in the second section we'll talk about some other ways to control microbial growth usually if we're talking about trying to kill microorganisms control the growth one of the first things that we talk about is heat cranking up the temperature is a way to kill microorganisms remember that heat is going to denature those proteins so if you worked in an industry and you were trying to validate things like autoclaves these are some of the terms that you might use so for example if you take a look at the last term decimal reduction time this is the time required to kill 90% of organisms at a particular temperature so for example if we had a dvalue 150° c at 30 minutes that means if we held the temperature at 150° C for 30 minutes we would actually kill 90% of the organisms that are present so these terms here thermal death Point thermal death time has to do with the temperature and the time points that you would use to kill these microorganisms using heat now what is the best way to heat things up to kill microorganisms there are different mechanisms the first one is dry heat and there are these big dry heat ovens um they're a little slower to penetrate materials uh they're really good for metal instruments or glasswar or even things like oils and powders that can't get wet um and this is using a dry heat at a certain temperature and certain amount of time one of my very first jobs out of college was working at B Lam making saline solution so in our pilot plant when we' made gallons and gallons of saline solution solution um the equipment that we used was metal and so we used dry heat ovens to actually clean our equipment the most common thing that we use in Labs is moist heat so we can use moist heat through an autoclave and what an autoclave does is it's going to heat things under pressure and using steam now this is good for things that can get wet if they can't get wet then you might want to use a dry heat oven or maybe some other um some other method to use so this is most often what we use in a micro lab is an autoc clay so the test tubes that we use in lab have actually been run through in autoclave they've been cleaned and if you've gone to the dentist's office and they' brought out the equipment to use um for your dental appointment it has been run through an autoclave so I've used these in microl laabs I've used these in Pharmaceuticals and there are different small tabletop and then there can be much much larger um machines and so this is probably the thing that we most often use in a micro laap early on we talked about Louis pasture and pasteurization pasturization does not sterilize things so it Heats things up for a certain period of time time it will reduce the microbial load but it won't necessarily sterilize so when you're buying milk at the store and it says that it's pasteurized that doesn't mean that it's sterile that there's no bacteria that pasteurization has actually reduced the microbial load in that dairy product and there's different forms of pasteurization depending on the methods and how long they're actually holding it for as well as the temperature I'm going to spend a little bit more time talking about the autoclave because this is something that is really common in microlabs so this picture is an autoclave in fact it looks very similar to the one that we have in the prep lab so our biohazardous waste for example is run through the autoclave our glassware is run through the autoclave even the media can be autoclaved and then cooled and then poured so this is the setup where you would would actually have an access for steam to come in and this is going to hold heat under a really high pressure and the door on our autoclave almost looks like the doors that you might find in a ship or a submarine um and you know it kind of has that that big circle that you kind of have to crank and this is what is maintaining that pressure it's almost like a pressure cooker basically is we have in the lab now this is what our autoclave looks like and so here are some other autoclaves that you might see so the tabletop I used quite a bit in Pharmaceuticals uh the previous picture was what I used to use in in microlab when I was working with um the microorganisms that I was characterizing so when I worked in Pharmaceuticals and I worked in formulations we made some very small batch drug solutions and so all of our glasswar everything would actually be put through one of those tabletop autoclaves and it would actually be digital so it would actually spit out a tape so that we would know exactly what the temperature and pressure was every minute of the Run um I used to hear horror stories about doors flying off because they weren't closed properly and so I will tell you I was always very paranoid um around autoclave so if I knew that my auto clave was in the other room um on one side I would actually move to the other side of the room just in case something happened with the run if you look at the picture on the right this is a big autoclave so this is something that I saw when I worked at balam so we might see this in an industrial setting uh we might also see this in um in a hospital so they can be varying sizes um if you go to the dentist office more than likely you're going to see a tabletop autoclave um when we would do our runs uh we would actually wrap them up in paper so the things that we were autoclaving and then we would seal it with autoclave tape and then once the Run was through when we would take out the package that tape would actually have stripes