this is chapter 7 the control of microbial growth so starting out with some vocabulary and terminology of microbial control looking at sepsis and asepsis so sepsis refers to a bacterial contamination so then asepsis a meaning without is the absence of significant contamination so just like in lab we practice our aseptic technique so we don't have any cross-contamination during our lab experiments aseptic surgery techniques are also used to help prevent microbial contamination of open wounds in the operating room sterilization refers to removing and destroying all microbial life so it completely destroys all forms of microbial life on the surface commercial sterilization to make sure we kill those botulinum endospores in canned goods so botulism bacteria disinfection involves destroying harmful microorganisms but not necessarily all the microbial life on the surface antisepsis is destroying harmful microbes from living tissues so basically disinfecting a living tissue so like an antiseptic mouthwash right destroys the harmful cavity-causing bacteria from the inside of your mouth degerming is the mechanical or physical removal of microbes from a limited area so an example would be just washing your hands so that is an example of degerming so you have to rub your hands physically together and scrub them with the soap to remove those microbes sanitation involves lowering microbial counts to save public health levels and minimizing disease transfer from person to person so like your lab when you have to sanitize your work area at the beginning and end of each lab so we're applying a sanitizer of approved use and we're applying physical mechanical force when we wipe the surface biocides or germicides are treatments that completely kill microbes bacteriostasis or bacteriostatic treatments don't kill the microbes but they inhibit them or stasis meaning kind of stable so it kind of freezes them in place so they can no longer multiply bactericidal is homicidal right toward bacteria so it's going to kill those organisms whereas bacteriostatic just prevents them from multiplying so if the chemical agent or antimicrobial agent is removed these bacteria could potentially start reproducing again so this is showing the effectiveness of different types of antimicrobial treatments so we have a lawn growth of bacteria on our petri plate and we look for these zones of inhibition so these are areas where there's no bacterial growth so this is showing that chlorine seems to be the most effective on these different bacteria it also shows that different antimicrobial agents have different levels of effectiveness against different types of microorganisms so in previous chapter we talked about exponential growth of microbial colonies when microbes die they also die at an exponential death rate so this is typically graphed on a logarithmic scale so we have that nice straight line so if we apply cleaner that has a death rate of 90 percent every one minute so every minute ninety percent of the population is killed so we were to graph this arithmetically and it wouldn't be a very feasible graph so the log scale just gives us a nice even line all right so we start with a population of 1 million cells after a minute 90 percent has been killed we're left with 100 000. another minute passes right we're down to 10 thousand right ninety percent of those ten thousand die we're left with one thousand ninety percent of those die a hundred ten and then finally one so this particular example our cleaner takes six minutes to effectively kill 99.9 percent of the bacteria so this is why it's important to make sure you read the label on your household cleaners to make sure you're leaving the antimicrobial substance on the surface long enough to reach that 99.9 percent so in this example if we only left it for two minutes right before we wiped it off the surface or cleaned it up right we would still have 10 000 of those bacterial cells remaining the effectiveness of our treatments are going to depend on a few different factors so one being the number of microbes so the higher your population load right the more cells you have to kill the longer it's going to take right to reach that 99.9 effectiveness so this example we start with a lower population right so they're all completely killed within three minutes but if we start with a higher population load it's going to take more time the environment can also influence the effectiveness of your treatment so presence of organic matter and how it reacts with the cleaner temperature presence of biofilms remember the biofilms can be a little bit more resistant to antimicrobial agents because they have those layers of bacteria so the cells on the surface would be affected but the cells in the lower layers would be protected time of exposure like we said so make sure you read the labels and make sure you're leaving your cleaners on long enough for them to be fully effective right so if we were to remove our cleaner after three minutes we still would have a relatively high population remaining effectiveness can also depend on microbial characteristics so different types of cleaners have different levels of effectiveness on different microbes depending on their own characteristics like cell wall structure or metabolism so there are three main mechanisms of action or plans of attack that these antimicrobial agents can take when they are killing these microbial cells so one action could be to alter or damage the cell membrane so the cell membrane is kind of like the skin of the cell so if we poke holes in that membrane now the internal cell contents can leak out and other harmful substances can now get in the cell some microbial control agents may target the proteins or enzymes of the cell so remember enzymes pretty much control and regulate all cells metabolic activities and those biochemical reactions and enzymes are just proteins and everything goes back to structure reflecting function so if we denature and change the shape