this is the lecture for chapter six microbial growth part two the focus for this chapter is on the requirements for microbial growth and what we mean by microbial growth is having the cells grow and go through cell division in order for this process to occur the cells need to be able to metabolize they need to be able to take in nutrients perform catabolic reactions on those nutrients to release energy that can be readily used by the cell and to produce precursor molecules that can be used in anabolic reactions to build larger molecules such as the macromolecules which can be used in cellular structures and also to help with cellular processes such as cell growth and cell division in order for the cell to be able to metabolize and perform all these functions the cell needs to have functional enzymes so any factor that would affect enzymes will affect the ability for the cell to grow as i discussed in the first part of the lecture the requirements for microbial growth are in two basic categories the first category are the physical requirements for growth and this is basically the type of environment that the microbes are placed into the second general category are the chemical requirements for growth and the chemical requirements for growth refer to the nutrients what sort of molecules and atoms are needed by the bacterium in order to grow in the previous lecture i went through the physical requirements for growth and these are temperature ph and osmotic pressure and if you want bacteria to grow you will need to incubate them at their optimal temperature or at least within their temperature range same with ph the environment needs to be at the optimal ph or again within the ph range and both of these things should remind you of the previous lecture in metabolism when i talked about enzyme activity because you will see similar characteristics for enzymes all enzymes have an optimal temperature and an optimal ph where they function at their fastest rate so the characteristics of the bacterium directly late relate to the characteristics of their enzymes osmotic pressure is a little bit different bacteria need to be placed in an isotonic or a hypotonic environment if they have a nice healthy intact cell wall if they are placed in a hypertonic environment then the cells will plasmalize they will dehydrate they will lose water and their proteins will denature because they will be unable to hydrogen bond and also they will not have available water for metabolism also in the previous lecture i went through the chemical requirements for microbial growth and i went over the macronutrients again macro means that the bacteria or the microbes need them in relatively high concentrations and i talked about the need for carbon nitrogen phosphorus and sulfur also i went through the micro nutrients again micro means that these are required but in extremely small amounts and the two types of micronutrients are the growth factors and the trace elements and the difference between these two is that growth factors are organic and trace elements are inorganic and the role they play in metabolism is that the growth factors are basically the coenzymes and the trace elements serve as the cofactors and if you remember from enzyme structure coenzyme or a cofactor is required to bind to the apo enzyme to create a functional holoenzyme and that active site is a combination of the apoenzyme and the coenzyme or the cofactor in going over the chemical requirements for growth you may have noticed that i missed one very important atom and that is oxygen and oxygen deserves its own place to talk about in terms of chemical requirements for growth because the relationship between living things in oxygen is a little bit complicated the relationship between living things and oxygen is a little bit complicated because in reality oxygen is actually toxic to living cells and to actually understand that relationship it's important to go back and look at the evolution of the first cell and how oxygen relates to the first cells and how cells adapted to oxygen early on in evolution when the earth was first formed about 4.5 billion years ago it's important to note that before life existed in the atmosphere there was no free oxygen there are many different types of gases like carbon monoxide hydrogen gas nitrogen gas water carbon dioxide and other gases but it's important to note that there was absolutely no oxygen present in the atmosphere so the first cells to evolve were most likely autotrophs they were photosynthetic cells because there was no source of organic molecules on the planet at that time so they had to be self feeders autotrophs autotrophs perform photosynthesis so you should be familiar with the equation of photosynthesis photosynthesis involves taking carbon dioxide and water using the energy from sunlight to create an organic molecule glucose which will be used for energy and a waste product of oxygen so if this was the first process metabolic process on earth it required carbon dioxide and water and if you look at the early atmosphere carbon dioxide was present and water was present and of course the sun was present so everything was there for photosynthesis so this is probably the first type of metabolism that evolved on the early earth so as all of these cells were performing photosynthesis to produce glucose which could be used for energy oxygen was released as a waste product and the oxygen started building up in the atmosphere and as the oxygen built up in the atmosphere it actually reached toxic levels because when oxygen interacts with water it creates oxides oxides are toxic to cells when oxygen interacts with water it forms different types of oxides and here is an example of some of these oxides so there are super oxides there are peroxides hydrogen peroxides and so on and these oxides can oxidize organic molecules and so this is an example of flow chart of what happens when these oxides oxides start oxidizing or causing oxidative oxidative damage to different molecules when these oxides interact with lipids they can cause chain breakage and they can actually have an effect on the membrane on the selective barrier that surrounds the cell also oxides can have an effect on