Hello everyone and welcome back to Microbiology. Today we'll be discussing Chapter 9, Microbial Growth. The lecture objectives for this is to understand the nature of biofilms, how microbes divide, what are the different growth factors and how they relate to microbial growth, identify a microbe based on its different growth requirements, how are microbes cultured, agar versus liquid media, how media is prepared, compare and contrast selective and different differential media and examples of both and how to measure growth and calculate cell concentrations. Previously we discussed the function of the glycocalyx, the sticky layer on the exterior of the cell wall.
When well organized and tightly bound to the cell it is referred to as a capsule. However, when loosely bound this layer is endearingly referred to as a slime layer. Who can recall a major structure that slime layers can lead to?
Hopefully some of you thought of a biofilm. This is an image of a slime layer. You can see it as this mesh network that is very loosely bound around the cell wall. The slime layer is a crucial binding agent for biofilms. About 90% of bacterial cells in nature exist in biofilms, so its structure and development is important to understand.
This allows the cells in the slime layer to attach to each other and services to form biofilms, and also increases their virulence by protecting them from removal or digestion or attack by our immune system. These are the four basic steps in biofilm formation. Biofilm formation begins with the attachment of planktonic cells.
Platonic comes from the Greek word planktos, meaning drifter or wanderer. Who can guess what purpose channels can serve within the biofilm? As the structure grows, microenvironments form, such as anaerobic environments, or waste products from one organism becomes a resource for another.
and channels and pockets serve to provide exchange of chemicals within the biofilm. So the question can be asked how and when do cells know when to stop development or to properly respond to biotic changes to their environment. As you can see in the steps listed here there's step one which is a reversible attachment of platonic cells.
This happens very quickly, so within a few seconds it's either going to stick or not. Then in step two These first colonizers become irreversibly attached. And this takes, again, on the order of seconds to minutes. Step three are continued growth and cellular division, which takes a time course of hours to days.
And then in step four, the production of EPS, which is exopolysaccharide. Essentially, this is what the biofilm is made of. in the formation of these water channels.
And in step five, attachment of secondary colonizers. So these are colonizers that come after this primary biofilm is produced. And dispersion of microbes to new sites.
And this can happen over the course of days or months. So eventually this biofilm gets to such a large extent that the microbes stop growing. And... Instead of just continuing to grow until all resources are depleted and it leads to cell death, something's going on here, something that is preventing them from taking it a step too far and leading to an entire collapse of the microbial community. Microbes do so with the use of something called quorum sensing, which is the regulation of cellular activities based on cell density.
Or put another way, the self-regulation based on the number of cells for a given volume of area. Quorum sensing is the sensing of microbial populations, a quorum, and modulation of the cell's processes based on such information. So how do cells collect this information? Autoinducers are the chemical compounds used to sense surrounding microbial populations, such as the type of microbes and the concentration of cells.
This enables the detection of other neighboring cells and critical thresholds of populations called quorums to be identified by a cell. The way it works is cells produce what are called autoinducers, which are small and diffusible molecules. that are detected by other cells and when they reach a critical concentration they can initiate a cascade of gene expression that affects cellular activity. This allows cells to predict the population concentration and in response change their behavior such as to stop growing or to stop using certain nutrients and allow for or change maybe their their cell state. Maybe they form endospores or maybe they leave the biofilm, etc., etc., allowing for the regulation of population size in relation to available resources or needs.
One of the challenges in science is the use of terminology. Some scientific terms have other meanings in our day-to-day lives, and so this can be a confusing situation. For example, the word theory, as you learned in your previous...
biology courses, or other science courses, theory is the highest level of achievement science can attain in modeling a phenomenon in our known universe. It is basically a highly tested, robust, and accepted explanation. However, in our day-to-day language, we use the word theory much more loosely to basically mean any explanation, even if it hasn't been tested at all.
Microbial growth Also, as a very specific technical term we're going to use throughout the semester, it specifically refers to the increase in number of cells or the cell concentration in an area. Microbial growth is not the size of the cells changing, merely the number of cells that are present. So you may say that humans can grow in size in our day-to-day language, but when you talk about microbial growth, you should be sure that you only mean the number of cells, not the size of the cells.
So let's take a look at what replication looks like for a bacterial cell. Bacterial cells replicate through binary fission, which is the splitting into two cells. The cell will need to replicate different components of the cell, like DNA replication. Next, the cell will elongate, so a caucasus cell will become more ovoid in shape.
a bacillus cell will become more elongated. At this point, the cell will start the process of separation by forming a division septum, the inward building of the cell membrane and cell wall. And let me point this out here. This is this little indent here that we've seen in previous images of bacterium. Eventually, when this process is complete, and the cell wall is fully built in, you can have separation of the two different cells.
Note that the parental cell does not remain, but instead turns into two new cells. The process of creating new cells is called cytokinesis. Cytokinesis is a complex process that involves a collection of proteins to synthesize new cell wall and membrane components. One of the most significant of these proteins involved in the divisome is FITZ-Z, which stands for Filamenting Temperature Sensitive Mutant Z. But you don't have to remember what it stands for.
FITZ-Z essentially assembles what is called a Z-ring. This is the beginnings of the divisome. You can think of it as the foundation of the new cell wall between the two cells, which is a membrane protein complex. of the newly forming cell wall membrane.
It establishes a protein complex and which is cleverly called the division, the divisome, sorry, and then activates the elongation of the peptidoglycan layer and cell membranes leading to cytokinesis. This is the beginning of cell wall development between the soon-to-be two brand new cells. Budding is another example for replication, which is another form of asexual reproduction. Reproduction which is exhibited by yeast cells and few but some bacteria in which reproduction reproductive division yields a parental cell and a smaller daughter cell. Here we can see the parental cell right and on the right and then a cell which is growing out of it an outgrowth.
which leads to budding off from the parental cell. Unlike binary fission, this does not create two equal cells. There remains a larger adult mother cell, or parental cell, and a much smaller daughter cell. Next to the GIF is an electron microscope image. If you notice these circles right here on the mother cell, these are what are called budding scars, where the process of budding has occurred before on this mother cell.
