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00:09 mm hmm. Mhm. Ah Welcome folks. Um So this

00:29 remember that we have first exam uh this week thursday and friday. Um

00:37 questions about content? Look at the review sheet. Um Got questions uh

00:45 office hours. Of course you can't officers can always arrange for another

00:49 it's fine. uh chapter five So I just did that before flat

00:55 uh weren't able to cover those on um unit exam or a quiz

01:03 So I just put up some there's maybe six or 7 questions um After

01:11 questions the answers were on on last , so it's not for Great or

01:18 but just so you can have some to go through the latest chapter five

01:23 . Um and that's in the unit one folder. Okay. Um Let's

01:31 . So remember also that although there's a couple of problems on the exam

01:37 are the tax relation type with the growth, you can't have a calculator

01:43 you. Not a cellphone calculator obviously a calculator of any kind, handheld

01:48 casa will be made aware of that that you won't be bugged because you

01:53 a plague on a calculator or Okay. Uh what else? Smart

01:59 . So this week there's no no quiz because we have the examiners

02:03 Smart work. Do sunday. There the The Chapter five due on Tuesday

02:09 nothing nothing next Sunday will transition to next unit. Um Next monday monday

02:19 start unit too. So in that six, 13 and 14. Okay

02:27 And that material will be up tomorrow . So if you're eager to start

02:33 that already, you can if you to. Um So they were gonna

02:37 up Chapter four. that's a that's mistake we're gonna talk about in those

02:43 at the beginning, the definitive check that's all we got left and then

02:47 for five as short as well. and so Wednesday we'll get through most

02:54 pretty chunk of five today. It's that long but it means that Wednesday's

02:59 they're not gonna be along. I'm going near for the whole period.

03:03 luckily I don't know, 45 minutes something like that. Uh to finish

03:09 the last of Unit one which is end of Chapter five. Okay.

03:14 let's see um Any questions. yeah, yeah. Formulas.

03:25 That formula. You need to memorize formula that that will be, there'll

03:28 a question saying blah blah whatever. then there'll be the equation at the

03:34 of it the here here here. . Um alright, so let's start

03:42 the question. Okay, so this into um Plympton who may or may

03:50 not talked about yet. Let's see you, can you answer this

03:55 Sorry, surface and the ability to our absolute requirements for which one of

04:07 ? Mm hmm. And do I've uploading the clicker points on blackboard.

04:14 if you see, don't see I'm Oh is is it giving you

04:25 on your on your and the Okay. One more time. What's

04:33 what's it doing to options? jesus, okay. All right.

04:45 then that yeah, I'll just give credit for it. So But what's

04:49 the what's the answer here? that's the that's biofilm formation. So

04:55 completely weird. Why don't you give two choices? All right. My

05:00 just give you credit for that So, um Alright. Hopefully other

05:04 aren't like that. Um Okay. yeah, so service. So also

05:12 , so just we talked about this the end last time or towards the

05:16 end of covered biofilm formation. So uh you know, it's it's

05:24 couple of things to remember. You , obviously the surfaces, the surface

05:29 required uh to be able to stick that surface and not receive having the

05:34 of a pillai, we're gonna be to be able to stick to that

05:40 uh initially um uh being reformed the . So remember the biofilm is not

05:48 just a random event? Uh It this material that kind of sticks everybody

05:55 . Right? The former film. it's it's a planned event if you

06:00 it's a gene um encoded event. genes involved. Um It's it is

06:08 but a random process. Okay. to be able to one thing when

06:11 remember the last time I mentioned you to visualize kind of in this diagram

06:16 that there is a source right of supplying this thing. Right? And

06:23 um whether it's a pipe, you look what's going through your or shower

06:28 maybe or something. You know there's there's a flow of nutrients which because

06:33 what drives it. So it's the of nutrients it has to be there

06:38 to create the favorable environment. And what the quorum sensing mechanism is

06:43 Is presumably if you have a lot members there then it is a favorable

06:49 because they aren't settling. They're they're dividing. And so that's how you

06:55 that threshold level of these chemical signals they get to a certain threshold then

07:01 triggers the process. And so The be well they wouldn't be gathering and

07:07 bigger numbers and getting to that threshold it work. It's an indicator that

07:11 a favorable environment. So that's 400 . Okay, so the chemical signals

07:17 as they are reached that threshold that themselves actually induce expression of. And

07:24 jeans the first of those being the party saccharine information. So that's a

07:29 Zachariah protein. It can be proteins there as well think of this kind

07:33 glue holding everything together. Um and of course growth proceeds from um on

07:41 surface and two dimensions to three dimensionally it gets of course massive with billions

07:47 selves um the environment in the You can have cell types in the

07:55 that may differ somewhat from those on interior. Okay. Um The ones

08:01 the next year can be more susceptible say antibiotics if it's a it's a

08:06 important biofilm, um a pathogen on catheter or what have you uh surface

08:14 a heart valve maybe or something uh uh antibiotics may work better against those

08:22 the outside of biofilm represented on the because they can't penetrate that well.

08:28 Maybe that sets up some kind of types inside that biofilm. So uh

08:34 you're pathogen biofilms can be quite um because that in certain trying to get

08:41 of it, uh the dissolution process kind of our film falling apart.

