this is the lecture for chapter six microbial growth part one this chapter is all about microbial growth and there are many reasons why we would actually want to encourage the growth of different microbes some of those reasons have to do with research on pathogens if we want to develop a treatment for the disease caused by the pathogen we need to be able to culture the pathogen or grow the ma pathogen in the lab so that we can research it and determine what treatments will be able to eliminate the disease other examples of why we would want to grow microbes is with biotech for example food if you want to produce some bread you need to have the growth conditions for the yeast to replicate and be able to form the bread for you also in terms of genetic engineering recombinant dna we will use that to produce different things like vaccines and different hormones like growth hormone human growth hormone or insulin to treat individuals so in that case we want those microbes that contain the recombinant dna to grow and replicate so that they can produce those protein products that we need in this lecture we are going to focus on bacteria and the requirements to get bacteria to grow and remember that bacteria are living cells and they are constantly metabolizing so what we want to do if we want the bacteria to grow is we have to provide the conditions to allow the bacteria to continue to metabolize and replicate the requirements for growth can be divided into two categories the first category are called the physical requirements for growth and the physical requirements for growth this basically involves what sort of environment are you going to put the microbes in in order for them to grow the second general category of requirements for growth are the chemical requirements for growth and when i say chemical think more about chemistry rather than caustic chemicals in chemistry what do we study we study atoms and molecules so the chemical requirements for growth are basically relating to the nutrients that the microbes will need in order to build up molecules and get energy the first physical requirement for growth that i want to go over is temperature every species of microbe has an optimal temperature and the optimal temperature is where it grows at its fastest rate in addition to an optimal temperature every microbe has a temperature range and that is the range of temperatures where you can get at least some growth occurring and with that temperature range you have a minimum temperature that is the lowest temperature where the bacteria can actually replicate and you have a maximum temperature that is the maximum the highest temperature where the bacteria can still replicate at least at a low level so if you are interested in encouraging the growth of a particular type of microbe you need to identify what is its optimal temperature and then incubate that microbe at that optimal temperature this is a graph that goes over the different types of temperature requirements for different microbes and temperature is always recorded in celsius and just to remind you that at 0 degrees celsius water freezes at 100 degrees celsius water boils and on the y-axis is the rate of growth with different microbes there is a wide variety in temperature requirements and in general scientists have divided them into five different groups or five different temperature requirements the psychrophiles the cyclotropes the mesophiles the thermophiles and the hyper thermopiles and for each of these groups there is an optimal temperature and then of course a temperature range where you can at least get some growth of those microbes starting with the psychophiles psychophiles are cold loving bacteria and cool or cold loving microbes and their optimal temperature is a little over 10 degrees celsius but their temperature range ranges from negative 10 to 20 degrees celsius so at freezing temperatures they are able to grow it's not their optimal temperature but they can grow fairly well and these psychrophiles are not organisms that you're going to run into very commonly because they are found in polar regions and in the cold depths of the ocean so these organisms are not something you will run into very often the next temperature requirement up are the cyclotropes and the cyclotropes their optimal temperature is about 22 degrees celsius which is typical room temperature and the cyclotropes their temperature range goes from about 0 to 30 degrees celsius and cyclotropes grow very well at room temperature that's about their optimal temperature but they can also grow at about four degrees celsius and that is important because four degrees celsius is the typical temperature of a refrigerator so cyclotropes are often known for spoiling food so while four degrees celsius is not their optimal temperature they can replicate at four degrees and so over time they can cause the spoilage of refrigerated food the next requirement up or temperature requirement is are the mesophiles mesophiles their optimal temperature is about 37 degrees celsius which is body temperature in these microbes their temperature range goes from about 10 to about 50 degrees celsius and these are the microbes that make up the human flora so these are the microbes that are found growing on the outside of our body which would be cooler could be all the way down to about room temperature and they're also found living inside our bodies and the mesophiles are also most likely to be human pathogens because once these microbes enter our body we are about at their optimal temperature so they can grow and cause disease in humans a little bit of a higher temperature requirement are the