And in general, you will note that there's an asymmetric dividing of these cells. You don't have a typical septosome that forms between the two proportionally sized cells, as did in binary fission. This cell is going to be much smaller than the parental cell as well.
So abutting also does not include the Z-ring, as in binary fission. There are several requirements for growth. Some of these are abiotic, with an A-Bio-Sorry, A- B-I-O-T-I-C, which means without life.
And so some of these are not related to life, such as, you know, nutrients may come from other life forms, like we eat plants and animals for our growth requirements. But oxygen is not based on life, right? It just comes from the atmosphere or in different.
materials, but it doesn't necessarily have to come from other living things. So that would be something that's abiotic. Some of them are biotic, like we discussed, some of the nutrients that microbes can obtain.
Our growth requirements that we're going to list out here are temperature, pH of the solution or medium they're growing in, the osmotic pressure, oxygen levels, and nutrients. Just as you can imagine, different animals need their own specific environmental conditions, so will each microbe have their own specific growth requirements. Many of these growth requirements can be graphed like the one you see on the right. On the x-axis is the growth condition, and this graph temperature. On the y-axis is the dependent variable, which will be the growth rate.
Start at the lower temperature on the y-axis. you will see that there is a minimum growth temperature beneath which no growth will occur for this particular organism which is E. coli.
As temperature is increased, growth rates of a microbe will slowly increase until reaching an optimum where a maximum growth rate will occur. After the optimum, the temperature becomes too hot and the cell growth rate falls off. rapidly. And so there is a fine line between the maximum growth rate and having too much of a growth of a good thing. Eventually a maximum temperature is reached where cells can no longer survive and that's seen here upwards above 45 degrees Celsius.
Take a moment to study this graph and understand the trends between the minimum and the maximum and the optimum. You do not have to memorize the exact numbers along this whole graph. This is just an example, remember, but there are some lessons to be learned by looking at the numbers. This is a graph for E.
coli. If you look, you will see this strain can grow optimally somewhere around 37 degrees Celsius. You can tell if you pay attention to the actual dots, which are the actual raw data that was collected.
The curved line is something that's just synthesized in Excel. And to be honest, it's not going to truly reflect the numbers. So when you look at graphs like this, it's always nice to understand that that line is something generated after the fact by a software program. It may not truly represent the numbers. We can see mostly all of these data points.
The growth rate is the highest, right around 37 degrees Celsius. And there are a few outliers here. but mostly it groups around there even though the line peaks around 40-41. So we can see that most of the data points collected circle around about 30, cluster around about 37 degrees Celsius and E. coli usually grows in an optimum of 30-37 degrees Celsius.
Take a moment to analyze what you know about where E. coli lives as was discussed in this core so far and why this is related to why it grows optimally at 37 degrees celsius Well, the reason is because it grows in the colons of mammals, digestive tracts. And since its host organism is mostly the digestive tract of mammals, and their internal temperature is somewhere hovering around 37 degrees Celsius, most strains of E. coli have evolved to grow best under those conditions.
Makes sense, right? We can now categorize organisms based on their minimum, maximum, and optimal growing temperatures. If given a microbe's optimal temperature, you should be able to categorize it.
The reason I keep defining root words is because they will continue to appear throughout your academic and career lives. Remember hydrophilic? And that means lover of water?
Well, psychrophiles, psychro meaning cold here, so we have microbes that are lovers of the cold. Psychrophiles can grow at the bottom of the oceans or other extremely cold places. and they can range in temperatures below freezing up to about 20 degrees Celsius with an optimum somewhere around 10 degrees Celsius. Miso, middle loving, is most organisms are mesophiles and most food spoilage microbes are mesophiles as well and this is why refrigeration works so well. So we can see mesophiles are optimal growth rate is somewhere around 30 to 40 degrees Celsius.
and you're going to refrigerate your foods much lower in temperature than that. Okay, next is the thermophile, which means heat-loving. And then we have even the hyperthermophiles, which grow in places like hot springs and hydrothermal vents. Actually, they can grow over 100 degrees Celsius is boiling temperature for water.
They can even grow in temperatures above boiling. Now, I won't ever ask you a question where you are given a maximum or minimum growth temperature and then have to determine is it a psychrophile, mesophile, thermophile, hyperthermophile, just because the overlap is too great in some of these areas. And I don't mean to confuse you. But you will need to be able to identify an organism if I give you their rough optimal temperature or where we're growing them. in a lab, which is going to be around the optimal temperature usually, you should be able to tell me what type of organism it is based on that information.
Next is pH. pH is a complicated concept, which is why we have prerequisites for the course, but we'll take a short review of pH. pH is a measurement of the amount of hydrogen cations. H plus is how they're usually written. And H pluses is also a proton. So all of those are synonyms to each other.
The pH scale typically ranges from 0 to 14. This will be counterintuitive at first, so pay attention. The lower the pH, so the lower the number of the pH, the more protons there are. And the higher the pH... Oh, and this is more acidic too.
And the higher the pH number, the lower the number of protons in solution. And we call this increasingly basic, a basic solution. And a pH of 7 is neutral, being right in the middle, so between 0 and 14. Why does pH matter? Because acidic and basic solutions can change the sort of chemical reactions and structure of chemicals in a solution or in the cell. pH affects the structure and function of all macromolecules.
Extremely basic pH, which means a low amount of hydrogen or protons there, means that the hydrogen bonds and nucleic acids, such as DNA, begin to separate. Lipids are important in their forming of membranes and intracellular organelles. and a higher pH will hydrolyze them. This chemical process is what is used during soap making, where fats or oils are combined with lye, which is a base.
In cells, this causes membrane instability. To make matters worse, remember we covered that ATP is made at the membrane? ATP is made, which is the energy, basically currency of the cell.
It is made similar to how electricity energy is made from a water dam. The cell creates an imbalance of protons on one side of the membrane. This is called a proton gradient. And then it lets the protons pour through a specific opening called ATP synthase that drives the synthesis of ATP. And we'll get into this later.
