Transcript for:
Chapter 4

all right we're going to jump into chapter 4 now and with chapter 4 we're going to learn about a topic that uh is going to overlap a lot with uh some of the concepts that we cover in the lab uh part of this course as well we're going to learn about the growth of proaryotic organisms um and first we want to define growth we see that growth is an increase in the number of cells um so when we're talking about uh a single cellled organism uh it doesn't grow by getting bigger it's still just one cell so it's not the cells are getting bigger it's the population is growing the number of cells are getting bigger so microbial growth is uh new cells being created um and the population growing in its number and what we see here is that generally proarotes are going to multiply through a process of binary fision binary fision is where one cell is going to double its DNA and then split to form two identical cells so binary fision fision to cut and binary in half so one cell splits forming two identical cells these cells are identical to the cell that we began with we see here that this is an exponential process we see that the rate of growth um you know gets faster and faster as time goes on because as one cell splits it becomes two the two cells in that same amount of time will split to form four the four cells in the same amount of time will split to form eight 8 becomes 16 become 32 become 64 become 128 become 256 and we see here that in the same amount of time that it took for us to increase the population by one cell later on we're going to increase the population by a million cells in that same amount of time so it's exponential growth and so the uh point that we just brought up there is the generation time the amount of time that it takes for the population to double in size that's something we want to take into account and um the generation time is um in the bacterial world is is often very short it can be as short as 20 to 30 minutes um between uh the moment that a cell is created and the moment that it splits to create two new cells so we can calculate um the approximate size of a population over time using this formula here uh knowing the current number of cells we are able to figure out um the uh I'm sorry this is the um the current number of cells that we're uh that we're estimating um we're going to figure out by the number of cells that we start off with multiplied by um the uh 2 to the n which n represents the number of uh divisions the the number of generations that have taken Um so this is helpful if we are working with complicated numbers we can use this formula here so for example um we have this scenario where we start off with five E.coli cells on a potato salad four hours later how many cells are there going to be if the generation time is 20 minutes uh well what we need to figure out then is that if the generation time is 20 minutes that means that the cells are going to divide three times for every hour for 4 hours that means that there will be 12 divisions if there are 12 divisions and we start off with five cells we're able to plug those numbers in here and see that five cells in four hours are going to become 20,000 cells okay so this uh shows you uh how that uh formula can be useful um but uh letting you guys know how we are going to use these concepts for an exam question um we're not going to require that formula but instead we're going to just work with uh the uh basic idea here um that we saw uh before that one cell becomes two two become four uh four become eight eight becomes 16 16 become 32 you should be able to do this math okay this is nice and easy each one of these arrows represents a generation time okay the amount of time that it took in order for one cell to become two cells and so on so the question that you would be asked on the exam to show that you understand binary fision and where generation times fit into this would be something along the lines of we're always going to start off with one cell um so starting with one cell um how many cells uh uh will there be in uh let's say uh 2 hours if the uh generation time is uh 30 minutes okay so in that case what you need to figure out here is that well if the generation time is 30 minutes then that means we're going to get two divisions in an hour and over two hours that means we're going to get four divisions so you need to look at um this right here and say okay well how how far are we going to get with four divisions well this is division one two three four that means that we will have 16 cells after two hours okay so be comfortable with doing something simple like that um so I could do another one let's say um you know starting with one cell uh we'll say uh how many cells uh will there be in uh you know one hour if the generation time is 20 minutes and in that case uh well generation times 20 minutes uh 1 hour means that we're going to get three divisions so uh our answer here how how many cells are we going to have after three divisions well one two three gets us to eight cells okay so so that's uh a test question you know don't expect to go into the test you know being like you need calculators and you know this a math course it's not this just came down to you showing that you understand um how binary binary fision works the number of cells that we get after each division and you understand how to work with the generation time okay what's really going to relate to the lab side of this course uh is this understanding of what we need to do in order to