08:47 you will, if nutrients cannot be , write the nutrient flow is shut

08:53 . Now members can't aren't surviving. it's like okay, time to go

08:58 . And the dissolution process occurs. so remember, we have 22 cell

09:05 or types that are kind of basically swimmers. And those that once they

09:10 on the surface, they tend to that. Uh so plant tonic for

09:16 cells versus uh sticker cells they call . Okay, so those are kind

09:23 the highlights really of the biofilm formation . Any questions about that. All

09:32 , So um So in those So next is our last thing to

09:38 about in Chapter four. Um So is just to illustrate uh if you're

09:43 a lab uh you'll look at these if you're in the 5 30

09:50 Um But or tomorrow's lab of course the uh we're actually doing a gram

09:58 . We're not doing a sports statement because you can look at you can

10:01 you can see sports not doing a fan and you can tell from this

10:04 here. So um they look like tile. Well they are refract tile

10:12 . So here's an endospore in in cell. This is probably a what's

10:18 a free in those pores been Okay. Um This is likely just

10:23 completely vegetarian called vegetative cell vegetative The functioning fully functioning healthy normal

10:32 Um we'll talk about that in a . But this is this is material

10:36 I think it's the one from the 250 million year old bacteria found in

10:42 , Sea salt needs Carlsbad. So the one below 40 million year old

10:48 found in the stomach of a be in Hamburg. Okay. So the

10:55 is in those spores spores are prevalent old. The kingdom's. Okay.

11:03 plant, What have you? Um forms are typically in the reproductive

11:10 Okay, fungal sports for example. . Um uh There are other types

11:16 bacterial sports that aren't like this that involved in uh reproductive aspect. Um

11:25 protozoa ones can form not necessarily sports they form a form called a

11:31 Okay, so it's a cyst or . These are all typically what we

11:37 dormant dormant forms viable. Okay. but not really actively reproducing.

11:46 So I think it was plant seed plant seed represents an embryo inside that

11:52 . Okay. That will not begin grow until favorable conditions. Right,

11:59 . Right. Um So these are similar that the as you can

12:05 these things remain dormant for millions of and then revived under the right

12:13 And so um so that's the thing the endospore is particularly unique. So

12:19 saying sport, but when you put window in front of it that raises

12:23 to another level of resistance. And uh frankly why we use the

12:30 in lab to sterilize media, we the autoclave to basically kill endorsed sports

12:37 may be present. Okay. They particularly resistant resistant to chemicals. Um

12:44 uh extremes of ph um temperature. , radiation. So they can be

12:53 uh um already formed. Okay. so and they particularly form under these

13:02 of stress conditions, lack of nutrients elevated temperature. What happened? Um

13:09 the endospore themselves? The endo spore are only in two groups of bacteria

13:16 and bacillus. So clostridium you're familiar the is tetanus bike system. This

13:23 caused by members of that group. anthrax was really the only dangerous one

13:30 in the bacillus group. Most bacillus pretty much benign soil, they're both

13:36 of soil inhabitants. Okay. Um clostridium group and they're both grand

13:46 And what really differentiates them both. there Um we'll talk about this as

13:51 start. Chapter five um behavior in of oxygen. Okay, so bacillus

13:58 typically aerobic like us. Um maybe aerobic, what we call micro era

14:06 . But Australians are obligate anaerobic, live in the presence of oxygen.

14:12 . Or very very low levels of . They don't use auction. Their

14:16 anna ropes. Okay. And uh I mentioned, there are a number

14:21 disease causing types in the group and for producing a plastic toxins.

14:27 toxin of botulism toxin, gas, causes tissue death in contaminated balloons um

14:36 so forth. So there we'll talk them in the context of diseases at

14:42 end of the semester. But but , those two groups facilities and frustration

14:47 these. And those sports which is unique, unique type of sport.

14:51 so two former sport is not a thing. It takes several hours.

14:57 number of genes involved. It too a is a organized process.

15:04 coordinated process ending up with a um , proceeded by D. N.

15:11 replication and then a what's called There's an unequal compartmentalization occurs or in

15:19 smaller compartment. The endless performs. in the process we formed this double

15:26 we form, we deposit a layer pepper look like. And in their

15:32 different types of what are called coat . Uh So it becomes a very

15:37 um resistant structure. Okay. And it used the terms mother cells,

15:43 floor. Those are the two unequal that form um the the removal of

15:51 . So water in a Sell you as well. My life is pretty

15:55 70% water. And when temperatures it's the increase in water temp that

16:02 does the damage inside of the Okay. Um so or if it

16:08 ice crystals form and cause damage So it's it's the just the amount

16:13 water present in cells that can actually the thing that kills them when extreme

16:18 of temperature may arise. And so taking water out, that actually helps

16:24 terms of viability. Okay. And when those four formation occurs, a

16:30 of water is taken out. So gotta go down from about 72,

16:33 20-30% of water. And that helps keep it viable when it's in this

16:39 dormant state. Okay. Um The process is really just coming out of

16:45 end of those four states into a cell. Okay. And so we'll

16:51 over those terms here. So When you look at a endospore former

16:59 culture, I'm just throwing it not for the purpose of getting into form

17:05 . But you're just growing it for reason, you'll typically see three types

17:10 selves. Uh we're looking under the . Okay. You'll see. Uh

17:15 these are what we call the completely is a gram stain, completely bluish

17:22 colored cells. This guy, this , this guy. These are completely

17:32 . So let me get hold There were these, Yeah, pretty

17:41 ahead of mark. Okay. Um again, you see on the

17:45 right vegetative cell, there's no speculation on. It's kind of going through

17:49 hole, normal growth phase, so divided etcetera. Um But then you'll

17:57 cells that have this fact out That's the end of the sport for

18:01 . Okay, so these cells are the process of and speculation.