It's not necessarily for you to remember, but you will later on when we talk about metabolism. But that's how ATP is created. And so when the membrane is disrupted, protons will freely leak through other. weak points in the cell membrane, and ATP production is impeded.
So proper pH is critical to maintaining cellular growth. Alternatively, if the pH is too high, nucleic acids and lipids can be degraded. Additionally, it can affect the folding structure, activity, and charge of proteins.
Proteins are the tools used by cells, and damaging them can profoundly affect the cell's ability to survive. As with temperature, each microbe has a minimum, maximum, and optimal pH. Acidophiles grow in pH ranges from one to five and a half. An example of acidophiles are the bacterium that produces and lives in yogurt. Neutrophiles grow in pH ranges from five 0.5 to 8.5. Alkylophiles have a bit of overlap with neutrophiles and can manage pH as low as 7.5 but can survive in pH as high as 11.5.
And if you can look at this graph here or this chart here on the right you'll see that our stomach acid, gatcharic acid, is down to one. So it's very hard for microbes to survive in this pH. Some can do it just barely. Some form endospores in order to survive. And some people can do it just long enough that maybe they pass through the stomach and make it, you know, to some safer pastures further down in the GI tract. But this is part of the reason why our stomachs are usually quite effective at killing microbes.
Additionally, if we look here at alkalophiles, again, most Microbes are going to be neutrophiles, especially pathogenic bacterium. This is why bleach is so effective as well as being a great disinfectant. Most bacteria are neutrophiles since most environments are closer to neutral in pH. Something I would like to impress upon students is that most microbes are human neutral or human friendly. Many valued microbes are acidophiles.
They have been important through human history in food preservation, such as fermented foods. These acidophiles create low pH food environments. Think of the conversion of milk to yogurt that is done.
This is done in part by the acidophile lactobacillus. It's useful in other areas to have a low pH as well, such as certain body parts of the human body like the vagina and stomach. which maintains a low pH, thereby regulating the type of microbes that can be present in those environments. Here's an example of food preservation in the conversion of malted barley water called wort in the brewing industry or brewing science into beer.
This graph shows a pH change over fermentation time. So this is time in hours and this is just called analyte level. which it actually had some numbers here, but it's just showing in general terms what's going on. So pH is going to be the round field dot here.
And so we're starting at a higher pH, right? And we're moving lower over the brewing process. So this graph shows pH change over fermentation time.
If you were to take wort, which is unfermented beer, it's basically the sugar juice from barley, and you were to add yeast to that solution. You can see what occurs over time in this graph. The yeast will actually change its pH over time through their metabolic activity. When the word is inoculated, they get rid right to work consuming sugars and producing acids as a byproduct, which lowers the pH. The pH gets lowered quite dramatically and very quickly and prevents other sorts of pathogenic or food spoilage microbes from growing. Additionally, we see alcohol percentage go up over time, which is another byproduct of yeast fermentation and can also act as an antimicrobial.
So we can see why this information is relevant to us. and the use of our knowledge of pH and microbes can be beneficial to both food science and medicine. Here is a list of foods and the common microbial acids that are produced from them. We start with yogurt, and by now you've heard of lactic acid bacteria that produce lactic acid.
Vinegar, which is a dilute acetic acid. If you've had kombucha, It has a combination of lactic acid and acetic acid. And finally, the main acidic constituents of beer include lactic acid and acetic acid. Osmosis is the movement of a solvent, in our case water, to equalize concentration across all areas. We saw this when we discussed hyper and hypo and isotonic solutions.
We also covered how this can damage the cell's shape, volume, or even lyse the cell. We call the force that puts putting pressure on the membrane as this occurs as osmotic pressure. Cells in hypertonic environments will lose water via osmosis.
And if the difference in solutes outside the cell is great enough, these changes can lead to the cells to plasmalize. Most cells need isotonic environments to survive. For example, 0.85% saline solution is a common carrier solution for bacterium. Using highly purified water can actually kill cells.
When I worked in the Arctic studying Arctic sea ice algae, we would collect ocean water samples to bring back to the lab. to experiment on. Before going out to sample more microbes and reuse those containers to prevent cross-contamination from the prior sample, before going out to sample again, we had a rinsing protocol with highly purified water which would kill remaining algae.
This is because those algae had high salt concentration growth requirements and this hypotonic solution caused osmotic pressure inside the cell. and cell lysis to occur. Checkpoint one.
Why does adding large amounts of salt to meats preserve them and prevent microbial growth? Let's now discuss oxygen requirements and the different categories of microbes that fall underneath their oxygen requirements. The first is obligate aerobes.
and they require oxygen to live. So can anyone think of an obvious example of an obligate aerob? I think one that comes to mind is humans.
Humans absolutely require oxygen to live. Facultative aerob uses oxygen when present. They prefer it. It's a great way to make a living.
But they can live without it with other... metabolic capabilities that allow them to survive without oxygen. Then there is the obligate anaerobe.
So they're obliged to not be in the existence of oxygen. And anaerobe means without oxygen. And they are harmed by oxygen.
And we'll get into this later, but oxygen can be damaging. Aerotolerant anaerobe is a microbe that cannot use oxygen but they can tolerate it when present. So they can't make a living on it but they can they have methods to deal with the toxicity of oxygen itself.
Those are the aerotolerant anaerobes which are often confused with microaerophiles. Microaerophiles require a small amount of oxygen. Now let's get into why oxygen can be toxic.
Oxygen comes in many different toxic forms. These toxic forms often are created accidentally in the cell due to all of the numerous chemical reactions happening within it. I'm sure some of you or all of you have probably heard of antioxidants.
Antioxidants are considered healthy because they are substances that prevent the toxic effects of some forms of oxygen. Now, a lot of antioxidants are produced by our own cells, or you can derive antioxidants from the foods you eat. You may have heard of oxidative damage and a variety of products marketed to you from the food or cosmetic industry in order to reduce oxidative damage. These forms of oxygen are toxic because they are highly unstable. Let me show them here.
And this is superoxide anion and hydrogen peroxide. So when... These forms of oxygen bump into biomolecules and macromolecules within or on the cell. They can react with them and turn them into things that are less functional. The first example that I'm going to list here is called superoxide anion.
