| 00:02 | Hello folks. Um Welcome. Let's here. Okay. You hear me |
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| 00:11 | back there ladder testing. Hello. Cat. Um Let's see here. |
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| 00:27 | today we're gonna finish up uh chapter . Got a couple more topics to |
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| 00:35 | about and um and there we That was really loud. Okay. |
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| 00:49 | Here. Mhm. Okay so a of things. So we got a |
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| 00:57 | for five, remember last time I it was last week? Uh checker |
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| 01:02 | was one of those class things but not so don't worry about that. |
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| 01:08 | we're gonna sacrifice ultimately short we'll finish up Tuesday. Right? So any |
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| 01:13 | these things that are called a catch day, right? That's where um |
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| 01:21 | um if I haven't finished everything up that point that I use that to |
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| 01:26 | of Pretty sure. Right? So so it should not so it shouldn't |
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| 01:31 | a full day on Tuesday, maybe minutes or something like that. But |
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| 01:35 | , so that's kind of what those are about to finish up whatever materials |
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| 01:40 | in that unit, there's always come before the next unit. So so |
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| 01:46 | start unit to next thursday. So week from the day. Okay, |
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| 01:52 | um that will be flipped, And that material unit two is |
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| 01:59 | Everything in there is available. What have you? Um The flip |
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| 02:05 | has the chapter six part one? . Okay. Which is mostly just |
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| 02:12 | in terms of the viruses. Here's we define it. Here's the structure |
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| 02:16 | it, we do life cycles in two. Okay um exam week from |
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| 02:25 | uh see if you haven't already signed for it and uh you don't wanna |
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| 02:31 | . So that starts tomorrow. That's little more comprehensive. I think there's |
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| 02:35 | 25 26 questions. Uh 45 minutes complete it. Um basically covering trucker |
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| 02:46 | and as much as five as we about today. Okay then that's that |
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| 02:54 | I think that's it. Um Any or anything? Okay so let's look |
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| 03:04 | this as a recap from last So we went through um right that's |
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| 03:11 | we started with you call it So we looked at um quantitative um |
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| 03:19 | cells grow a couple of different practice there involving coming from generation time and |
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| 03:27 | using that in a couple of different to find out how long it takes |
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| 03:32 | X. Numbers to get to this or whether it was to calculate generation |
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| 03:37 | . Okay if you are kind of comfortable with those equations just go through |
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| 03:43 | practice problems around blackboard. They are worked out. Right? So hopefully |
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| 03:50 | a logical way so that if but you have some questions about it, |
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| 03:55 | me know you will be able to a calculator, handheld calculator during the |
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| 04:01 | uh costs of people will be made that you're allowed to have those in |
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| 04:06 | so you shouldn't be hassled about Okay um So uh after the after |
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| 04:14 | the quantitative quantitative stuff we looked at batch growth curve. Right so all |
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| 04:22 | , all the living things, we're have some kind of a growth |
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| 04:24 | Even humans. Okay. Um It's that we can track the growth curve |
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| 04:30 | bacteria rather quickly right within the span a day or less. Um In |
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| 04:35 | case we're going to see all these phases. Right. The only difference |
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| 04:40 | be is how it will vary. So we may have we may have |
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| 04:50 | a pencil working. Okay we may , hold on. Okay one more |
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| 04:57 | if this works or not let's try . Okay so you may we're gonna |
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| 05:02 | all four of these. Okay but what will vary is will the lag |
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| 05:07 | maybe it's extended. Maybe it's Maybe the inflection of exponential growth is |
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| 05:17 | as steep or more like that. the duration of stationary phase this far |
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| 05:23 | is it shorter? So all these of variables can occur depending on micro |
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| 05:28 | , depending on what you're growing it depending on the physical and chemical growth |
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| 05:33 | that are conditions that are going. all these things will vary um can |
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| 05:39 | these different parties but the bottom line you're still gonna get four of these |
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| 05:44 | . Okay. And so we can batch growth so remember match growth is |
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| 05:51 | , take samples to monitor growth. it let it go its whole |
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| 05:56 | Okay if you want to do more that. Right? We can if |
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| 06:00 | interest is to get lots of higher yield than what that gives us. |
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| 06:05 | add food. Right? So fed allows us to do that. We |
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| 06:11 | um nutrients right to Our culture. ? Carbon pretty much the biggest |
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| 06:18 | And that allow us to increase cell . Okay, remember that. We |
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| 06:24 | add everything if we know that. grand will give us so you'll be |
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| 06:29 | we typically can't add it up We have to add it at intervals |
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| 06:35 | the growth cycle because adding an up is too much right? Osmolarity issues |
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| 06:41 | highest values. So you have hypertension outside the cell. The cells fighting |
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| 06:48 | instead of growing. Right. So can't really do those kind of |
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| 06:51 | You have to add it as it's . Okay. Um if you really |
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| 06:56 | to control everything, we talked about uh bioreactor where you control all the |
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| 07:03 | . Okay. For the role of parameters uh all that stuff and of |
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| 07:15 | they can grow crazy. Okay. they do. And then we think |
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| 07:20 | finished kind of with if you don't are the resources you have to do |
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| 07:26 | types of studies? Well do you a computer controlled reactor that does everything |
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| 07:32 | you program or are you reduced to this? Okay, shake flask. |
