okay guys so we're officially starting unit two now for lectures so this will include chapters six seven and eight so starting with chapter six here looking at microbial growth so the first unit looked at just kind of an introduction to microbes an introduction to how we study them things like that this unit we're going to look at now how microbes grow how they function what different things they can do how they handle getting energy and producing food so just like every other species including humans microbes require a constant influx of things from their habitat whereas humans eat food different organisms are going to bring in different sources of energy so for example some protozoa bring in food from a termites intestines some bacteria dying on inorganic sulfur just depends on what species we're looking at the reason that they do this is because there are certain elements that are needed by all living things and there's several but some of the big ones are carbon hydrogen oxygen phosphorus nitrogen things like that and the list goes on now what's going to vary is how they get these elements how much of these elements they get things like that so source form and then the amounts of each element so when we're looking at these different nutrients we'll call some of the macronutrients and micronutrients but these are all grouped into one big category called essential nutrients which is just something that has to be brought in to an organism in order for it to survive so it's something that has to be provided so the two categories macro and micro just refers to how much of that element is needed how much of that nutrient is needed macro means large micro means small so macronutrients are ones that are needed in large amounts these are things like carbon hydrogen oxygen and then micronutrients sometimes called trace elements these are ones that we really only need in small amounts usually things for like enzyme functions maybe protein structure whereas those macronutrients are used for like the really important things like metabolism and cell structure and these are kind of lower on that list we saw in the previous slide things like zinc nickel so on so forth another way we can look at these nutrients is based on whether or not they contain carbon so this breaks down into two groups inorganic and organic molecules organic molecules are going to be ones that have carbon kind of another way to think of organic molecules is you can think of them as kind of quote unquote living things these are generally things that come from something that's living or products of living things things like carbs like lipids proteins methane nucleic acids versus inorganic molecules these are ones that can have going to have no carbon in them these are sometimes referred to as non-living sources so these are usually things that are more found in like the atmosphere in different bodies of water sometimes in the earth as mineral deposits so these are going to be more things like metals salts gases water things like that so when we're looking at cells of course this is a huge group of cells because we're talking about all microorganisms at once but for the most part most living things are made out of mostly water for example humans depending male or female is a little different but we are about 60 70 percent water at any given time the cells for these microbes are no different they're mostly going to be water and then they're going to have proteins in there some other organic compounds like carbs lipids nucleic acids and then all of those are going to be made up of mostly six elements carbon hydrogen oxygen nitrogen phosphate and sulfur and then they'll have some of those trace elements as well now again this is kind of lumping all microbes together so there are some variations but we'll just try and make our lives easy and say that this is for all microbes so a bit of a closer look here looking at those six main elements starting with carbon usually organisms bring these in through those organic molecules either carbs lipids proteins and nucleic acids hydrogen is used for a couple different things one of the biggest ones is help maintaining ph which we'll talk about here in a little bit it also forms a type of bond called hydrogen bonds so of course we need hydrogen for that it also serves as an energy source in respiration which we'll see in a later chapter in this unit usually this comes from inorganic compounds things like water right h2o several salts and gases as well sulfur we need for a lot of different vitamins and amino acids that are used to make proteins and this comes generally from rocks or sediment like the minerals in the ground nitrogen is generally used to build proteins sometimes nucleic acids as well and this comes usually from nitrogen gas phosphate is the main source for phosphorus which is one of the big elements that we need and we use that then to make nucleic acids several enzymes and part of the cytoplasmic membranes the cell membranes and we generally find this in phosphoric acid as well as several mineral deposits again in rocks and then finally oxygen this is found in most organic compounds not all but most so again those carbs lipids proteins nucleic acids these are used for a lot of structural functions as well as enzymatic function just means um kind of reactions starting and causing reactions generally these are found in inorganic things like water right again h2o as well as some salts and then of course oxygen gas and then a couple other important nutrients again there's a lot out there we're kind of grouping some together at the end there but these are kind of the main ones that we will look at potassium we need this for protein synthesis and then as well as keeping the membrane functioning properly sodium we need for pretty much every type of cell transport that we'll see calcium we need to stabilize the cell wall and it's also used to produce and then kind of keep the endospores for bacteria that can make them alive kind of keep those dormant i should say magnesium this is used to stabilize membranes as well as make ribosomes and keep those functioning irons needed for pretty much every protein that's used in cell respiration which is how organisms bring in and then use energy and then zinc is needed for dna replication and then these other ones grouped together copper cobalt nickel molybdenum magnesium sorry manganese silicone iodine and boron we're all just going to lump these together and say that we do need trace amounts of these technically they're used by some microbes but not others so we're just going to again lump them together okay now we're going to delve into what microbes eat so we know that they need these different elements these different nutrients but how do they bring those in so this is going to depend on one of two things actually both things carbon source as well as an energy source so first we'll look at the carbon source and for this there are two categories an organism will either be an autotroph or a heterotroph an autotroph is sometimes called a self feeder this is an organism that uses an inorganic carbon dioxide co2 as its carbon source which means it's not dependent on other living things it doesn't get carbon from a living source it just takes it in as this gas from the air usually or from water if it's in a aqueous environment a heterotroph on the other hand is an organism that gets their carbon from an organic source usually this means that they are consuming another organism so like a carnivore eating a you know a smaller prey animal or even things like prey animals eating the grass or leaves or bugs or whatever they're eating okay so autotrophs don't use living things heterotrophs use living things then if we look at the energy source we again have two categories phototropes or chemotrophs phototrophs are organisms that can photosynthesize photosynthesis if you don't know is the process of making energy from sunlight so this is how plants generally and as well as some bacteria are going to get their energy from the sun chemotrophs on the other hand are organisms that get energy from chemical compounds which is just a fancy way of saying that again they generally eat other organisms they can't just go out in the sun and be like i'm full they have to take in some kind of food source so generally when we're talking about carbon sources or energy sources you merge these two types into a single word just for convenience so for example a