thermophiles thermophiles thermo heat file loving these their optimal temperature is about 60 degrees celsius and their temperature range goes from about 40 to 70. and these are found in hotter areas often in hot springs then the last group are the hyper thermophiles hyper means high heat loving and their optimal temperature is somewhere between 90 and 100 degrees celsius so the main point of this is that 100 degrees celsius boiling temperatures they are very happy that's very close to their optimal temperature and their range goes from about 70 little less than 70 to 110 and hyper thermophiles are kind of like the psychophiles because you're not going to run into them very often they prefer extremely hot environments like boiling water and so you can find these also in the hot springs but also down on the ocean floor at the hydrothermal vents where the water is heated to boiling by volcanic activity so these are the five different classifications or the five different temperature requirements that microbes can fall into the second physical requirement that i want to talk about is ph and just to remind you that ph relates to liquid water sometimes in liquid water some of those water molecules will spontaneously disassociate that's the fancy word for meaning that it will break in half so sometimes a hydrogen ion will break off with a water molecule leaving a hydroxide ion and ph is a measure of the relative concentration of the hydroxide ions compared to the hydrogen ions and for ph you should be familiar with the ph scale the ph scale the numbers on this refer to the inverse log of the concentration of hydrogen ions so it's an inverse log which means that the lower the number the higher the hydrogen ion concentration also it's a logarithmic scale so that means each number is 10 times more or 10 times less than the next number so for the ph scale ph scale basically goes from 0 to 14 and at 0 those solutions are considered acidic and they have a high relative concentration of hydrogen ions so there are more hydrogen ions compared to hydroxide ions neutral is around seven it's between about six to eight and neutral is where the concentration of hydrogen ions is equal to the concentration of hydroxide ions then as you reduce the amount the relative concentration of hydrogen ions the solution becomes basic and those are the higher numbers of ph living things tend to prefer a neutral ph the vast majority of living things prefer a neutral ph this graph shows different ph requirements for growth and similar to temperature every organism has an optimal ph where it will grow at its fastest rate and it also has a ph range so that is a range of ph values for which you will get at least some growth as i mentioned before the vast majority of living things prefer a neutral ph so those are neutrophils and so their optimal ph where they grow the best and function the best is about seven but they can survive in phs from about 5.5 to about 8.5 besides the neutrophils there are some extremophiles who prefer very extreme environments in terms of ph acidophiles are acid loving and so their optimal ph is about three but they can survive all the way in a ph of about one up to a ph of about 5.5 on the other extreme you have alkyophiles in alkyophiles they love an a basic ph so their optimal ph is about a 9.5 but they can survive down at a 7.5 and all the way up to an 11.5 so there are three basic classifications for ph requirements for growth acidophiles prefer lower ph's neutrophils prefer moderate phs and alkylphils prefer higher phs and again the vast majority of living things are neutrophils the last physical requirement for growth is osmotic pressure and osmotic pressure relates directly to osmosis so i'm going to take this opportunity to review osmosis for you so you'll better understand how it can affect the osmotic pressure for the microbes first the definition of osmosis osmosis can be defined as the diffusion of water across a membrane and this is a really important definition because it gives you all the important parts to osmosis first thing is that this is a process of diffusion and if you remember diffusion is a type of passive transport no energy input is required and also in diffusion molecules from move from an area of high concentration to an area of low concentration so they are moving down a concentration gradient and the molecules will continue to move towards that area of low concentration until equilibrium is reached and in equilibrium there is no concentration gradient there is no area of high concentration and area of low concentration so osmosis is referring to the diffusion of water and it only is talking about water molecules so when you are referring to osmosis you are looking at the diffusion of water and the last important part is that it involves a membrane so osmosis involves the diffusion of water across a membrane with osmosis what you are doing is comparing two solutions to each other these two solutions are separated by a membrane and just to review the vocabulary that's involved in solutions every solution is a combination of a solvent and a solute and a solvent is the major dissolving complex so in biology the solvent is always water in inorganic chemistry it can be different types of liquids like alcohol and things like that but in biology it's very easy the solvent is always water the solutes are all the things that are dissolved in the water so that could be ions different types of ions it could be sugars it could be proteins it could be all sorts of things but the solvent is water the solutes are all the things dissolved in the water and solutions are always expressed as a