study organisms and learn more about um their growth characteristics um and in order to study an organism we need to make sure that we have just that single species that we are studying um you know if I was trying to study um a bacteria that I found in the human gut you have to remember that that uh cell is is in a space that's got over 400 species of bacteria mixed around with it so I need to find a way to get that one species separated from those other hundreds of species and I need to obtain what's called a pure culture pure culture is a bacterial population that is just one species okay in order to do that we are going to um start with a single cell and separate that single cell from other cells and therefore um by separating it from the other cells and growing it by itself every cell that comes from that will be identical to it they'll all be the same species and pure cultures are going to grow as colonies so uh when we look at like this we see that um each one of these dots is an area of bacterial growth that came from just one cell and we call those little circles colonies now the uh disadvantages of working with a pure culture in order to learn more about a bacterium the first is that some organisms behave differently in nature than they do on a petri dish okay uh so what it does inside of the human body um under the conditions of the human body is going to be different than what it's going to do when it's just growing on a petri dish um and we also talked about how in in the environment um based on uh changes to the environment u bacteria did different activities like for example forming that fruing body when conditions were bad so they don't behave the same way all the time under all the same well under all conditions but this is a big one right here only about 1% of organisms can be grown uh in in a lab setting on a on a petri dish by themselves uh so organisms uh form relationships with one another in the environment and they become dependent upon one another so we can't grow uh all these organisms that exist in the world on a petri dish by themselves uh but we do see here that fortunately um the organisms that do cause disease in the human body generally are able to be grown on a petri dish uh so the the organisms we really want to be studying to learn more about disease those ones we we we can study easier in a lab setting so that's good news um so how do we get a pure culture how do we get only one species of bacteria to study well first of all it's going to require a solid medium um so uh in order for these cells to be separated from one another they have to be in one spot in space we will not be able to separate those cells from one another in a medium that's not solid such as a liquid medium we can't separate cells from one another in a broth okay so we need a solid medium we need a container that can be kept aseptic uh so kept with um without any exposure to um uh the organisms that are in the air remember pasture's experiment how he had to create the swan neck flask that allowed for air to come in but none of the cells that are in the air so we need a container that works like the swan neck flask then we need a method to separate the cells so we have a space to separate them on we have an environment that's aseptic now we need to actually pull the cells apart from each other so they can grow as individual colonies and all this requires an aseptic technique which we have a whole PowerPoint about aseptic tech techniques um are procedures that minimize the likelihood of contamination um as we uh uh do both uh exercises in research and healthcare so the first requirement what do we do to have a solid medium for cells to grow on well what we do is we work with augar agar is a very special material um that is this material that's in the petri dish right now the agar is uh unique because it is a material that is not digested by the bacteria the agar is what holds the um the the um surface together and if that was digested then that would mean that um instead of being solid it would liquefy as it got digested and we just said we needed something solid the agar is not digested this is confusing for people because there are nutrients in the agar that are digested the nutrients are needed for the bacteria to grow but understand that the agar is not the sugar and the amino acids the agar is the material that makes this solid and that is not digested in addition the agar is not destroyed under high temperatures so we can uh uh put the um augar at extremely high temperatures uh to sterilize it um and then let it cool down and and it will uh solidify um so we don't have to worry about the high temperatures um ruining the molecule of the agar and we see that the augar stays solid up to 95 degrees C this is great because we are going to grow organisms generally at high temperatures um close to body temperature oftentimes because um let's say it's an organism that infects people then that means it grows best at human body temperature and we want u the agar to stay solid at human body temperature 37 degrees C um let's compare agar to something else that solidifies think of the gelatin that we use to make Jell-O um the problem with gelatin you know it would make the material solid but Jell-O doesn't stay solid at at high temperatures right you take Jell-O out of the fridge and starts to liquefy again so agar is special for these three reasons it is absolutely important to understand why we work with agar um and and not other materials when it comes to making