proteins they can alter the structure of the protein and when you alter the structure of the protein you alter the function and often when this happens to enzymes the enzymes are inactivated and if an enzyme is inactivated then the cell won't be able to metabolize and that can lead to cell death also oxides can oxidize dna and when dna is oxidized that can rel result in strand breakage which which can directly relate to killing the cell or it can cause mutations and those mutations can change the enzymes that are produced and if those enzymes are altered in a way that makes them non-functional that will also cause death so these oxides are very toxic and they can cause cell death very easily because they have an effect on the lipids the proteins and the dna and in early cell evolution when the concentration concentration of oxygen got to be about 20 percent in the atmosphere there was a huge extinction event because most of the cells at that time did not have any way to protect themselves from the toxicity of the oxygen toxicity of the oxides and most of the living cells at that time were killed off oxygen wasn't only toxic to those early evolving cells oxygen continues to be toxic to cells now and we actually know that they can be toxic to our cells because when oxygen interacts with water even today it still forms those oxides and those oxides can still damage dna it can cause dna to break and it can mutate dna and if our human cells if our human dna is mutated that can possibly lead to cancer and so in order to protect ourselves from cancer it has become very popular to talk about something that we should be eating and drinking and those are anti-oxidants and the reason those are so popular now is because those antioxidants are able to neutralize the oxides which will protect our dna from being mutated and hopefully prevent cancer but antioxidants are not the only way we can protect our cells all of our cells actually have enzymes and several different types of enzymes that can detoxify the oxides and one such enzyme that's very common in eukaryotic and prokaryotic cells is catalase catalase you know this is an enzyme because it ends in ace and catalase will bind hydrogen peroxide and it will catalyze it break it down into its components oxygen and water and then of course the oxygen will be released out into the air so even the cells today that are surviving in an environment that has a lot of oxygen in it they need to have these enzymes like catalase to protect them from the toxic effects of the oxides so when determining the oxygen requirements for living things you need to keep in mind two things the first thing is do the cells actually contain the enzymes to detoxify the oxides that are produced by oxygen if they have the enzymes then they will be able to survive in an aerobic environment the other thing to keep in mind is do the cells actually use oxygen as part of their carbohydrate metabolism so remember the three pathways for carbohydrate metabolism are aerobic respiration anaerobic respiration and fermentation and aerobic respiration is the only pathway that requires oxygen these are the five different oxygen requirements and they are obligate arab facultative anaerobe obligate anaerobe aerotolerant anaerobe and a microaerophilic obligate aerobes obligate means they are required arrow refers to oxygen so these are organisms that require oxygen and the reason they require oxygen is because they can only perform aerobic respiration and when you grow them they can only grow where oxygen is present so of course because they require oxygen they will have the enzymes to detoxify the oxides facultative anaerobes these are organisms that can perform aerobic respiration and fermentation so when they are grown in an aerobic environment they can grow well when they are grown in an anaerobic environment they can also grow but what you will see is more growth in an oxygenated environment because using aerobic respiration they can produce up to 38 atp per glucose molecule whereas in an anaerobic environment they can only perform fermentation which gives them only two atp per glucose molecule so they grow much better when they are in an aerobic environment obligate anaerobes obligate they must live without oxygen so these organisms do not have the enzymes to detoxify the oxides so they are restricted to living in an anaerobic environment they're restricted to living in the anaerobic environment so of course they cannot use oxygen for their metabolism and most of the time they use anaerobic respiration aero tolerant anaerobes these are organisms that only use anaerobic respiration but they do have the enzymes to detoxify the oxides so what you will see is even growth with or without oxygen they do have the enzymes because they're aero tolerant so again they can detoxify the oxides but the presence of oxygen is not a benefit that's the difference between the facultative anaerobes and the aero tolerant anaerobes both of them have the enzymes to detoxify the oxides but having oxygen present is a benefit to the facultative anaerobes because they will produce more atp when oxygen is present the last group are the micro aerophiles micro means small arrow means oxygen file is loving and these are organisms that use aerobic respiration but they only have low levels of the enzymes to detoxify the oxides so they're still very sensitive to high concentrations of oxygen so those types of organisms are restricted to areas or environment environments with low levels of oxygen and they must have some oxygen because they require it for their metabolism all organisms can be classified by their source of energy and their source of carbon and these classifications are called the nutritional classifications and this is a flow chart that goes through those different types of classifications and the first way to divide them is by energy source and there are only two potential sources of energy and that's either from chemicals which when i say chemical i mean molecules so either from molecules or from light and so we'll focus on the side of the chart with the chemicals so if an organism uses chemicals molecules as their energy source they're called chemotrophs next we