| 58:31 | problems. It was like five. the first page. The answers are |
|
| 58:36 | the second page. So you can it yourself if you choose not to |
|
| 58:38 | the answers but your answers are all out just like this. So so |
|
| 58:46 | it's just, you know, always do this part here, even though |
|
| 58:49 | may seem very basic, you just but the parameters you're given, |
|
| 58:55 | plug them in where they fit and , you know, just work it |
|
| 59:02 | . It should be not too Okay, um you know, these |
|
| 59:09 | of things you do under one like maybe say you're looking at what's |
|
| 59:14 | effect of antiseptic disinfectant or something, you might go, okay, |
|
| 59:19 | I get it. You might see , you get quantitative differences. So |
|
| 59:24 | gives you just data to kind of what you may be seeing. So |
|
| 59:30 | there's different context to be used in data. Okay. Um Alright, |
|
| 59:37 | let's look at this question. so this is a nutrient driven phenomenon |
|
| 59:48 | by a lack or depletion of nutrients there's lots of stuff we'll learn this |
|
| 59:55 | where that produces the different phenomena lack of or abundance of sequence. |
|
| 60:07 | but lack of can cause this. a lack or depletion. So I |
|
| 60:46 | this could happen during stationary phase. certainly a depletion of nutrients. |
|
| 61:09 | Okay, Countdown 3, 2. hmm. Yeah, it would be |
|
| 61:18 | those poor formation. Yeah, so one trigger for it. Okay, |
|
| 61:27 | Any kind of stress, uh temperature, radiation depletion of nutrients, |
|
| 61:35 | chemicals etcetera can induce in those four . Um what about this 1? |
|
| 61:45 | , a surface and ability to Our absolute requirements. Four. |
|
| 61:55 | it's not in those four formation. . Okay. Camp down 321 |
|
| 62:43 | Okay, biofilm formation correct? So will be our first biofilms. Didn't |
|
| 62:52 | those boars. Okay, so uh couple of things. So as mentioned |
|
| 62:58 | question surfaces. It's all about Okay. Um and two it's all |
|
| 63:07 | lots of girls. Lots of Okay. And so going clockwise from |
|
| 63:16 | one here. Okay. Where we just a cluster of cells, what |
|
| 63:20 | call might call a micro colony. . Not yet visible to make it |
|
| 63:25 | um very quickly becomes an aggregation of and millions of cells that are on |
|
| 63:34 | surface in two dimensions. But then um emerges up off the surface in |
|
| 63:42 | what's called a biofilm tower. Right three dimensions. Okay. As you |
|
| 63:47 | see, certainly beginning here and then here like mountains just rising above right |
|
| 63:55 | of cells, so to speak. And that in itself here here creates |
|
| 64:02 | can create different different micro environments in film. Okay. Some cells are |
|
| 64:08 | interior than exterior. Maybe perhaps create properties in those cells. Okay. |
|
| 64:16 | They are um from a medical standpoint are becoming or have been for a |
|
| 64:23 | time a very uh problematic if not situations. Uh They're very persistent biofilm |
|
| 64:30 | be very persistent. Um But one the hallmarks mobile film is it sticks |
|
| 64:36 | stuff that sticks to services. Okay things from a medical standpoint, things |
|
| 64:42 | catheters that are inserted. Um medical , hip replacements, um these heart |
|
| 64:51 | , he's all for it surfaces upon potential biofilm formers can attach. And |
|
| 64:58 | disease um teeth right then that's obviously than being gross. It's it's uh |
|
| 65:08 | that yellow standards that actually colored it enhance the biofilm itself. But you |
|
| 65:13 | that sticky feeling in your mouth is a biofilm. Okay. Um and |
|
| 65:19 | you think of uh you know bacteria your mouth right there? There's all |
|
| 65:22 | of fermentation going on in their production saccharin material that serves the kind of |
|
| 65:35 | the uh And so stay at the here with sugar snake. That is |
|
| 65:45 | some a I've encountered in convenience stores fast food restaurants where we have a |
|
| 65:55 | can go get your drink from a fountain dispenser, right? Um You |
|
| 66:00 | their lines going to it right from canister of the syrup and then the |
|
| 66:05 | to thingy. Right? And so they go through the lines to the |
|
| 66:11 | the dispenser um they can become clogged to this material. This in particular |
|
| 66:17 | one that actually produces material from the that are in the line and creates |
|
| 66:25 | document called sugar snap. Right? um what happens is of course it |
|
| 66:31 | up with spencer. And for the of the place the problem becomes when |
|
| 66:38 | , people don't really care much about but when it starts to smell then |
|
| 66:43 | care. Alright so that's um so have to come in and get rid |
|
| 66:47 | this stuff. My life wait for . Yeah but it is fire. |
|
| 66:54 | now what? But don't get the bio sam was just a random uh |
|
| 67:03 | blob of all kinds of different Okay because it's not that at all |
|
| 67:09 | actually very orchestrated, phenomenal. Okay is species specific. Okay um And |
|
| 67:19 | of the factors that's most important. of the more important ones, the |
|
| 67:24 | former is the the presence of february in that by also informative. To |
|
| 67:33 | knowledge there's always not fire somewhere but my knowledge most not all bios reformers |
|
| 67:39 | that. They have to figure I pillai because it tackled to the surface |
|
| 67:44 | an important thing. Okay. And what facilitates that. Um So it's |
|
| 67:50 | process that forming a biofilm. And of course the way all cells |
|
| 67:57 | two different chemicals. Right? So are involved in the production of the |
|
| 68:02 | . Okay so we have these stages initiation attachments, maturation maintenance, dissolution |
|
| 68:12 | . So let's um let's just vote the diagram. Okay so we have |
|
| 68:19 | call plank tonic versus uh sticker Celso versus stickers. Stickers. The |
|
| 68:28 | So swimmers are the ones that are of the scouts if you will reconnaissance |
|
| 68:35 | for environment favorable to land on um environment. So we have to get |
|
| 68:43 | driving force for this is abundance not of but abundance of nutrients. |
|
| 68:51 | Or at least a steady supply of because remember this thing's gonna blow up |
|
| 68:56 | the Brazilian cells and that's happening then they've been supplied. This is certainly |
|
| 69:02 | it's the opposite of nutrient deprivation. , this is a new trick. |
|
| 69:07 | okay. And so and many of Yeah phenomenon, balding A that um |