18:07 form the end of sport. You have so they have completed that

18:10 will stay in the south. It's a free end of sport at that

18:15 at that time. Okay, so the end of the process. That's

18:18 happens. Okay, so um the of sports, I don't don't worry

18:27 memorizing these things down here. I wanted to mention it because with these

18:33 groups of endospore formers, you can the fact that they species will form

18:40 how this force formed and how they . And you can use that as

18:45 way to identify members of those And so the endospore itself can cause

18:53 and it can kind of blow up swell the cell like you see here

18:59 here here Or it may not it just you know not swell like

19:04 A non sworn numbers are swollen. position of the sport, is it

19:08 one end is a kind of toward end. So sub terminal, this

19:13 terminal. Is it central in the ? So you can have three

19:17 Can be swollen, hands swollen. Something maybe club shift is typically what

19:22 call that. But anyway, the being you can identify these members based

19:28 their sport and those four characteristics. . Um Now the cycle.

19:35 so speculation begins with um DNA replication so the chromosome replicates but it forms

19:46 elongates alright. Taking up the whole of the self and that's what we

19:52 an actual filament. Okay. And it will uh copy goes into each

19:59 . So now we get the compartmentalization unequal in size for the mother cell

20:06 than the four sport for sport is the end of the four formation will

20:11 occur. Okay, the mother cell uh function is really to direct the

20:21 chelation process. There's a bunch of involved. And so the mother cell

20:26 have proteins being expressed and those are proteins that are acting on the force

20:34 DNA and directing the the process. . And so once the mother cell

20:41 kind of started that process then the sport kind of just does its

20:47 And what happens is that the mothers actually engulf that forced board to form

20:55 double membrane. Right? So you the double membrane here and then that's

21:00 people like him will be synthesized inside . And that double membrane. Okay

21:08 you form a type of light can within there. You have some other

21:12 being deposited there as well. What called coat proteins. Okay. Um

21:17 unusual molecule called diabetic Olynyk acid or . P. A. Is thought

21:25 be involved in binding to the chromosome the chromosome calcium. Mines are thought

21:32 have some process in the water extraction of the forming spore. Um Also

21:40 in nature. So calcium gets lines deposited in there and so were forming

21:46 what they call ex operandi um um Spork hoped eventually is the at that

21:54 that we're pretty much done. And so so from here up to

22:00 from up to here throughout I'd say stage here. So all these stages

22:09 here where we see that the like recall the gram stain the cells with

22:19 endospore in the middle right? The cell containing an endospore. That's what

22:25 see at these stages. That's what cell would look like. Okay.

22:30 like real quick beep beep that would like this. Okay so it's just

22:40 don't know by looking at the cell a microscope. We don't know which

22:45 of these is not identifiable with light . Which which of these stages in

22:50 . It is. But that's what would look like under the microscope.

22:54 . And finally, the free sport be the seller vegetative cell kind of

23:00 around it in the end those Those four. Okay. Of

23:04 In that state uh that we've seen can stay like that for thousands of

23:13 . Okay. Um More typical of is uh is not that long but

23:20 know, it can be as long it takes until there's a favorable

23:24 Okay. And they'll they'll germinate. . Um so that's in the nature

23:30 the end of this is only with windows. Because you hear the terms

23:34 a lot. But this this is for this for this for this

23:38 These two groups. Okay. Um questions about that. Yeah.

23:46 So And that concludes chapter four. . And uh Let's move on to

23:57 . And so we're only covering a of this which is focused on what

24:02 call zero tolerance, which is bacterial if you will in the presence of

24:09 . Okay. Uh it's it spans spectrum of you know how the growth

24:16 you'll see. Okay. And then talk a little bit about physical chemical

24:21 uh control of bros. Okay, first with oxygen. Okay.

24:29 we haven't yet. Yes, we'll this in unit two. Uh talking

24:36 respiration and all what's involved with But um in a aerobic world that

24:44 live in. Um number one oxygen is a very reactive molecule.

24:53 . The molecule that likes to grab . Okay, and become reduced.

24:59 a powerful oxidizing agent. Okay, course we use it to help us

25:05 . Right with the old. If recall the uh auction at the end

25:10 our electron transport chain. So you auctions the terminal except er right,

25:17 worry about that yet. I'm just to you its role here. And

25:24 in the in the process of we then form water. Right,

25:27 picks up electrons are reduced to Right? And so that's how we

25:32 that to breathe. And so um oxygen being very reactive can also interact

25:42 molecules enzymes involved in the process. . And the results of that are

25:50 side reactions that occur. Okay, these species you see here super

25:56 hydroxide, radical hydrogen peroxide all reactive themselves. Okay, Especially super

26:04 So um the uh and that can damage. So those molecules will interact

26:11 proteins create acids uh causing that. . Uh same in our own

26:18 Same thing happens. Okay. And too, so the cells would have

26:24 as a result of the damage You have to have protection.