photo autotroph would be an organism that can photosynthesize and brings in its carbon through that inorganic means right just bringing in that carbon dioxide that co2 gas a chemoheterotroph on the other hand would be an organism that eats other organisms both for energy as well as for a carbon source so looking now at the autotrophs and their energy sources so we said we generally combine these so autotrophs then usually have two main groups photo autotrophs and chemoautotrophs photoautotrophs again are we said photosynthetic organisms they get energy from sunlight and then they use that for metabolism they also bring in that co2 gas so they again don't rely on any other organisms these are generally the ones that if you've ever seen like a food web or a food chain these are sometimes called producers because these are kind of the base for all food webs the kind of starting organism on all food chains chemo autotrophs then these can be broken down further into two types chemo-organic autotrophs and then litho autotropes chemo-organic autotrophs these are ones that specifically use organic compounds for energy and then specifically use inorganic compounds as a carbon source so organic remembers the quote unquote living things so generally they eat other organisms for energy but then they have a carbon source that's something that's not living so like co2 gas something like that litho autotrophs on the other hand they rely completely on inorganic materials for carbon and energy so they don't eat any other living organisms and then our heterotrophs again broken down in two main groups photoheterotrophs these are ones that use sunlight as an energy source but to get that carbon they do take in some kind of organic molecule chemoheterotrophs on the other hand they get both carbon and energy sources from organic compounds and there are generally two types saprobes which are sometimes called decomposers you can pick your favorite um or parasites saprobes decomposers these are generally free living microbes that feed on detrious or decaying material from dead organisms so these are things like fungus like mushrooms they're growing on like fallen dead decaying trees things like that versus parasites these are microbes or organisms that get their nutrients from cells or tissues of a living host so they're feeding on something that's living most of these are pathogens that cause disease so regardless of what type of microbe we're just talking about whether it's chemotrophs heterotrophs autotrophs doesn't matter phototrophs doesn't matter whatever type of nutrient that microbe feeds on or gets energy from they have to have a way to bring nutrients into the cell they also need a way to get things out of their cells specifically waste products because cells living things produce waste they have to get rid of them because most things if they build up they become toxic so the transporting of things into and out of the cell occurs across the cytoplasmic membrane or the cell membrane even in organisms that have cell walls it's the cytoplasmic membrane that does this and so this is going to be driven by two things atomic movement as well as molecular movement so atomic movement is kind of weird this is kind of like a physics-y nonsense but basically it's this phenomenon where atoms and molecules are in constant motion and it's random motion even the air around you right now is filled with molecules that are bouncing bumps bouncing bouncing bouncing bouncing bouncing around and that has to do with a lot of things air movement as well as like heat and temperature concentration differences things like that so you have all these little things bouncing around all the time that's atomic movement then molecule movement or molecular movement this is where atoms and molecules move in a gradient which basically just means that things don't like to be crowded so just like us if you go to a party where it's like wall-to-wall you can barely move and it's like so so full at some point you're going to want to step out like maybe step out on the patio or like you know go into another room take a breather when you leave you're like oh my god i can move um same thing if you go like in line at the grocery store there's a line of like 14 people and then a line of like two people guess guess which one i'm choosing right i'm jumping over kids like knocking over old ladies i'm going to that short line right it's the same thing with molecules they go from areas of what's known as high concentration to areas of low concentration they're going to spread out they like their own space just like we like our own space and this is a process known as diffusion so atomic movement molecular movement molecular movement specifically is this process of diffusion going from high to low concentration so we can take that a little bit further and look specifically at osmosis osmosis is the diffusion of water across what's known as a selectively permeable membrane so this just means remember diffusion is movement of something from high to low concentration so this is just the movement of water from high to low concentration across a membrane selectively permeable just means that this membrane blocks the movement of some things but not all things usually water can freely move across membranes but big things like proteins big things like sugar molecules generally aren't just free to come and go as they please so this is how cells deal with various solute concentrations in watery environments or aqueous environments because they know water is always going to move from high to low concentration right water is always going to want to spread out through this process of osmosis until it reaches equilibrium or basically until it's balanced so this is kind of how they get around this you know really salty environments or environments that are not salty enough or don't have enough sugar too much chemicals whatever it is in the water so just showing this image here so we have a little flask filled with water and then inside it we have this little balloon of say sugar water or salt water whatever it is and you can see the little blue molecules those are water molecules and then we have the little red molecules that's the solute either the salt the sugar whatever so this little membrane this little sac that these things are sitting in is the selectively permeable membrane most things are not going to be able to freely move so those big molecules those big red sugar molecules are not going to be able to move across that membrane but water can move freely so water is going to slowly move from areas of high to low concentration now this kind of screws with people because you need to think of the concentration of water in terms of how much percentage there is of water in a space so for example in the beaker if you take that little sack out that's 100 water right there are only blue molecules in it okay but in that little sack you can see there's a mix of red and blue molecules so say that sac is like a 50 salt solution if it's a 50 salt solution that means the other 50 percent is water right so if water wants to go from high to low concentration water is only concerned with itself so it knows there's a hundred percent of me in the beaker but only fifty percent of me inside this little sack even though there's salt or sugar or whatever it is in there water doesn't care all it sees is the other water and it's like oh man look at all the space in there i can go from like a two bedroom apartment to a three bedroom two bath house boss so it's going to go into that little sack which you can see in the little kind of blown up image you can see the one arrow kind of pointing that little blue uh blue molecule in right there's technically three arrows one is going out but two are going in so you can see there's more movement of water into the sac so we're looking at this osmosis we're looking at what's known as the osmotic relationship which just means the concentration of things of solutes right inside and outside the cell basically on either side of that cytoplasmic membrane so if solute confuses you basically just think of how much stuff and how much water is on each side of that membrane if there's more stuff that means there automatically has to be less water right because there's only so much space okay but if there's less stuff that means there has to be more water right if there's 100 water that means there is no stuff if there's 50 water that means