of those proteins they're no longer functioning so those cells can no longer undergo their usual metabolism another angle of attack would be to damage the nucleic acid so the dna or the rna so the dna is kind of the base source of information on how to make these proteins and enzymes and how to assemble and make new cells so if we damage the genetic blueprint or the instructions then the cell will no longer be able to make the proper proteins and undergo its usual metabolism and growth there are two main categories of methods we can use to control microbial growth physical or chemical so one of the most basic types of physical methods would be heat because heat is going to denature those enzymes and essentially make the cell unable to undergo its metabolic reactions when we look at a thermal death point we're talking about the lowest temperature at which all the cells in a liquid culture are killed in 10 minutes so 10 minutes is kind of our baseline right so what temperature does it have to be for all the cells to be killed in that 10 minute time frame so this figure is showing that most all of the pathogenic bacteria and harmful insects in this particular soil will die between the 60 and 80 degrees celsius range in about 10 minutes so this would be their thermal death points thermal death time is the minimal time for all a bacteria in a liquid culture to be killed at a particular temperature so instead of presetting the time we're presetting the temperature and then seeing how long it takes at this temperature to kill all of those cells so in this particular example we're starting at zero and by 45 minutes we've killed all of the cells in the population decimal reduction time or drt is the minutes it takes to kill 90 percent of the specific population at a given temperature so related to the thermal death time but we're only looking at 90 of the population killed we can also use moist heat sterilization for microbial control moist heat works by coagulating and denaturing those proteins the boiling is one method we could use to sterilize surfaces and objects we can also use free-flowing steam so if steam's hot enough for the water to evaporate those high temperatures can denature the proteins of any microbes in lab to sterilize our lab equipment and media we use an autoclave which utilizes steam under pressure so it's about 121 degrees celsius at 15 pounds pressure per square inch for 15 minutes so that high pressure steam kind of washes over our objects we put in there and sterilizes them moist heat sterilization is good for killing all organisms and endospores so not all methods will kill endospores as easily but the steam has to contact the item's surface for it to be effective the larger a surface area an object has is the longer sterilization time it will require so again we said the steam has to make contact with the surface of the object so if we have more surface to cover right it's just going to require more time to make sure everything makes contact so this example showing just your standard test tube right we can sterilize in about 15 minutes but a larger flask or bottle right in the thousands of milliliter range right could take half hour or more to reach that full sterilization sometimes we can use test strips to indicate if the objects have been sterilized so if we left it in the autoclave long enough we'll have our indicators or we'll know if we need to put it back in for a little while longer but also keep in mind not all objects can withstand moist heat sterilization so some things will melt under those high temperature and pressure conditions so remember louis pasteur developed the process of pasteurization to reduce spoilage organisms and pathogens in beverages so we still use this process today so it utilizes high temperature for a short time so 72 degrees celsius for 15 seconds so it's not necessarily long enough and hot enough to kill and break down all of the cells right so we don't want to change the taste and overall texture of our food product in this example our milk we apply just enough heat to denature the enzymes of the bad microbes we don't want to spoil our milk for that 15 seconds and then we rapidly switch it over to a cooling phase so then those enzymes are kind of frozen back in that denatured state and ultimately unable to function however it doesn't kill all of the microbes in the product so some organism still survives these thermoduric able to survive those high temperatures so this is why even though a lot of food items are pasteurized they don't have an indefinite shelf life there's still some slow growing spoilage bacteria in those dry heat sterilization kills by oxidation so this is essentially just an open flame so like a bunsen burner or accelerators in lab right when you flame your loop you're just incinerating and burning any microbes on that surface hot air sterilization is basically like a oven sterilization oven but still using the same principle of dry heat sterilization like we said not all substances can withstand those high heat and temperature methods so another method we could use a physical method is filtration so passage of a substance through a screen-like material so this will be used for those heat sensitive items hepa filters are very efficient at removing microbes and particulate air as small as three micrometers so hepa stands for high efficiency particulate air filters if we need to filter even smaller microorganisms like viruses or proteins we can use membrane filters so any microbe or particle right would get trapped in this filter and then we're left with our sterile filtrate so so just as heat was a physical method of microbial control low temperatures and freezing can be another method however whereas heat had more of a bactericidal or bacterial killing effect low temperature is primarily a bacteriostatic effect so your standard refrigeration deep freezing and freeze drying so even though we put our food in the refrigerator it still go bad in a few days