Hello, welcome to chapter seven. In this lecture, we're going to talk about different ways of controlling microbial growth. And one of the assumptions that many of us make is that we often want to kill every single microbe on a particular surface or tissue or something of that kind. And when we are talking about killing every living microbe, we're talking about sterilization.
But oftentimes that's not necessarily the goal for a number of reasons. If we wanted to completely sterilize something, it might be detrimental to the surface that it's on. Think about tissue. We don't necessarily want to destroy the tissue as well as killing all of the microbes.
And so often it's going to be a balance between the constraints that you have and what you're trying to preserve and what you're trying to get rid of. And so sterilization would be the most stringent of methods, in which case there's no microbial life left. And this would mean destroying endospores as well, which we've discussed in our...
pretty hardy and hard to treat. So you can imagine that if you're getting to the point where you're also destroying endospores, you might also be destroying food items and or living tissue that you might want to be preserving. So certain situations, we may need to sterilize, but not, again, to the point that we are negatively affecting something that we want to keep, like a food item.
So in the case of commercial sterilization, we're interested in eliminating spores from Clostridium botulinum, which causes botulism. But to sterilize the food would be too much and negatively affect the quality of the food item. However, we can use this slightly.
less stringent method, a commercial sterilization that's good enough at killing endospores from Clostridium botulinum, but it may not kill endospores from other microbes. And as we've learned in the previous chapter, not all microbes grow at room temperature, not all microbes grow at body temperature. They may need higher temperatures or colder temperatures.
And so it turns out some of the endospores that might be found in food items don't necessarily grow at temperatures that the food is stored at typically, which means that they're not really of an issue. And while they may cause food spoilage, they themselves don't cause disease in humans, unlike botulism, which for botulinum, clostridium botulinum, which causes a toxin, produces a toxin that is dangerous to humans. And so in this case, killing of endospores for the killing of Clostridium botulinum endospores is sufficient even if there might be other microbes in the food. In other instances, we may want to destroy potentially pathogenic microbes, in which case we might be referring to disinfection or antisepsis. Both of these are pretty much the same.
The only difference here is that you're destroying vegetative pathogens on inanimate objects. So something like a table, you would disinfect a table, right? Surgical instruments and the like.
Whereas if you have a tissue from a patient that you're transplanting or a wound. what you would be doing would be antiseptics, wounds, living tissue, etc. And again, the goal in this instance is not to kill all microbes, such as sterilization, but rather to kill pathogenic cells or bacteria. You also have de-germing.
In this case, this is more like what you would do before you give a patient a vaccine shot. You would use a little bit of alcohol and swab their arm or the side of the injection before inserting. the syringe, which could potentially introduce pathogens or microbes into the tissue.
But by de-germing, you eliminate that or reduce that risk rather. And so with de-germing, it's more about reducing the number of microbes in the particular area that you're de-germing. So you're not disinfecting. There might still be some. but you're greatly reducing the number of microbes.
And then lastly we have sanitization, which is the least stringent of all of these methods. And in this case you're trying to remove or reduce the number of microbes, but typically from some kind of non-living surface such as, you know, utensils for eating, plates. and the like. And as we go through this lecture, and especially when we get to some of the methods for controlling microbial growth, whether it's physical or chemical in nature, you're going to come across several terms, or these two terms, microbicidal and microbial static. Cidal means killing and static means pausing, right?
Not growing. So microbicidal situations would result in the killing of the microbe, whereas static, microbial static, would not kill, rather they just inhibit the growth. So the bacteria are kind of just in timeout while that particular physical or chemical agent is present and if it were to be removed the bacteria might then resume their growth and replication.
An interesting aspect of factors that control microbial growth or rather that are microbial is the fact that when you apply, let's say, the bleach or some other disinfectant or sterilizing agent, what tends to happen is that, or at least the way many students think about it, is that the minute you add it, they all go up in flames and get destroyed. But the reality is that it's the rate at which the microbes die is going to be constant. And so if you start with let's say, a million microbes and you apply the treatment for an X period, X amount of time, right, some time, what you're going to do is you're going to reduce the population by about 90%. So only about 10% are going to survive that first interval.
