this is chapter 6 on microbial growth microbes just like all living things have certain physical and chemical requirements for life the physical requirements may be things in their environment so things like temperature so all organisms have an ideal temperature range at which they're able to live same thing with ph there's an acceptable range of ph that certain organisms prefer and osmotic pressure so those hypertonic isotonic hypertonic solutions so remember we said the hypertonic is a more concentrated solution so it's going to draw the water out of the cells isotonic is the same concentration on both sides so everything's stable and balanced hypotonic is less concentrated than in the cell so inside the cell is more concentrated than the solution so water will go into the cell to try to dilute that concentration causing the cell to swell and potentially burst chemical requirements so things like nutrients and chemicals that cells need for their metabolism so things like carbon right so all organisms are carbon based some other nutrients like nitrogen sulfur hydrogen oxygen and some organic growth factors like proteins and amino acids so when we talk about microbial growth we're talking about an increase in the number of cells so normally when you think about growth you think about right a child growing up or a puppy maturing into a dog so the organism gets larger microbes being single-celled organisms obviously can't get larger but they can increase in the numbers in their colonies so colony is defined as a group of cells large enough to be seen without a microscope and generally they're derived from a single cell so in lab when we looked at our plates for microbes in the environment and we looked at the different colonies that grew so one important physical requirement for microbial growth is temperature so all organisms have their own minimum optimum and maximum growth temperature so kind of the lowest temperature that they're able to tolerate an optimum temperature where they grow the fastest and are the healthiest and maximum growth temperature so remember temperature is important for metabolism those biochemical reactions and those enzymes and their function so we can classify some organisms based on their temperature preferences so your psychophiles are your cold loving organisms so they prefer things in the freezing range or even below freezing so these are the organisms responsible primarily for food spoilage so we can preserve the shelf life of our food a couple of days by refrigeration but there are still some spoilage bacteria that can survive in the refrigerator temperatures so your leftovers from dinner the other night are probably only good for a couple of days and then you might want to throw them out so these psychophiles always think of them as the psychos that love the cold weather so my minimum temperature would probably be around 50 degrees mesophiles meso meaning kind of in the middle so these are moderate temperature organisms so around our body temperature range so most of the pathogenic bacteria and bacteria that we deal with in our bodies and in our lab and in this class would be the mesophiles so they live at the same temperatures as we do so we're going to interact with them a lot more the thermophiles are the heat loving so these organisms prefer the higher temperatures cyclotropes or these psychos as i call them grow between 0 degrees celsius and 30 degrees celsius 0 degrees celsius is 32 degrees fahrenheit so that's the freezing point so again these are the organisms responsible for your food spoilage so some bacteria can still survive and may grow in these refrigerator temperatures so this chart is showing the surface area or the amount of food in a container and its effect on cooling and chance of spoilage this larger container takes much longer to reach the refrigerator's ambient temperature than the smaller more shallow container so this darker band shows kind of the danger zone range so the longer our food sits in this temperature range the more the bacteria have a chance to grow and spoil our food so the smaller container only spends less than an hour maybe half an hour before it reaches that cooler temperature whereas our larger container is in this danger zone for several hours thermophiles optimum growth temperature is between 50 to 60 degrees celsius so 60 degrees celsius is about 140 degrees fahrenheit the human body temperature is 37 degrees celsius so 98.6 degrees fahrenheit so these thermophiles in the 140 degree fahrenheit range are found in places like hot springs and organic compost the hyper thermophiles found in even more extreme temperature ranges nearing 200 degrees or more ph is another important physical requirement for microbial growth most of our bacteria prefer to grow between ph of 6.5 and 7.5 molds in yeast prefer a more slightly acidic environment ph of 5 and 6. the acidophiles or acid-loving organisms grow in the very acidic environments osmotic pressure is another physical requirement or environmental factor for microbial growth so in hypertonic environments where we have a higher solute concentration outside the cell than inside the cell will cause plasmolysis