our petri dishes now we need to have a aseptic container and that aseptic container is the petri dish the petri dish basically works a lot like the swan neck flask um we uh will put the petri dish uh into an incubator upside down it has a loose fitting lid and then you have the bacteria growing on the lower portion of this uh so the agar would be right here upside down and what this does is this creates an environment here where air can come in and reach the cells uh but if there was any contamination in the air the contamination would not reach the cells so I'll call this bacteria in the air so this is working like that swan neck flask the agar is exposed to air but it is not able to be contaminated by bacteria that are in that are in the air so that is what's uh special about using a petri dish and the benefit to the petri dish we see it's not airtight it allows for air to reach the cells uh but it avoids any contamination with uh organisms in the air our next thing to consider is uh a method to spread out the cells this is what's called a streak plate method the streak plate method uh we see the procedure outlined for you here and this is explained in the lab as well um the streak plate involves um taking a sample of cells and spreading them out over one portion of the petri dish importantly now you flame the loop you clear off any extra organisms and then you just take a small amount of material from here this is before you've allowed it to grow so you streak here you see nothing you streak from this spot and you go back and forth and you spread those cells out that you took from here flame the loop so you have a clean loop again and then streak from here to pick up some cells and spread them out over the last section what we did is we took our initial um sample of cells that included hundreds of thousands of cells and we spread hundreds of thousands of cells out we scoop through a little bit of that area to pick up uh hundreds of cells and we spread out those hundreds of cells here we then scoop through here to just pick up a couple dozen cells by picking up a couple dozen cells we're able to spread them out so they end up on the petri dish in their own specific spot this was one cell that landed here and it grew to form a population now of millions of cells that we call a colony and that is a pure culture all of the cells in that spot are the same species so if I sampled from here I have no idea what bacteria I'm picking up i could be picking up dozens of different bacteria here same thing here but when I sample from one of these colonies I know that every cell in that colony is the same species a pure culture so that's how we begin doing research we need to purify the population of bacteria so when I'm doing research with EC coli I know that what I'm working with is E.coli and not any other uh random bacteria that might have been mixed in with it okay so now as we grow um bacteria in a lab setting uh we have to understand that generally we're growing bacteria in what's called a closed system on that petri dish there is a limited amount of nutrients and waste is going to accumulate as the bacteria grow in that place so a a petri dish is only going to be good for uh so long before eventually the bacteria on its surface are going to die because um there's not enough nutrient for them to continue to grow and the waste is starting to wear them out and so what happens is we get a growth curve uh on a petri dish and we see the descriptions that I'm about to give here are all written out for you but I'm going to use the visual um as I use those descriptions so when we put a bacteria uh population onto a petri dish uh we initially enter what's called the lag phase this is the moment where the bacteria has to pick up nutrients from its environment before it can start making new cells so there is a lag there is no growth at first but once the bacteria have established themselves on the petri dish and have begun to pick up nutrients now they will start growing rapidly like we talked about at the start of this chapter we'll see the exponential growth where one cell is going to become millions of cells over a matter of you know uh hours to days okay so the log phase is the period of rapid growth um the population increasing in size dramatically but then we hit this point called the stationary phase the stationary phase is where the amount of nutrients has been reduced a lot and waste products have started to accumulate and so cells are dying just as quickly as new cells are being created we see that described here the rate of growth is equal to the rate of death so we no longer have any increase in the population even though we're making new cells our population is not getting any bigger eventually we hit the death phase which is where the rate of death now is faster than the rate of growth nutrients have been depleted so severely that uh the cells are are dying off rapidly and finally we enter what's called the prolonged decline in this case we see that the strongest cells the most fit cells are the ones that are going to stay alive the longest when many members of the population are dying um so this is where we see the fittest cells surviving the longest and we see that uh you know we get down to a very small number of cells that just hang around um you know things like for example the endospores the endospores don't need nutrients they are just dormant and surviving when the environment is not good enough for other cells to survive okay we're going to apply that