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| 07:41 | . Still do some things with that we talked about. You can control |
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| 07:44 | you control um Aaron put to a right? By doing things like increase |
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| 07:53 | shapers um have a flask that has kind of indentations in it. Right |
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| 08:00 | more turbulence. More mixing of right? Um at a ph indicator |
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| 08:05 | we can visualize when it's right so can do those things just very tedious |
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| 08:11 | on your part rather had better to something like this that you don't have |
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| 08:17 | keep the cost of. Anyway these issues these are issues for you biotech |
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| 08:26 | because these kind of things that you be working. Okay so um and |
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| 08:31 | course using this information from here to this kind of information. They're so |
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| 08:40 | these and what um any questions about . Okay so let's so this next |
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| 08:50 | is actually two for one. Right is gonna get us into the last |
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| 08:55 | of this chapter four. Okay so question both questions will use the same |
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| 09:03 | . So this is the first This is often a nutrient driven phenomenon |
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| 09:09 | by lack or depletion of nutrients. not the only thing that will do |
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| 09:17 | . E depletion nutrient, there's other that will cause this state. This |
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| 09:23 | a common one, counting down five . Okay alright let's um I'm |
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| 10:09 | The next question I'll come back to . So let's look at number 2 |
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| 10:18 | . This is often I'm gonna say that this is a nutrient driven phenomenon |
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| 10:28 | a surface and the ability to attach and oh let me do that. |
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| 10:41 | . You can yeah. Okay. down from 1312 54 21. |
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| 11:21 | Yeah that is bio for information. ? The giveaways here are surface and |
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| 11:29 | but it is nutrient driven biofilm. anything gonna boil it down to the |
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| 11:35 | ? It's um have a surface cells able to attach that surface and then |
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| 11:43 | because you're gonna you're going to a represents a lot of cell growth. |
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| 11:48 | ? And if you're gonna sustain a or even initiated and then sustain |
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| 11:53 | you're gonna need a constant flow of . Okay? So food surface |
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| 11:59 | Okay. The basics of a Okay. The previous question that was |
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| 12:06 | formation. Okay So that one was okay and that one was d Alright |
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| 12:14 | the depletion of nutrients. So among things so endospore represents a dormant |
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| 12:21 | Think of maybe now it's still plant . Plant seed doesn't produce a plant |
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| 12:27 | you put it in the ground and water to it. Alright, Germany |
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| 12:31 | in those four represents kind of a animation. Maybe um it's it's viable |
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| 12:39 | that it was for but it's just growing right? But it's a very |
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| 12:45 | form. Okay. And they form a result of nutrient deprivation radiation temperature |
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| 12:56 | , Osmotic stress. Uh Any kind stress can potentially induce the deformed handles |
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| 13:04 | . Okay so these are the last things we'll talk about Chapter four. |
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| 13:08 | those four formation and biofilms. And we'll start with biofilm formation. Okay |
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| 13:15 | biofilm formation um like I said represents lots of cell growth. Right? |
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| 13:22 | you can see here From starting at and going clockwise right? So we |
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| 13:29 | in its first two panels here. little bit of gross. Right? |
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| 13:33 | of course it's all occurring on the . Right? So it begins with |
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| 13:36 | surface so it's attaching to the surface then beginning to grow. Okay now |
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| 13:44 | you really get beyond uh this stage right, approximate this stage here. |
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| 13:53 | . And go forward. It depends um this is where the communication comes |
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| 14:01 | . So if my film is not you might think to random assortment of |
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| 14:06 | and just kind of comes together and as a single unit or something. |
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| 14:11 | . It's completely orchestrated. It's gene um cells are communicating with each other |
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| 14:20 | allow it to happen or not to . So it's far from a random |
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| 14:25 | . It's a species specific thing. . It's yes as a bathroom forms |
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| 14:34 | to the actual particular species certainly because in an environment it's out in the |
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| 14:41 | . The microbes are there. Yeah of some other bacteria can stick on |
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| 14:46 | maybe become a transient part of But it is a species specific |
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| 14:52 | Typically a future you have to have bio for those that are lacking mutants |
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| 14:58 | biofilm Formers that mutant lack of february follow by. Oh, okay. |
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| 15:05 | course. For attachment. So it's about getting enough cells on that |
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| 15:13 | . Right threshold left. This is we call a cell density dependent |
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| 15:21 | You have to have enough cells present the process to occur. That's what |
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| 15:26 | called quorum sensing. Right? There been a part of a government entity |
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| 15:32 | you have to have a quorum you , you have to have enough people |
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| 15:35 | to vote. Right? So similarly have to have enough cells there to |
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| 15:41 | to make a bio. Think about . Right? If you do then |
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| 15:46 | can proceed on through Okay. To next steps. And so what's the |
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| 15:52 | behind that kind of a process? , it's really about the viability of |
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| 15:58 | whole of that species because presumably there's cells collected on the surface is because |
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| 16:05 | are is a favorable environment, there's there for them to grow. |
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| 16:11 | And so that's what you need if gonna initiate and maintain a steady supply |
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| 16:17 | food. Okay. So presumably more more cells are gonna be attractive |
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| 16:23 | You know, there's chemical signals going there being attracted to it but they're |