26:29 And the protection comes in the form three different enzymes. Okay, so

26:35 call these three reactive species not surprisingly oxygen species. Again, this process

26:44 . We just happen to looking at cell. That is an aerobic inspired

26:49 , same molecules Okay. Are generated the organism uses oxygen or not.

26:59 . Because auction can still wrapped in molecules. Okay, so you don't

27:05 this could be a a bacterium that doesn't use oxygen at all. But

27:10 will still be affected by it and still get these species form and it

27:14 damage the cell Again unless there's Okay, in the form of these

27:21 ourselves have all three of these. . If you've ever poured hydrogen peroxide

27:26 the cut, you may have had your finger or leg whatever. Well

27:30 as a bubble. Right? The is due to the catalyst that those

27:35 have. Okay, neutralizing the effect the hydrant profit. Um anyway,

27:42 three of those those three molecules Okay, afford protection against damage.

27:50 , so here is how auction can with this is a component that's in

27:57 respiratory chain over here. Okay, it's also present in other other cells

28:02 don't use auction. It's it's it's an uncommon molecule. And so the

28:09 is the formation of super oxide Yeah, now that's one step.

28:14 , so how do we neutralize Well, S. O.

28:17 Okay. Super oxide. Disney taste S. O. D. For

28:21 . Okay, that will combine with to form hydrogen peroxide still reactive but

28:29 peroxide is not as bad as super but still bad. Okay, but

28:33 the catalysts and paroxysms can take care that. Okay, the end result

28:39 basically to form water. Right? that's not harmful obviously. So so

28:45 catalyst and proxies do their job to the um reduction of these reactive

28:53 Okay, so um so when we at how bacteria behave in the presence

29:02 air I. E. Oxygen. . They will have various responses.

29:09 basically five types of responses. We'll . Okay, there's those it's all

29:15 correlates to the protection they have or have for level of protection. So

29:27 may have some protection. Maybe have or more of the enzymes. Maybe

29:32 have all three but they don't have at maximum levels. Right. So

29:36 gonna be variation. Okay, but behavior they have in terms of growth

29:42 in the presence of their directly relates what do they got in terms of

29:48 . Do they have all of Some of them not not the maximum

29:52 or do they lack it completely because do not have any of it.

29:58 , and that's gonna tell you what patterns will will be. Okay,

30:03 let's look at Okay, this Well, not yet questions coming up

30:08 this. Like Okay. How do detect these patterns? Okay, so

30:14 have a medium that will has a of options hide the top or the

30:21 . Okay, so we create that this medium formulation called fluid dialogue

30:30 Okay, so it contains a dye there that turns pinkish when oxygen is

30:37 , less pink when there is less present. So so you see how

30:41 tube is dark red, dark red pinkish at the top, and less

30:44 you go down because the levels of are going down from top to

30:49 Um It's a it's a um it's jelly like matrix. It's not completely

30:57 and it's not completely solved. It's between, it's kind of a jelly

31:00 matrix. Um There's chemicals in there can bind the pockets. Um When

31:07 make this you um of course boil put in the autoclave that will drive

31:13 any gas in there. Okay? then as it cools down and air

31:18 to diffuse back in it. Um because it's a jelly like matrix air

31:27 and slowly. Right, so go it sets up a grading because those

31:31 chemicals kind of bind up oxygen. the net effect is uh to create

31:37 grading. And so um the um when you annihilate this medium,

31:46 you're gonna take a wire loop. ? And you will cede, you'll

31:52 take some binoculars and you'll just go the whole length of the tooth with

31:57 . Okay, so what you wanna is basically seed that entire tube with

32:04 through the whole length. Okay, what you're doing. So then what

32:09 wanna do is incubate, allow themselves grow. Okay so then what you're

32:13 is asking is which in which area the tube? One of the

32:20 I see it begin to replicate because at the they're at the part of

32:24 tube they like to be at from best growth. Okay. So is

32:29 going to be here? Is that only place we'll see growth? It

32:34 be there, we'll be there or it be throughout the whole too?

32:39 right. All the pimps, Goes back to what the protection it's

32:44 . All right. Um That will where it will grow in that

32:48 Okay. Um because even though you the whole the whole thing top to

32:55 itself, we're only depending on what what its capabilities are. The only

33:03 cells that you see that that will begin to replicate will be where it's

33:07 for. Okay so uh so with in mind now we can get the

33:14 . Okay so um let me reset . Okay that does it give you

33:23 answers again, jesus christ Hold on me see if I can fill it

33:28 that here, just real quick mm . I I don't I can't I

33:41 do it from here to go back the other software. So alright you

33:45 there's another three questions to try. . Alright so this is rude

33:50 five strains are grown medium uh to the growth pattern. So based on

33:59 results, which strain has S. . D. Catalysts and proxies but

34:06 reduced levels of these or maybe missing or two of the enzymes.

34:13 Um So mm hmm. What would your guess on that one? What

34:22 be your guess which strain? Based these patterns? Which strain A.