concept of the curve in a moment here but before we do um this is not always going to be the case um you don't always work with a petri dish and let your population of cells die i mean that's not a great idea to let your um cells that you're researching die off so the opposite of a closed system is an open or continuous system in which we add nutrients along the way and remove waste and in that way you're able to keep the population alive and growing so that you can do years of research using that same um strain of bacteria you just keep it alive by keeping it nourished so let's apply the growth curve to um the bacteria that we are growing on a petri dish um we're going to have to go and pick bacteria up off of a petri dish as a source of cells to study but there is a technique to this what we see here is that all the cells in the colony are not equal to one another if I take this petri dish here and I say I'm going to sample cells from this colony here it matters where I touch on this colony when I'm picking up cells we see here that the cells in the center are the oldest cells because this is where the growth began from you had a single cell sitting here and then it grew to make new cells and they made new cells and they made new cells and they made new cells and so the innermost area is the earliest and now oldest cells those cells have used up the nutrients are burdened with waste and those cells are going to be in the death phase so you could imagine if if I gave you a wire loop and said "Go pick up some bacteria from this colony," your instinct would be to jab it right in the center but interestingly that's the worst place to go that's where you're going to have the cells that are least likely to be alive outside of that are the cells that are in the stationary phase where they are still growing but also dying at the same rate the best place to get cells from the outer part of the colony because these are the newest cells these are the cells that have expanded to a new area on the agar they are getting new nutrients and they are not burdened with the waste of the cells around them so the cells in the outer part of a colony are the ones in the log phase all right so let's take on that for a moment as we move on to uh our next concept here um the um environmental conditions that are going to influence um bacterial growth uh bacterial growth is going to be influenced by the same factors that affected our own human body remember in uh A&P we talked about uh homeostasis and the importance of maintaining a constant internal environment in order to stay healthy and function normally well we have the same thing going on with bacterium they're going to grow best at certain temperatures under certain conditions of oxygen pH and salt concentration and um what we're going to introduce here is a suffix called file and file uh means to love and so if you put a prefix in front of it something file like for example hydrophilic meant to love water um it's going to tell you that the organism grows optimally under that particular condition so we'll see that applied here in a moment but another uh suffix that we're going to use is tolerant tolerant means that the organism doesn't love the environmental condition but is still able to grow with that environmental condition it's not negatively affected by it we'll take the example here of hallow tolerant hallow refers to salt so something that is salt tolerant it will grow happily with no salt around it is not haloilic it does not love salt to the point that it needs it for growth it's just able to grow if salt is around it's not slowed down by the salt so tolerant means that it doesn't hurt the organism but the organism also doesn't love it either so our first condition to consider is temperature um just like humans uh organ microorganisms are going to have temperatures that they uh thrive with and then that uh on either end are going to be too extreme for them to survive and thrive um our first temperature range uh is described with this term here cycrofile a cyrofile is an organism that grows I at its fastest rate in these extremely cold temperatures okay so this is an organism that you would not find growing in the tropics it doesn't like those temperatures it likes extremely cold temperatures this would be an organism that you'd find you know growing in uh you know in the the uh around the uh Arctic region uh ideally uh cold temperatures below freezing okay um meopilic is this temperature range here that's uh between room temperature and and above body temperature our body temperature is 37° C so meopilic um describes uh an organism that uh infects the human body uh because the human body is in this temperature range okay so most organisms that we are going to learn about and study are going to be messilic because the reason why we care about them is because they're organisms that grow in people okay or they're organisms that at least grow in our environments in our environments uh around room temperature up to body temperature thermophile we start getting really hot here uh optimal temperatures well above uh 100° Fahrenheit but then this is the crazy one hyperthermophile hyperthermophile ideal temperatures are above 70° C um these are the organisms that can grow in boiling water we see the ari are brought up here again we said that these were organisms that lived in extreme environments with the example of the extremely high temperatures of um the waters around an underwater volcano and what's special about these organisms