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| 16:28 | there because of the favorable fire. . And they can then begin to |
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| 16:33 | . And so in panels 12 and . Pretty much just growth on a |
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| 16:40 | surface. Two dimensions. Right now can see this thing growing right out |
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| 16:46 | off of the surface, right, over here. So it's growing in |
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| 16:50 | dimensions. So obviously represents a tremendous of so gross going on as this |
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| 16:56 | happening. Okay, so uh so these are taken on campus, there's |
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| 17:03 | I left brick wall, that's if goes through SnR one heading toward |
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| 17:11 | you walk out and look to the . Um that brick wall there, |
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| 17:17 | this pipe coming out, I'm not it's coming from but uh they used |
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| 17:22 | be a wooden board sitting here And it was just dripping on that |
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| 17:27 | board, that wooden board was just green biofilm on it right now, |
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| 17:32 | just kind of popping down to the below. Okay, but that massive |
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| 17:37 | , that's the for me. uh this is like from last |
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| 17:41 | I'm not sure if the freeze killed or what's going on with it right |
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| 17:45 | , This is from STL building, can see the bow from rolling |
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| 17:50 | So obviously similarity is we got, have a surface where this is occurring |
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| 17:57 | these windows are very uh a lot water that drips down on them. |
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| 18:01 | you're gonna have certainly supply of nutrients way. And of course a pipe |
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| 18:06 | is dripping down material, it's gonna organic material in there that's gonna sustain |
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| 18:12 | and formation of biofilms. So pipes notorious for having biofilm buildups in. |
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| 18:18 | , so uh medically important types uh staph staphylococcus warriors which is a common |
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| 18:28 | skin uh pathogen that can cause things oils and different types of rashes. |
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| 18:35 | There are types that are bathroom forms so these you often see in the |
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| 18:43 | what are called hospital or more turned as healthcare acquired infections, infections acquired |
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| 18:51 | the setting of a health clinic or hospital or something. Um And that |
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| 18:56 | often come from medical devices like a shown here um breathing ventilation tubes, |
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| 19:04 | parts of people's bodies like knee hip replacements, heart about these are |
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| 19:10 | certain to write surfaces can get contaminated these these these pieces of equipment becomes |
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| 19:18 | right? But the person handling it maybe it's not wearing gloves or something |
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| 19:23 | otherwise contaminates it. Staff is found your skin is found in your deepest |
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| 19:28 | your nose and they can contaminate in person. Right? And now they |
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| 19:34 | introduce that pathogen. Right? Um on that this case. Catheter bio |
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| 19:45 | on that catheter, lots of cells produced very high cell density as you |
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| 19:50 | here. And in a biofilm gets big um There's gonna be different |
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| 19:58 | Right? So and not all the in a biofilm will behave the same |
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| 20:03 | ? You have some more on the part of the biofilm that are more |
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| 20:08 | in nutrients and and air if that . And investigated more maybe more interior |
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| 20:15 | aren't growing as much getting as much and then you begin to see some |
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| 20:19 | in terms of. Right? That's if you have a biofilm former that's |
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| 20:25 | infection like this They can be very to get rid of. So it's |
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| 20:31 | a 10 day to write about We're talking like weeks and months trying |
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| 20:36 | get rid of this thing. Okay could be quite serious. Um and |
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| 20:40 | this is not just on catheters but types of medical devices as well. |
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| 20:45 | so it's something to to definitely be of if you are going to health |
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| 20:50 | so because these are not a not thing. Okay. Um Yeah so |
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| 20:58 | go look at basis of biofilm So we're gonna have, this is |
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| 21:03 | examples biofilm So t the right Now typically a biofilm forming um This |
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| 21:12 | down here this you see in um food restaurants, any kind of uh |
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| 21:21 | gas station. Uh We get the of thing where you have the soda |
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| 21:26 | where you fill up your drink because things have tubing that goes to it |
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| 21:30 | a cylinder C. 02 with the to make the soda travels through the |
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| 21:37 | that comes out and fills your cup . That tubing. Get environment that's |
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| 21:43 | and sugars in there. Um You form these kinds of polymers that you |
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| 21:47 | here clogging up the tubing and when gets clogged up overs occur and then |
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| 21:55 | person running the restaurant is like So they get called in to try |
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| 21:59 | fix the problem. But again, of you to buy from pipes are |
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| 22:03 | for different pipes tubing can get uh can get biofilm formation in those. |
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| 22:09 | , so the process of this. , so look at it and kind |
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| 22:15 | five step. So yeah, this a creative process is not random |
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| 22:21 | There's a stepwise part of this step uh scheme to this. And so |
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| 22:30 | gonna go through different a couple different types. Those that are kind of |
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| 22:35 | and those that are sticking to the . Okay, so called plank tonic |
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| 22:41 | are your swimming types? Okay. . And then you're what they call |
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| 22:48 | types. Right. Are the ones expressed and allowing attachment to the |
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| 22:54 | Right. So you're from swimming too . Um and then it's in this |
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| 23:01 | phases right here where um you have course chemical signals going on. |
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| 23:10 | Some cells have migrated down to the and are throwing out these chemical |
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| 23:17 | It was a favorable environment certainly in beginning to grow multiple. And that |