on it so that was a clue to us that okay that had actually been autoclaved you might see similar type things at a dental office when I worked at ban La when we had these huge autoclaves um we had teams validation teams that would validate our sterilization methods um and so that was their job to make sure that this equipment was doing what it should do now in our lab we have our autoc clay validated so it is checked on a yearly basis but if you're unsure if your autoclave is actually perform forming as it should you can actually use these little ampules from time to time and our lab technicians they will actually put these in an autoclave run occasionally just to make sure that our autoclave is doing what it's supposed to be doing and this is taking these little ampules that have media and they also have a little Spore strip and so because this autoclave is under pressure when this ampule is put into a run the pressure will actually squeeze the tube and break the internal glass tube and it allows the media to actually um go into that Spore strip so after your run you would actually take this out and then you would put it in an incubator if you saw no growth then that means that your autoclave was doing its job in sterilizing and it killed those spores if you saw growth and it was cloudy that means that your auto clave really wasn't working as it should so it wasn't a high enough temperature and pressure in order to kill those um those spores there are some other methods than heat that we can use to slow down bacterial growth Refrigeration now we know this with our kitchen so if you have leftovers you want to put them back into the refrigerator because that lower temperature is going to help to slow microbial growth so if we want to preserve some specimens um sometimes if I'm trying to save PL plates I'll put them in the refrigerator now these are living organisms that sometimes when we put them in the refrigerator uh they don't always look and behave the way that we want them to but this is something that we certainly do in our own homes put it in the in the refrigerator and that can help uh extend the life of your uh extend the life of your leftovers freezing is another thing that we can do so we can freeze our leftovers you go to the store and you get some chicken and then you can actually put it in the freezer and now it can be good for months we can actually freeze things down to a very low temperature so that we can actually preserve our microbes so this is something that they can do in microlabs just to make sure that uh that there's going to be a supply of microbes anytime that you take the water out of something drying that means that it's going to inhibit microbial growth remember though that our endospore forming bacterial friends they can actually survive that drying so may not necessarily be the best option for everything and then freeze drying this is drying something after it's frozen also called lyophilization if you've ever had space ice cream where you have those little pellets that are coold the little ice cream pellets that's basically um lyophilized ice cream one thing that we will be testing in lab is is radiation so UV light can actually kill um microbes so it can damage the proteins can also damage the nucleic acids as well UV light is one of those things that certain wav blanks can actually get rid of viruses and bacteria so we're going to be testing UV light in lab we're actually going to be using a microorganism that has a pigment and that pigment actually helps to absorb some UV light so it's going to allow us to actually see different time points and how UV light exposure affects that microorganism uvite is good at getting rid of both viruses and bacteria because it damages nucleic acids however it's not so great at getting through things so it's useful for decontaminating the air so I mentioned our bios safety cabinets obviously when we were working in those cabinets we used visible light but at the end of the day once we were done and we cleaned it we actually flipped on another light that was UV light so it actually killed anything on the surface UV light is not so good at getting through things so if you were to take a plate of growth and put the cover on it or even a piece of paper and expose it to UV light it's not going to be able to get through so it's not going to be able to do anything to that organism it's the same principle of why we wear like sunglasses so those sunglasses help to protect our eyes from UV light because it's not so good at getting through things gamma radiation is another thing that they will use um not only a different industry um like Pharmaceuticals um mettech devices but they will also use this for food as well I've also seen it in the um makeup industry as well so this uses x-rays gamma rays um that actually creates ions and it dislodges electrons this uses Cobalt 60 um many Industries are actually trying to find some different ways of sterilization because there's from what I understand a world shortage of Cobalt 60 this is very useful for getting rid of bacteria and viruses in Foods um different products medical devices so my husband works for a medical device company and they actually design equipment that you would use in surgeries he actually designs the packaging and so um once that device is packaged up they will actually send it through gamma radiation and that will kill anything in there so that it's actually sterile when it gets to the surgical Suite I've also seen it used in foods um many