there's 50 stuff if there's 25 percent stuff means there's 75 water okay so when we're talking about this we're basically going to end up with three possibilities in terms of concentration we're either going to have an environment be hypertonic isotonic or hypotonic a hypertonic environment is the solution remember on we're looking at two sides of a membrane the hypertonic solution is going to be the side of the membrane that has more stuff more solute and thereby less water compared to the other solution so i always think of hypertonic solutions i always think of like hyperactive kids they're all hopped up on sugar they have too much sugar right isotonic solutions then you're going to see if you have the same concentration of solutes and water on both sides of the membrane in which case both of those solutions are going to be isotonic hypotonic then is going to be the solution that has less stuff more water in it so just remember again there are two solutions that we're looking at so one on either side of the membrane so if you have one side that's isotonic that means that both of these have to be isotonic because we're comparing them right so it can't be equal if the other one is something else and then same goes for hyper and hypotonic if you have one side hypertonic that means the other side has to be hypotonic and vice versa and so the reason we care about this is again because that water is going to move in these different environments so for example the left side there it's showing two different types of microbes the top microbes with the pink are ones with the cell wall the ones on the bottom are ones that don't have a cell wall they only have that cytoplasmic membrane either way the two images on the far left there are in an isotonic solution which means the concentration inside and outside of the cell are the same and you can see with the blue arrows there it's showing that water is moving equally in both directions so there's no kind of mass movement of water this isotonic solution are is a cell's happy place and this goes not just for microbes but our cells as well right this is kind of the goal that cells want to live in then these other two hypotonic and then hypertonic solutions so in the center we have those two cells living in a hypotonic solution remember hypotonic means that there's less stuff more water and you can see in both images there's very little solute there's only two little little blue molecules outside of both cells but there's a lot inside the cell so outside the cell is hypotonic which means if you have one you have to have the other inside the cell is hyper tonic so in this case again water is going to move where there is more stuff right where there is less water high to low concentration so water is going to push into the cells in this case now we run into an issue with hypertonic solutions and hypotonic solutions in hypotonic solutions if water is pushing in again there's only so much space in a cell right so cells that have a cell wall that's used for protection that's used to kind of help prevent bursting but if you look at that bottom cell there in the center you see these little holes it looks like water is rushing out that's because at some point and this is true for our cells as well because we don't have cell walls at some point so much water is going to rush in that it's going to make the cell pop or lyse that's bad that means the cell just died okay so hypotonic environments are bad for our cells now look at hypertonic solutions so again this means now we have a cell that's put in a solution that has a lot of stuff very little water compared to inside the cell so in this case the outside hypertonic inside hypotonic right again water is going to go where there is less water which means more stuff so in this case water is going to rush out of the cell which you can see by those arrows and again this is a problem it's a little bit better with cell wall but you can see the little shrively inside the cell wall there organisms that don't have a cell wall like our cells they don't have that cell wall the cell will shrivel up shrink kind of like a little raisin through a process called crenation and again it can get so bad that they can die so hyper and hypotonic solutions are bad for ourselves at least up to a point at some point it for sure will kill ourselves but even a little bit can make a big difference so happy place isotonic even concentrations so that was the movement of water how it can freely move in the cell based on or into and out of the cell based on solute concentrations most things though cannot freely move across that membrane remember it's selectively permeable most things cannot move so for this we have to have different transport processes and there are several different types two big categories passive transport and active transport and then a couple types of each we are only going to look at two types of passive transport simple diffusion and facilitated diffusion and then active transport we're just going to lump into one category so passive transport is basically just some kind of transport that does not require any energy input because the molecules will just naturally want to do this because passive transport is going to move things from high to low concentration so this is again just a form of diffusion right which is why both of the two types of passive transport are named something diffusion so simple diffusion is going to be solid solutes just going directly through the membrane walls it's basically i always think of like the kool-aid man which some of you are young and you may not know what i'm talking about like those old kool-aid commercials with the big dude big like jug of kool-aid he like through the walls oh yeah right that's basically what molecules are going to do in this case they're going to go straight through the walls of these cells nothing can stop them that's simple diffusion facilitated diffusion on the other hand is still molecules moving from high to low concentration right still passive transport but they're not just going to burst straight through the walls they're going to have to go through some kind of opening a channel or a pore basically they have to use a doorway like us at the end of a class or at least in a normal class when the bell rings in school or whatever happens teacher dismisses you there's a lot of you in class then you start facilitating diffusion you start leaving through the doorway right most people wouldn't just run through the wall of the classroom at least preferably let's not do that okay so that's passive transport no energy required things go from high to low concentration the only difference between the two simple diffusion facility diffusion one uses a doorway one does not simple diffusion straight through the wall facilitated uses the doorway active transport on the other hand this is going to require energy because now we're going to force molecules to go where they don't want to go because we're going to force them to go from low concentration to high concentration we're going to force them to get crowded and they don't like that so just looking at these again so we have our two types of passive transport on the left there and you can see the little blue molecules that's uh simple diffusion or just diffusion in this image here but simple diffusion just going straight through that cell wall and then facilitate diffusion going through that doorway high to low concentration active transport on the other hand we see atp our little energy molecule it is now forcing those green molecules to go from low to high concentration which they don't want to do so we have to force them that's why we need energy okay so active transport passive transport that base is movement depending on what energy is used and what type of doorway is used or if a doorway is used we can also describe the movement of things in terms of exocytosis or endocytosis which basically just means which direction are they going are they coming into the cell or leaving the cell so exocytosis is things leaving the cell sending things out or exiting the cell endocytosis is bringing things into the cell now there are several types of each we are going to make it very simple we are only going to have two types of endocytosis so two types of bringing things in and then we're just gonna have exocytosis says okay things leave so the two types of endocytosis we're gonna have phagocytosis and pinocytosis so this