is that their proteins are heat resistant they do not dene they do not unfold and lose their functions under conditions of such high heat um whereas uh uh humans and any organism that is a messophile we would never be able to survive uh in in those temperatures because our proteins would not function so this table shows you those terms and reminds you that file means optimal so it doesn't mean it's the only condition that they can grow in the meopile we see here grows optimally you know at or around human body temperature but it is still growing uh you know below body temperature it is still able to grow for example in the refrigerator it just isn't growing as fast its rate is much slower and its fastest rate of growth is when it's infecting a person you know that ideal temperature for them to grow their fastest this slide brings up just how specific the temperature can be um for an organism we see here that these two organisms mcoacterium lepra which is the uh organism that causes uh leprosy uh also known as Hansen's disease and trepidma paladum the organism that causes syphilis these organisms while they infect the human body do not infect the core body they don't want to be at your core temperature of 37 degrees C instead they prefer the regions of the body that are cooler than core body so we see them infecting in the case of leprosy the affected tissues are the extremities ears hands feets finger oh feet fingers um in the case of syphilis uh the uh you know uh external genitalia in males are kept cooler than uh core body temperature and then we see uh portals of entry that once again are exposed to the outside temperature and kept cooler than the core body temperature so um so this shows you just how specific an organism can be it's it's not like oh okay it's an organism that infects people so it infects anywhere in the person the region that it affects is based upon its tolerance to the temperature this builds on that idea we see that syphilis was actually treated by infecting people with malaria because malaria induced a high fever so while you your body was fighting the malaria the high temperature uh was high enough to kill off the uh the bacterium that was causing syphilis and this is something we'll relate back to uh when we learn about the immune system we'll talk again about the importance of a fever and how that helps us combat bacterial infections the next thing to consider is oxygen requirements um what we see here is a range of requirements from having uh having to avoid oxygen this is the the organism requires no oxygen all the way up to the organism requiring lots of oxygen and each step up increases the oxygen needs okay so we're going to start with obligate aeroblate like an obligation this is a requirement it requires anorob anorob means uh no oxygen so it requires no aerob we see here these organisms are actually killed by oxygen uh because oxygen um when it goes into watery solution uh becomes molecules like super oxide and hydrogen peroxide these molecules are created in all cells your cells bacterial cells all cells um that are exposed to oxygen end up with super oxide and hydrogen peroxide in their cytoplasm but we are not killed by this because our cells contain enzymes that uh that get rid of these molecules and in the bacterial world we see that bacteria use enzymes like super oxide dismutase and catalase as uh the uh tool to convert super oxide back to water and oxygen and hydrogen peroxide back to water and oxygen therefore getting rid of those dangerous molecules okay but what we see is that claustrdium uh is going to be our genus of bacterium that lacks those enzymes so claustrdium is an obligate anorob this organism does not grow when oxygen is around because the oxygen would damage and destroy it okay so memorize this today we're going to learn about four um species of bacterium that are in the genus claustrdium all four of them you should know immediately beginning today that since they are claustrdium they are grandpositive they are spore formers remember claustrdium made endospores and they are obligate anorobes so we're going to learn about these two organisms for the first test but for the fourth test we're going to learn about claustrdium perfringens for example and that one um you don't need to be um you don't need to be taught what it is you know because it's claustrdium that it is grandpositive spore forming obligate anorode okay so these organisms need an environment without oxygen and that relates to then where they grow so um botulism uh this uh it affects uh canned foods uh so canned or jarred foods it grows in a space without oxygen tetanus uh you get tetanas with a puncture wound puncture wounds are particularly dangerous because um they do not expose the injury to air the puncture closes up and it leaves the bacteria in a space without oxygen okay so our next level of oxygen requirement is the aerrow tolerant anorobes anorob means again no oxygen but in this case they are aerero tolerant they tolerate oxygen they don't use it they do not use it but they are able to grow if it's around okay so they don't use it but it's it's fine if it's there so here's streptocous piogynes this is an organism that causes strep throat if it's in your throat it's of course exposed to oxygen but it doesn't use oxygen for its growth okay facultative anorob is the next step up this is an organism that does not need oxygen it is an anorob it does not need oxygen however what uh we learned about back in uh uh A&P or general