or the cell to essentially shrivel up and die so the water will be drawn out of the cell toward this more concentrated solution due to that high osmotic pressure however some organisms prefer these super concentrated or hypertonic environment so extreme or obligate halophiles require that high osmotic pressure or high salt environment facultative halophiles can tolerate high osmotic pressures if necessary so sometimes when we talk about obligate versus facultative obligate means that they are obligated to be in those high salt environments right so if you're obligated and there's no way out of it you have to do it so always use the example i'm obligated to spend thanksgiving with my in-laws every year it doesn't matter if i want to or not i have to go facultative means that it's flexible so if the situation arises right where we encounter a high osmotic environment right in the cell can tolerate it and be okay one of the most important chemical requirements for all life and microbial growth is carbon so all living things are based on carbon chemistry so if you ever take an organic chemistry class it's nothing but carbon based chemistry so carbon forms the structural backbone of all the organic biological molecules so proteins sugars fats everything is based on carbon some organisms like chemo heterotrophs use organic molecules as energy so we acquire this carbon from a food source and reconfigure the carbon into other biological molecules autotrophs use carbon in the form of co2 for photosynthesis so here we have glucose a sugar so it's a six carbon sugar amino acids that form proteins or carbon based fatty acids are just chains of hydrocarbons atp adenosine triphosphate the primary energy molecule so carbon's important for organic chemistry because it's able to form large complex and diverse molecules so when we talked about chemical bonds in anatomy and physiology remember that octet rule or the magic number that all atoms want to have is eight valence electrons so they interact with other molecules or atoms to reach that goal of eight so carbon is able to form four separate bonds and share up to four electrons so it could form four single bonds so this carbon here's has a bond to another carbon to another carbon into two hydrogens it could form two single bonds and a double bond so now we have kind of this kink in the chain not shown here it could also form a triple bond it would be triple bonded to one atom and single bonded to another so we have all these various shapes and functions of biological molecules nitrogen is an important chemical requirement for proteins dna and atp some bacteria use nitrogen for nitrogen fixation in their metabolism so all of the bases the letters in dna the atcg are sometimes referred to as the nitrogenous bases because they contain nitrogen so nitrogen is a vital component in dna nitrogen is also important for the production of atp it's an all-important energy molecule sulfur is used in some amino acids like methionine cysteine and some vitamins like thiamin and biotin some bacteria like your purple sulfur bacteria are able to decompose protein for the sulfur from these amino acids phosphorus is very important chemical requirements used in dna rna and atp as well as form the basis of the cell membrane remember the cell membrane is a phospholipid bilayer phospholipid has a phosphate or phosphorus attached to that fatty acid chain all right it's also going to form the p in atp so adenosine triphosphate has those three phosphate groups trace elements are things like vitamins and minerals or inorganic elements that are only required in small amounts so most of these are going to function as your enzyme cofactor so things like iron and zinc so remember the cofactors or coenzymes we said allow an enzyme to function so without the coenzyme our substrate won't fit on our active site so we need the coenzyme to allow that enzyme to function well oxygen is an important chemical requirement for us obviously we cannot live without oxygen but different microbes have different tolerances to oxygen so obligate arabs so again obligate meaning they have to so these can only grow in the presence of oxygen so oxygen is required so we humans as well are obligate air we have to breathe oxygen or we will die so these are noted in tube growth so they'll grow near the top or the surface so that's this is where the oxygen is in the test tube facultative anaerobes remember facultative meaning they can adapt they can tolerate different scenarios so these microbes can tolerate both aerobic and anaerobic but they grow greater in the presence of oxygen so when we look at these in the test tube you'll see most aggregated near the top near the oxygen but there are some down below in the aerobic environment that are still able to grow a little bit obligate anaerobes can only grow without oxygen so oxygen is actually kind of a toxic gas to these obligate anaerobes so you'll see these grow at the very bottom of the tube farthest away from that oxygen layer aerotolerant anaerobes are similar to facultative but they don't really have a preference the facultative prefer to have oxygen but they can deal without it aero tolerant