is sometimes called cell eating versus cell drinking which if that helps you great if it doesn't don't worry about it um but phagocytosis is basically the process of bringing in really large things or solid matter versus pinocytosis is bringing in some kind of liquid so you don't have this big like clump of things you're just basically like slurping in so if you look at phagocytosis on the left side of that image there you can see the cell kind of reaches out with what's called pseudopods i don't care you know the name just want you know they basically reach out grab this thing and bring it in versus pinocytosis you can see instead of reaching out they basically just kind of like slurp in it causes the cell membrane to kind of indent process called invagination i know but kind of comes in then pinches off and then you brought in this little sack of liquid so sort of kind of cell eating versus cell drinking phagocytosis pinocytosis okay this next section is going to now look at environmental factors that are going to influence microbes and there's a bunch we're going to start with temperature things like how hot how cold certain gases the two we'll focus on are oxygen and carbon dioxide but there are several then we'll look at ph osmotic pressure radiation atmospheric and hydrostatic pressure and then also how other organisms can influence whatever microbe we're specifically looking at starting with temperature so there are three kind of ranges of temperature or three kind of main temperature bases that we're going to look at with different microbes a minimum growth temperature an optimum and a maximum growth temperature the minimum growth temperature is going to be the lowest temperature that an organism can survive at doesn't mean they're growing well but it means they're still living versus the maximum growth temperature is the same thing it's just the highest temperature that an organism can survive at and then the optimum temperature this is going to be the best temperature that an organism lives at where it not only survives but thrives so in terms of what kind of temperatures we're looking at there are lots of different groupings of organisms based on what temperatures they prefer so starting with psychophiles these are organisms that prefer like really cold temperatures any anywhere from 0 to 15 degrees celsius which is very very very cold so these are ones that generally live in the antarctic or the arctic deep oceans where it's very cold very little sunlight things like that then as you gradually make your way up we go next to cyclotropes psychotropes are cold tolerant it means they don't mine cold they can survive at cold temperatures but it's not as cold so these are generally where generally in between 15 to 30 degrees celsius so these are the ones that live in like rivers and lakes then we have mesophiles these are kind of the middle loving range these are anywhere from 10 to 50 degrees celsius this is most organisms including us mammals things that live on the surface of the earth or up high in the surface water where that sunlight from the sun kind of warms the environment then we get into our groups that prefer hot environments starting with thermophiles these are heat loving these are 45 to 80 degrees celsius these are ones that live in the topsoil where the sun kind of beats down on it silage compost piles things like that and then finally into our hyper thermophiles or our extreme heat loving organisms these live in those temperatures that are 80 to 120 degrees celsius which if you don't know i know we work off of fahrenheit but just to kind of give you guys a more generalized look at temperatures zero degrees celsius is the temperature of freezing so that would be like you know where water turns into ice and then 100 degrees celsius is boiling so for example psychophiles they prefer temperatures that are about freezing hyper thermophiles they prefer that boiling or above boiling temperatures places like hot springs places like hydrothermal vents they've even found some living in um like in and around volcanoes okay so very hot temperatures so one thing to note when we're talking about these ranges just because an organism can survive in that range doesn't mean it's their ideal temperature so for example let's look at mesophiles we said most organisms are mesophiles though those are that kind of middle range organisms and they have that range of like roughly 10 to 60 degrees celsius whatever that was but if you notice there's this quantum coat in this image here because we're talking about bacteria we call it the danger zone because bacteria are bad for us but for the microbes that's kind of the prime time is the very center of that range really only like 20 to 50 degrees celsius versus they can still survive a little above and a little below below that right they can go down to 10 degrees they can go up to 60 degrees celsius but it doesn't mean that they prefer it there right they are still growing just not as well and that's true for any of those ranges for example the psychrophiles the ones that like they're really really cold temperatures they can survive up down to that freezing that zero degrees celsius but they might prefer a little bit warmer like five degrees celsius all right so that's it with temperature now let's move on to gas requirements so we're only going to look at oxygen and carbon dioxide o2 and co2 so starting with oxygen this has its greatest impact on microbial growth far more than co2 does because it's one of the most important gases in terms of respiration and it's also used as an oxidizing agent which we'll talk about in a later chapter so in terms of requirements for oxygen microbes are going to fall into one of three categories microbes that can use oxygen and detoxify it microbes that can neither use oxygen nor detoxify it and then those that don't use oxygen but they can detoxify it so one thing about oxygen is when it's broken down in this cell respiration it can turn into this toxic material which is something that organisms need to either break down or get rid of so that's what we're talking about in terms of detoxifying it so if they use oxygen they have to be able to break it down or if they bring oxygen in but don't necessarily use it they can still break it down make it non-toxic so those that use it detoxify it those that don't use it don't detoxify it those that don't use it but can detoxify it so he said that once oxygen goes into cell respiration it's turned into toxic products so there are generally four options or four possible toxic products that are made either singlet oxygen a superoxide ion hydrogen peroxide or hydroxyl radical all of these are generally fairly reactive and toxic to cells some of them so much so for example hydrogen peroxide is actually used as a disinfectant which means it can kill things we've like put it in products that's why if you've ever poured like hydrogen peroxide into like a cut or something it starts bubbling right that's actually it breaking down any like debris that's in the cut trying to clean the cut so of course we don't want that type of material inside cells and microbes are no different so in order to break down these byproducts of oxygen we generally have several processes that are handled by enzymes enzymes we'll talk about in a later chapter are basically these specialized little proteins that start or speed up reactions so we're going to have a two-step process that requires two enzymes one for each step so the enzyme name is in the little blue boxes so step one you have one of those toxic oxygen byproducts there you're going to add hydrogen and you're going to use this enzyme called superoxide dimutase it's going to turn that toxic oxygen into hydrogen peroxide and just oxygen gas so hydrogen peroxide is h2o2 so we said that's still one of those toxic byproducts right it can be used as a disinfectant so we're not done yet it's slightly less toxic it's a little less reactive but it's still not good if it builds up so then we go into step two where we take that hydrogen peroxide h2o2 we use our second enzyme catalase catalase is going to turn that into water h2o and more oxygen gas both of which are non-toxic and in fact cells generally need both of those so when we look at oxygen usage and tolerance patterns there ends up being kind of five groups of microbes aerobes speculative anaerobes anaerobes aerotolerant anaerobes and micro aerophiles so remember anytime something