Just recall an anion is a molecule or an atom that has a charge. An anion specifically means that it has a negative charge. A cation would mean that it has a positive charge. But an anion has a negative charge, and this form of oxygen is extremely unstable and dangerous because it wants to react with other molecules so that it can become more stable. It's highly unstable.
Shown here, it can actually react with another superoxide anion and a hydrogen. to create molecular oxygen. Molecular oxygen is just O2. Molecular oxygen is not an ion, so it has gotten rid of its extra electron in this process and become neutrally charged. Molecular oxygen is a much more stable oxygen, which is the form we breathe and is the most common form in the atmosphere.
However, the other molecule formed in this reaction is a hydrogen peroxide, or H2O2 is its formula. This reaction is catalyzed or moved along by an enzyme called superoxide dismutase in the cell. So this enzyme drives this reaction forward, and this is great because H2O2 is a little bit more stable than superoxide anion, and a little bit less harmful.
However, you've probably heard of H2O2 or hydrogen peroxide, and it is a toxic compound we use as an antiseptic. It can be used in order to try and kill foreign cells to prevent infection on wounds. So it is still a relatively unstable and harmful compound. Some microbes can catalyze the following reaction here using an enzyme called catalase, where they convert H2O2 into into water molecules and an oxygen molecule, which are two very stable compounds in the cell. Now, don't worry about balancing out the equations here or anything like that, but you need to know about superoxide anion.
It's incredibly unstable and damaging. It can be converted into molecular oxygen and hydrogen peroxide. through the enzyme superoxide dismutase and hydrogen peroxide can be processed even further into h2o and molecular oxygen which can be catalyzed by an enzyme called catalase and so if we're thinking about organisms that exist with or without oxygen these enzymes are going to be incredibly crucial Now we know that oxygen is a necessary component for some organisms to grow, such as humans, but some of its other forms are quite lethal, and dose makes the poison.
If the cell can convert it to less damaging forms, it can tolerate oxygen a lot better. This will be incredibly important to classifying microbes. Some microbes need oxygen, some don't. Some don't use it, but can tolerate it because they have the enzymes to process its dangerous forms.
while other microbes don't use it and do not have the enzymes to process the unstable forms. So they can only exist in the complete absence of oxygen. And again, the presence of oxygen is called aerobic, and the absence is called anaerobic.
Here are five different categories of microbes based on their oxygen requirements. We can test for what type of microbe category an organism is by where it can grow in a column of liquid broth. The top of the broth is... There is oxygen exposure, where oxygen exchanges with the atmosphere and broth surface and diffuses into the broth medium.
In the middle, there are micro amounts of oxygen as the oxygen diffuses deeper. And at the bottom, no oxygen has made it down that far. The first category are obligate aerobes.
They need oxygen in order to survive because it is part of their metabolic requirements. It's how they obtain energy. Therefore, we find them growing only at the top. Because they live in the presence of oxygen, they have evolved a full suite of enzymes to handle the unstable forms, including both superoxide dismutase and catalase.
Next are facultative anaerobes. As you can see, they grow best at the top where oxygen is the highest, because oxygen is very good at... helping the cell produce energy.
They can grow without oxygen too though. So they have a very complex metabolic chassis where they can do it, exist without oxygen and still manage to survive. But oxygen makes them grow better. And so that's why you see the most number of cells at the very top. And they too have superoxide dismutase and catalase.
Obligate anaerobes do not use oxygen for growth. and have not evolved the enzymes to neutralize the unstable forms. And so therefore, you only see growth at the bottom. Aerotolerant anaerobes, they do not use oxygen in their metabolism to make energy, but they can tolerate its presence. And so they do have the superoxide dismutase, but it's not necessary for them.
to have the catalase because they don't actually handle a lot of oxygen so they just have the superoxide dismutase lastly and this is the category that often is confused with aerotolerant anaerobes are the microaerophiles and these organisms require a small amount of oxygen but they don't they don't quite they're not able to deal with the the lethalness of oxygen entirely and that's because they lack the enzymes to process the toxic forms. So those are the microaerophiles and you find them growing right in the middle here at this special zone where there's not that much oxygen down further in the tube but not so low that they're completely deprived of oxygen. Now a couple things to note in this graph another or in this table another confusion that students often have is they confuse aerotolerant anaerobes with facultative anaerobes. Look at these two tubes and how do you tell the difference between them? The answer should be that with facultative anaerobes, they like to use oxygen and they're capable of using it.
And so the highest density of cells you will find at the top, but you'll find cells throughout the entire column. The other thing that can confuse students is sometimes as a if the culture tube gets too old even though you might have the cells growing in the top as the cells die they can fall out of solution and fall to the bottom so you have to be very careful with old tubes it's best to look at tubes fresh. This is an example of that exact problem but we'll get into it here so this is Based on the growth pattern, what are the oxygen requirements of the microbes in tubes A and for tube B? So I want you to give me the category they are. And for tube A, the living cells are found here.
And it's a little bit of an old tube, so there's some dead cells down here I need you to ignore. And for tube B, the living cells are found here and growing out down through here where there's no oxygen. and they cannot grow as high as the microbes in tube A, where there's this little bit amount of oxygen present. So what category is A and what category is B?
Let's switch our focus to nutritional requirements. We can start with that there are some basic elements that all organisms need. Carbon is involved in basically everything.
It is involved in all the major biomolecules of life, defines what an organic compound is. Organic compounds are compounds found in living things that are made in part with carbon. As you know, nitrogen is essential for amino acids. All amino acids have nitrogen in them. And so nitrogen is going to be incredibly important as well and required for all living organisms.
The amine group in amino acids is a nitrogen group. Proteins are made of amino acids, so we need nitrogen to make proteins, and all cells need proteins. Sulfur is also a compound that's in some of these essential amino acids, and so it's important for protein synthesis.
Phosphorus is a component of ATP, which stands for adenosine triphosphate. As you know, phosphorus is also an important element in lipids. And DNA and RNA also have phosphates, as well as nitrogen and carbon.