So that means we're going to be brought down to 100,000. So let's say it was treatment with this compound or sorry, this agent. for one minute.
So after one minute, we would have 100,000. After two minutes, we would have 10,000. After three minutes, we would have 1,000 and so forth. And so the point here is that simply applying the disinfectant, the sterilizing agent, doesn't completely kill them all at the exact same time.
And this is important. especially when you're in clinical situations and you want to disinfect or treat surgical equipment, or sorry, if you want to disinfect maybe a patient room where patients are being seen, or if you're trying, more importantly, if you're trying to sterilize surgical equipment, you can't just put it in something really hot for two minutes and consider it sterilized. You have to consider the fact.
that not all microbes are going to die. And so you then need to apply the appropriate treatment for the appropriate amount of time. So the time here is the key component. And to illustrate these points, we have these two graphs here that give us a little bit different information. So on the first graph, we have on the x-axis time in minutes, and then on the y-axis, we have the number of surviving cells.
So if we start with a known quantity of bacteria, we can see and measure how many bacteria are alive at a particular time after exposure. So at T0, they haven't been exposed and we have all 10 to the sixth bacteria living, so all million bacteria living. And these different lines are for different treatments.
Okay. sorry, the lines are different organisms for one particular treatment. So in this case, let's say it's autoclaving, the technique or a method that we're going to talk about in a little bit. So autoclaving would be what we're using to sterilize and kill these microbes. And we have microbe A, microbe B, and microbe C, right, three different species of microbes.
And what we see is that we get this gradual decline, and it's not from a million to zero, right, after one minute or two minute or whatnot. There's a slope to the line, right? And it's a constant slope. And we can see, right, or I guess rather I'll pose a question of the three organisms, which is most susceptible to this treatment and the conditions that are. under the conditions that this test was done, right?
Whatever they might be, as that's kind of irrelevant, right? But we can see that A has the steepest slope, so A is the most susceptible, right? It takes a little over 20 minutes, maybe around 25 minutes of treatment with this particular agent before, or with sterilization before these microbes die. Whereas in this hypothetical situation, this microbe takes about 60 minutes.
And this one takes much longer, so it's less susceptible, it's more resistant. So microbes may react differently to the same exact treatment because of differences in the microbes, between microbes. Another important consideration is that the population size or density is also going to play a factor.
So in this graph we have on the x-axis time again and then on the y-axis we also have the number of viable microbes and in this case we have the exact same microbe, exact same species, but we're starting with a high population, right, and a low population for high density and a low population density. So high numbers and low. And we can see is that because the rate is constant it's going to simply take longer for this population to reach zero. then if we started with orders of magnitude fewer microbes, it would take considerably less time to get to a place where we've, if we get to zero, where we've sterilized whatever it is we're sterilizing. And so in this part we're going to talk about the physical methods of microbial And these physical methods are going to be the ones that are listed here.
And they include temperature, filtration methods, desiccation or drying out, osmotic pressures, and radiation. One of the most common ways of controlling microbial growth is through the application of heat. Heat begins to denature proteins. and other macromolecules. And the reason that heat is going to denature these proteins is because it starts to destabilize intermolecular interactions that are facilitated by hydrogen bonds.
So if you recall back from chemistry, hydrogen bonds are weak intermolecular interactions between partially charged molecule on one side, there's a molecule with a partially negative, and here's another molecule on this side with a partial positive, and because of this, that would not be a positive, right? That would be a positive. These differences in charge, in partial charges, you have this weak interaction.
Electrons are not being shared, electrons are not being stolen, rather it's simply an interaction between obviously charged molecules. So these hydrogen bonds help stabilize protein structure, secondary protein structure. tertiary protein structure, and if the protein has it, quaternary structure. In DNA, we'll also see that the base pairs, so we have two strands of DNA that are complementary, and the nucleotides are here, right, there will be hydrogen bonds that help stabilize and keep the double-stranded DNA molecule together. when you apply a treatment, what you're doing is you're going to be disrupting those hydrogen bonds.