percentage and often you are referring to the percentage of solute so if it is a five percent sodium chloride solution then that means five percent of the solution is the solute the sodium chloride and 95 percent is the water because of course every solution will equal a hundred percent when talking about osmosis there are three important terms you need to understand and those are isotonic hypotonic and hypertonic and these are terms that we use when comparing two solutions to each other so these terms are actually comparison terms and an analogy to this another example of comparing something is height so let's say that i tell you this person is shorter if i refer to this person as shorter your first thought should be compared to whom and so if you compare him to one of his teammates he is definitely shorter but if you compare him to a different teammate he is taller so just referring to him as shorter is not enough information you need to know whom i am comparing him to because sometimes he is shorter and sometimes he is actually taller so with isotonic hypotonic and hypertonic if you have a be a solution in a beaker and refer to it as hypotonic your first question should be compared to what other solution and with osmosis questions we are usually comparing the cytoplasm of a cell compared to the environment so usually the situation we are setting up is an environment solution so the solution a could be the environment and there's a cell in the environment and the cytoplasm is solution b so isotonic hypotonic hypertonic are comparative solution comparative terms when you're comparing two solutions to each other now i want to go through these three terms in more detail and as you remember isotonic hypotonic hypertonic is in our comparative terms where we are comparing two solutions to each other the environment compared to the cytoplasm of a cell and just to remind you of the analogy using height these are comparative terms so if you say a person is shorter it is really important to define whom you are comparing that person to so with isotonic hypotonic and hypertonic it's very important to know what solution you are comparing to what other solution so the first term isotonic it's very important to go over the roots of the word iso means same and tonic means solute so isotonic means same solute and solute remember are all the things that are dissolved in the solvent dissolved in the water so if a solution is isotonic that means it has the same solute concentration as the other solution so if i tell you that solution a is isotonic then that means a and b have the same solute concentration and if they have the same solute concentration they will have the same water concentration so if they have the same water concentration then there is no concentration gradient so there's no diffusion so it is at equilibrium so solutions that are isotonic to each other will not have any osmosis occurring now to go back to the analogy of height if i tell you he is the same height as somebody then it's done he and the other person are the same height but if he is not the same height as someone then one of the people has to be taller and one has to be shorter so for solutions if they are not isotonic to each other then one has to be hypotonic the other has to be hypertonic and now the terms hypo hypo means lower like if a person is going through hypothermia their body temperature is lower so hypotonic lower solute so the hypotonic solution will be the solution that has the lower solute concentration and conversely it will have the higher water concentration the hypertonic solution on the other hand is going to be the solution that has the higher concentration of solute like a hyperactive kid has a higher level of activity so hypertonic higher solute and if it has higher solute concentration it's going to have the lower water concentration so if for example solution a has the lower solute concentration let's say it's five percent sodium chloride then it would have the higher water concentration which would be 95 percent water so if solution a is the hypotonic solution then solution b has to be the hypertonic solution and a hypertonic solution would be anything that is higher than 5 percent so we could say solution b has a 10 percent sodium chloride concentration if it has a 10 sodium chloride concentration then it is going to have a 90 percent water concentration and then you have a concentration gradient the hypotonic solution is going to have higher water the hypertonic solution is going to have the lower concentration of water so diffusion occurs from the area of high concentration to the area of low concentration so you will have osmosis occurring so water will always move from the area of high concentration to the area of low concentration so osmosis always goes from the hypotonic solution to the hypertonic solution so as a review isotonic refers to the solutions having the same concentration of solute and water so if they have the same concentration of solute in water there will be no osmosis if these two solutions are not isotonic to each other then one must be hypotonic and the other one must be hypertonic and osmosis goes from the hypotonic solution to the hypertonic solution osmosis is an important concept not only for microbiology but also for anatomy and it's really important to be very comfortable with the idea of tonicity the isotonic hypotonic hypertonic and so i've put three practice problems for you to try so this is the first practice problem a cell that is filled with a five percent sodium chloride solution is put into distilled water what will happen to the cell second practice problem a cell that