But without phosphate, there's no DNA, there's no RNA, there's no ATP in most lipids. So it's important for synthesis of all of those molecules. Lastly, there are many of what are called trace elements that are needed in smaller amounts, like iron.
copper, zinc, etc. And they're used for enzymatic reactions. They help enzymatic reactions take place, so they're called enzyme cofactors.
Now that we have the base understanding of growth requirements of microbes, let's study how they are cultured. Microbes are grown in culture media. This is simply the material they're grown in or on. It can be solid or liquid.
The media should contain all of the nutrients or materials to grow the microbe. And here we have different examples. We have an agar plate, which is a solid.
We have an agar deep tube, which is agar poured into a tube here. And we have agar broth, so this is a liquid, or sorry we have liquid broth, there's no agar in this. And then agar slant, which is another solid tube, but it's just made at an angle here. And what you'll see in common is all of the solid medias have agar in them.
In order to make a culture media, the nutrient material upon which microorganisms grow can be either a liquid or a solid form, but it must be initially sterile, which means that it contains no living microbes. In that way, you can inoculate it with your particular wee little beastie. and have a tube that is only growing what you want it to grow. Liquid media will be our broths that we have seen pop up time and time again throughout the semester and we can add an agent to that broth to turn it into a solid medium and this chemical is called agar. Agar is a complex carbohydrate dried from kelp or algae and it is used as a thickening agent to produce solid media agar.
from liquid media. It is essentially the jello of microbiology. Jello uses gelatin, which microbes unfortunately love to consume and therefore degrades over time.
Agar, however, is not easily broken down and is generally not degraded by most bacteria and is relatively low in toxicity, making it a great source for hardening different sorts of nutrient broths in order to make a solid structure. We can see here this person is pouring some hot agar into a dish, and this is called a petri dish. And it comes with a kind of plate to pour into the basin, and then this lid that he's holding, this person is holding, to protect it from the outside air, contaminating, bringing in unwanted microbes that could grow on top of this.
In this way, we can grow just the microbes of interest on the top of the surface. If any of you have ever used gelatin before, it acts and behaves quite similar to how jello is made. So here's a pile of agar on the left side. It comes in this dry powder form and it's mixed with your nutrient broth.
And that's not far enough yet. First, we have to do something special to it in order to get our agar plate made. The solution has to be then heated and usually this is through a pressure cooker.
and we'll talk about that later, but it's heated to above 100 degrees Celsius. And once it's heated to 100 degrees Celsius, then the little solid granule agar particles melt to create a liquidy gel-like substance. Scientists will cool this down to a temperature so it's easier to handle, usually around 55 degrees Celsius, and it remains liquid at this temperature. So the scientist is pouring an agar plate after letting it cool down. to around 55 or so.
Once it goes into this dish, it's going to be allowed to cool down to around 37 degrees Celsius or so, or cooler, put in the fridge. And that temperature below the temperature of basically the human body allows it to become, go from a liquid to more of a hardened gel. The fascinating thing about agar is it doesn't turn into a liquid again. until you heat it all the way back up to 100 degrees Celsius. So if you took this plate, you warmed it up to 55 degrees Celsius, it wouldn't melt, even though it was a liquid beforehand.
So it would have to be reheated back up to 100 degrees Celsius. So it has to be quite warm, quite a ways up, before it turns into a liquid again. And this allows scientists to grow microbes at a variety of different temperatures on a solid medium without worrying about the solid turning into a liquid again.
where the microbes eating is solid and degrading it. This heating process is a great prelude into talking about sterilization techniques, which we'll get into a lot more in the next chapter. Medium needs to be absent of any residual microbes, so that the only microbe that grows on it is the one introduced purposely for study. In order to sterilize the medium, scientists often use an autoclave, pictured here, which is essentially a large pressure cooker. that kills everything.
In order to accomplish this, scientists need to heat the solution up to 121 degrees celsius for 20 minutes. So in order to get it up to 121 degrees celsius, it needs to exceed the temperature of boiling water, which is 100 degrees. In order to do this, it's heated under pressure, 15 psi to be exact. so that liquid doesn't just evaporate away and stays in liquid form as it gets up to those higher temperatures. Again, the medium has to be heated up to 121 degrees in temperature, and this is the internal temperature, not just the temperature of inside the container.
So just like if you're baking a cake, it might take a while for the inside of that cake to get up to temperature. So it's held at this internal temperature for about 20 minutes. There's other alternatives to this.
There's also dry heat, which is effective. And this can only be used when sterilizing lab equipment that's absent of water. This obviously isn't for medium, which must have water in it.
Without the presence of water, microbes are just more susceptible to heat and damage. It's recommended to heat the lab material to 160 to 170 degrees Celsius for... two to four hours. It takes a lot longer here to accomplish the same thing. This process of dry baking is very useful for items that you may use to handle or carry microbes such as glass labware.
Sometimes scientists need to get rid of living microbes but they don't want to heat a solution. The way around this is through filtration. The filter has small enough microscopic holes in it a solution can pass through. but the size of the pores of the filter can prevent microbes from getting through.
The most common filter hole size is usually 0.2 micron. Think back to our early chapters and analyze why 0.2 microns make sense. The reason for this is even the smallest of bacteria should not be able to fit through a small pore opening of only 0.2 microns. Media can be categorized as either chemically defined or complex.
Complex media is far and away the most common type of medium. It can vary quite a bit from batch to batch. It is made up of things like meat, yeast, or plant extracts, and the quality and chemical makeup can vary between such batches. Chemically defined recipes use exact concentrations of chemicals instead of extracts. So exact amounts of a refined sugar, salt, trace elements, vitamins are added and then mixed together.
Two techniques can be implemented with growth media. One is to make the growth media selective. Selective media is designed to not allow any type of bacteria to grow on them. They specifically select certain microbes that are allowed to grow and suppress the others. So some steps are taken to limit the breadth of microbes that will grow on this media and suppress the growth of others.
Differential media is designed to distinguish between different types of bacteria growing on the same plate, so the appearance of the culture will be different depending on the microbe present. Media can be selective, differential, or neither. Media could also be both selective and differential, all on the same plate.