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| 23:24 | the level of the chemical signaling. , if it were an unfavorable environment |
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| 23:31 | those shoes are there are likely not to be multiplying so very slowly. |
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| 23:36 | you're not going to build up the signal. Okay. Uh So that's |
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| 23:42 | you want to happen through that micro it's a favorable environment then. Yeah |
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| 23:47 | more will come to be attractive and form the biofilm. If not well |
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| 23:52 | you shouldn't have whether it's coming there it's not something that's going to sustain |
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| 23:57 | . So find another more favorable So if it does kick in then |
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| 24:03 | get into what's called policy saccharine or they secrete this. So um this |
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| 24:10 | basically the glue that holds the biofilm . Okay. And um it's the |
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| 24:18 | when that threshold level is reached, enough cells in the chemical signal that |
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| 24:23 | induces the genetic program to produce the saccharin. So it becomes forms and |
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| 24:32 | the biofilm begins to grow. And then of course it grows initially |
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| 24:36 | the surface two dimensions and then eventually off the surface it's called biofilm |
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| 24:43 | Okay. Now it would certainly be these biofilms are there's a finite lifetime |
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| 24:52 | them although they can last quite long there's a steady supply of nutrients whether |
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| 24:56 | tripping water nutrients tripping through a pipe can sustain it or but maybe |
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| 25:03 | maybe it ends. And so you're have a large massive growth if you |
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| 25:08 | have in the future it's applying they're not gonna be sustained so be |
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| 25:13 | to break apart this solution it's called then you have so revert back to |
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| 25:20 | plant tonic state so now they want detach right and go elsewhere to find |
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| 25:25 | better environment. So they have to back into the swimming mode. Okay |
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| 25:30 | we can kind of see this all . Okay so so again this phenomenon |
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| 25:37 | harm sensing. Okay so that's the in this case you see this and |
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| 25:43 | isn't exclusive to biofilms. You see sensing in various different phenomenon. We'll |
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| 25:51 | it again in the context of uh transformation uh three transformation of the DNA |
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| 25:59 | the environment and certain types only do , there's enough cells present. So |
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| 26:06 | you see this in various different phenomenon bacteria. Um So as mentioned then |
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| 26:14 | have these plant tonic swimming cells. will then come down to the surface |
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| 26:22 | lose the uh and then uh attach you can have twitching motility, right |
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| 26:33 | you can get that kind of motion then growth right then of course the |
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| 26:41 | gets thrown off um And then themselves more and more attracted to that |
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| 26:47 | Then you're gonna get more signal. you'll re threshold and you initiate biofilm |
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| 26:53 | . So supply satellite production and growth from the surface and the biofilms. |
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| 27:00 | , so as mentioned cells they're here the surface, right on the surface |
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| 27:08 | . Okay well there'll be some differences those and those more on the |
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| 27:14 | Okay just there separated away from nutrients oxygen so likely going a little slower |
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| 27:23 | . Um And so you can see of these differences within your biofilm |
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| 27:27 | And again you can relate to differences antibiotic resistance. So that's where issues |
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| 27:33 | come in if it's a medically important . So but again this obviously represents |
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| 27:41 | lot of cell growth. Okay to that you better have nutrients flowing |
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| 27:49 | Sorry, depends on where you have rely on my finger to do |
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| 27:53 | So let me try this again. . Um Okay so you're relying on |
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| 28:02 | just will not work. Okay well know what I mean? Okay, |
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| 28:08 | are flowing in. Okay that can the biofilm but if it goes down |
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| 28:13 | drops out then no disillusion will And stickers will become swimmers. Look |
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| 28:21 | more favorable environment. It's kind of cycle of the biofilm. But they |
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| 28:25 | be maintained for quite some time if a steady supply. Um Many questions |
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| 28:32 | that. Yeah. Yes, Yes. Yes. Any other |
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| 28:56 | Okay so um Alright so in those , so in those fours represents a |
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| 29:06 | of differentiation um are in a initially a growing vegetative state vegetative cells kind |
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| 29:18 | a normal metabolically functioning cell form. ? Uh If you become stressed in |
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| 29:25 | case of industry information it can then into this different form. Okay. |
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| 29:31 | form that allows it to remain essentially and dormant for how long each month |
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| 29:39 | , thousands of years, Hundreds of of years, millions of years. |
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| 29:45 | ? So these are a couple of of the extreme millions of years on |
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| 29:50 | from fossil evidence um and revived. are viable. And those boards that |
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| 29:57 | able to be revived in the Okay. And in this section are |
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| 30:03 | number of them. So you see is actually a vegetative cell vegetative cell |
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| 30:14 | , yep. Here. Okay. that's the actual endospore forming inside |
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| 30:23 | Right, This would probably be over . These are actual free. And |
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| 30:29 | four. So when this like for , this cell right here is done |
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| 30:33 | me, the endospore then this part released from the cell and then you |
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| 30:39 | something like this or this. this one right here is one that's |
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| 30:47 | completely vegetative self is not making it those four. Okay. And you're |
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| 30:52 | see all three types. If you're at a culture of the type that |
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| 30:56 | form in the sport, you're gonna all three of these in there. |