|
| 69:21 | the product of lots of cells. this corn sensing mechanism? This is |
|
| 69:29 | in different context. We're going to this again actually in uh cells that |
|
| 69:35 | I think they do. That sensing also a part of that for some |
|
| 69:39 | . Okay. Because this like that also a cell density dependent thing that |
|
| 69:45 | have enough cells present. Alright. those of you guys have worked in |
|
| 69:51 | . All right, in a quorum do your thing. Right, So |
|
| 69:56 | maybe five people so you have to at least a minimum to be able |
|
| 69:59 | get anything done right, staying So how do they do sales since |
|
| 70:08 | ? They're giving up. Alright. a threshold level of those capitals which |
|
| 70:13 | only happen. Ideas. Okay, that's what the form sensing thing |
|
| 70:18 | It's about cell density. That's Right, Because there's enough cells and |
|
| 70:24 | is enough chemicals that have come together induce this laughter information. Right? |
|
| 70:32 | electronics sells right will become stickers, to speak, Okay, actually lose |
|
| 70:38 | to gel. Okay. And now the surface, so surfaces in Portland's |
|
| 70:45 | horizons and service. And so micro formed their um and they can have |
|
| 70:53 | twitching motel. Alright, remember that's surface dependent pillai on the surface. |
|
| 71:01 | . And so the Canada twitching motility on here. And so micro |
|
| 71:05 | these are colleagues but not yet Looking at. That's um Again, |
|
| 71:12 | signals coming up. Right. So this point is where the biofilm either |
|
| 71:19 | produced or not. Okay, the or nothing. And so it's all |
|
| 71:24 | getting of chemical signals which depends on themselves to the surface presumably if you |
|
| 71:31 | threshold is because this surface can sustain . Okay. And can sustain more |
|
| 71:39 | . Right? So presumably it's a environment which will that lead to this |
|
| 71:46 | , actual policy saccharine formation. So is this is the glue that holds |
|
| 71:50 | together, so to speak. So it's obviously material can be a |
|
| 71:55 | of material um it's a june career . Okay. Uh the whole biased |
|
| 72:06 | is in june okay. Um and again, important things for grad a |
|
| 72:14 | reaching a threshold forms sensing the production extra polish sakura. So when you |
|
| 72:21 | a threshold level then you get expression the genes for this production. Okay |
|
| 72:28 | you go from two dimensional growth on surface to three. Now we're really |
|
| 72:35 | in terms of in fact it's very to see this and that in pipes |
|
| 72:40 | well, pipes problems your surface. ? And a pipe generally has liquid |
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| 72:45 | and the liquid generally has nutrients. it's like it's continuously fed. Yeah |
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| 72:51 | you want to say about the film walk out that door and it was |
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| 72:56 | there. There was on that as come out of SnR 1211 stairs and |
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| 73:04 | look outside and there's like a bored there second green stuff on the bottom |
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| 73:08 | liquid. Um And so it without you can get lots of growth |
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| 73:18 | Now what happens is you create these environments where cells on the surface, |
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| 73:25 | . Versus sells more interior. There's difference is there? Right. So |
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| 73:31 | in terms of nutrients and so you to create holds right at the towers |
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| 73:36 | kind of makes through material flows through around everybody gets bad. Right? |
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| 73:42 | in terms of the aspect of saying , worry if this were a |
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| 73:47 | Okay, the pathogen bacterial after. . Well that could be an issue |
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| 73:55 | well because these they can be hard penetrate. Yes maybe the ones on |
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| 74:00 | surface are more susceptible but those in interior not so much that they can |
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| 74:05 | resistant. Okay, that's the thing these medically important biofilms, they're harder |
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| 74:11 | win. And so very often these what we call the hospital acquired infections |
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| 74:19 | get in the hospital you don't you you don't get to go to the |
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| 74:24 | result of having a biofilm infection. you acquire it while in the hospital |
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| 74:29 | you're getting something else done like your scope or something. And then in |
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| 74:33 | process of using those medical devices these are introduced and they grew up right |
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| 74:39 | so they're already grip and they can in some cases baby. Okay um |
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| 74:45 | staph infection 10 10 many of those of so um anyway so bottom line |
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| 74:55 | the biofilm uh surface surface attachments. things like uh the quorum sensing being |
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| 75:04 | celebrated and then use the process um apply satellite information and doing world America |
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| 75:10 | growth in three dimensions. Now now nutrients do become an issue and not |
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| 75:18 | then yeah the Canon is off right . The sticker self becomes swimmers again |
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| 75:24 | find another character. Right? She by itself. Okay so um did |
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| 75:35 | actually the first thing thursday we'll finish legal sports and they go with the |
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| 75:40 | . Thanks books. |
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