They won't be holding the molecules in place, whether it's a protein, nucleic acid, or other biomolecules. And so you begin to denature. Think about PCR and the technique that we use for or for example for COVID testing and many other tests, diagnostic tools.
One of the reasons we use high temperatures in PCR reaction is because it helps to denature the DNA that's going to then be useful for the annealing and the amplification process of the PCR reaction. So temperature is a really good way, an effective way, of disrupting molecules, biomolecules, that are very important to life. and as such can be very useful for controlling microbial growth if applied at the right temperature and for the correct amount of time.
So the temperature that you use and the exposure is going to be important. Another important consideration when applying heat is the use of moisture or no moisture, right? Is moisture present or is moisture not going to be a component? And you can imagine that under certain circumstances, you may want moisture.
But under certain circumstances, the material you're trying to sterilize might not do well with moisture, might get ruined. And so you may not have the option of using moisture, even though you want to use heat. And we'll talk about that in a minute.
So the amount of moisture. And as it turns out, the presence of moisture makes heat treatment. that much more effective because it's much more effective at transferring heat and so it's going to be that much more effective at denaturing proteins and other biomolecules. And here are some important terms for you to also recall the thermal death point and the thermal death time, but I won't go over them just make sure you do review them. And so one of the most common One of the most common ways that we use heat that also includes moisture is through autoclaving.
Autoclaves are the instruments that are used for autoclaving. Really what they are is a chamber that is pretty heavy duty. The reason it's pretty heavy duty is because once you seal it, the chamber is filled with steam, which provides the moisture, and then the temperature is increased, and the pressure is also increased. And so it's this high pressure, high temperature chamber that is going to then sterilize your products of interest, whatever it is you might be interested in. Auto cleaning is great for sterilizing.
Why? Because again it's got high pressures which results in these high temperatures. They're also relatively easy to use. They're not that expensive and they don't produce much toxic waste given that what you're really using is just water vapor.
But again, an important consideration when you're using an autoclave is whether the material that you're using is sensitive to moisture or not. So you want to make sure that if you're going to be autoclaving or using an autoclave, you are using it for the appropriate materials. In addition to, or one important consideration when using an auto-eclave is also to pay attention to the pressure that one needs to use. In order to achieve sterilization, you need to reach sterilizing temperatures, which are 121 degrees Celsius, 121 degrees Celsius.
This can be achieved by applying pressure. And so at certain pressures you're able to achieve a boiling temperature of water, a certain boiling temperature for water, right? So at sea level water boils at about 100 degrees, which is not going to be hot enough to sterilize.
So we apply pressure. And if we apply this amount of pressure, we then are able to achieve temperatures that are going to be sufficiently high enough to sterilize whatever instruments we're applying. And so this is not what you don't really have to worry about this when you go into clinical settings, because the institution... a hospital or clinic that you may be working at that has the autoclave will know what settings to use depending on where you are.
So if you're at sea level, you have to apply certain pressures. If you're at other, maybe, you know, mile high city, you may have to use higher PSIs given that the pressure is higher at those altitudes. But again, that's something to consider if you're using an autoclave. Another thing that you have to be mindful of is that autoplays work by having steam, right, the hot vapor or water vapors making contact with whatever it is you're trying to sterilize.
So if you're trying to sterilize, let's say, a pair of scissors for surgical procedures and they're metal. they would perfectly withstand or they would you know easily withstand the 121 degree temperature so that's fine but what would you but what you would need is to have the steam make contact with the scissors which is fine right you would place your scissors in here but then when all is said and done you open this up and if you just place your scissors in here and you take them out again We've learned that microbes are all around us and what would happen, right? They would be sterile once you put them in here and you've reached 121 degrees for an x amount of time, right?
Maybe usually something around 20 minutes and then it would be sterile. But the minute you open this and bring it out it would no longer be sterile. So these instruments need to be wrapped in some fashion.