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| 30:59 | just the proportions will vary depends on of what stage they're in. |
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| 31:04 | so obviously in those sports represent a um they're very resistant. Okay, |
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| 31:13 | to chemicals radiation, um temperature, any kind of stress they're resistant |
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| 31:21 | Okay. And so there are lots things that former sport across the whole |
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| 31:30 | animal, the pro cario groups, of things from scores we're all familiar |
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| 31:37 | fungal spores. Right, mold Um There's there's also cysts which are |
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| 31:45 | dormant form. Um cysts spores. But the endospore, right, When |
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| 31:51 | put those four letters right in front sport, you're creating something that's like |
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| 31:57 | level in terms of resistance. Yeah. Other types of spores and |
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| 32:02 | can have a certain level of resistance so than a just a vegetative |
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| 32:07 | But in those four is again another . I don't see anything like |
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| 32:12 | That's this resistance. Okay. That's we have to use an autoclave sterilized |
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| 32:16 | kill and those spores. Right? And so the two majors only but |
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| 32:23 | only two groups on earth that I of that do this. These two |
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| 32:30 | bacillus clostridium. That's it. Um Both gram positive. Both |
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| 32:38 | Uh but very different metabolically. The um are obligate anaerobic. Right? |
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| 32:45 | can't live in the presence of The silvers are mostly um are |
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| 32:52 | maybe microcephalic, but they use Okay. And so the types, |
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| 33:00 | probably familiar with the clostridium tetanus, , gas, gangrene. There are |
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| 33:05 | lot of toxin producers in that Um number of pathogens in that |
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| 33:10 | But so those are more or less uh anthrax of course. Is that |
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| 33:17 | exception. But they're both soil Okay. So um and they're gonna |
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| 33:24 | this characteristic shape. Right? When forming indoors for uh you see the |
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| 33:29 | club shape at the end. They it. That's the end of the |
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| 33:32 | forming little refract I'll bodies over These are in those floors forming. |
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| 33:38 | um so when we look at sports general, So this is a highly |
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| 33:46 | genetic program to do this, It takes several hours for it to |
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| 33:50 | uh complete And so you have as cell is undergoing is you have compartmentalization |
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| 33:59 | in the south. Okay. It's you have the application then you have |
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| 34:04 | formation of two compartments and they're after when the sport formation occurs. |
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| 34:10 | Um the ability to be very resistant endospore to be resistant. It has |
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| 34:16 | do with a large part with the amount of water in the cell. |
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| 34:22 | cells that are, you know, body uh that undergoes extreme heat, |
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| 34:30 | ? It's the water in the cell is the most damaging, right? |
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| 34:34 | water and in vegetative cells. So going to destroy protein function, |
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| 34:41 | If you can withdraw a lot of water desiccated itself. Not completely. |
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| 34:48 | goes a long a long way into it very stable. Okay. That's |
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| 34:53 | happens in speculation, remove a lot the water in the process. |
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| 34:58 | And so um so as you go this, we're gonna see. |
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| 35:04 | I've mentioned this before Cell right? just think of that as a doing |
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| 35:13 | thing. Um And then as mentioned the population of a sport former in |
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| 35:18 | four former gonna see vegetative cells, producing a spore and free in those |
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| 35:23 | . Okay. Now again, if seeing if you look at the sample |
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| 35:27 | the microscope And you're saying 99 of cells. You see 99% of these |
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| 35:37 | it's really not it's just growing it's not anything stressing it. Okay But |
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| 35:42 | you see uh 50% 60% 80% of forms then yeah it's clearly undergoing a |
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| 35:55 | relation process. Something is stressing There's nutrients lacking what have you. |
|
| 35:59 | going through that you can engage and state of the of the cells by |
|
| 36:05 | proportion of the types of C. um Now the um form of the |
|
| 36:17 | . Okay so these these this is specific. Right? You can actually |
|
| 36:24 | the bacillus species. Actually can help identify what type it is by what |
|
| 36:29 | of sport forms. Okay so it be different in location at one end |
|
| 36:36 | the middle maybe between the middle and end. That's those three. That's |
|
| 36:41 | we call terminal sub terminal central. it can be swollen And also be |
|
| 36:48 | the three different locations. So basically different and those warmer pathologies and again |
|
| 36:54 | are species specific. So you can it as an identification tool. Okay |
|
| 36:59 | Now the process uh so we'll Okay that's undergoing some kind of stress |
|
| 37:12 | temperature stress chemicals chemical stress radiation. happened? Okay then that begins a |
|
| 37:21 | where first duplicates D. N. . Okay um And then we get |
|
| 37:29 | in the cell. Okay so one goes to the mother's, sorry goes |
|
| 37:37 | the mother cell and one goes to fourth sport. Okay So the force |
|
| 37:44 | is ultimately where the end those four from. So this guy that's gonna |
|
| 37:55 | the maturity. Okay now the mother . Okay its role is to um |
|
| 38:06 | express jeans that are pretty much what call transcription factors. They will travel |
|
| 38:13 | the fourth board in direct expression of genes to form the endospore. Kind |
|
| 38:19 | controlling controlling what's going on to the of different proteins. Okay that are |
|
| 38:26 | to the fourth board saying turn this and that on what happened become a |
|
| 38:33 | . And so um next what happens that mother cell compartment right begins to |
|
| 38:43 | the force for. And so this now the beginnings of how you get |
|
| 38:48 | very resistant coat around the endospore. so we form a double membrane. |
|
| 38:55 | and inside there we produce pepper look , okay so don't mistake this for |
|
| 39:04 | a now it has two membranes and in the middle. It's now a |
|
| 39:08 | negative. No it's not that okay not this isn't like an out of |
|
| 39:14 | . It's not it's not that okay bacillus apostles. But this in those |
|
| 39:20 | this is how the coke forms. called we call it cortex is |
|
| 39:25 | Okay so um we then add this called acid D. P. |
|
| 39:35 | For short. Okay and then uh buys the D. N. |
|
| 39:41 | Bias the D. N. Kind of helps protect the D. |
|
| 39:44 | . A. In the state calcium deposited into the membrane to kind of |
|
| 39:49 | it. Um Also water is All right so we're gonna remove from |
|
| 39:56 | water. All this kind of helps serve keep that a ultimately viable cell |
|
| 40:02 | this windows for for okay but of you can see how the D. |
|