grocery stores might label this and actually even put it in a little different display cabinet so I was actually going to a grocery store one time to buy some burgers to cook on the grill and they had the regular burgers and then in another case they actually had preformed burgers that had been hit with gamma radiation now this does nothing to the food it does nothing to the people that are eating it it just has to do with killing those microorganisms so if you haven't gathered I am a germaphobe so I was happy to buy those Burger burgers that I knew had been run through gamma radiation so you might see this in different Industries microwave radiation this can also sterilize things um only useful if it contains water and strong visible light um can also affect bacterial enzymes um certain dyes can actually um be more effective with strong light on microbes so here is a picture of the effect of UV light and bacteria which we will be testing in lab and so here we have um a lawn of growth and it's been exposed to UV light so you can see the growth you can see those colonies before the UV light was actually centered on this petri dish before this organism was was exposed and then after you can see that it actually killed the bacteria we are going to be testing different time points and how long um it's going to take for that UV light to actually kill all of the organisms and what happens with the exposure time again we're going to be testing this on a special microorganism serum maresins that produces a red pigment and that red pigment helps to absorb some of the UV light so that we can actually see those um the different effects uh with the time points some other methods um ethylene oxide gas or etto this is an alkala agent that disrupts cell metabolism um they can actually use this for uh for breathable packaging so if can actually get in there again it's not going to do anything um to the equipment it's just going to sterilize it get rid of the microbes ebeam or electronic beam um this is something that they've been using more of because of that shortage of Cobalt 60 this uses beta particles to inactivate microbes it is faster than gamma radiation but it doesn't penetrate as much and then chlorine dioxide gas this is something that's been around for a while but again they've started to use this more it's a strong oxidizer so they can actually use this for food Public Water Supplies medical devices as well now you might be thinking wait a minute chlorine it actually does not chlorinate um so it's it's not what you would typically think of and so this is uh another method that that they've been using more and more this last uh one of the last things that we'll talk about is filtration so if we need to sterilize a liquid but we can't autoclave we can't heat it up because it will damage it then we may want to use filtration and filtration is using these filters with tiny tiny pore sizes it basically filters out any infectious agents now I told you about my bacteria they were halophiles so they like salt so I used a media that they liked that was food for them that would allow them to sustain their life but every time I added salt to it and then autoclaved it the salt would actually precipitate out into a solid so I was not having luck preparing my media in an autoclave so I ended up having to go to a filtration system so I filtered my media to sterilize it because I did not want to contaminate my bacteria and those pore sizes were small enough to filter out any infectious agents and contaminants so I've also used this in Pharmaceuticals so I mentioned formulating drug solutions so we would autoclave our glassware but when we were formulating our drug solutions we could not heat them up because we would end up destroying the drugs so we use these very tiny filters now when you buy these filters they can be very large for large scale production or very small I mostly use the small ones so you can see on the right is a very small filter that's actually attached to a syringe so in my biosafety cabinet I filtered my drug formulation through this filter and then collected it in my sterile glassware and then sealed it up we could not heat our drug solutions because they would have been destroyed what you're seeing on the top left those are the containers that I actually used uh to sterilize my media so this came all wrapped up in sterile and it actually came with a sterile collection uh collecting vial and I would do this in the hood so I would pour my media in if you notice that nozzle I could actually attach a hose up to a vacuum system I would turn on the vacuum system and when I added my media to the top it would be sucked through that filter and filtered out any contamination so that I had um sterile media that then I could cap and I could store and I could use for a period of time um so again there can be they can be very small they can also be very large for large scale filtering the last thing that we've already talked about in some previous um chapters this is osmotic pressure so this has to do with the tenic so the amounts of solutes and water we have on either side of the membrane and I mentioned how these high concentrations of sugar and salt will basically draw the water out of the organisms if the water leaves by by bacteria it's not going to be able to live and so this is something that they've done to preserve food for years and years so in like salted Meats jellies jams because of that osmotic pressure it draws the water out and then the bacteria can't live