And we'll get into some examples on the next slides. This selective media is called bismuth sulfide agar. Bismuth sulfite inhibits the growth of ground positive bacteria, and only salmonella typhi can grow in the presence of bismuth sulfite, so it's used to isolate S. typhi from fecal samples.
Why is this such an important medium? You can imagine fecal samples would have a whole slew of different microbes in them, but here we see in this image we're able to identify S. typhi in the mix of bacteria.
Because we have their growth on bismuth sulfite bacteria, if this was a non-selective media, we wouldn't be able to see the presence of S. typhi alone due to all the other bacterium competing for space. And they probably look very similar at this level.
We should take a moment to note that the dots you are seeing are visible to the naked eye. So could these be single bacterium? The answer you gave should have been a resounding no. Remember, bacteria are usually about one micrometer wide, and a 12-point font period is a whopping 500 micrometers. So these dots did come originally from a single bacterium that landed there, but from which multiplied into hundreds, if not thousands, of bacterium until they were large enough to be seen by our naked eye.
Blood agar is an example of differential media. This media is actually made with blood, which is one of the components. So you can decide if it is a complex media or chemically defined media. Species that are able to destroy these red blood cells, they can, so there are species that can actually destroy red blood cells, right, pathogenic bacterium, for example, some of them can destroy red blood cells. And by destroying the red blood cells and lysing them open, that changes the color of those red blood cells.
And so the color goes from this rich red color to a clearing of the red color, which we call a halo effect. This destruction of red blood cells is caused by certain pathogenic bacteria and is called hemolysis. An organism that's not capable of hemolysis will not change the color and degrade the red blood cells.
And so this is an example of a differential media. We can tell the difference between organisms that are capable of hemolysis and those that are not. Here's an example of a differential and selective media. Manital salt lager, also called MSA.
Within MSA, there's a high salt concentration, which makes the plate selective. Only salt-tolerant genesis, like Staphylococcus, will be able to grow on MSA. Staphylococcus is an organism that's present on your skin. And you can imagine your skin has a lot of salt.
So we can get Staph or Staphylococcus to grow on this plate. The ingredient mannitol allows the plate to be differential. Manitol is a type of sugar and added to this plate in addition to it with a compound called phenol red, which is a color indicator.
Phenol red turns yellow when the pH drops below 6.8. And if you remember in that image I showed you of the wort being turned into beer during fermentation, as the sugars get consumed during fermentation, acids can be produced by certain microbes. Manitol. is a sugar that only some organisms are able to degrade.
And the acids from its fermentation change that phenol red from a red color, which you can see in the uninoculated example in the upper left third. into a yellow color. So the medium is selective due to its salt content, restricting growth, and also differential because we can tell what type of Staphylococcus is growing. S. epidermis cannot ferment mannitol, and we can see it doesn't change the mannitol to a yellow color.
However, S. auris can. This is very important because S.
epidermis is one of our friendly bacterium, while S. auris can cause disease. Maconchi agar is also differential and selective.
It contains crystal violet and bile salts and these two compounds suppress the growth for gram positive bacteria. We would then say it selects for gram negatives. It doesn't select for gram positives because gram positives aren't the ones growing. It selects for gram negatives.
It has a pH indicator as well called neutral red and this neutral red turns a hot pink color at low pH. It uses the same trick where we add a sugar that only certain microbes can ferment or break down and creates acids from and that sugar is lactose. Lactose is added to the medium and bacteria that can ferment it will lower the pH of the sugar and make this hot pink color. E. coli and salmonella are both gram-negative bacteria so they can both grow on this plate but only E.
coli can ferment lactose. Therefore would you expect that the top or the bottom bacteria streak to be a coli. Checkpoint three. EMB agar plates contain eosin and methylene blue.
Eosin is toxic to gram-positive bacteria. Methylene blue turns black in the presence of bacteria that produce lactic acid. What kind of medium is EMB agar? Here are a couple of artistic agar plates. To the left is EMB with E.
coli. A freshly made plate will be reddish and E. coli makes a metallic green sheen. The second plate is MSA with Staphylococcus.
Can you remember what Staphylococcus would be responsible for the color change we see in this plate? Some microbes are fastidious. Fastidious means they are picky in their nutritional requirements.
An example of this would be Neisseria gonorrhea, which requires media with blood or hemoglobin, special amino acid additions, and special vitamins added in order to grow. In fact, most organisms are so fastidious they can't even be grown in a lab. If you remember back to the first lecture we covered, that over 99% of microbes are non-culturable in the lab. An example of a microbe that's not fastidious would be our E. coli.
No conversation about microbial growth could be complete without talking about generation times and doubling times and the population density throughout a growth curve. So as discussed, bacteria go through binary fission, and the rate this takes for it to happen is somewhere between 20 minutes to 24 hours usually. depending on the species and the conditions. Generally speaking, pathogens tend to multiply quite rapidly on the scale, so closer to the 20-minute side, where some other microbes that exist out in nature, maybe soil microbes, etc., tend to grow a little bit slower, so the generation time is a little bit longer.
Generation time is the time it takes for bacteria to go from one cell to a pair of cells. So a synonymous term for this would be called a doubling time, right, because you double the number of cells. You've gone from one to two. You've doubled the number of microbes. If we look at this little graphical depiction of bacterial cells dividing, we can see that there is a pattern that emerges.
We go from one to two cells, and now we have two cells. that can each go from one to two cells. So we go from one cell to two cells to four cells to eight cells. How many cells would then occur in the next generation?
You should have said 16 cells, and that is what we mean by doubling. So each one of these doublings is called a generation. If we start with our founding population, we call this generation zero. This is the first bacterium on the scene.
like imagine you have a broth and a single bacterium falls in. This is generation zero. The symbol for generation is lowercase n. So you see in the table here in the top right, number of generations n, the founding generation is generation zero, and then we go up from there.
We can draw this pattern as a formula. Up above here, start at the very top, and we just start with our generation 0. Write down the number of cells in that generation 0. In our example, it's going to be 1. Now we're going to experience a doubling of that with n equals 1, so 1 times 2. We're trying to calculate how many cells there are, and so on and so forth. So we can have 1. to three generations, and this means we take our founding number of cells, times it by two, by two, by two, three different doublings.