can only have anaerobic growth but they can tolerate oxygen so they'll still grow in the presence of oxygen and microarrow files have only aerobic growth but they require lower concentrations of oxygen so they're not completely anaerobic at the very bottom of the tube but they're not completely aerobic where they need the full force of the oxygen in the air so they grow about halfway down where the oxygen just starts to dissipate a bit so here's just a fun fact and maybe a potential bonus question on your next exam so there was no free oxygen in the early earth atmosphere so when life first evolved the first cells evolved in their metabolism evolved there was no oxygen in the environment so there was no oxygen based metabolism so this is why we still have some anaerobic organisms today why wasn't there any oxygen we have lots of oxygen now so where did all of this oxygen come from the answer is photosynthesis so free oxygen in the early earth atmosphere did not accumulate until the evolution of photosynthesis so once those photosynthetic bacteria started to photosynthesize remember oxygen is a waste product of photosynthesis all of that oxygen started to accumulate in the atmosphere and originally it was considered kind of a toxic poisonous gas to these cells they had no way to deal with it but through evolution the cells adapted and figured out a way to use the oxygen to their advantage organic growth factors are organic compounds obtained from the environment or other organisms so basically the building blocks for the biological molecules amino acids and vitamins nucleotides so amino acids are the building blocks for all proteins so we can make every protein imaginable by arranging these 21 different amino acids in different configurations so a lot of these building blocks are recycled so when you eat a steak for dinner right you're essentially eating muscle tissue from a cow which is protein so your digestive system will break down that protein in the steak to the basic amino acids and then your body will use those amino acids to make your own muscle tissue protein whatever other protein is needed by the cell biofilms are microbial communities so we said before microbes don't generally live in pure isolated colonies they live in these communities with multiple microbes all kind of living sharing the same space so these biofilm communities form slime or hydrogels that help them adhere or stick to hard surfaces once the cells reach high enough density so there's enough cells in the population they can start to communicate via quorum sensing so in quorum sensing essentially the bacteria can talk to each other so if you only have a few cells right there's not as many recipients for your signals or your messages to go to we have lots of cells in the same space right it's much more likely that your signal will get spread to more cells so these bacteria will secrete an inducer or a signaling chemical to help attract other bacteria cells to the community they basically put up a house's for sale sign so these communities kind of work together and they can share nutrients and they help to shelter other bacteria from harmful environmental factors so overall these biofilms can be more resistant to treatments like antibiotics so the cells deeper in the biofilm and the lower layers would be more protected against any antimicrobial agent we try to apply to it this is actually a figure showing different types of bacteria on a normal tooth surface so the reason we have to brush our teeth every day is because these biofilms can start to form so when your teeth get that kind of furry fuzzy feelings you need to brush your teeth that's that biofilm forming so we have some early colonizers that are kind of the first on the scene to kind of set the stage for the new community and then we have some later colonizers so if we don't brush our teeth quick enough we have enough time for new bacteria to move into the neighborhood so when biofilms get large enough they can form kind of a primitive circulatory system so they'll form these channels in different clumps of the biofilm so as fluid or the water current flows through can deliver nutrients and remove the bacterial waste products so again essentially kind of like a very early primitive circulatory system they can also send off new migratory clumps to start new biofilms elsewhere culture media is what we use to grow bacteria on in the lab so these are specific nutrients prepared for microbial growth culture media should always be sterile meaning it should not have any living microbes on it when we first use it when we inoculate a sterile culture media we're introducing microbes into that medium so a culture would be the microbes that grow in or on our culture media so when we do lab and you do your inoculations you have a culture so a broth culture or pictures on petri plates that you took a piece of or a sample from and then inoculated or introduced to a new media solid culture media uses auger which is a complex polysaccharide harvested from red algae it's used as the solidifying or the gelling agent for our petri plates our slant tubes and our deep tubes it's good to use the auger from the red