34:27 . C. D. Or Has these protective enzymes but but not

34:34 maximal levels or maybe missing one or . Okay. What respond? Which

34:41 can you eliminate? Start there. ones can you say? It's not

34:46 at all, can't we? Okay. Um See see because she

34:55 what complete an arrow? Right. . Stick at the bottom of the

35:03 . But there's no oxygen. that's the only place that grows.

35:07 . Remember we actually we seeded this tube. Yeah. Right. But

35:17 but only the ones down here are ones that actually continued to grow.

35:22 . So yeah, I wouldn't be it. Um You wouldn't think it

35:27 be a right because that's up there the highest levels of oxygen. The

35:32 levels of oxygen are at the top the tube. Okay. So that

35:36 has the full complement of enzymes and normal amount. Okay, so um

35:46 B. And C. Okay. two our um grows throughout.

35:55 And but there is a they may the same but is there a difference

36:03 B. And D. Yes. the difference at? That's right.

36:10 is more even distributed. Be tends have more cells up here. All

36:15 . It grows throughout like need does there's more cells at the top.

36:18 the more cells at the top because why or auction it means more because

36:26 means more different. Well, because you're on the right track.

36:31 more opportunities more growth and there's more because not more nutrients. But it's

36:41 has to do with what we haven't about it yet but it has to

36:43 with um an oxygen metabolism. so an oxygen using the tablets and

36:49 his 90 PS more https generally always more. Okay. So that's why

36:57 indeed differ. You distinguish those two looking at the top because B is

37:04 we call a faculty native animals. . Like an E coli just like

37:10 . Um If there's you know in where there's oxygen present, okay.

37:15 will grow uh and it will grow a little bit more because it's just

37:20 nature of an oxygen using the tablet you more a little more https.

37:25 De is what we call a and is on the next slide anyway.

37:31 d is an aero tolerant and a . zero tolerance being the operative

37:37 Okay. You can tolerate oxygen because has a protection but it doesn't grow

37:44 at the top because it does not oxygen at all. They're typically fermented

37:51 . Maybe they're anaerobic or aspire ear's , but they don't use oxygen.

37:56 there's really no difference for it at top compared to a faculty of

38:00 which can use oxygen. Okay. B Is the micro era file?

38:10 . And um I would say that probably what the answer would be to

38:16 one. Okay. Um It has it uses oxygen. Okay? Um

38:24 because it has reduced protection. It's going to be sensitive to high

38:30 of oxygen. So like In the it's 20% 02 atmosphere conditions,

38:37 Um It likes 10%, Something like . Okay. And so uh so

38:46 me just flip to the slide so can break these down. You

38:52 I break them down kind of this . A robe and a robe,

38:55 , faculties can go either way. . Um So Arab types, you're

39:02 . Arabs like us. That's what are. Um And the micro era

39:08 . Okay. So the key thing the era of is they use oxygen

39:14 respond. That's the only way they Okay, Must have it. It's

39:20 that one type is a little more to the atmospheric concentrations. The micro

39:26 . Okay. And to be in a precarious world, most things

39:32 really micro aero files or anna Rome's caves, sheer numbers. That's many

39:41 that's kind of the categories they fall . Okay. Um You're gonna robes

39:49 not. So in contrast to the do not use auction. All

39:55 Be the ferment and or inspire and , which we'll talk about later,

40:01 they don't use auction. Okay. so now the difference between these two

40:07 the protection, right? Obligation. don't have any protection against those

40:11 So there that's toxic to them. , So they're the ones that grow

40:15 the bottom of the tube. Uh paws treaty is in that

40:20 Okay. The aero tolerant and Arabs not use auction, but it's not

40:26 they have the protection against it. have the S. O.

40:29 The catalyst etcetera. Okay. So can go throughout the tube.

40:36 Um but they just don't grow more the top because they can't use

40:40 Okay. Your faculties of types can can use 02. If it's

40:45 you don't have to use it. was not available. They can grow

40:49 way. Okay. E coli is faculty in general. It can grow

40:55 with oxygen. It can grow, can ferment, no auction can and

41:00 can also respond to an honorable thing do all three of those things.

41:04 know what it does, how it , depends on what's available to it

41:07 what the conditions are. Okay. um so so of course it has

41:13 protection has a complete protection against the effects of options? Okay. Um

41:22 questions. Yeah, but it's Do you still want to take

41:30 No, because I'm gonna get free you only had two choices on your

41:33 . So it's not working right? got if you're here you got you

41:40 golden. No problem. It's it's to me to do that. Figure

41:45 what's going on with this. But not penalized for that, so,

41:50 hmm. Yeah. Okay. So you have so if it has the

42:04 the serotonin and a robe and effective a robe, you would guess would

42:10 the full complement of protection because they throughout the whole two top to

42:14 Right? Highest levels of 02 to . Oh, too. Right.

42:19 uh the Albuquerque and a robe doesn't any protection. Right? The arab

42:27 the top. So you're gonna think gonna have it should have the max

42:30 , right? All three enzymes at the high levels. Right? So

42:37 what's that leave us with the micro have protection, but not the maximum

42:44 . So that tells me, well, it can use oxygen or

42:48 lower levels of oxygen the time. it wouldn't be at the top.