divide them by their carbon source and there are two potential sources of carbon it can be from organic molecules or inorganic carbon dioxide from the atmosphere if an organism gets its carbon from organic molecules then it is called a chemo heterotroph hetero other feeder it has to ingest other living things to get its carbon and then within these chemo heterotrophs we can divide them into groups by the final electron acceptor they use in their carbohydrate metabolism if the final electron acceptor is oxygen then they are using aerobic respiration and that is pretty much almost all the living things that we can think of the animals the fungi the protozoa and a large number of the bacteria if that final electron acceptor is not oxygen then it's either an organic compound or an inorganic compound if the final electron acceptor is an organic compound then they are using fermentation to get their energy from those organic compounds and those are some types of bacteria if instead their final electron acceptor is an inorganic compound that is not oxygen then they are using anaerobic respiration to get their energy from glucose and some examples for this are those anaerobic obligate anaerobic bacteria like clostridium going back up to the carbon source if we instead go down the line of the inorganic carbon from the air these are called chemo autotrophs so they are not ingesting other organisms to get a source of carbon and for this these are some unusual types of bacteria that we don't really come in contact with very often all right going back up back up to the energy source now we look at organisms that use light as their source of energy and they will be called phototropes now all organisms need a source of carbon and for those phototrophs it can either come from organic molecules or inorganic molecules and organisms who use light for energy and get their carbon from organic compounds they are called photoheterotrophs and these are certain types of unusual bacteria if we look at those organisms who get their carbon from carbon dioxide they are called photo autotrophs and these photo autotrophs use photosynthesis to generate their organic molecules and this is just a detail on photosynthesis that you don't have to really pay attention to but basically all of these organisms are all the photosynthetic organisms the plants the algae and different types of photosynthetic bacteria and these are photosynthetic bacteria also in this flow chart you need to know the four terms chemoheterotroph chemoautotroph photoheterotroph and photo autotroph you should know where these organisms get their energy source where they get their source of carbon and examples of each now that i've gone through the different requirements for growth both the physical and chemical requirements for growth i want to talk a little bit about how we culture bacteria in the lab when culturing microbes in lab we use media and the media is going to provide all the chemical requirements or the nutrients for the microbes in addition to the chemical requirements it will also provide the correct ph and the correct osmotic pressure and there are several different types of media that you should know the first type of media is called chemically defined and we refer to it as chemically defined because we know every component that is in the media and exactly how much so chemically defined media would start with distilled water and then the researcher would put in exact amounts of certain types of sugars amino acids and other components and this chemically defined media is used to grow chemo autotrophs and photo autotrophs complex media on the other hand is complex and this is often used from extracts or it's made from extracts kind of like chicken broth to make chicken broth you take a chicken you put in water and boil it to extract the nutrients from the chicken and when you do that you don't know exactly what amino acids are present or what amounts so complex media is often made from extracts and that is what we use to grow chemo heterotrophs reducing media you've already used reducing media reducing media is used to grow obligate anaerobes because it reduces the diffusion of oxygen into the media and the example of that was thioglycolate selective media selective media has compounds in it that suppress the growth of unwanted microbes so it's going to select it's going to allow certain microbes to go and grow and to prevent other microbes from growing differential media differential media has different compounds in it that will change color depending on the type of microbe that is grown so this allows you to tell the difference between different types of microbes and the last type of media is called enrich enrichment media and this is media that has extra growth factors trace elements extra nutrients that are a little unusual and this is usually used to grow fastidious bacteria and fastidious bacteria that is the name we give the bacteria that have very particular nutritional requirements so often they're very difficult to grow for a little more detail on some of these different types of media reducing media is used to grow obligate anaerobes an example of this is thioglycolate media which impairs the ability of oxygen to diffuse through the media and also thioglycolate contains an indicator which will change color where oxygen is present so you will have high concentrations of oxygen at the surface and oxygen is present until here and then you have an anaerobic environment there so what this creates in thioglycolate is a gradient of oxygen going from an area of high concentration to an area of low concentration when you inoculate bacteria into thioglycolate you can observe the patterns of growth to determine the oxygen requirement for the bacteria obligate anaerobes will tend to grow only where the oxygen is not present obligate arabs will grow where oxygen is present and facultative anaerobes or aerotolerant anaerobes will be able to grow throughout the tube selective media is a type of media that inhibits the growth of certain types of bacteria and allows the growth of other types an example of this is pea agar so it's phenol ethyl alcohol agar and this is comparing the agar to a regular tsa plate