is filled with a 5 sodium sodium chloride solution is put into a 10 sugar solution what will happen to the cell and if you notice this is the same cell in a different type of solution and then the last practice problem when a cell is placed in a sugar solution it gains water explain what happened now that i have reviewed the basic principles of osmosis we will come back to the physical requirement for growth that involves osmotic pressure so what this is referring to is the type of environment that the bacterial cell needs in order to function so the first option in terms of the environment is having an isotonic environment so the environment is isotonic to the cell that means they have equal concentrations of solute equal concentrations of water so there is no osmosis no net movement of water and in this case a cell a microbial cell bacterial cell can survive very well the second type of situation is putting a cell into a hypotonic environment so the hypotonic environment means that there is less solute outside in the environment compared to the cell so the environment is hypotonic the cell is hypertonic and in this case there will be osmosis in osmosis the water will move into the cell now most of the time the bacterial cell will be able to function fine because the bacterial cell has a rigid cell wall and that rigid cell wall the whole purpose of the cell wall prokaryotic cell wall is to protect from osmotic lysis so most of the time a hypotonic environment is fine for a bacterial cell the only time when it is not fine is when the bacterial cell wall has been weakened or damaged so if the bacterial cell wall is weakened or damaged then the bacterial cell will go through osmotic lysis the last potential situation is to put the bacterial cell in a hypertonic environment so the hypertonic environment means there's more solute outside of the cell compared to inside the cell so in this situation the environment is hypertonic the cytoplasm is hypotonic so you have a concentration gradient so water will osmose outside of the cell and in this case the cell is going to lose water and the cytoplasm will shrink so the cell is going to dehydrate lose water and this process is called plasmolysis and when this happens this will cause the death of the bacterial cell so the bacterial cell needs water so that the proteins have the normal three-dimensional structure so they can have correct hydrogen bonding to hold their structure and also the bacterial cell needs water for metabolism because remember in dehydration synthesis and hydrolysis water is an essential part of those two types of metabolism so if you put a bacterial cell in an isotonic solution or a hypotonic solution as long as the bacterial cells healthy and has a normal cell wall it will be able to function in both and metabolize in both if you put bacterial cells in a hypertonic solution they will not be able to stop the loss of water to the environment they will go through plasmolysis and that will cause their death when thinking about these three physical requirements for growth keep in mind that the whole purpose is to keep the bacteria metabolizing if the bacteria can perform its catabolic reactions and anabolic reactions then it is able to get energy it's able to reproduce its molecules and go through processes like cell growth and cell division and this is all dependent on making sure that the bacteria has functional enzymes because if the enzymes cannot function then the bacteria cannot perform the biochemical reactions and if it can't perform these biochemical reactions it cannot survive so the physical requirements for growth two of them temperature and ph relate directly to enzyme activity so they relate directly to enzyme activity while osmotic pressure making sure the bacteria is not in a hypertonic environment relate to facilitating metabolism by maintaining the shape of the proteins and also providing enough water for catabolic and anabolic reactions when growing bacteria in lab like we have done in labs so far we take into account these physical requirements for growth in terms of the media when we are producing the media we make sure the media has the correct ph so in general it is at a neutral ph and we also make sure that it has the correct osmotic pressure and actually most of the time the media is slightly hypotonic compared to the bacteria so the media is taking into account the ph requirements and the osmotic pressure requirements and then of course most of the time we put the media into an incubator that takes into account the temperature requirements the second category for growth requirements are the chemical requirements for growth and this involves the atoms and the molecules that are needed to make sure that the bacteria can metabolize so again another way to think of these is these are the types of nutrients that the bacteria need to be able to build larger macromolecules that they need such as the proteins that will act as the enzymes or to replicate the dna when the cell goes through cell division and also for catabolic reactions and mainly the catabolic reactions are used to release energy the chemical requirements for growth are divided into two basic groups the first group are the macronutrients and macronutrients basically means that the microbes need large amounts of these nutrients and basically when we go through the macronutrients we focus on the elements like the periodic table of the elements we are focusing on the different types of atoms that are required and these are the main atoms that are required in relatively large amounts for a bacteria to function and some of these i'm going to go through in a