And they were typically placed in these little baggies that are half paper and half plastic. And if you've ever wondered why that is, and you've seen these in the laboratory, and sometimes they're even just paper on both sides, right? One of the reasons that they're either paper on both sides or half paper, half plastic is because plastic would not allow for the moisture to penetrate, right? So if you place your scissors or yeah, your scissors in this little plastic baggie, the moisture would not touch the scissors.
So the contents of your bag would not be sterile. The outside of your bag where the water vapor touched would be sterile. So by placing your scissors or surgical instruments in these little bags where one side is plastic and one side is paper, the paper is permeable to the vapor, the water vapor, and would thus be sterilized. And the same thing would go if your bag was paper on both sides. The great thing is that the paper is porous enough for the water vapor to enter, but not porous enough for microbes to pass.
And so it would be able to go in, make contact with the surface of the scissors, sterilize it, and when you remove your little bag from the bag, it would be very easy to get the water vapor out of the bag. the chamber, it would still be enclosed in the wrapping and would remain sterile until you open. Another important consideration is that Sometimes people use aluminum foil to wrap their materials that they're going to auto-clean.
The downfall or the error in that, again, is that the water vapor needs to make contact with your instruments or whatever it is you're sterilizing. And so if you've sealed it in aluminum foil, the aluminum foil, again, will not allow the penetration of the vapor. the water vapor and so the contents would not be sterile. So it's important to properly use an autoclave.
Another important consideration when using an autoclave is that you won't be able to see things that are sterile, right? You put in your instruments, you push some buttons, stuff happens, you come back 30 minutes later, you open it up and you retrieve your items assuming that they are going to be sterile. But there's no way of visually seeing if they have been sterilized properly or not.
And while if everything's operating fine, it will be sterile. However, it's a machine that is capable of having malfunctions. And if the temperature, right, if the pressure isn't the appropriate pressure isn't achieved, then you won't achieve proper sterilizing temperature. Similarly, if there's an error with the vapor and maybe water vapor or steam is not pumped in, then the proper temperature might not be achieved.
Or you might be doing a heat, a dry heat, which would not be able to sterilize into that amount of time. So there might be several things that go wrong with the instrument and you would have no way of knowing. So one thing that's...
pretty important whenever using autoclave is to use some kind of paper or a tape that has some compound on it that gets charred when it reaches the appropriate temperature and it turns black. That way when you insert your contents into your autoclave you put a little bit of that sticker onto the contents right and when you retrieve them they should all have that little charred look to them. indicating that they had been sterilized.
And so you might see that in clinical settings. So what we've just discussed is using heat in the presence of moisture. But sometimes, as I've alluded to, use of moisture is not appropriate or not possible.
And so you would then need to use some other method. And so dry heat would be useful. And the way that this is going to work is by simply desiccating or removing water from the environment.
This takes a lot longer so it's often not good for things that are going to be too sensitive to the heat. So there are some considerations when... you have to use dry heat.
You have to be able, your instrument that you want to sterilize needs to be able to withstand the temperatures that are needed when using dry heat. Another method that uses heat is pasteurization, which is invested by Louis Pasteur, or named after him, and it's a way of preventing food spoilage. We don't, we don't put them, we don't put food in an autoclave, for example, or use dry heat because that would make the food items not palatable or ruin the quality of the food item. But we can use enough heat to kill most organisms, most microbes, to help minimize the number of microbes in the food item.
But it might, it will not be sterile, but it would reduce the numbers. enough to help reduce food spoilage. On the other end of temperature is low temperature. So we've been discussing as very high temperatures which can be which are microbicidal but on the other side of the spectrum we have low temperatures which can be either microbicidal or micro static.
depending on the duration and or the time. So one of the reasons that we have refrigerators at home is because it slows down, the lower temperatures slow down metabolic activity in the microbes, and therefore they grow slower. So when you have something that's pasteurized, which already has a low number of microbes, and then you put it in your refrigerator, you're also helping to reduce the growth of the microbes. which is going to prolong the life shell of your food. You can also freeze items and that can be microbicidal as the water when it freezes forms crystals that can potentially freeze, sorry, rupture cell membranes.