This tells us how many cells we have. One times two times two times two equals eight. Another way to write this is with exponents.
So one times two to the third, which also equals eight. The trick is that not every founding population starts as 1. So maybe a cluster of cells is introduced into that new broth tube. So maybe we have 3 cells in our n equals 0. The equation would be revised then to be 3 times 2 times 2 times 2, which equals 24. 24 cells after 3 generations. So how to estimate the final number of cells after a given amount of time? We've kind of gone over this here in the previous slide, but we're going to break it down here.
Take a look at the following example. A culture was started with four cells. The microbe has a generation time of 20 minutes and is allowed to grow for one hour.
How many cells will there be after one hour? Well, we know that there are three 20 minutes in one hour. 3 times 20 is 60, and there's 60 minutes in an hour.
So we're going to be looking at three generations. We can write this out as 4 cells times 2 times 2 times 2. So that's 8, 16, 32. 32 cells there. A better way to write this is 4 times 2 to the 3rd, which equals 32. And so the general formula... is going to be the starting number of cells times 2 raised to the power of the number of generations that will equal the final number of cells.
And the symbols for how this formula is written out is capital N equals number of cells lowercase n equals generation number. So the equation is the number of cells at generation zero times two to how many generations you have equals the number of cells at whatever generation you have. So in this example, n sub zero at naught is four cells, the number of generations is three, and it's going to be equal to capital n at three generations, which would be 32 cells.
Let's do another example here. Alright, so again this is our general equation. We want the final number of cells. We're going to take the starting foundation generation, multiply it by 2 to the number of generations.
The example is two Escherichia coli cells. Two E. coli cells were transferred from the cook's hands into a warm Thanksgiving gravy. If E.
coli doubles every 20 minutes in the gravy, how many will there be after 6 hours when it is served? Well, we know that for every hour there's 60 minutes. So 60 minutes divided by 20 minutes means there's 3 generations an hour.
And there's 6 hours. So 6 times 3 generations per hour equals 18 generations. And so if we plug this into our formula, N naught, number of cells at the beginning, is 2. and we're going to multiply that by 2 raised to the number of generations, 2 to the 18th, and that's going to give us a total number of about a half a million E.
coli cells in that gravy. Written in scientific notation, it would be 5.24 times 10 to the 5th cells. You don't have to write it in scientific notation. It should be something you get familiar with if you take any chemistry courses or further science courses.
Basically, you count the decimal point over. So it's 1, 2, 3, 4, 5. And you put the period there, the decimal point there. So it's 5.24 times 10, and then you write how many times you move the decimal point over. 5.24 times 10 to the 5th cells.
You can see it only took 6 hours to get to a half a million E. coli cells from only 2 founding cells. And that's why it's important to make sure that you're not leaving things out in a warm environment, you know, around 37 degrees Celsius for a long period of time. It should be hot.
served and then cooled rapidly. But be careful not to put a hot food in your fridge because you could warm the food around it and then everything's warm for a longer duration. So be careful about that.
So I'm going to post a practice slides with several practice math questions that are worked out that you can go through. Many of the math problems in this unit will be based upon that power point. So please go ahead and take the time to review that. So we saw how quickly E. coli can grow in a short amount of time.
However, there are some microbes that can grow very, very slowly. So this paper right here shows that there are long-lived bacteria that are reproducing only about every 10,000 years that live in rocks deep in the soil. about one and a half miles below the ocean floor.
And they can be as much as 100 million years old. So microbes have a diverse array of survival strategies. And so in certain environments where nutrients are not always the best or environmental conditions aren't always the best, they just grow slower rather than dying. You know, if they're evolved to do so. Of course, you're not going to stick in a coli 100. 1.5 miles below the ocean floor and it survived.
It would die instantly. But there are microbes that evolve to live in those conditions. Checkpoint four, if an infection starts with a single bacterium that undergoes binary fission every hour, how many bacteria will there be after five hours? So what we're trying to get at here is to show how the bacterial growth is exponential.
If we put this on a graph where the x-axis is the time that has passed and the y-axis is the number of bacteria, this just shows the insanity of what an exponential growth curve looks like. So a species that doubles every 20 minutes like E. coli can go from 1 to 4.7 times 10 to the 21st cells within a single day.
Now let's discuss the curve of growth itself. See, bacteria are commonly grown in batches. A batch is a method of growing in which no additional nutrients are added during growth and no waste products are removed, kind of like the culture tube.
You make a tube of media, you add the bacteria into it, you let the bacteria grow up, and eventually it'll die out. And one way scientists measure growth is by culture density. Culture density is defined as the number of cells in a milliliter.
In a batch culture, distinct phases occur throughout the growth of the culture. Here are the different phases of growth. And remember, when we are talking about microbial growth, we are discussing the population size, not the size of the bacteria.
So first, bacteria are introduced into the fresh medium, and this begins the lag phase. In this phase, bacteria are just getting used to their environments and they're going through an intense activity of preparation for growth but no increase in population is really occurring here. Eventually they transition into the log phase which stands for logarithmic growth which is the area where reproduction is occurring at a rapid rate through binary fission. or budding through or mitosis in other words with yeast is another common example this is called the log phase and it's an exponential growth in the population so this is the number of bacteria here okay eventually we reach the stationary phase and this is a period of equilibrium where the number of microbial divisions equals the number of deaths so the population is stable through here And this is a period where the concentration of microbes are so high that the nutrients are starting to become a limiting factor here.
Or it could be the oxygen available in the medium, whatever it might be that's available there. Eventually, resources get exhausted and we reach the death phase where the population is decreasing at a logarithmic rate as well. Now, the way this is usually graphed.
is on what's called a semi-log chart. And so this axis is just a normal axis where there's an equal increments of zero through 10. A log chart is a logarithmic chart. So the higher you get, the quicker it progresses through the numbers.