little bit more detail some of them like carbon nitrogen oxygen phosphorus and sulfur first chemical requirement for growth that i want to go through is carbon and carbon is an atom that is extremely important for life on earth because all living things are carbon based and in fact that is the definition of organic organic molecules are molecules that contain a high concentration or a high amount of carbon and carbon is important because it makes up the basic skeletons of all the macromolecules that are in all living things and carbon is extremely variable and it can create a wide diversity of different types of molecules so it can those molecules can vary in length they can also vary in the types of bonds there can be double bonds and single bonds which can change the characteristics of the molecules also carbon skeletons can be unbranched or branched and they can also be arranged in rings in all of these including double and single bonds so carbon is extremely important because it forms the basis of all the organic molecules all the macromolecules in all living things the second macronutrient that i want to go over is nitrogen nitrogen is also a type of atom that is found on the periodic table of the elements and nitrogen is essential for some of the macromolecules the organic molecules so if you remember the four types of organic molecules carbohydrates lipids proteins and nucleic acids and nitrogen is essential for the structure of nucleotides and you should remember that nucleotides are the building blocks for the nucleic acids for dna and rna and every nucleotide has a phosphate group a sugar group and a nitrogenous base so the nitrogenous base has a lot of nitrogen atoms in it the second type of macromolecule that requires a lot of nitrogen are the amino acids the amino acids and the amino group actually refers to a nitrogen containing group amino acids are the monomers that make up the proteins so nitrogen is essential for forming nucleic acids and performing proteins the next macronutrient is phosphorus phosphorus is also a type of atom that is found on the periodic table of the elements and phosphorus is also important for nucleotides remember nucleotides have a phosphate group a sugar group and a nitrogenous base well the phosphate group has a phosphorus atom and the phosphorus is important in the sugar phosphate backbone of dna so the sugar phosphate backbone it alternates sugar and phosphate in its structure also atp i don't know if you're aware but atp is actually a type of nucleic acid so it has a very similar structure to a nucleotide it has a nitrogenous base adenine it has a ribose and it has three phosphate groups so phosphorus is very important for forming nucleic acids and atp the last macronutrient that i want to go over is sulfur and again sulfur is a type of atom and sulfur is not so common in the macromolecules but it is found in two types of amino acids in cysteine and methionine so cysteine is the cis on the genetic code and methionine is the met so sulfur is a very important part of these two amino acids and even more importantly sulfur is involved in disulfide bonds that can form in protein structure so disulfide bonds two sulfides binding together and this is really important for the tertiary structure of proteins so disulfide bonds are very strong bonds and they help prevent proteins from denaturing and often these sulfur containing proteins are found in bacteria that live in thermal vents or in hot springs so in hot springs the temperature of the water is fairly high and high temperatures can break hydrogen bonds but it's much harder to break disulfide bonds so a lot of the proteins that the bacteria produce that live in areas of high temperature like in hot springs their proteins have many disulfide bonds and you can tell that because if you go to a hot spring there is often a smell of sulfur and that self sulfur smell smells like rotten eggs the last type of chemical requirements for growth are the micronutrients and micronutrients are nutrients that are needed in very small quantities but they are essential they are required but in extremely small quantities and the first group of micronutrients are the growth factors and growth factors are organic molecules so these are organic meaning they are carbon containing and here are some examples of some of the organic growth factors that are required by some microbes and again they are required in very small amounts but they are essential the other group of micronutrients are called the trace elements and trace elements are inorganic so they are not carbon containing and often these are different types of metals so the trace elements are different types of metals inorganic metals that are required in very small amounts and the purpose for these growth factors and trace elements is to serve as the coenzymes and cofactors for the enzymes in the bacteria so if you remember the coenzyme and cofactor are responsible for binding to the apoenzyme and together forming the active site on the holoenzyme which is the active enzyme and remember that coenzymes are organic so these growth factors are coenzymes and the trace elements are inorganic and they serve as the cofactors so in order to have functional enzymes the bacteria need growth factors and trace elements they need very few but they are essential and actually humans need them too we need coenzymes and cofactors for our enzymes but we don't call them growth factors or coenzymes instead we call them vitamins and minerals so the whole reason you need vitamins and minerals to keep you healthy is because they act as coenzymes and cofactors to keep your enzymes functional