has an a or an an in front of it and sciency terms it means no none or not so when we say aerobe versus an anaerobe aerobe is something that uses oxygen or it can handle oxygen at least anaerobe is something that doesn't use air doesn't need air so when we're looking at arabs which are sometimes called obligate aerobes these are organisms that require oxygen speculative anaerobes on the other hand these are ones that prefer oxygen which means they don't have to have it they ideally use it but if there's no oxygen they're okay right anaerobes sometimes called obligate anaerobes these are ones that not only do not use oxygen but they're in fact killed by oxygen so they want nothing to do with it then we have aero tolerant anaerobes these are ones that still don't use any oxygen but they're not killed by it like the obligate anaerobes are and then finally micro aerofiles these are ones that require very low levels of oxygen these are very small range here and we'll see these when you are doing different labs that look at this the image here kind of shows nicely what you end up seeing with these different types of microbes so for example if you have an obligate aerobe one that needs oxygen if you're trying to grow bacteria in a broth you'll see all of the bacteria up at the top of that broth because the farther down you go in that media the less oxygen there is right so they're going to be up by the air speculative anaerobes these are ones that prefer oxygen but don't need it so you'll have microbes all through that broth like an image be there but you'll have more of them at the top because again they prefer oxygen anaerobes those obligate anaerobes those are the ones that not only don't use oxygen but are killed by it right so you'll find all of those at the bottom of a media aero tolerant those are ones that don't care they can live anywhere so they're pretty much all through that broth again though they prefer no oxygen so you'll have a little more at the bottom and then finally microarrow files they need low levels so you won't find them way up top you won't find them way at the bottom but you generally see a line of them kind of in the center of that broth then moving on to carbon dioxide co2 so all microbes require at least some amount some form of co2 for their metabolism and there are different types so for example capnophiles these are ones that grow best at really high levels of co2 generally higher than what's normally found in the atmosphere these are important for really only a couple species in terms of microbiology that cause disease right because we had a lot of trouble first recognizing them because again it's not something that normally happens in the atmosphere so it took us a long time to figure out okay how do we actually isolate these in a clinical situation in a lab so for example the microbes that cause gonorrhea cause certain kinds of fevers pneumonia meningitis these are all capnophiles ones that need that higher level of co2 all right moving on to the ph scale so if you don't know just kind of real quick breeze through of the ph scale this is measuring basically how acidic or how basic or alkaline something is so basic and alkaline mean the same thing so we're looking at a ph scale it tends to throw people off to start with because when we think of a scale of something like on a scale of one to ten you know we think okay one side is bad one side is good that's not the case with the ph scale the phdl goes from 1 to 14 or 0 to 14 and basically the middle of that range right at 7 is considered quote unquote good that's where we have neutral this is like pure water neutral scale it's not acidic it's not basic and then the farther away you get away from that seven is the more acidic or the more basic depending on which direction you go so the further away from seven you go closer to one right or the smaller the number gets the more acidic something is so for example our stomach acid or gastric juice right is anywhere on a ph of like one to two right so it's very very acidic versus if you go above seven the farther you go up on that ph scale the more basic something is the more alkaline something is so for example like bleach is about a ph of 12 or 13 depending on the brand okay so 7 neutral closer to 1 acidic closer to 14 basic so when we're looking at groups of organisms based on ph we're going to have three groups neutrophils acidophiles and files so neutrophils are going to be those ones that prefer that middle ph range the neutral range generally they grow anywhere from like a six to an eight slightly acidic all the way up to slightly basic acetophiles these are ones that are going to prefer acidic environments for example a lot of fungi like molds and yeasts are going to prefer a ph of like five to six versus you have some like the bacterium hylopector by pylorae it actually lives in stomach acid it prefers stomach acid so it prefers that really acidic like ph of two and then alkyl files these are ones that are going to prefer that alkaline or those basic conditions anywhere above seven osmotic pressure this goes back to diffusion and hyper and hypotonic solutions like we talked about earlier most organisms are going to prefer equal osmotic pressure which just means that there's the same ion concentration on either side of the membrane so on either side of like the cell wall or the cytoplasm membrane whatever it is which means there's no overall net water movement or there's equal movement at least most microbes again prefer this that isotonic some of them prefer slightly hypotonic solutions but there are some microbes known as osmophiles that prefer hypertonic environments remember when we mentioned earlier when a cell is put in a hypertonic environment generally it means that there is more stuff less water outside the cell right so that causes water to leave the cell causes it to shrivel up and die not for these guys these guys like those environments so for example one specific type of osmophile is known as a halophile these are ones that specifically prefer high salt concentrations and there are two types we either have salt lovers or salt tolerant so obligate halophiles are ones that need high levels of salt speculative halophiles are ones that if the environment gets too salty for whatever reason they can handle it they won't shrivel up and die real quick so this is just looking at that difference again so on the left here we have an isotonic solution that that cell is living in where there's equal water movement or no water movement cells just live in its life it's good versus on the right there we have a cell put in a hypertonic solution where there is more stuff less water outside the cell water is going to diffuse go from high to low concentration so water is going to push out of the cell causing that cell to shrivel up go through that process of crenation so radiation and pressure we're not going to do a whole lot with but for radiation there are some microbes typically phototropes that use visible light rays as an energy source right they use sunlight for energy but most microbes are damaged by light rays and then on top of that there is that non-visible light like ultraviolet light uv light ionizing rays that we use for like x-rays and cosmic rays that are even more damaging for example uv rays are what cause sunburns right and skin cancer in humans so of course those are damaging some microbes can tolerate higher levels of radiation some not so much most not so much then looking at pressure again just barely touching on this most organisms have a certain amount of pressure they can stand but there are some that like pressure and these are known as barophiles barophiles these are ones that generally live in really deep downs in the ocean where there's a lot of pressure of the water pushing in on organisms and in fact some of them are so adapted to that high pressure that if they're taken out and put into normal atmospheric pressure like if they're pulled up through the sea they'll actually rupture when they reach that normal atmospheric pressure because they have so much kind of push back inside their bodies this is also why if you've ever gone like scuba diving if you've ever watched um anything about like like a movie or something or like trying to think of that relatively new movie with mandy moore and she was in a shark cage and shirt cage fell and all that nonsense whatever um but if you've ever heard of ben's or basically if you're