it's not going to last indefinitely in the fridge because of those psychophiles or those psychobacteria that like to live in those cold conditions and again it's primarily bacteriostatic because if we take the food out of the fridge and it's left at room temperature those bacteria will start to multiply again desiccation is the absence of water so all living cells require water so without water they cannot undergo their metabolism or grow and divide but again this is more of a bacteriostatic effect because once water is re-added so once we rehydrate that substance then the microbes can continue to grow so freeze drying is just kind of an extreme form of desiccation so things like coffee some meats and vegetables osmotic pressure can also be a physical method of control by using high concentrations of salts and sugars to create those hypertonic environments remember hypertonic is where the solution is more concentrated than inside of the cell so water via osmosis always wants to go where the solution is more concentrated because it wants to dilute it out so water will be drawn out of the cell into that solution and causing plasmolysis so things like jams and jellies beef jerky have longer shelf lives because of those high osmotic pressures however there are some of those halophiles like staphylococcus aureus that can still grow in these high salt conditions radiation can also be used to control microbial growth so ionizing and non-ionizing radiation pretty much everything below the visible spectrum can work to control microbial growth by damaging dna causing mutations microwaves can also kill some microbes but it's more so by heat by generating heat in the food not so much from the radiation itself and attacking the dna structure looking at some chemical methods of microbial control and effective disinfection so one important component of the effectiveness of your disinfectant would be the concentration so again it's important to read the label on your disinfectant bottle different disinfectants work better at different concentrations so you might have to do two parts water to one part disinfectant or three parts water depending on what it is so make sure you follow all the label directions the presence of organic matter can also influence the effectiveness of disinfectant so for example with chlorine right if there's certain organic matter present it can react with that as well and cause some carcinogenic byproducts ph is important in the effectiveness of a disinfectant so in this example chlorine is most effective between 5.5 and 7.5 ph so this is important if you have a pool right you also have to maintain certain ph level or your chlorine won't be effective at keeping your pool clean and then finally time time is another important factor in your disinfectant's effectiveness so remember read the label and keep the surface wet for that required contact time so remember if it kills 99.9 percent but it's decimal reduction time right to kill 90 of the population is one minute we started had that example with the million cells that took six minutes to kill 99.9 percent right so we would have to make sure our surface stayed wet that entire six minutes if we wiped it up or cleaned it up before that six minutes we would leave a lot of bacterial cells behind right so in order for them to work and be most effective make sure you're following that contact time the disk diffusion method evaluates the efficacy of different chemical agents so your filter paper would be soaked in different chemicals right or we could also do this with antibiotics and we're just going to look for that zone of inhibition or that clearing around the discs so this means there are no bacterial cells growing or present because the chlorine has killed them and prevented them from growing and multiplying and again showing that different chemical agents can have different levels of effectiveness against different types of organisms so a quat disinfectant has moderate effectiveness against staph aureus but not really any against pseudomonas phenols and phenolics operate by injuring the lipids of that plasma membrane right so we're going to attack the membrane and we said there were three basic plans of attack using the membrane the proteins and enzymes or the dna so these specialize in attacking the membrane essentially causing all the cell contents to leak out bisphenols are essentially just two phenols linked together common one found in your everyday antibacterial hand soaps is triclosan and these also work by disrupting and attacking those plasma membranes chlorhexidine is a biguanide that's used in surgical hand scrubs it also attacks the cell by disrupting the plasma membranes halogens are everything in column seven on the periodic table iodine is commonly used for microbial control as either a tincture or an iodophore tinctures are just the iodine solution mixed with alcohol iodophores are iodine combined with organic molecules they are going to work by impairing the protein synthesis and altering the membrane so it could attack the rna or the dna of the cell so it's not able to make those proteins and enzymes to carry out those metabolic reactions so betadine is a commonly used one used to clean skin surfaces before surgical procedures chlorine is an oxidizing agent that's going to shut down those cellular enzyme systems essentially by attacking the chemical bonds in those enzyme proteins ultimately denaturing the protein and causing it to lose its function alcohols are used for disinfectants and antiseptics they work by denaturing proteins and dissolving the lipid membrane very effective at killing bacteria and fungi however they don't really have an effect on endospores or those non-enveloped viruses so an envelope is just basically kind of like a cell membrane so it's a lipid membrane around the viruses so if it dissolves lipids but there are no lipids on these viruses then it's not going to be very effective viruses that do have envelopes like