42:53 . Wouldn't be throughout the tube. ? You would think would be somewhere

42:57 the middle. That's kind of the process. Yeah. Makes sense.

43:05 . Um Okay, so we just about that. All right. Um

43:15 , so now talk a little bit get into physical chemical control methods to

43:24 microbial growth. And so we talked So number one we we might be

43:30 whole batch growth curve? So the here we're talking about now we're focused

43:38 the death death rate, death the phase or stage of the curve.

43:45 this is all about what kind of can we use to control microbial

43:50 Um The and of course there's different for that. Obviously whether it's not

43:57 setting, hospital etcetera, four million restaurants, right? Or other services

44:04 you know um can be a situation people can get infected. Okay.

44:13 you always obviously when you're testing different and seeing how they control growth,

44:21 target pathogens but of course everything is to be reduced and leveled.

44:26 But you specifically you're trying to target for the most part. Okay so

44:31 you're testing your treatments, whether it's disinfectant and accepted will have you you

44:38 have a set of target pathogens, ? And they're pretty much common among

44:44 the manufacturers of these things. This just one example where you see the

44:49 virus uh cold virus, um salmonella . Coli. So and you always

44:57 test among bacteria, gram negative gram because they will you you'll see differences

45:02 the nature of the cell envelope. , so um but what they all

45:08 do as well our state as well the house effective. Right percent killing

45:16 they produce? Okay 99% 99.99 9.99 happened? Okay. So what what

45:26 that really mean when we talk about one of the parameters they're used to

45:37 two evaluate or two uses the metric cell death. That's what we call

45:45 logs of death. Okay So when look at for example an area that

45:51 um a million cells. Right? when they do these things they typically

45:56 like a little especially with the surface . Ah They'll have like a little

46:02 that's like two inch by two and open in the middle right put on

46:07 surface and they'll take a sample and like a cell count and then take

46:13 area and then maybe spray with disinfectant then let it sit there and then

46:18 another cell count kind of how to these things. And so if we

46:22 like a million cells and then we disinfectant for example. And that disinfectant

46:28 well 99.9% killed. And then we're off 990,000 cells in that area.

46:36 so certainly uh significant. Okay so we're down to 1000 viable cells.

46:45 ? Um and we can keep going in that instance we've we've done three

46:49 of death. Right? And so talked about this thing called the

46:53 Value shortly and the D. Value how how long to get one log

47:00 death? Okay. So of course want to have a time element in

47:04 to evaluate as well because you might how long it takes? Three days

47:08 to get three logs of that to five minutes to take. Three

47:13 Okay. So time is always gonna ported Hellman here obviously. Okay.

47:20 And this can vary. Other manufacturers a metric called 12 and then we'll

47:26 how long it takes. 12 blocks death. Okay, so, um

47:31 , so let's look at this Well, let's throw out an answer

47:36 me because obviously the quicker you only two choices here. So don't

47:42 this goes back to I think I this before about my pet peeves is

47:47 bad use of the word sterilization. is sterilization pasteurization? Yes. Yes

47:56 no. No, of course Is sterilization is disinfection? No,

48:03 is an exception. Sterilization results in final reduction of the microbial population by

48:10 0.9%. No, it would be . Right? 2 0. So

48:18 means destruction of all cells viruses, sports spores. Okay, what

48:27 No detectable signs of life unless you something. Okay. Yes, there

48:34 um an autoclave can certainly do But you can't autoclave. Everything.

48:40 and there are what we call sterility . Mhm oxide is one. We'll

48:47 about gas. Uh certainly gamma or rays can be a sterilizing agent.

48:53 there are certain agents, Even liquid that can be a sterile and

48:59 mm hmm. Pretty damaging. But still it's still considered disinfectant.

49:07 Uh it's there are things that are to bleach. I mean, it's

49:12 . Certainly good. But it's a it's not a sterilizing agent. Um

49:18 see. Okay, so this we already have a disinfection exceptions.

49:23 the difference, terrorists. Inanimate disinfectant, walls, countertops uh and

49:31 this tissue. Living tissue. Um for that reason, of

49:38 disinfectants can be um higher concentration that be more harsh if you will.

49:45 . Um and accept antiseptics. Of not because you're playing to living

49:50 You don't want to damage the Um, sanitation is a little bit

49:55 . So the disinfection and accepts Um of course target pathogens.

50:02 Um, sanitation is more overall reduction microbial numbers through sanitary hygienic practices.

50:11 , example. That example I give uh Mhm. Fast food place or

50:18 restaurants, whether you're a server or worked in the kitchen especially grew up

50:23 the kitchen. You know, you , what do you do? What

50:25 have to have, sharing it right? You have gloves on.

50:31 you do practices to minimize transmission of pathogens, right? You wash your

50:43 when you go to the bathroom right , we all know there's a sign

50:47 the door before you get out. said if you work here, wash

50:49 hands okay. Before um uh in the kitchen leaving uh storing food at

50:56 temperatures. They leave it out at temp for a prolonged period of

51:00 Um Free cleanliness, you know, the floor clean and countertops clean things

51:05 that. These are all hygienic practices can do himself reduces overall numbers.