biology we learned that aerobic respiration makes more ATP than uh fermentation does than uh anorobic uh u uh uh ATP production does so what that means is that this organism if oxygen is around is going to use the oxygen and grow faster because it's going to get more ATP uh by doing aerobic respiration than it would by doing fermentation this is going to be actually most organisms like a lot of uh bacterial species can grow when there's not a lot of oxygen around um but they thrive when oxygen is around we have E.coli as an example this infects your intestines your intestines don't have oxygen for them to grow so they're able to grow when there's no oxygen around but if I take E.coli and put it on a petri dish there's lots of oxygen and it's going to grow very fast okay so think of a facultative anorob as being very similar to to humans where we can make ATP uh through lactic acid fermentation we can do it without oxygen but it's not as good and the result is is far less successful microerafile break the name down file means to love aerero oxygen micro small amount so this loves a small amount of oxygen so microerophiles need oxygen to grow but they don't want to be overwhelmed with it we see an example of helicoacttor pylori which causes uh the ulcers of the stomach and beginning of the small intestine in that area oxygen is available we've swallowed air with our food and beverage so oxygen is available for the bacteria to grow but it's not like it's infecting your lungs it doesn't want a lot of oxygen it just wants a little bit and finally the obligate aob these are organisms that have an absolute requirement for oxygen to grow uh these would be organisms that uh ideally infect the lungs uh because they want to be in a space with lots of available oxygen okay so a lot to take in so far i hope you're sticking with me there's a lot of material in this lecture um we're moving on to our next um uh factor to consider is the uh pH um once again pH is something that's so important uh to even human health and and certainly then microbial health as well we see here that most microorganisms are going to live in a range that's near uh neutral and most of them are going to grow best at a pH of 7 at neutral so we call them neutrofiles preferring a neutral pH of 7 however we do have examples of microorganisms that that grow best in acidic conditions acidtoiles prefer acidic conditions and alkaloiles prefer alkaline or basic conditions okay now we have the interesting side note here that helcobacttor pylori that infects the stomach is actually not an acidophile it doesn't infect the stomach because it likes acid it actually uh hides itself from the acid we see here it neutralizes stomach acid in its vicinity by releasing the enzyme urase uh so it is a neutrfile that just happens to live in an acidic environment by burrowing into your epithelial layer and then protecting itself with the uras enzyme finally we have salt concentration to consider in general we see here that high salt concentration pulls water out of cells we saw in our last lecture the process of plasmalyis where you suck the water out of the cells and what that does is it puts cells into a dormant state so they can't grow because they're dehydrated however some organisms have the ability to resist water loss in those high salt environments and those organisms are referred to as hallow tolerant they tolerate or resist the presence of high salt concentration with the example here of stafllocus arius this is an organism that is uh present on your skin and your skin is very salty on its surface and yet this organism is not bothered by the high salt concentration so hopefully you're following along all right and you won't be drawing a panda on your test because you're keeping all of this straight um so we're getting close to the end here of factors to consider for growth the next one that we see is that uh the organisms are going to have certain nutritional needs um we see that uh these are the essential components for making the organic compounds that uh that all organisms use in addition proarotes are going to need a carbon source they're going to need something to make organic compounds from our first term that goes along with this is autoro an autoroe is an organism that makes its own organic compounds by using carbon dioxide an inorganic form of carbon so that's what's done with photosynthesis you take carbon dioxide and you use it to make glucose that is what an autoroe does heterotroes on the other hand in order to make their organic compounds they have to start with pre-existing organic compounds so if you want to make let's say um uh let's say you want to make fructose you have to consume glucose and turn one co carbohydrate into another carbohydrate another thing to consider is a source of energy um energy can come from light and so we see photoroof refers to an organism that uses light as its energy source once again think of photosynthesis where light is the energy source and then carbon dioxide was the carbon source whereas a cheotroof is an organism that uses organic compounds as their energy source this is what we do we need energies uh so what do we do we eat organic compounds we eat carbohydrates proteins lipids we are cheotroes just like all the organisms that are infecting our body our body is being infected because we are a source of organic compounds or energy but more specifically when it is organic compounds we refer to it as a chem organote because the chemical energy is from organic molecules