| 40:06 | . A. And the mother cell kind of just disintegrating. It goes |
|
| 40:11 | . Okay and so we're then left ex operandi um That's kind of a |
|
| 40:20 | structure. I don't really worry that about it but then it goes into |
|
| 40:23 | sport coat right mature sport coat. and then free sport. Okay so |
|
| 40:29 | here two Back to here let's say here these stages here. Okay. |
|
| 40:45 | . Okay that's where it looks like . Okay this form here. Okay |
|
| 40:52 | vegetative cell with the end of So when you're looking at that it's |
|
| 40:56 | one of these um four stages here 1234. So one of these |
|
| 41:05 | Okay that's what it looks like under microscope. Okay and then finally you |
|
| 41:09 | the free free sport released right as rest of the self disintegrates. Okay |
|
| 41:14 | again this this can hang around weeks years thousands hundreds of thousands millions of |
|
| 41:22 | . It all depends. Okay but can germinate. Okay think of germination |
|
| 41:29 | you're taking a seed completely earth and . It grows to a plant and |
|
| 41:33 | what's that's what's happening in those poor germinates into a completely the vegetative form |
|
| 41:41 | stuff. Which is the one that grow and reproduce and et cetera. |
|
| 41:47 | . Um The but you know again are very resistant in the sport. |
|
| 41:54 | why uh on a claim um produces under under steam under pressure and it |
|
| 42:02 | temperatures that are very high. 20 2 b. sub centigrade. Um |
|
| 42:09 | that moist heat is the key. can penetrate the those four code kills |
|
| 42:17 | . Okay and so on the page very effective at doing that. |
|
| 42:22 | Um Any questions Yeah the lack of is what drives it. So it's |
|
| 42:34 | it's not the only thing lots of can be radiation increasing the temperature but |
|
| 42:41 | deprivation of nutrients is a stress. yeah that too. Any other |
|
| 42:51 | Um All right so I think now that's chapter four. Okay we're gonna |
|
| 42:59 | gears a little bit. Of course been talking about growth. So just |
|
| 43:03 | than an extension of that. We'll first with the chapter five. Kind |
|
| 43:08 | two parts. Right? We don't a lot of the first part. |
|
| 43:13 | But the first part is basically uh environmental influences that's the first part. |
|
| 43:22 | second part is this the control So and that's what these for relate |
|
| 43:32 | environmental influences. Okay. We know when we want to grow microbes, |
|
| 43:38 | ? You supply supply C. O. M. P. |
|
| 43:41 | Right? You put them together in right forums, you know, the |
|
| 43:46 | type of the organism is you provide correct nutrients, then you provide the |
|
| 43:54 | temperature ph right. Osmolarity. These all combined to allow it to |
|
| 44:01 | And so so in this chapter um we read about temperature ph optimal |
|
| 44:12 | you'll see that um for most life earth, right? Where most life |
|
| 44:20 | earth is in the moderate ranges, ? A temperature between 15 and 40 |
|
| 44:29 | say centigrade. Right? That's where life lives. A ph of around |
|
| 44:34 | . That's where most living things Okay. Uh Osmolarity. If you're |
|
| 44:39 | marine environment, maybe 3% salt uh terrestrial environment 0.9 point 9 to |
|
| 44:47 | Okay, those are the normal Okay, that's where most living things |
|
| 44:52 | . But you know, we got on the fringes, right, Archaea |
|
| 44:57 | others can live extremes of ph temperature . Okay. Um but regardless of |
|
| 45:10 | , the thing about temperature ph similarity keeping it optimal. Whether you're in |
|
| 45:18 | middle range for most things are already on the extremes, it's about keeping |
|
| 45:24 | proteins in those bodies, whether you're seller something bigger. Keeping the proteins |
|
| 45:31 | . Right, So recall basic protein . Right, tertiary structure of protein |
|
| 45:37 | in a particular way and what keeps folded hydrophobic interactions. Charge attraction, |
|
| 45:44 | cobiella bonds, di sulfide bonds. uh And so osmolarity temperature ph can |
|
| 45:54 | all that. Right. So an optimal conditions of those parameters will keep |
|
| 46:01 | proteins happy. That's what extreme of right, can live that way because |
|
| 46:07 | have adaptations allow them to keep their happy at these extreme conditions. |
|
| 46:15 | So it all boils down to Alright. Whatever the optimum condition is |
|
| 46:20 | particular organism, it's it's a ph and clarity. It's because it's proteins |
|
| 46:27 | optimized for those conditions, right? them in the right shape and |
|
| 46:31 | Okay, so um we oops we're gonna focus on 02. |
|
| 46:41 | so there's gonna be different responses. , um Atmospheric levels of oxygen. |
|
| 46:52 | more or less go to. Um Lots of things are quite happy |
|
| 46:58 | those conditions. Lots of things aren't in those conditions and many are can |
|
| 47:04 | either way. Give me option. give me auction. I don't |
|
| 47:08 | I'm fine. Okay, so there's be a way then to kind of |
|
| 47:12 | know, what are the issues with ? Okay, well that's what we'll |
|
| 47:17 | . So kind of we're just gonna more at this aspect of the department |
|
| 47:23 | influence. Everything else here is about do we kill them basically? How |
|
| 47:29 | we kill them? How do we them fast? Right. So there's |
|
| 47:32 | terms. Well, look at here and in terms relating to the effects |
|
| 47:40 | antimicrobials and then examples of different types ways to kill them both physical and |
|
| 47:46 | . Right? Most of this we leave Tuesday. Right, so let's |
|
| 47:53 | on oxygen right now called zero Okay. So um if one is |
|
| 48:01 | in oxygen world, whether they use or not, we use oxygen as |
|
| 48:10 | of our metabolism, many things Right? But they still live in |
|
| 48:14 | oxygen world. Right? And so oxygen by itself can be very |
|
| 48:20 | Okay, so too is simply just powerful oxidizing agent. Okay, Very |
|
| 48:27 | . Okay, so, um but has a very special property that we |
|
| 48:35 | on, right? And we'll get . We'll get into this in year |
|
| 48:39 | . But it's a terminal electronic right? It's at the end of |
|
| 48:45 | metabolism, right? Of our Very important. You know, the |
|
| 48:51 | to elect very powerful. That's what call a reduction potential has the highest |
|
| 48:58 | potential, Right? And for So for that reason, you |
|
| 49:03 | it allows us to keep electron flow this way. Okay, so we |
|
| 49:08 | food. We oxidize it get electrons it. Then we shut them down |
|
| 49:14 | oxygen because oxygen is very powerful that the electrons. It goes right to |
|
| 49:19 | . It keeps the whole system It as a result of all this |
|
| 49:23 | this process here. It's not really is you get lots of a |
|
| 49:30 | P as a result. Okay, you going. Having an oxygen keeps |
|
| 49:36 | flow going allows you to produce lots 80 p. Okay, now, |
|
| 49:41 | consequence of that is oxygen can also with other components. Okay, so |
|
| 49:47 | can form what is called reactive oxygen . Okay. And that's where the |
|
| 49:54 | effects them. They can interact with . You take acids and damage |
|
| 50:00 | You've probably heard of food labels. foods with antioxidant properties like blueberries or |
|
| 50:09 | like that, among others. That's kind of properties they have. They're |
|
| 50:13 | to so something to mitigate the damaging of these molecules in yourselves because I |
|
| 50:20 | that that's what contributes to aging and . Okay, so and so ourselves |
|
| 50:29 | are in the same boat. We to have protection for that reason. |
|
| 50:34 | . And so how do you do ? Well, it comes through different |
|
| 50:38 | help protect. Okay, so the of them are cattle a's. And |
|
| 50:46 | peroxide eyes and S. O. . Short for super oxide. Disney |
|
| 50:51 | . Okay, so these are what us. Your cells have these three |
|
| 50:57 | . Okay. Um among the bacterial , it varies varies. Okay. |
|
| 51:05 | so the generation of super oxide radical . So F. A. |
|
| 51:09 | Is a component in up here in aerobic respiration process. So it can |
|
| 51:17 | and form this super oxide radical that can be neutralized by the effect of |
|
| 51:24 | . O. D. Okay, two hydrogen peroxide and then by catalyst |
|
| 51:32 | peroxide eyes to water. Okay. of course is harmless. Okay, |
|
| 51:39 | still fairly reactive. So you have have a way to deal with |
|
| 51:43 | It's less reactive to peroxide. But have cataracts and proxies to take care |
|
| 51:48 | that. Okay so those two Right? So again if you're using |
|
| 51:56 | , if you're an aerobic programs use in your metabolism like us, |
|
| 52:02 | Or if you don't don't use Okay. But you still live in |
|
| 52:08 | world of oxygen, you're gonna need , right? So it's irrelevant whether |
|
| 52:13 | actively use the option or not. exposed to it and these these can |
|
| 52:20 | produced so you're gonna need protection from . Okay that's really the essence of |
|
| 52:26 | behaviors bacteria Archaea will have the presence of oxygen is what do they have |
|
| 52:34 | terms of protection? Okay um do have it? Do they not? |