algae because it's generally not able to be metabolized or broken down by any microbes that we deal with red algae there is also used for some asian desserts and a thickener in soups and ice cream a chemically defined media is one where we know the exact chemical composition in that media so in this chemically defined media example right so we know exactly how much of each nutrient or component is in defined medium complex media includes extracts or digest down products of things like yeast meat or plants so depending on the type of media the type of culture we're trying to grow chemical composition can vary from batch to batch there's two basic forms for complex media so we can have a nutrient broth which is just the liquid portion without the solidifying agar or a nutrient r so we do have that red algae auger added right to solidify it kind of like jello right so we can pour it on a plate and have a large surface area for growth we could have a solid or a deep tube right when we stab down into to check for motility or the auger slant just kind of a smaller version of the plate just to see it grows on a solid surface we're also able to culture some anaerobic bacteria with special types of growth media and methods so anaerobic growth requires a reducing media so it contains special chemicals like sodium theoglycolate that will combine with oxygen to deplete it so this is a theo tube so it has this indicator here to show um that it has not been exposed to oxygen this is an anaerobic chamber sometimes called a brewer's jar so we would put our plates in this jar this chamber i'm gonna have special co2 packets that will cause this to be a anaerobic chamber and if you want to get really fancy we have airlock painters with the special arm ports for these anaerobic environments so depending on the biosafety level of the type of organism you're working with we can use different special culture techniques so bsl1 is kind of the most basic the safest level so no special reprecautions just basic teaching labs sl2 you need lab coat gloves and eye protection so this would be our level in our lab everything higher than that you need more specialized equipment biosafety cabinets to prevent airborne transmission or special hepa filters to filter the exhaust air for certain airborne diseases we can use different types of culture media to select for the growth of certain bacteria or to help differentiate between different bacteria selective media is think of in terms of natural selection so the natural selection of this media is going to suppress or select against the microbes that we don't want and it's going to encourage the microbes that we do want so sometimes these will contain certain chemicals that inhibit or suppress growth of every other type of organism the one type of selective media that we'll see in lab is emb stands for eosin methylene blue so this is selective for gram-negative bacteria methylene blue dye the same one that we used in our simple stain in this media is going to prevent the growth of gram-positive bacteria so e and b is selective for gram-negative so this is e coli on an emb plate so these are really cool they grow this really pretty metallic green on emb differential media allows us to differentiate or distinguish between colonies of different microbes on the same plate some media can have both selective and differential characteristics so this is an example of a blood agar plate so it's enriched differential medium so it allows us to distinguish between organisms based on how they undergo hemolysis so how they react to the blood cells on these plates so streptococcus pyogenes can be identified by a beta hemolysis so just a complete clearing of the blood cells so it's rupturing the blood cells right so that's why we have this clear zone streptococcus pneumoniae is characterized with alpha hemolysis so we have this kind of greenish color to the auger intracoccus ficalis has no hemolysis so it has a gamma classification right so it doesn't break down the blood cells it's able to grow on them but it doesn't break them down another selective and differential media is msa or mannitol salt agar so it's so it's selective because of the salt right so only staphylococcus species can grow in this high salt media environment it's differential because staphylococcus aureus will ferment the mannitol sugar in the media and turn it yellow staph epi does not ferment mannitol so the auger will stay pink and then this is a non-staphylococcus species so it's selected against and it cannot grow on this plate so remember we said sometimes a scientific name of an organism can be kind of descriptive or some of its characteristics all right so staphylococcus are those spherical shaped cells and clusters and arius means gold so staphylococcus aureus turns our msa plates gold enrichment cultures just help to encourage the growth of a desired microbe so we kind of pinpoint what's its favorite nutrient right and we increase those particular components to kind of tailor it to the taste of our microbe of interest so we want to grow those microbes up to a detectable level when we talk about pure cultures we're talking about only one species or