like proteins lipids carbohydrates but there are organisms that actually don't use proteins lipids and carbohydrates as an energy source instead they use inorganic compounds and we call these organisms chemoliththotroes they use things like uh iron and sulfur as an energy source when we put these together we get um this uh combination of um of factors um into a single term so we can combine the energy source with the carbon source to create a term that describes the metabolic activity of an organism more completely the two most common ones are chem organo heterotroes they use organic chemicals for energy and organic chemicals uh as a source of their own organic molecules so this is us this is uh the bacteria that infect us we take in organic compounds and we use it for energy and to make the the uh molecules we use to build our cells whereas a photoroof is a photosynthetic organism so think of algae plants and photosynthetic bacteria they use light for energy and then using that energy uh they convert carbon dioxide into organic compounds so we're able to put that all together this is a photosynthetic organism and then this is basically a consumer okay so our final point here when it comes to bacterial growth in a lab setting is that we need to be um selective of what media we use to grow the organisms because they have different functions um the first thing that we want to consider is what the source of nutrients is um our first type of media like augar or broth for example our first source is what's called a complex media and in a complex media we don't have an exact chemical composition we basically for example let's say with blood augar we put blood into the augar we don't know how much glucose was in that blood we don't know how many amino acids were in that blood we just are using the blood as a nutrient source so complex is not clearly explained what specific nutrients are in it um we could do the same thing with let's say milk like uh we can use skim milk as the nutrient source but that milk we don't know exactly how much of each um nutrient is in there versus a chemically defined media is one where um the media is created with a specific quantity of each uh chemical so that we're able to uh do more um uh specific studies on the nutritional requirements of an organism so this augar that I created I know that it does not have any let's say proline and so when I'm growing bacteria on it I'm going to find out if the bacteria can make their own proline or if they aren't able to grow because the the augur is lacking it so chemically defined you know down to every nutrient what is in that augur the next thing we want to consider are augars or broths that are um selective or differential a selective medium is one that um it only lets certain organisms grow uh we see the example here that uh if you use an an agar that contains antibiotics then the only organisms that can grow will be antibiotic resistant so a selective media only lets certain organisms grow and other organisms cannot grow on it another example of that is manitol salt agar that uses high concentrations of salt to kill off to prevent the growth of any other organism except for those that are hallow tolerant so manitol salt agar is actually really useful for growing staffarius because it is hallow tolerant and you don't have to worry about other organisms growing on it that you don't want selective it selects what can grow on it okay whereas differential media we learned about differential staining in differential staining everything got stained but they just got stained differently with Graham staining they either got stained purple or pink they all got stained just differently same thing here with a differential media everything is able to grow but how they grow is going to look different depending upon the particular organism we see blood augar is a differential agar we have three possible outcomes when bacteria grow on blood agar some organisms are going to do nothing to the blood cells we see them grow on the surface but not change the blood cells so this would be like an organism like E.coli going to look like this we see here that some organisms they're going to destroy the blood cells but leave behind the heem within the hemoglobin that makes this green color and other um organisms such as strep piogenes are going to not only destroy the cells but they're also going to destroy the hemoglobin completely and leave behind nothing just clear agar so they grew differently if I showed you this and said "Do you think that this is E.coli?" You would be able to say "No E.coli looks like this ecoli is gamma hemolytic not beta hemolytic." Uh if I showed you this and said "Do you think this is strepiogynes?" You'd say "No strepiogynes is beta hemolytic not alphaolytic they have different outcomes based on their uh metabolism as far as how they grow okay so we'll learn about um augars that are both selective and differential in the lab as well the final point that we want to make here with this chapter is um methods for us to measure the rate of bacterial growth and what you guys want to get out of these three tables is first of all are we measuring living cells or a mixture of living and dead cells so the first table here is showing us living and dead cells okay with a microscopic count if I go and I count bacterial cells with a microscope I can't tell the difference between the cells that are alive or those that are dead so if I'm using a count using a microscope I am getting a total number of