up until now we've been talking about how bacteria grow the conditions that they like we also discussed how viruses replicate now we've reached what I call the kill everything chapters now that we know how these culprits grow how is it that we can actually get rid of them how can we kill them so chapter 13 is on sterilization and disinfection some of these methods we are going to be talking about in this chapter we're actually going to be doing in lab so there's a lot of vocabulary along with this chapter there's just a few terms that I want to point out I'm also going to post a little cahoot game just to kind of go through the vocabulary just to kind of test your knowledge a little bit couple of things that I always like to point out with this vocabulary the difference between steril ization and disinfection sterilization means that you're getting rid of all microorganisms in material or on an object disinfection means that we're reducing the number of pathogenic organisms so we're reducing the amount of bacteria now I know when I clean my bathroom as much as I'd like to think that I'm sterilizing it I know that I'm only disinfecting it if you are working in a hospital into a surgic IAL Suite you want to make sure that all of that equipment is sterilized when you go to the dentist and they're taking out all of their tools you want to make sure that those tools are all sterilized the other term that I like to point out is bacteria static versus another term that we often hear bacteria side bacteria side means that it kills the bacteria outright where bacterio static means that it prevents the microorganism from growing so slows down the growth a little bit I remember when taking microbiology and I thought oh well surely we're killing everything and then as I learned more I found that we don't necessarily kill everything but we can kind of slow down the growth a little bit there's a lot of specific terms to whether or not we're trying to kill um spores or fungi as well as getting rid of viruses so as I said I put a little cahoot online just to kind of keep the vocabulary straight so if we're talking about getting rid of everything sterilization there's three principles and when you look at these principles it really is common sense so number one a definite proportion of organisms die in a given time interval so given the amount of time that that organism is exposed to that method we're going to end up killing off a certain portion the other thing that kind of makes sense is if there are fewer organisms that are present it's going to take a shorter amount of time to actually get rid of all of them to achieve sterilization if we have more organisms it's probably going to take longer the other thing and we're especially going to be seeing this in lab is that depending on the microorganism they can actually differ in their susceptibility to that particular antimicrobial agent and as long as we're talking about vocabulary anytime that you see antimicrobial agent that means that we're trying to kill that microbe so it's anything that is going against that microbe we've talked a lot about different organisms and different organisms in lab so gram positive versus gram negative spor forming bacteria versus acid fast bacteria so they are all going to be susceptible to different agents and agents are going to work differently on different organisms we know that if we're trying to clean our bathroom or even if we're trying to clean um surgical equipment we know that there could actually be a variety of microorganisms so that means that we're going to have to do something that can basically zap all of those microorganisms so most of the time in lab we really tend to reach for chemical antimicrobial agents and there's a few things that we can say about these in lab first off their effectiveness can actually be increased by high temperatures think about what happens to molecules when things heat up they start moving around quickly so they are going to work a little better if we actually heat up the system a little bit we also know that the microbes can be affected by the concentration of the agent we're using so is it really concentrated or is it really diluted um the ph and how long we're actually exposing that microorganism to that chemical agent that antimicrobial agent if I spray some cleaner on my bathroom counter and I immediately wipe it away opposed to if I left it on for 15 minutes and then came in to wipe it away leaving it on longer I'm probably going to up my chances of killing more microorganisms another chemical that we actually use in the lab as an antimicrobial agent is alcohol um we tend to use ethyl and isopropyl alcohol so when I worked in Pharmaceuticals and we worked with biosafety cabinets these actually had clean air be circulating around but after we finished our work and even before we started working one of the things that we would do is that we would actually wipe it down with alcohol um you probably don't want to use 100% um ethanol because that can be very damaging to our lungs so that could be kind of a risk to us um and if you are working with isopropyl alcohol which is usually what we tend to use in lab it's actually better to work with a 70% solution the reason for this is that with a little water added to it that water is necessary to denature bacterial proteins so a lot of these antimicrobial agents will actually denature those proteins that the bacteria have so if you remember denaturing a protein means that it basically falls apart it's not maintaining that structure and it can't do its job I don't like to use alcohol in um Micro Labs especially around an open flame so I tend not to use them a lot especially if there's an open flame if there's not an open flame I will use um isopropyl alcohol the reason for this is that I had a friend that worked in microbiology and somebody mislabeled a um an alcohol container she thought it was water there was actually alcohol in it um and so when she was using it thinking it was water um along with the flame she ended up going up in flames so I tend to be very cautious when I'm using alcohol in the lab so how can we actually test these chemical agents and we're actually going to be doing one of these tests in lab so the first thing that you're probably going to notice sounds a little