|
| 52:39 | don't have it? You have to a different strategy to survive. Okay |
|
| 52:45 | let's um look at how you kind figure this out. So there's a |
|
| 52:53 | type of growth medium you can Okay, fluid flag like flight. |
|
| 52:59 | and so this medium has components in that allow you to create a medium |
|
| 53:06 | an oxygen gradient. Alright so we from high to know. Okay so |
|
| 53:13 | a nutshell it's kind of a semi medium um It has chemicals that will |
|
| 53:20 | up oxygen and when you compare it put it in your table course, |
|
| 53:26 | it kind of boils it. So drives gas out. Okay, as |
|
| 53:31 | take it out and then allow it cool, the gas will kind of |
|
| 53:34 | to seep in, but because it's jelly like matrix, the diffusion of |
|
| 53:41 | gas is somewhat resisted. Okay. what happens is you get a gradient |
|
| 53:49 | auction forming in that tube. And so what you then do and |
|
| 53:54 | red color is due to a dye turns red in front of the |
|
| 53:58 | So you should have bright red at top and none at the bottom and |
|
| 54:02 | gradient in between. Okay, so do you do with this? Well |
|
| 54:07 | you do is you take your wire , right? Here's your plate of |
|
| 54:13 | , Pure culture you want to Right? You take that, don't |
|
| 54:17 | just go like this in the right in and out. It's gonna |
|
| 54:23 | inoculation. Right? And so what basically doing is seeding the length, |
|
| 54:29 | entirety of that tube with itself? now? What you're trying to figure |
|
| 54:34 | which in what location of this tube the cells actually grow? Right? |
|
| 54:42 | they grow? Will it be those the ones that grow? Will it |
|
| 54:48 | these guys in the middle? Will those at the top? Will it |
|
| 54:52 | throughout? It all depends on the document. And did they use |
|
| 55:01 | So you're basically looking at the growth that's gonna tell you in what class |
|
| 55:07 | of zero tolerance idiots. Okay, let's start with a question. So |
|
| 55:16 | it relates to the protective enzymes, to protective enzymes they may have or |
|
| 55:21 | lacking. Okay. How they So let's look at this question |
|
| 55:28 | This up. Okay, this Alright. So we've got five strains |
|
| 55:37 | on that medium I just described. , we knocked out of them and |
|
| 55:43 | looking at the growth pattern results. ? So based on the results which |
|
| 55:50 | A through E. Uh is not not lacking these protective enzymes. So |
|
| 55:57 | has them but big but it has levels of these enzymes or maybe is |
|
| 56:05 | one or two of them. So they've got they have some |
|
| 56:09 | Just not the full complement. So might something like that grow? What |
|
| 56:15 | would it show you? Okay. . Remember how the Grady goes? |
|
| 56:26 | auction to none. Mm. Hi . Okay counting down 10 9 |
|
| 57:24 | Right? Yeah. Okay. Um , so let's go through if you |
|
| 57:34 | answer. Okay. Who answered As an eagle? Why'd you have |
|
| 57:41 | eat? Okay. Oh right. , like business. Yes. |
|
| 58:00 | So correct. So e. Is correct answer. So e uh so |
|
| 58:05 | a reminder here. So let's So there was 02 max 02 at |
|
| 58:12 | top. Right? And then 002 here. Right, so it has |
|
| 58:21 | protection. Okay, so it means can it won't be able to grow |
|
| 58:26 | here. Right? So it's gonna at some level below. Okay, |
|
| 58:30 | it's not gonna be at the Right? This guy is that's the |
|
| 58:34 | . And a rope, right? actually kills it. Can't even grow |
|
| 58:39 | auction is present. Okay, So let's go through different types of what |
|
| 58:44 | are. Okay. E is what call a micro aero file. |
|
| 58:48 | So we'll break this down in terms uh, Arab and Arab faculty. |
|
| 58:57 | , So you're a robe. Um use oxygen. Is partner metabolism. |
|
| 59:05 | aerobic respirators like us. Okay. so there's two types. There's |
|
| 59:12 | We're out of the Arabs. And then there's micro arrow files. |
|
| 59:18 | , So auction is toxic to them atmospheric levels. They have to grow |
|
| 59:23 | like 10%, 1%, 5% or like that because they don't have the |
|
| 59:30 | protection if you have lower levels of protective enzymes or missing one or |
|
| 59:36 | So, they can't handle the full . About two that you get at |
|
| 59:41 | levels. So, the micro era . Okay. The so the other |
|
| 59:47 | to remember is, oh, to . Okay, get you more |
|
| 59:54 | Right, So everybody capitalism translates to ADP translates to more heroics. |
|
| 60:02 | so as we look at the ana . Okay, They don't use oxygen |
|
| 60:08 | there fermenters, they could be what called anaerobic. There's fires There reads |
|
| 60:13 | things other than 02. Okay. they can be obligate anna rose where |
|
| 60:19 | toxic. So they can only live areas where there's no 02. |
|
| 60:24 | Um The aero tolerant. Arab that's one that's probably the most confusing. |
|
| 60:31 | , so this guy does not use but it has the enzymes to |
|
| 60:38 | It actually has a full complement of to protect it. Okay. So |
|
| 60:44 | you look at the faculty type, , you call it is calculated. |
|
| 60:50 | . You can live with without Yeah. And if oxygen is |
|
| 60:57 | it can grow like crazy. So it will grow throughout the |
|
| 61:01 | Both the faculty native and the aero grow throughout the tube. Right? |
|
| 61:08 | there's one key difference. Right? right here. Is that alright, |
|
| 61:14 | at the top? So go to lose oxygen. Using more energy. |
|
| 61:23 | growth. Right? That's why there's growth here at the top compared to |
|
| 61:27 | air a teller and a rope. . And so that's the difference between |
|
| 61:32 | two. They're both grow throughout their against it. But the faculty can |
|
| 61:39 | oxygen and where its highest at that different interface, more gross is more |
|
| 61:46 | less homogeneous throughout the whole to write more growth here, is there because |
|
| 61:53 | doesn't use oxygen. Okay. Um that's that's those five types. |
|
| 62:00 | Any questions? Yeah. Yeah. it? The answer is because there's |
|
| 62:15 | be less auction in the middle number . Right. It still has the |
|
| 62:21 | that there's it has a protected some the protective enzymes that allow it to |
|
| 62:28 | auction. So that tells you it's going to be at the max |
|
| 62:32 | Okay. And so that will enable then it has to use remember where |
|
| 62:38 | file use the auction. So it's have to have some source of |
|
| 62:41 | Just not maximum and it's gonna go below the top level because they they |
|
| 62:50 | have oxygen to grow. They're they're like us except they just can't tolerate |
|
| 62:57 | levels of oxygen. So they have option to grow without it. They |
|
| 63:00 | grow if we don't have any capability ferment or respond without oxygen. |
|
| 63:07 | Um So I mean in the microbial , in the bacterial archaea world, |
|
| 63:15 | . To be honest, there's there's of these types of Arabs in fact |
|
| 63:21 | is in fact the types of anything really. Right. Anaerobic and aerobic |
|
| 63:26 | aerobic metabolism is not is not is the majority. Right? It's typically |
|
| 63:36 | faculty actively anaerobic aero tolerant. If anything, probably MicroAire files are |
|
| 63:42 | prevalent than than Arabs in the bacterial world. Okay. We have to |
|
| 63:48 | of you know when you think of that's anaerobic, you might think, |
|
| 63:52 | . It's gonna be in this really off environment. Right. Where the |
|
| 63:57 | can't even get into it at That's not the case right there. |
|
| 64:01 | . Right. You have to think micro environments because they're in your |
|
| 64:05 | All right. You have to open mouth because fermentation goes on your mouth |
|
| 64:10 | cavities. Right? So they're kind occupying the nooks and crannies like in |
|
| 64:14 | guns or you know where there's gonna less less oxygen. Okay. |
|
| 64:20 | uh micro environments can vary widely in around your body. Right? Where |
|
| 64:26 | less auction in some places, more others. So, and there was |
|
| 64:30 | environments you can have and it is Okay. Obviously your gut is one |
|
| 64:36 | one huge in a robe factory. . So anyway, uh the so |
|
| 64:43 | gears here a little bit just for second, a couple of minutes. |
|
| 64:47 | uh this is kind of the second of Chapter five. This is kind |
|
| 64:50 | the how do we kill to get of it? Okay, so basically |
|
| 64:55 | on that part of the growth curve death phase. Right? So, |
|
| 65:01 | remember, death can occur just as almost as long growth. Okay, |
|
| 65:08 | that's what we're trying to emphasize here controlling growth with different types of physical |
|
| 65:12 | agents. And so this is just you, you know, if you |
|
| 65:15 | your favorite disinfectant, right. A disinfectants are gonna have um they're all |
|
| 65:23 | of similar in terms of how they're , but there's a pretty much a |
|
| 65:29 | set of microbes that you test Right. Different types of viruses? |
|
| 65:34 | even fungi showing different bacterial types. you see the oldest ones here. |
|