scuba diving and you go down so far you can only go down so far so fast and same thing when you come back up you can only go up for a little bit then you kind of have to stop let your body equalize go up equalize go up equalize if you just shoot to the top your body just basically can't handle it and your brain basically ruptures and bleeds out which is not ideal this is also why we have to have like these really crazy thick crazy um kind of detailed plants to make things like submarines and anything that's going to go deep in the ocean because again this huge pressure can actually crush most things it can crush like a human skull like a tin can right if we just went down there with no protection all right then the last group of factors we'll look at are how other organisms affect whatever organism you're looking at so different types of partnerships so there's going to be two types symbiotic and non-symbiotic relationships that we'll look at and then we'll break those down a little bit further this is a nice kind of slide overall when you come back to study i would refer back to this one kind of just a nice overall image so first we'll look at the symbiotic relationships so symbiosis is just a term that says that two organisms live together with some kind of close partnership in which case each member of that symbiosis is known as a symbiont or one of the organisms in that partnership and there are three main types of symbiosis mutualism commensalism and parasitism mutualism is where organisms live in an oblique obligatory but mutually beneficial relationship or basically they both get some benefit from the relationship right it's mutually beneficial so for example microbes that live in our intestines we all have them just how it is um there's a benefit to some of those microbes now some of them are pathogens that make you sick but for the most part we do have a lot of microbes that live in our bodies at all times and we kind of need them and it's a mutualistic relationship because the microbes get a nice warm safe place to live they get lots of food from us and then meanwhile we get something from them as well for example the ones that live in our intestines they help us break down food sources we normally wouldn't be able to eat for example a lot of plant materials we actually can't digest fully on our own we need microbes to help us to get the nutrients out we also have some microbes that produce vitamins like vitamin k which helps us create blood clots and things which we need so if we don't you know if we get it cut we don't bleed out that's a mutually beneficial relationship commensalism that's where one partner gets some kind of benefit but the other partner is unaffected it's not harmed but it's not benefited either skin microbes is not a great example i'm not going to lie because we actually do get a benefit to these i originally had it as oh they get a nice safe place to live but they don't really hurt us but that's not true because they do kind of help us because they help fight off invaders coming in a better example would be like birds nesting in a tree so the bird gets a nice safe place to live high up away from predators gets a home but the tree for the most part isn't really benefited it's not really harmed as long as it's not something like some huge bird that's breaking branches or like woodpeckers that are stabbing the tree you know to get bugs things like that for the most part they're not affected and then last one paracetation parasitism so this is where one organism is benefited and the other one is harmed so this for example is pretty much every pathogen they get in they get food they get nutrients and then we get sick so those were the three symbiotic types of relationships now let's look at the two non-symbiotic relationships starting with synergism so synergism is a relationship between two organisms that benefits them but it's not necessary for survival it's basically just something that they can do but they don't have to do so for example they can sometimes do something that normally they wouldn't be able to do uh like the lichens we talked about in the first unit the fungi and the algae or the fungi and the cyanobacteria that live together can make this home produce their own food and then they can live wherever we have algae that lives by itself we have fungi that lives by itself so they don't have to get into this relationship but they can and it gives them a benefit another good example is biofilms biofilms are things that we see in a lot of bloodstream infections we also see it in a lot of gum disease and dental caries where different types of microbes kind of clump together form this kind of film this plaque basically that is kind of a protection for these different microbes that they wouldn't normally have and so this just look i know we've looked at biofilms before but just mixed communities of these microbes where one kind of starts this little slime layer and then more add in as more add in it's harder and harder to get rid of that biofilm which is bad for us good for them one thing that's kind of interesting is that because there are different species in this biofilm they have to communicate in kind of unique ways because it's just like us living with you know like a fish tank or you know cats or dogs or whatever we can't communicate with them the same way so we have to use different ways to communicate for biofilms this form is known as quorum sensing and this is used by bacteria basically for them to communicate with those different species in that biofilm as well as their normal species but essentially what they do is they just send out chemical signals instead of communicating like they normally would they basically send out like these little flare guns to be like hey food over here hey something's trying to hurt us so it's kind of interesting so another non-symbolic relationship is known as antagonism so this is where free living species are ones that don't live inside you know another organism associate but it's not a benefit to either one in fact it generally happens when they're competing for for resources so this is similar like if you remember like an english class like an antagonist as one that's working against like the main character the protagonist that's kind of what this is they're working against each other because they're competing for the same space or the same food source whatever it is the reason we care about this in terms of microbiology is because some organisms have developed really unique ways to compete for resources including antibiosis antibiotics is the production of antibiotics or inhibitory compounds to basically stop other microbes from coming into their space so the way we first discovered penicillin is this one type of bacteria penicillium was pumping this antibiotic out into the soil around it where it lived and it was the only microbe able to grow there because everything else came in the antibiotic it made killed everything else off so we were like hey that would be great to put in our bodies very human response but this was important for the microbe because that one that's doing the antibiosis had that competitive advantage because it was able to increase its space thereby increasing the nutrients that were available to it because if nothing else could come in and compete you had all this space all right now moving on to some reproduction um so review remember the way most prokaryotes reproduce is through that binary fission one cell splitting and becoming two where the daughter cells are identical to that parent cell remember this is a form of asexual reproduction which just means it takes one cell to make new cells you don't need two like you do for sexual reproduction like humans have and the overall steps again we don't get too complicated with these but elongation and chromosomal replication interceptation and then finally cell separation so generation time also known as doubling time this is the amount of time it takes to complete one cycle of binary fission so this is the time it takes to get from one parent cell to two daughter cells one generation or against the doubling time so this is going to mean that the population is going to increase by a factor of two or basically it's going to double so if you have one cell you'll end up with two daughter cells if you have two cells you'll end up with four daughter cells four cells eight daughter cells so on and so forth and this doubling is going