51:11 you do use disinfectants can be a of that process when you're cleaning off

51:16 , um washing your dishes right? go throughout high temp kind of process

51:24 sanitize. Sanitize. Okay, so for the purpose of minimizing transmission of

51:31 food borne agents. Okay. And what sanitation is. Okay. And

51:38 yeah, it says reduced numbers to to what level. And those are

51:43 that are are specified by. Typically county health Department will specify the level

51:51 then they'll audit we all know I'm sure you probably heard the friday

51:56 reports on on the local networks and that's what they're doing. They're checking

52:01 into restaurants, checking to see that are following proper protocols, etcetera.

52:07 . Um All right. Alright. a little bit about terminology as it

52:13 to killing of microbes bacteria, static bacterial politic. Okay, so these

52:24 are kill they obviously they killed kill actively kill cells viable cap. So

52:36 we don't go into the technique a account. But what you're doing is

52:43 we take an environmental sample. You to know what the bacterial county

52:47 Okay. Let's say we have and worry about writing all this down because

52:52 just for explanatory purposes only. So we have a sample,

53:00 Bacterial cells. What you do is take this and you dilute right?

53:07 may do it several times. And you put them in little test

53:14 . Okay? And then you could them out far enough, okay?

53:21 you take a sample and you'll get on plate. You're trying to get

53:29 between 30-300 on the plate. What call a countable number of copies.

53:35 been done for decades. And so worked out the math that statistically to

53:39 accurate to be in that range. . And so um And what you

53:46 is you count the colonies. So you have 42 climbs on the

53:52 . Well then, you know that know the number of delusions you took

53:55 get to that 42 cars, Gives you the what's called a viable

54:04 viable because what you're counting on the living sounds right. And each the

54:11 is that each colony Arises from one . Okay. So in that

54:18 you're counting 42 cells. Originally there just 42 cells and they made each

54:22 column. Okay. That's why you to divert these out so much because

54:26 cells are super tiny. Right? if I just took a sample from

54:31 very concentrated broth like say right there no delusion and I put them on

54:37 plate it's just gonna be a a because we call it a wand.

54:42 there's no it's just a lot of right? You can get a bible

54:47 from that so you have to keep looting amounts. So the cells are

54:51 apart far enough that they form individual and that takes you know doing them

54:58 pretty far. Okay But once you that you get a plate with accountable

55:03 and you can do the back calculation you can estimate what's called C a

55:10 . Okay per mail see if you calling the forming unit. Right?

55:14 none of this stuff I'm gonna You can do this and laugh and

55:17 later just to show you how do get a viable can't. Okay and

55:23 what you do is um so the is when you've added okay Added in

55:31 three treatments here we've got a growing to which we've added a some kind

55:39 antimicrobial. Okay at the arrow. let me see. Okay what's happened

55:43 we've done that? Okay so the solid line is a viable camp so

55:51 can see that with bacteria seidel materialistic counts drop. Um But you're a

56:03 agent is one where it's inhibiting Alright it's not actively killing cells but

56:10 not growing Okay once you see the . Okay now the dash line.

56:17 so the dash line is total Okay? So in this protocol,

56:24 you can do that by looking under microscope. Okay. Is there any

56:30 ? And you'll see sets obviously. if you're looking at looking at samples

56:36 the microscope from out here. All , up here, you see that

56:46 and dead cells look the same, ? For And you can't distinguish.

56:53 , under the microscope. Under the . And maybe the library that sells

56:57 the same they're dead. But you count them as a as part of

57:02 total can't. Right. So that's here in total headcount, it's

57:08 Right? They're not they're not growing ? The dead cells and live cells

57:12 basically pretty much people. Okay. no debt increase in the cells.

57:17 in here. Back to the Okay. Number one electrolytic bachelor recital

57:26 counts going down. That's a common . Right? But then the difference

57:32 is a total count flat total Okay, so again, it's about

57:40 in dead cells. Okay. It a bacterial lyric agent. What's about

57:46 catalytic agent Doing license is the operative there. It's blowing up to

57:53 Okay, so a live cell you distinguish. So the only cells that

57:58 visible are live cells under the Okay, well in bacteriological agents are

58:05 down because bacteriological agent is blowing them . Okay. So you don't see

58:12 . Right? Don't don't see anything everything's going down. Right. Numbers

58:17 actively going down because the dead cells actually blowing because you can kill the

58:22 and leave it intact but you can kill a cell and blow it

58:26 Yeah there are eight. There are that work on the membrane,

58:32 resolve the membrane cell falls apart. agents kind of work inside the cell

58:38 be blocked from the synthesis and Children that way but they don't blow it

58:42 . Okay so back to the silo lyric agents. Right? The difference

58:47 those two um the you know with bacterial agents and I'm not sure that

58:54 are even used uh in a medical because well certainly if it's a gram

59:01 infection, remember the gram negative Nintendo ? If you give it back to

59:06 agent then you're blowing up and then in the toxins being released. Okay

59:12 part of something you wouldn't want to the gram negative infection. Certainly depending

59:16 severity of infection. Um but you really the difference in metro side

59:23 they both kill one just killed by themselves. One killed by not doing

59:27 . Okay, the material side of , they're not lowering the viable count

59:35 they are inhibiting growth. Okay. and so just just like, nope

59:43 here, this here is just meant reiterate that difference in how they look

59:51 the scope. Okay so here you and wrote A versus robi.