And in fact, in laboratory settings, both clinical and academic, we often store bacterial samples in freezers and then when you need a little bit of bacteria to grow up to do some experiments you can go to your frozen stocks take a little bit and put them in some culture that helps them grow and then they they do just fine. So it can be microbicidal but depending on certain parameters and how you freeze them it can be microbic static. A nifty thing that you can do at home and maybe some of you already do with clothes and if you have stinky shoes is that you can take your clothes or fancy jeans or something.
You can put them in a bag just so that your clothes aren't touching all your other food items in the freezer. Put it in a bag, put it in the freezer for like a day, take it out so it can thaw, put it back in the freezer and you do this back and forth. letting it freeze and then taking it out fine and freezing it again.
And this repeat freeze-fying is what's going to help kill every microbe acyl. And the added benefit is that your stinky jeans or your stinky shoes will not be as stinky anymore because you'd be killing off the microbes that are causing the smell. Another way of controlling microbial growth is through desiccation, which is the removing of water. And this method is going to be micro-static, and if water were to return, you would then, the microbes would be able to reanimate and continue to grow. So it may not necessarily kill the microbes.
Another method that's routinely used, especially for for materials that are liquid in nature is filtration. And this is going to be able to sterilize, assuming that you use a pore that is smaller in diameter than most microbes. So the standard is to use a filter paper where the pores, these pores here, the diameter of these is 0.2 micrometers.
maximum. It could be smaller but at a maximum, sorry, at a minimum they are 0.2 micrometers. And the idea here being that you add your solution that is, you know, not sterile, you filter it into a container that is sterile and the filter will trap the microbes on one side and your solution passes on to the other side and then you would have sterile solution.
So this is going to be a really good way of sterilizing without having to use heat, but again it would be only applicable for liquid solution. Another way that can be used to control microbial growth is through the treatment or is through the use of radiation. whether it be electromagnetic or particle radiation.
And depending on the radiation, it may be microvastatic or microvacidal, and also depending on the duration. But the general consensus is that they're going to be used in such a way that they're going to be bactericidal and help sterilize. And so the way that these are going to work is through oxidizing molecules, or at least ionizing radiation, are going to remove electrons from biomolecules.
So they are going to be oxidizing agents. And if you remember from our discussion in metabolism, they're... the molecule, so we have a molecule here that then uses an electron, right?
There's the electron, and then that electron is gone, right? It's going to have a charge associated with the proton maybe floating out over here. And so what's going to happen is the ion, or sorry, the x-ray or gamma ray, will have oxidized this molecule, which means that it is going to have lost an electron. These free radicals are then going to cause problems for the organism. The non-ionizing forms radiation, namely UV, are going to work slightly different.
They don't have enough energy to remove the electrons, but instead what they're going to do is cause DNA damage. One of the most common ways that they cause DNA damage is through thymine-thymine crossing. So if we go draw a piece of DNA double-stranded, right, and we have our faces and we have an A, a T, a T, and C or something, we would have a T, an A, an A, and a G, and so forth, right?
With UV, what's going to happen is We've discussed how there would be hydro bonds that are stabilizing the interaction between this nucleotide and this nucleotide this way, right? So there's hydro bonds between these complementary bases. However, in the presence of UVU radiation, what happens is the formation of covalent bonds between the thymines, adjacent thymines, right?
This is going to cause damage to the DNA, which makes it so that the DNA cannot be replicated, the cell cannot be reproduced, and if it doesn't get repaired or too many of these happen, the genetic integrity of the cell is lost and the cell will then die. And we're going to have a lab later in the semester where we talk about a beach where they had a big problem with sewage runoff and fecal bacterial contamination in the middle. in the beach.
And one of the, or the way that they helped minimize that is through the use of UV radiation treatment plant, the use of a UV radiation treatment plant to help reduce the number of microbes in the water before being released into the ocean.