to keep happening as long as the environment remains favorable so it'll keep doubling constant rate every single generation population will double so the amount of time it takes to have one complete generation renew or the length of generation time is known as the growth rate of an organism so on average generation time is anywhere from 30 to 60 minutes for microbes but it can be as fast as 10 minutes or as slow as several months for example mycobacterium leprade the bacterium that causes leprosy has a generation time of 10 to 30 days significantly longer than the average for microbes versus like e coli generally has a generation time of about 12 minutes so environmental bacteria tend to be the ones that have these longer generation time measured in usually months versus pathogens the ones that have to get into another organism those are the ones that usually have really short generation times and the reason for this if you think about it is because if you're getting into a living thing usually living things have some sort of defense against that microbe coming in like humans we have our immune system anytime a microbe gets in our immune system starts going to work trying to kick the ass of whatever pathogen it is trying to make a sick so they need to be able to grow really really quickly if they take a month for that one microbe that got in to start growing and reproducing we would never get sick which would be awesome for us not for the pathogen so when we're looking at the math of population growth basically what you can do is you can take the cell population size and represent it by the number two with an exponent so that exponent that little number up high above the two that's going to increase by one every generation so that little number that exponent is basically the generation number okay so it's basically for example if you look don't worry about time and hours on the image here but if you look at number of cells you start with one cell then you get two cells right that's one generation complete then if you go further you have two cells exponent of one that's the first generation if you go into the next generation you can do either four because it's still easy to do that math right or you can take two to the second power for these lower generations especially when you have a small population size to start with that's that's just as easy either way but if you get to these really large population sizes it's easier to say 2 to the 8th power rather than doing the math to figure out okay what would be the number of cells we have at the eighth generation if they double every single time um and then so on and so forth the further on you go you can see down at the bottom there 2 to the 20th power for the 20th generation would be you know over a million microbes so this growth pattern is known as exponential which just means that the population numbers are expressed in a math term called logarithms or exponents so we have those little numbers above the two there so when we're looking at this exponent don't worry about the other stuff just yet but look in the bottom right hand here when we're looking at these exponential growth right what we end up seeing is what's known as a j curve in the red line there where you have really small numbers that grow very very suddenly and continue growing very very quickly again that's important to note that this is only as long as the environment remains favorable then if you look at the image to the left there that kind of breaks down the number of cells generations the exponent that you would use and then the logarithm again it's kind of nice if you were struggling with kind of keeping up with that i know this is a bio class you're like what the hell is math doing in here this sucks but just kind of breaks that down so for example if you have one cell right that would be the very first there wouldn't be a full generation yet so that would be zero for the generation number which means if you change that to that exponential number it would be two to the zero power then if you go further say let's go we have eight number of cells that would be the third generation you could represent that with two to the third power and that would be that three as the logarithm or as the exponent so very rarely when we're doing research for microbes or any living organism do we know the population size immediately upon looking usually we have to kind of guess so in order to kind of figure out that population size we have an equation nt equals n times 2 to the nth power where nt equals the total number of cells in a population right the population size the little t represents a certain point in time so say like okay in you know the third year of a study or in you know 2020 whatever and then in the parentheses there that's the starting number of cells that was in that population the exponent n the little n up above the two that's going to tell us the generation number so then 2n altogether is going to be the number of cells in that generation so that sounds very complicated so let's do an example real quick so let's say you have e coli and e coli has a 20 minute generation time let's say that we start out with a hundred cells how many cells will there be three hours later so for this we're going to use that equation and t equals n times n sorry times 2 to the nth power and we have to do a couple of things first here we first have to figure out our n that's in the parentheses right our initial number and we have to figure out that 2 to the nth power so we already know n because we said n is going to be the starting number so that's going to be 100 the number of cells we started with now the 2 to the nth power is a little trickier because i gave you 20 minute generation time but i'm asking you three hours later so you basically have to figure out okay how many times does 20 minutes occur within the three hour period which is going to be nine so that two to the n is going to be 2 to the ninth so this is what we end up with n to the t equals 100 times 2 to the ninth power do some math and some nonsense with a calculator you end up with 100 times 512 so you end up with at that point in time at three hours right the population size is going to be 51 200 microbes so you can see just with this little quick equation here even though we just made it up you can see how quickly the growth of bacteria especially can get out of hand because they have those really short generation times they can grow very very very quickly which is why they're so dangerous when they're pathogens and things like that and they're in biofilms and whatever else affects us so exponential growth and the equation nt equals n times 2 to the nth power is all well and good but very rarely in real life in fact so far never is that exponential growth a long term thing enclosed systems which is pretty much every environment whether it's in the lab whether it's out in the real world there is at some point going to be a lack of resources so at some point you're going to run out of space you're going to run out of food waste products are going to start building up whatever it is and that's eventually going to cause basically cells to stop dividing and start dying instead so that exponential growth rate is kind of their maximum growth rate so when we're looking at true systems or closed systems or batch cultures when we're looking at real life what we'll end up seeing is known as a growth curve this is basically a pattern that's going to be seen in bacterial populations and real life systems as well but you know for other organisms but we of course are in microbiology so we'll look at mostly bacteria and one way that we can kind of map this growth curve is through a standard what's called plate count technique where you put a small number of microbes into some sterile broth you incubate it for several hours and at regular intervals you take some of that broth you plate it and grow whatever is on that media then once it's all grown up you count the number of colonies which sounds more complicated than it is so here's basically just a visual image of that where you take this broth you put some microbes in it and you can see at the top there the time intervals every hour they incubate it and then every hour they sample it put it on a plate and grow it and you can see in that third row there with the little plates that very first sample at an hour there was no microbes there and then you get more and more and