familiar is carbolic acid carbolic acid this is goes along with the phenol test um and if you remember we talked about some of those early scientists that were working to kill microorganisms and one of the things that they used was carbolic acid so this is what's called the phenol coefficient so one basically means that it's as effective as phenol so kind of using phenol as a gold bar standard if it's less than one that means the disinfectant is less effective if the phenol coefficient is above one that means it's more effective than phenol now different chemicals can have different phenol coefficients on different organisms so let's say that we had a 0.7 phenol coefficient on staf orus that means it's going to be less effective than phenol on that organism but we could have a 1.2 phenol coefficient on eoli that means that chemical is going to be more effective than phenol so this Co efficient can differ based on the chemical can also be different with different organisms I'm going to kind of skip the filter paper test because that's actually the one that we're going to be most familiar with and talk a little bit more about the use delusion test which is a little more sophisticated so what you would do is you would take a metal tube you would um coat it with bacteria then you would dip it into different chemical agents with even different dilutions and concentr ations then you would take that tube and put it into a media and put it in an incubator to see what grows if there's a lot of growth that means that that particular chemical was not effective um if you don't see a lot of growth then that means that it was effective that's a little more sophisticated setup than what we will do in lab and this is the filter paper test so with a filter paper test you take a little disc of filter paper soak it in a chemical agent and then you put it on a lawn of growth anywhere that you see clearing after we incubate the plate that means that the bacteria has been killed so if it's clear around that filter paper that means it was effective if there's growth all over that filter paper disc that means it's not effective so I'm a visual person so I think it's a little easier to actually see with a picture so the the top two you can see the delusion test there with that that metal rod there and the tube and then there you can see the um the chemical um phenol underneath that you can see the two plates so there's three filter papers they've been treated with chemicals they're put on a lawn of growth and then the plate would be incubated and if you take a look at the left picture there's growth everywhere and so that means that that chemical did nothing to that bacteria wasn't able to kill it so obviously that chemical is not effective if you look at the picture on the right you can see the filter papers but there's clearing every place that it looks dark that's actually the clearing and the bacteria has been killed so in this case this chemical was effective at killing that bacteria so I want you to take a minute and think about some of the household products that you have and when you go to the store and you're buying a cleaning agent so think about some of the things that you look for when you're buying a disinfectant so you can even pause the video just to kind of jot down a few things and to think about some of those things that that you're looking for so chances are you probably came up with some things on this list so what are we looking for in a good disinfectant well I'll be honest with you that I told you that my mother was a nurse and I always tended to use a lot of the products that that I grew up with um and then as new products came out I sort of Diversified and thought oh you know what this sounds like a good idea maybe maybe I'll try that um so these are some things that we should be looking for with a good disinfectant so number one it should be fast acting if if you've got company coming over and you are cleaning your bathroom you don't want to have to spray it and then leave it on for 4 hours you want it to be relatively quick so you want it to be fast acting second is that we want it to do its job so we want want it to be effective against all types of bacteria think about all of the many bacteria that we could actually have growing in our bathroom so we certainly want it to be effective against a wide array of infectious agents we also don't want it to be harmful so many people look to make sure that it's non-toxic so that we want to make sure that it's not harming us we want to make sure that it's harming the microb this is what we call Selective toxicity that we'll be talking a little bit more about we don't want it to harm us we don't want it to harm our pets we also don't want it to harm our kids as well the other thing is that we don't really want this to be damaging the material that we're spraying it on so if you've got a chemical agent and you spray it on your counter and it turns your counter Orange um that's not such a a great product the other thing is that it's got to be stable now some people will actually buy concentrated versions and they will prepare it themselves I'm all about convenience so that's probably not something that I'm going to opt for but some people do because you know they can buy a good quantity with a low price and then they can make it up as they use it um but regardless if you are making it up yourself or if you're buying it you want to make sure that it's stable you don't want it to expire 2 weeks after you actually buy it the other thing is cost so I don't know about you but I'm certainly not buying something that's going to break the bank um I want it to be convenient I want it to be easy to use I tend to use a lot of Clorox wipes just because they're so convenient um and so that's what I tend to go for um so I know we all have our preferences um that's just one of those preferences that I have the other thing is smell this is often the first thing that people say is that they're like it's got to smell good well some people want it to smell like lavenders other people want it to smell like a hospital so it smells like disinfectant so this is one of those things that's really personal preference um I'll tell you a little story that um recently I I've always used pledge as um to dust um my house and so I accidentally