coronavirus which is a enveloped virus so alcohols like found in hand sanitizer would be effective at killing those ethanol and isopropanol are the most commonly used and the most effective at 70 percent remember we said concentration of the disinfectant was also important in its effectiveness so alcohols work kind of as a two-prong approach so we attack the membrane lipids we dissolve those lipid membranes and we denature and destroy their proteins heavy metals and their compounds work by oligodynamic action so it only really takes very small amounts of these heavy metals to have an antimicrobial effect most commonly used ones are silver mercury copper and zinc so we use silver nitrate on newborn babies those eye drops to help prevent ophthalmia nenatorum which is basically conjunctivitis in newborns we have mercury additives in paint going to prevent mildew and microbial growth in paints um copper sulfate used in algaecide for pools of pool chemicals um if you have a pool if you ever see the word blue like on the label for the pool chemical that means it has copper in it um zinc chloride found in mouthwash so this is showing those zones of inhibition or lack of bacterial growth where these heavy metal coins are making contact with the auger surface and fun fact brass door knobs automatically disinfect themselves in about eight hours right surface active agents sometimes refer as surfactants or just detergents right so they're going to work on the surface of an object to remove microbes so things like soap work by degerming and emulsifying the microbes and oil globules acid anionic sanitizers contain anions or those negatively charged ions that react and disrupt the plasma membrane quaternary ammonium compounds sometimes called quats are cations those positively charged ions that can actually kill the cells so they denature the proteins and disrupt the plasma membrane so a standard type of soap or surfactant agent would be like just your dawn dish soap right so or any hand soap really they all work about the same so they work as surfactant so they're lipid based right so remember lipids dissolve lipids like dissolves like all right so we have this lipid based soap that's going to emulsify or form these fat or oil globules so these are basically like little uh i think of phospholipids so we have a hydrophilic head and a hydrophobic tail so the tails will make contact with the in this example a stain or just say oil globule or with some microbes on it or some dirt on your hand so they'll surround the oil on your skin right with the microbes but it's not going to detach on its own right so we have to have that mechanical agitation you have to rub your hands together right for at least 20 seconds right to lift all of that oil and microbes off the surface and allow the surfactants to form these globules right and once they're lifted from the surface we can then wash them away with the water so comparing the effectiveness of various antiseptics so different types of antimicrobial agents will have their own kind of specialization or plan mechanism of action and different disinfectants will have different levels of effectiveness against different types of microorganisms so your bigonides phenolics and your quads primarily target the plasma membrane some aldehydes radiation we said targeted the dna because this cause mutations um halogens metals phenolics alcohols can also target the proteins those enzymes so this is showing a percentage of particular type of bacteria surviving different treatments right so with just soap and water right we didn't really remove that many microbes at all so the most effective was our iodine with 70 ethanol so because our modern lifestyles have given rise to high levels of food production more mouths to feed and a greater demand for food shelf life we have also increased use of food additives these chemical food preservatives so these are things to help preserve flavor or improve taste and appearance in a food as well as a shelf life the most commonly used are your benzoates and nitrites sulfur dioxide can prevent wine spoilage so spoilers bacteria and so the benzoates benzoic acid nitrites nitrates work to prevent endospora germination antibiotics are proteins that are produced by one microorganism that inhibit the growth of another we use some antibiotics to prevent spoilage of dairy products as well as livestock and meat production so we can see the effectiveness of different antibiotics on different bacteria by using the zone of inhibition plates so we'll do this in lab later in the semester just to see which bacteria or which antibiotics are most effective against certain bacteria aldehydes work by inactivating those proteins by denaturing them so this is used for preserving specimens and medical equipment so things like formaldehyde that we use to store our to preserve our dissection specimens our specimens for dissection so different type of microbes have different degrees of resistance to our standard antimicrobial treatments so some of the least resistant are your viruses with lipid envelopes your gram-positive bacteria some of the most resistant are the endospores the mycobacteria and they have those thick outer layers in their cell walls and prions we'll talk about prions later but prions aren't really cells they're just proteins so a lot of our antimicrobial agents target you know either the cell membrane or the dna well prions don't have either of those things so this table is showing the effectiveness of different types of chemical agents against these highly resistant endospores and mycobacteria you see not all antimicrobial treatments are effective for all bacteria so alcohols were pretty poor against endospores but relatively good against mycobacteria whereas chlorines were fair against both so again just showing that different types of antimicrobial agents have different levels of effectiveness against different types of microorganisms depending on their own individual characteristics