more of these cells starting to grow so this is more what we do in or would do in face-to-face labs you'll have an online lab that kind of looks like this but we would certainly have done more of this in face-to-face classes so when we end up kind of analyzing these different growth curves we end up with a very standard rate of growth for almost every microbe the time length may vary but the overall shape of this growth curve is almost always the same and remember this is important to note that this happens only in closed systems because the amount of resources deplete and eventually wastes and things that are toxic end up building up so you end up having four phases lag phase a log phase sometimes called an exponential growth phase stationary phase and then a death phase or a logarithmic decline phase so first looking at the lag phase so this is the amount of time where you don't really see any new bacteria growing like in the image two slides ago that very first plate after an hour you didn't see any colonies on that first plate this is the amount of time it takes bacteria to basically get used to their new environment to acclimate so they're not really dividing yet they're just kind of like look at my new home right decorating and nonsense then you go into the log or the exponential growth phase this is where the bacteria start dividing and because it's a fresh environment they're doubling every time they go through a generation so this is that maximum growth phase this is basically that exponential growth which is why sometimes called the exponential growth phase where they're growing very very quickly at least during this time stationary phase then is when the bacteria have divided so much that at some point they start competing for nutrients they start competing for space they start competing for food and so some cells in this case are going to win and some are going to lose so what happens is you still have bacteria growing you still have bacteria dividing but the same time you typically have the same number of cells start dying off so basically the number of bacteria start to stabilize and then eventually no matter what microbe at some point they will eventually go into that death phase where toxic waste products start building up nutrients are gone so now bacteria begin to die off more quickly than they start growing so the numbers start going down and so here's just showing again an image of this same thing lag phase exponential growth stationary death phase in that lag phase you can see there's very few live cells because they're acclimating then in exponential growth you get lots and lots and lots of cells then in that stationary phase some start to die off but you still have some living cells so the total number of cells doesn't change then in death phase that population number starts to decline because you have more and more cells dying so this is important to know for us for a number of reasons for example we need to know some of this about microbial control food microbiology as well as culture technology for example we know that during this exponential growth phase where microbes are growing fastest because they're actively growing they're more more vulnerable to things like antimicrobial agents and heat so it's easier to kill them because when they're actively growing they're more vulnerable to their cell metabolism getting broken up binary fission being stopped all these different things to kind of stop them which is very important for pretty much any time you're trying to control microbes for example if you're trying to keep food from spoiling like pasteurization is one of those things that we talked about in the first unit where they heat something for a certain amount of time to kill microbes but not destroy the product like they do with pasteurized milk that heat because the microbes are growing still kills off the microbes because they're so kind of weak at that point not weak but they're vulnerable at that point so that's important to know same thing for uh like medical microbiology trying to keep like ourselves healthy we know that's a good time to you know use some kind of chemical agent to kill them we also know for medical microbiology that a person that's actively shedding bacteria in those early stages is far more likely to spread it than someone in those later stages because in that early stage they're growing so quickly as you pass it to the next person they're already growing so it's easier to pass that infection versus if they're in the death phase at the end of like your antibiotic cycle or whatever they have given you you can still pass it but you're less likely to pass it because a lot of those microbes are dying off so let's say we have a bacterium like e coli that has a generation time of 20 minutes if this was allowed to grow exponentially for 48 hours it would produce a population that weighed 4 000 times more than the weight of the world just let that sink in for a second let it marinate right that's a ton roughly of microbes okay so why haven't bacteria taken over the planet because remember an exponential growth is not a real world situation at some point they run out of nutrients they run out of space they run out of resources and toxic things start to build up and kill them off so luckily our planet is a closed system there's only so much space for any living thing right including humans right and that's actually one thing that scientists are very worried about the past couple years because there are so many people on the planet we're worried that we have ourselves reached our kind of um height of our exponential growth and they're worried that we're going to go into that death phase soon if we don't do something about it to either increase the carrying capacity of our planet to increase the number of people we can have or to slow our growth rate because at some point we will run out of space we will will run out of food just like microbes do okay and then the last little tidbit i want to cover is different ways that we can analyze population size without having to culture without having to do plates or broad things like that so starting with turbidity this is basically just a fancy term for how cloudy something is so instead of having to make a culture having to plate out whatever you're looking at a lot of times if you don't care the specific number if you just need to know if something did grow a lot of times they'll just look at sterile broth and see if it has any kind of cloudiness to it because sterile broth is normally clear so if you put microbes in it and you want them to grow if you pull the culture out the next day or you know 48 hours later if that broth is cloudy now it means that the microbes grew and the cloudier it is generally the bigger the population size we use this a lot if we were in face-to-face lab face-to-face labs i would use this every day that we went into lab i'd go check is it cloudy yep okay we can use it is this not nope all right i have to figure out something totally new for us to do today we can also use counting which sounds easier than it is but basically if you know how many cells are in a certain amount of fluid you can use some math nonsense and multiply that to determine the number in a total volume of that same fluid and there are different ways you can do this counting so you can do mechanically which involves basically putting a itty-bitty amount of this culture on a slide and then getting on a microscope and actually counting the cells on this little pre-measured grid which is like in the image to the left there problem with this is it's not very accurate because on a microscope there's no way to tell live from dead cells so you could be you know in that death phase where half of the microbes are dead but all you see is just their little body like their little carcass not quite the same or you can use automated versions these are more accurate but far more expensive and they can actually tell the difference between live and dead cells depending on how fancy you get we can also use genetic probing so this is kind of a variation of polymerase chain reactions that pcr technique called real-time pcr basically this allows for quantifying or counting microbes in an environment or a sample without isolating them without having to culture them that's really all i care that you know for the moment because we'll actually do more of this later on in the semester as well so why talk about it twice