bought like an orange pledge now I grew up with a Lemon Pledge and so that orange just did not sit right with me I ended up going out and buying the The Lemon Pledge that I was used to so this is one of those things that's personal preference and you'll probably notice um this is also a marketing tool as well so when you go to the store there's many different fragrances that you can buy no matter what agent you go for and the smell I think we all want it to be easy to use coste effective and number one it's got to do its job it's got to be effective so how do these chemical reagents actually work well as I mentioned proteins many of them will denature proteins so if you change the protein shape it can't do its job so that's going to affect that protein function so some of those reagents if they're denaturing proteins they can either be bacteria static where it kind of slows down growth or they could outright kill the bacteria where it's bacteria cidal it can also affect the membrane so break down the membrane so that it can actually penetrate and get into that cell and disrupt how things go across the cell um it can also affect metabolism so the enzymes and how that bacteria makes en that bacteria can't make energy it's not going to be able to survive it also can affect how it replicates its nucleic acids so these are things that we often talk about with the characteristics of living things so any way that these agents can disrupt that organism and how it lives then it's going to do damage to it one of the things that we often use in our house and in industry and hospitals are detergents and detergents and we know that washing with soap is really our best defense um washing our dishes with detergents our clothes with detergents is a great way to clean things and to get rid of things so how is it that detergents actually work well these detergents form my cells and this is actually what you are seeing in this picture so with this my cell you actually have like these non-polar regions and then these polar regions on the inside so this almost like traps the dirt or that microorganism in a little bubble these are my cells recently we were watching television and one of these commercials came on for one of these my cells facial cleaner and my husband said what is a my cell and I said let me tell you what a myella is it's basically a cleaning agent so when you see those fancy words it's basically a marketing tool to say o we've got this fancy new method yeah it's basically soap that is doing this so with this my cell that bacteria or dirt is actually trapped in that my cell and then it can be washed away I did post a little video on my cells and detergents um it's a short little five minute video just so that you can see how it works um to clean our hands and also you know when we're washing our clothes so what is the best detergent to buy well just like the stains that we talked about using in lab cat iic or positively charged detergents end up working better think about why this is why is it that it's better to use those cat ionic dyes in lab that's because that bacterial cell wall is going to be negatively charged Opposites Attract so those cionic detergents are actually going to work better anionic are going to be less effective now how would you know if your detergent was actually cationic or anionic um you'd probably have to do a little research but most of the detergents that that we're using and the Soaps that we're using um are going to form those my cells and they're going to be able to capture those bacteria the last thing that I'll talk about are some other agents that inhibit microbial growth uh first up acids and bases so these are great cleaning agents um one of the best cleaning agents that I have in my house is vinegar it has a very low PH sometimes I'll I'll dump it down my drain we can also use these as food preservatives as well heavy heavy metals so Silver Mercury can also inhibit microbial growth these could also be hazardous to us so certainly not something you know that you would be using in your house but certainly something um in certain industries um you'd want to use caution with halogens these are going to be things like chlorine iodine bromine um so we know that not a lot of things can can live in the presence of chlorine iodine is sometimes um a good cleaning agent that they may use um you know to to sterilize skin something like that alcohols we already talked a little bit about that they're going to denature proteins we need a little bit of alcohol um they can dissolve lipids and affect the cell membranes of bacteria interesting to note though they don't always sterilize although alcohol is a good way to actually clean things like skin so taking an alcohol wipe before you give somebody an injection or an IV or if you know you're you're hooking up a pore to actually wipe that off with alcohol very easy to use very convenient with those alcohol wipes um so that's often a good thing to use in the field phenols we already talked about that it can denature proteins cell membranes denature enzymes oxidizing agents so oxidizing agents like hydrogen peroxide can disrupt disulfide bonds and proteins so again affecting how that protein can function hydrogen peroxide is a great cleaning agent so the 3% hydrogen peroxide that we can buy in the store works great at cleaning that was one of the things that we would actually clean our bios safety cabinets with is alcohol and an added protection of hydrogen peroxide I remember when I first got my ears pierced um I actually cleaned um my ears with hydrogen peroxide to to kind of prevent any infection alkala agents they're going to disrupt proteins and nucleic acids uh something like fahid this is something that we would have to take caution working with because it can also cause some forms of cancer the dyes that we use in Labs so things like um methylene blue or Crystal Violet now when we use those dyes because we Heat star slides the bacteria is already dead but they can actually affect bacterial growth and then some plant oils they can also inhibit microbial growth I'm sure that you've probably worked with many of these things you know even in your house or um in your in your job um but there's a lot of different microbial antimicrobial microbial agents that we can actually use