cells that are on the slide but I don't know if they're alive or dead the culter counter uses an electrical um potential that's that's run through the cells and then measures how much resistance there is to the flow of the electricity the more there is resistance the more cells there are and so this device allows for us to calculate how many cells are present based upon how much electricity is struggling to get through but once again a dead cell is going to resist electricity as much as a living cell so we're using electricity to count the number of cells but we don't know if they're alive or dead a flowcytometer uses light as a way to measure the number of cells based upon how cloudy the solution is so if you have bacteria in a broth and you shine a light through it the more bacterial cells that we have the cloudier the broth gets the less light will pass through it so this is measuring the cloudiness of the broth and once again a dead cell is going to uh block light just as much as a living cell does so understand the general concept microscopic count you're using a microscope to count cells culter counter you're using electrical current to count cells low seytometer you're using light uh to count cells but in all three cases you're counting both living and dead cells the next one is the viable cell count we're counting the number of cells that are alive in this case we start off here with the plate count and the plate count is really the only one of the three of these that's a real like you new idea here the plate count is let's say I found out from the direct microscopic count that there were a thousand cells but I put those cells onto a petri dish and I get four colonies that grow that means that of the 1,000 cells that we counted only four of them were alive they only formed four colonies so growing an organism on a petri dish allows for you to count the number of cells that were alive in a sample okay because if I put a thousand cells on a petri dish but 996 of them are dead they're not going to grow i will never see them but the four that are alive will grow and form four colonies and so I know that there were four living cells membrane filtration precedes the plate count we see here that um we can use uh membrane filtration to uh filter the air or water collect samples from the air or water then place them on a uh a petri dish and count the number of cells that grow so it's the same thing as the plate count it's just we're starting with first getting a sample of organisms from the environment from the air or a water source and then finally the most probable number is just a statistical method of estimating the number of living cells we see it's not precise we're not directly counting and saying there are four we're saying there's likely based upon the information four to 10 you know a range of cells that's more than likely accurate finally we have methods of measuring biomass so instead of trying to um count the number of cells what we're trying to determine is how fast are the cells growing and the way we can figure this out is how quickly does the mass of the population double okay if I have one gram of bacteria and I want to know how long it takes to double the size of the population well then one gram of bacteria when it doubles its population will become two grams of bacteria i don't care how many cells there are i care about how long it took for the population to double this is a way of measuring rate of growth so in the case of turbidity this is going back to the flowcytometer we can measure the amount of light that's blocked by the solution of bacteria and basically when that value doubles when we find out that there is twice the amount of cloudiness that means that there is twice the amount of cells so we can use turbidity or the cloudiness of broth and a flowcytometer to measure how long it takes for the population the number of cells to double because the cloudiness or turbidity has doubled likewise we can use weight uh this is obviously more tedious because weighing bacteria how are you going you know how are you growing the bacteria in a spot that you can then transfer it to weigh it and everything not really a great way of doing this not really practical as much as you know bacteria growing in a broth that you just put into a you know keep in that same tube and run light through it but this one allows for us to once again just uh count the num well count the um the the amount of time until this population is doubled based upon the weight of the population so once again if one gram of the organism um is present at at 1:00 and then four hours later at five o'clock there are two grams you say it took four hours for the population to double okay so with this table you do want to study this in preparation for the exam but please don't get bogged down on the little details i'm not asking you the fine details it's if you know the general idea of what the device is doing like uh culter counter using electricity flowcytometer using light um and you know what's being counted living and dead cells versus only living cells versus not counting the cells but counting the mass or the the size of the population in general um if you understand all that you'll be ready for uh test questions relating to this handout okay so that's the the table that's referred to here on this slide and that handout is in your handout section on Blackboard all right so we are done we can look back on this horrible experience of drowning in that material and laugh at it right