and p e a agar inhibits the growth of gram negative bacteria so if you compare this relates to the gram reaction this is a gram negative bacteria there is much less growth on the pea agar than you see on the nutrient agar and conversely gram positives grow well on pea agar so this is the amount of growth you see on the pea and this is the amount of growth you see on the regular agar so pea is a selective media gram negatives won't grow very well and the gram positives will differential media is media that will change color or cause the bacteria to change color which will enable you to tell the difference between different types of bacteria one example of differential media is blood agar and this is called blood agar because sheep red blood cells are mixed in with the agar so this is the uninoculated agar with the red blood cells in it when staphylococcus epidermidis is grown on the blood agar there's no significant change to the agar but when staph aureus is grown on the agar the staph aureus can secrete an enzyme that breaks down the red blood cells so what happens is that the agar then turns yellow so this is an example of differential agar differential media when staph epi is grown on it there is no change in the agar but when staph aureus is grown on it it will turn yellow when bacterial cells are put into the optimal growth conditions that means they have all the physical requirements for growth and the chemical requirements for growth then their enzymes are able to function and the cell can metabolize and eventually it can divide the process by which bacterial cells divide is called binary fission and binary fission is a very simple process so when the bacterial cell is ready to divide it will duplicate its chromosome so it will make a copy of its chromosome and every bacterial chromosome is actually attached by one part to the cell membrane now bacterial chromosomes don't have much of a cytoskeleton so they can't cannot go through the process of mitosis where you have the spindle fibers forming and pulling apart copies of the chromosome so the only way that the bacteria can separate these two copies of the chromosome is to elongate so the bacterial cell just physically grows longer once these two copies of the chromosome are physically separated then the bacterial cell divides so binary fission is a very simple process the cell duplicates its chromosome then it elongates grows longer and once the two copies of the chromosome are separated it divides the cell when bacteria are given all the physical requirements for growth and chemical requirements for growth then they will divide by binary fission and a population of bacteria will grow logarithmically that means every generation they will double double their number so where you started with one the next next generation you'll have 2 then 4 then 8 16 32 and so on and so that is a logarithmic curve where you get a doubling of the numbers every generation and for bacteria most bacteria will double their numbers every 20 minutes when they are in an optimal situation when they have all the physical and chemical requirements that they need so what that means is that if you start with one bacterium in about seven hours you will end up with one million so bacteria are able to increase their numbers logarithmically if the environment and the nutritional requirements are presented when a population of bacteria is placed in a conducive environment where all the physical and chemical requirements are met that population of bacteria will go through specific phases of growth and those phases of growth are called the bacterial growth curve and this is an example of that growth curve so on the x-axis is time and on the y-axis is number of microbes so the first stage that the bacteria will go through is a lag phase so this is from time zero when the bacteria are introduced to the environment and the lag phase you will not get an increase in the overall numbers of bacteria so the numbers of bacteria will stay relatively the same and that is because the bacteria is adjusting to the new environment it's adjusting to the temperature the ph the osmotic pressure and all the nutrients that are available assuming that the environment is optional optimal then the bacteria will enter into the log phase and that's where the bacteria are replicating at their fastest rate and so they're increasing their numbers logarithmically and that's the log phase after the log phase they're going to enter into the stationary phase and the stationary phase is named this because the bacteria are still dividing but now you have a significant number that are dying and the reason this is happening is because as these bacteria during the log phase were metabolizing they are using up the nutrients so the amount of nutrients is decreasing during stationary phase and also waste products are being produced and often the waste products of metabolism are acidic so what is happening during the stationary phase is that the ph of the environment is decreasing and you should remember that changes in ph can have an effect on enzyme function and having that effect effect on enzyme function will have an effect on the bacterial survival so after stationary phase then that population bacteria will start into the decline phase and the decline phase this is where you have bacteria dying so more bacteria are dying than are replicating and the reason for this is the same as the stationary phase nutrients are being used up waste products are building up and the ph is decreasing so with these numbers you can see the total count of the bacteria and then the viable count the viable count would be the bacteria that are alive so all populations of bacteria will go through a lag phase log phase stationary phase and a phase of decline now when we inoculate the bacteria on tuesday that's time zero and when you look at them 48 hours later they're about here in log phase so when you are observing the bacteria on thursday they are still very healthy and very viable if you were to inoculate the bacteria on a tuesday and not look until the following tuesday you would probably be in the decline phase and so a lot of the bacteria you would see would not be viable bacteria