| 65:40 | . Uh, usually medically important Right. And so, um, |
|
| 65:46 | , you read the label and it you this and that this affects hundreds |
|
| 65:50 | services. This is marketing stuff. , but uh, my interest here |
|
| 65:56 | that they always have typically a big lettering, 99.9% killed, 99.999% |
|
| 66:05 | killed. Right? Or microbes So what does that actually mean? |
|
| 66:10 | that's really what somebody's we're looking for are we counting these things how much |
|
| 66:15 | how fast? Right. So we look at and when they do these |
|
| 66:18 | of studies, they typically have a template that's like maybe too much by |
|
| 66:23 | and square and put it on the or something. And they'll swab it |
|
| 66:28 | see how many bacteria actually there. many microbes are there. And then |
|
| 66:32 | take another square and apply disinfectant and see you know what percent has been |
|
| 66:39 | . Right? And so if you a million cells and then you kill |
|
| 66:44 | as Lysol claims to do, Then should have killed 999,000 of them. |
|
| 66:51 | ? So the point is you still cells left on your little area |
|
| 66:56 | You're not sterilizing the service when you this as a disinfectant. You're reducing |
|
| 67:02 | numbers greatly. Okay, you still have viable cells there. Okay and |
|
| 67:08 | we begin to talk about you know disinfectants and antiseptics work and kill its |
|
| 67:15 | of death. That's what manufacturers of things. That's the parameters typically use |
|
| 67:21 | of death. How how long to One longer death in many. And |
|
| 67:26 | parameter varies from manufacturer to manufacturer. rely on 12 blocks. Uh |
|
| 67:31 | And what time frame? Okay. the point is here this is what |
|
| 67:35 | trying to do. Kill them and them fast. Right? So um |
|
| 67:42 | let's look at this. You're you're aware of my pet peeve of misuse |
|
| 67:48 | the word sterilization. So we had similar to this before a few weeks |
|
| 67:55 | , see if we remember that. And so as we go through this |
|
| 68:02 | more so next time. Yes. any of these treatments. Whether it's |
|
| 68:09 | acceptance disaffection, whatever you are reducing numbers of everything. Okay but obviously |
|
| 68:17 | looking at, are you affecting Right? Because you know certain that's |
|
| 68:22 | you want to try to reduce levels . Okay so uh the time around |
|
| 68:34 | . Yeah I predict 98%. I'll it right, 99%, Right? |
|
| 68:46 | the lifestyle can says okay That's Not correct anyway. Alright. |
|
| 68:59 | Not quite Alright even close. sterilization is none of the above this |
|
| 69:07 | . Okay so what is sterilization? is destruction in my little two inch |
|
| 69:15 | there by sterilizing there would be no sell spore or virus in that |
|
| 69:22 | 0 99.99990 is what it would Okay. Um disinfection and acceptance where |
|
| 69:32 | know what those are ready. That's has to do with what you are |
|
| 69:38 | it to applying it to a living . That's anti sepsis, inanimate |
|
| 69:44 | Walls, bench top, that's Okay. Um so by you know |
|
| 69:51 | can infer from that that disinfectants can be harsher. Right? Think bleach |
|
| 69:58 | as a problem alcohol which is a harsher chemical but it can be quite |
|
| 70:04 | disinfectant. Um And so of course and sepsis. Yes, all microbes |
|
| 70:13 | gonna be affected by this. They're be reduced a number. But |
|
| 70:16 | trying to see what what about the are they the ones being produced? |
|
| 70:20 | are the ones that are human health obviously. Okay, sanitation is kind |
|
| 70:25 | a little different. It is an reduction micro but more surely as to |
|
| 70:33 | you carry out your practices. Think of restaurant reviews, right. |
|
| 70:39 | you have used to be a guy Houston or if you remember him maybe |
|
| 70:44 | . His thing was slime in the machine. So the restaurant reports and |
|
| 70:50 | first one that started this in Houston . So you know food at improper |
|
| 70:55 | um messy floors with food around uh food out at the wrong temperature. |
|
| 71:03 | kind of things are all practices that lead to foodborne illnesses. Right. |
|
| 71:08 | you do things like where the loves clean up, you have proper refrigeration |
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| 71:13 | and that. These are hygienic You do to reduce numbers. |
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| 71:17 | But what numbers but the numbers you have to get to this is typically |
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| 71:22 | by your county or state Health Department that they're the ones that come around |
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| 71:28 | check on these things. Okay, that kind of, what is what |
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| 71:32 | is? Okay, um Any questions that? Yeah. Okay, pasteurization |
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| 71:41 | um is a temperature process to reduce in foods and beverages to relate to |
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| 71:50 | and beverages. Pasteurization talked about that Tuesday. Okay, let me uh |
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| 71:57 | since something is coming up on I have to finish with this question |
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| 72:01 | . Okay, so I realized that is a football fan. So there |
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| 72:10 | no right answer. Whatever you you get credit for, Right? |
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| 72:17 | assuming everybody knows what Kansas city home is that refers to. Okay, |
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| 72:49 | see what I think it's gonna E it's a favorite answer, I |
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| 72:54 | my guess or see maybe it's c , so well there's no Chiefs |
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| 73:08 | I see that. Eagles Texans. . Okay, Texans. Yes. |
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| 73:16 | put any money on that one. , seven. We can see |
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| 73:34 | you can put a title on the . What about the protection? You |
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| 73:41 | put yeah, they should be there on the video points lecture they put |
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| 73:48 | stuff in there. Yeah because there something wrong with your video points |
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| 73:55 | So they fixed it. So I it and so I posted it today |
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| 73:59 | you should see the version. That's should be the right version. |
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| 74:04 | Yeah you will now because it's gone the right thing now. So you'll |
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| 74:10 | . Yeah. Yeah. Yeah they're they can't sexually reproduce right or. |
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| 74:21 | they can. Yeah itself. No will begin to then come out of |
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| 74:26 | state under favorable conditions would be the of nutrients. No stress. Yes |
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| 74:42 | . Whether it's nutrient deprivation or favorable which will be anything that will enhance |
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| 74:50 | growth. Yeah. Question. Well the the basically the D. |
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| 75:05 | . A. Of the of the becomes engulfed in a sport coat is |
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| 75:11 | around it. Okay so when it's then then that then the into a |
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| 75:20 | form social by. Yeah. Yeah is a it's a version of |
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| 75:30 | But yeah it is different. That's right. Sure let's see. |
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| 75:42 | . Okay. Yeah I wanted to you something. I would say grill |
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| 76:02 | and look at the day one lecture 17 January and there was like a |
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| 76:08 | minute or 10 minute section in That explains exactly that question. So |
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| 76:14 | a combination of stuff it gives you little here's here's how here's what to |
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| 76:18 | . Um I would say give that look and you have questions, let |
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| 76:22 | know, look at that first and that makes sense. And then uh |
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| 76:27 | then um if you you need just let me know. Okay. |
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| 5999:59 | |
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