60:01 The total accounts. This will be total capital received many of these

60:04 Okay. Mhm. No change in head. Um It's kind of flat

60:13 then robi you see self disappearing as look at them under the scope which

60:20 indicates a bacteriological kind of effect right . Okay. Effect his sons are

60:36 blowing up so to speak back to . Um uh Any questions about

60:44 Okay. Yeah. Really? Yeah is again he's back to definition killing

60:56 . You know sell spores, A sepsis is um is a reduction

61:03 is a reduction in in microbe Especially pathogens. So yeah a substances

61:09 the is still in the same categories antiseptic sis Okay, A sepsis means

61:16 reduction. Yeah. Um aseptic technique kind of similar terms. It's meant

61:23 do things in a way that minimize . Any other portion. Yeah.

61:36 . Um with the back through um can be depending on the agent and

61:43 are you going actually down to It could be yeah but but not

61:48 not always it depends on the term means you're killing themselves by licensing

61:55 It could lead to a sterilization process sure. Yeah. Or I mean

62:14 terms of how they're being killed it again it depends on the agent that's

62:18 students sterilization so some may completely obliterate that way or maybe not. It

62:24 depends. Is it another question. . Yeah criterias you have to get

62:32 viable count on that because we're just at it just basically think considering you're

62:37 under the microscope that's your view of . So yeah you have to get

62:40 viable count to say is that bacteria that bacteria static? Right.

62:46 Any questions? Yeah. All Mhm. I I'm not gonna say

62:55 don't I'm going to say that if me I gotta I have to assume

63:00 if it was a gram negative infection you have to be aware of that

63:05 and and so it's you know how is the infection is a septic blah

63:09 blah blah. So yeah I'm sure do but just you have to have

63:13 back your mind. Yeah another Okay. Alright so let's look at

63:22 okay so this is a D. here. I mentioned that. So

63:27 all you're doing here is again focused the you know killing him fast.

63:33 ? How fast can we kill Obviously we're looking at the line that's

63:36 negative flow. So we've added a to Uh culture that started out at

63:42 to the 8th here. Okay. um uh and steadily dropping. Okay

63:50 what do you do is you pick couple of points? So for her

63:53 value is is it's one log one difference. Okay one log drop.

63:59 so we picked two points arbitrarily 10 to the 5th and 10 to

64:04 . Okay then just extrapolate down And so remember that that one log

64:10 death, you know, was 90 . Okay. And so and so

64:19 this is about a minute you said new value here is one minute.

64:23 mean that's how long it took to one log of death with this,

64:26 the treatment was. Okay. And one of the things that can

64:30 you know, how well treatment may . Um one can be. How

64:36 how many microbes are you dealing Okay. Um Organic loads. So

64:42 this refers to think of that as of the the dirtiness of the surface

64:49 say you're trying to clean okay if you want to disinfect the surface and

64:54 surface is heavily soiled okay with dirt grime or whatever. It's best to

65:02 do a regular cleaning first before you disinfectant. Okay. Because maybe that's

65:08 that organic material. That dirt if will grime is the thing that's absorbing

65:13 disinfectant. And it's not really even to the microbes. So uh it's

65:19 common to clean the surface first and disinfect uh the the types of microbes

65:25 dealing with. And you don't believe gonna know that specifically. But um

65:31 try to pick maybe uh an agent will be worked well on both.

65:36 negative gram positives, et cetera, kind of thing? Um The agent

65:42 , you know, is it very to the surface? Like are you

65:46 on stainless steel or something or are putting it on another type of uh

65:51 with a composition that's totally different. you know the stability of the of

65:55 compound. Is it uh light sensitive that may trigger something? Is it

66:01 How volatile is it? Alright if spray it on the surface and it

66:07 away evaporates right away, is that or not? So good? So

66:11 kind of all these factors play a and ultimately how effective it will

66:18 Um And so this log arrhythmic Right? So remember that a couple

66:22 the batch growth curve right? We lagged log stationary death. Right?

66:28 so the changes in numbers in log death are are exponential. Okay.

66:34 are log rhythmic. Okay. But never a scenario where you'll see something

66:40 that, okay. Where everything dies all at once. Okay. There's

66:47 that some rate whether it's a very rate but it's at some rate and

66:52 because it's all about members of the . My well very slightly.

67:00 And they will accumulate damage at a rate. Okay. And once it's

67:06 the threshold and cell dies that's gonna somewhat from cell to cell. Especially

67:12 it's densely packed. You know, in the periphery may be more susceptible

67:15 ones in the middle in the Okay so there's always gonna be some

67:20 of a rate at which killing You won't, again, we're not

67:25 see that. Okay. I'll fall the cliff at one time.

67:32 Um, okay. So let's, , I, I don't, I

67:40 want to do another question where you use your clickers again. So,

67:45 , what's that for this? Let just check. Yeah, I was

67:47 stop there anyway, so that's anybody any questions, So, okay,

67:53 , thanks. We'll finish up. don't have a whole lot more to

67:56 . So we'll finish it up on . Okay. So I'll fix this

68:04 , whatever this is, hopefully. hmm.

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