strain so a colony is a population of cells that arise from a single cell typically on our petri plate sometimes you see the colonies referred to as cfus or colony forming units because technically there are millions of cells but they were all derived from a single unit cell so we use the streak plate method in lab to help isolate these pure cultures so if you have a mixed sample right so we have two bacteria mixed together so when we do the streak plate you streak your first streak then you sterilize your loop or you get a new loop and you streak again so we're taking some of the cells right from these streaks here and we're just kind of pushing them over to this side of the plate now so then we would sterilize our loop get another loop and then we take some of these cells a couple cells from here and push them over there and spread them out so ultimately we'll see a progressive thinning of our bacterial growth until we have these single isolated cells so now we're able to distinguish between the two different species the red ones and the yellow ones we're up here when we have a lot of cells a lot of growth it's hard to kind of separate and see what's what so we spread it out thinner and thinner and just streak it out to get those isolated colonies bacteria divide by binary fission meaning they essentially split in half and form copies or clones of themselves so again remember microbial growth we're talking about an increase in the number of cells not the size of the cell so the cells will stay pretty much the same size so just increase in their numbers so the process of binary fission begins with replicating the dna in the nucleoid right so we have to make a copy of the dna to give to our copied new cell um then the plasma membrane and the cell wall will start to kind of pinch off in the middle here start to form a new cell wall so eventually the new cell wall is completed so we've separated these two cells and now they separate so because bacteria undergo binary fission they can increase their numbers very rapidly and exponentially the generation time is the time it takes for a cell to divide so different so this generation time can range from 20 minutes to 24 hours and again the binary fission is going to double the number of cells every generation so because after just a few generations we start to deal with exceedingly large numbers growth curves are usually represented logarithmically so in this example we have one bacteria cell right that starts our colonies or our founder all right so it multiplies into two cells two cells multiply into four four becomes eight eight becomes 16. and so on and so forth right so after only 20 generations right we're up to a million cells so if a bacteria's generation time is say one hour so in less than a day less than 24 hours we already have over a million cells so we tried to graph this on a standard graph we have this really sharp uptick in this exponential growth so it would just get really hard to keep graphing it this way so that's why we use the log scale so a true exponential curve on a logarithmic scale would be a nice linear slope so another potential bonus question on your next exam a little riddle about exponential growth so lily pond starts with a single leaf every day the number of leaves will double so two leaves on the second day four leaves on the third day eight leaves on the fourth day so on and so forth on day 30 the pond is full there's no more room for any more lily pads so on which day was the pond only halfway full on the 29th day so remember we're doubling every day so the first right almost three weeks or more right we're just dealing with small numbers so two double is four so both are small numbers but you get increasingly larger numbers once you start to double a large number it becomes a larger number and then we have kind of this exponential growth is kind of shooting up so it's not until the 29th day that the pond is only half full most bacterial populations follow a standard kind of pattern or phase of growth so the first phase is the lag phase so during this phase so looking at just the shear number the log number of bacteria in our population right it's pretty low right there's no real increase in population the new settlers have arrived they're kind of getting their bearings they're preparing for their population growth getting all their nutrients and enzymes in order so the log phase or the logarithmic or exponential phase is where we have this kind of taking off in the population numbers so they shoot up very rapidly right exponentially the stationary phase is the period of equilibrium where the line is kind of stationary meaning it doesn't go up or down so during this stationary phase the number of microbial deaths is balanced by the production of new cells so cells are dying at the same rate that they are being reproduced so we're just replacing the ones that die right but we're not increasing our overall numbers and finally the death phase so now the population will start to decrease so now cell death is outpacing cell reproduction and it will also decrease at a logarithmic rate so at that same kind of exponential rate just in the other direction