So this looks like a linear growth chart. But in reality, it probably looks more like this, where it's just continually the rate of increasing population gets steeper and steeper but because they graph it on what's called a log chart basically the higher up you get the faster it's going through the different changes in units here it's kind of hard to explain but it's called a semi-log chart to make it easier to graph it all here and you get this nice clean linear look to the growth you won't be tested on that. There are many methods for quantifying or measuring microbial growth and we are going to focus on two main ways which are plate counts that are used for serial dilutions and then the direct microscopic count.
The most common method for directly quantifying microbial growth is actually referred to as plate counts. where you actually count the number of what are called colony forming units on a plate. Each one of these colony forming units is not just one bacteria.
Remember how big this plate was in relation to the person's hand that was pouring the plate earlier? What these represent are on average a single bacterium that lands on the spot of the plate. So right here imagine one bacterium landed there. You can't see it at the time but eventually it grows into thousands and thousands of bacterium. and forms a colony which can be seen and so this is called a colony forming unit because we know that about one bacteria landed there to form that colony and they're called cfus for short so if you were to count all of these bacteria you counted 67 colonies you would assume that for whatever volume you poured on this plate there's about 67 bacteria in that in that unit of volume so if you poured one milliliter here You counted 67 bacteria or colony forming units.
You would assume that in that original milliliter, there was probably about 67 bacteria per milliliter. There's a problem with this though. Bacteria can be so numerous so quickly that it's very easy that if you were to pour something out of a broth tube and spread it around on a plate, that you would...
create what's called a lawn on the plate. So unlike that previous image where you could see nice and neatly individual CFUs, here those colony forming units are so close to each other that they don't form separate colonies, that they just form one giant mass in the plate called a lawn. And individual CFUs cannot be distinguished and counts cannot be made. So we cannot know how many bacteria were in the original liquid culture. That's no big deal though because scientists have a workaround for this called serial dilutions.
So instead of taking this original inoculum and putting it right on a plate because they know it's going to be too concentrated, they'll put it they'll dilute it in a tube. So we'll take one milliliter and they'll put it into a tube of nine milliliters and make 10 milliliters. So they've diluted it one tenth and then they'll take one milliliter of that tube and they'll put it on a plate.
See if they can count individual colonies. They don't stop there though because you want to do many of these plates all at once so that you can let them all incubate together. So they'll do several of these. So now they'll take one mil out of this tube as well, put it into another fresh nine milliliter broth tube.
So now there's one mil and a total of 10 mils of solution. They'll mix it and then plate one milliliter again. And you see it's a little bit more, you can see more of these individual colony forming units, but it's not good enough.
And so these are called serial dilutions. They're done in serial. See, each milliliter is taken from the previous tube.
And so each tube is going to be more diluted. This was 1 in 10. And because you took 1 milliliter and put it in this one, which had 9 mils plus 1 mil equals 10 milliliters, you did 2 1 to 10 dilutions. and that's actually a 1 in 100 dilution.
We'll talk about that later. And the next one is going to be 1 in 1000. The next one will be 1 in 10,000. And the final one is 1 in 100,000. Now which plate to count is kind of difficult.
You want to try and avoid plates where there are cells. See if he's touching each other. because it might be hard to tell if there's two CFUs there, colony forming units, or three. So it's nice to get good spatial separation, but you don't want too few or else it could be sampling error. Three is kind of low here.
Usually what we're looking for is between 30 to 300 CFUs to count on a plate. Now, Once you count the number of bacteria on a plate, the mathematical question is how do you find out what was in the original inoculum? And that's going to be using a process called a dilution factor.
So a 1 to 10 dilution here, if you could count all these microbes here, you could figure out what was in the original inoculum by timesing it by 10. Because you know the original inoculum was 10 times as strong. Now if you take a mil out and you put it into a new solution, now you've done two 1 to 10 dilutions, which is a 1 to 100 dilution. So you times the number of CFUs counted here by 100, and that would tell you how many bacteria were in the original concentration.
So on and so forth. That's called a dilution factor. And really briefly, it's the inverse of the dilution.
So 1 to 10. That means you take 10 and times it by the number of bacterium to get the original inoculum. Now that's really confusing, so let's go ahead and take a step back and simplify the process by using a different example. Let's make some coffee.
So dilution factors are used to calculate the original concentration of something. A dilution factor is defined as the reciprocal of the dilution. What do I mean by that? Well let's get into it. I want you to intuitively understand how dilution factors work.
So in this example here we've made a very simple dilution of our espresso. We've taken one part espresso and added two parts steamed milk in order to produce a latte. Let's think about this intuitively at first and then we'll understand how dilution factors work mathematically. So first of all, What is the percent concentration of espresso in the latte? You should have said 33%.
There's one part espresso and there are two parts steamed milk. So it's not 50%. It's not one and two. It's one out of three, right?
That's 33%. You have to figure out the total volume here. And the total volume is three parts.
So it's one part out of three parts total. Alright, so what is the fraction of this dilution? Well, I just said it's one-third.
To find the dilution factor, all we need to do is follow the definition. The dilution factor is the reciprocal of the dilution. So the dilution is one-third. The reciprocal is just the inverse of that. You flip those numbers over.
So it's 3 over 1, or 3. To find the original concentration of espresso, take the current dilution in the latte, which is 1 third espresso. and multiply it by the reciprocal fraction. What is the original concentration of the espresso? One-third times three, the dilution factor, which equals three over three, or 100% espresso.
Now this is an overly simplistic example because the espresso didn't come from a diluted espresso already. We didn't dilute things by much, nor did we do serial dilutions. Let's take a look at another example, but you can see why taking the current concentration of something, which is in this case the concentration in the latte, and in microbiology it's the CFUs on the plate, and then multiplying it by the dilution factor can give us the original concentration.
Let's see how this would play out in chemistry next to further our understanding. In this example, Instead of making a latte, we are going to make a diluted solution of ethanol here in the middle. Taking a 50% solution of ethanol, mixing it with the pure water solution, we've taken 5 mL of both into the new solution flask B. Now, we know we are just halving the concentration of ethanol, so the new concentration will be half of the original. It will be 25%.
However, What if we did not know the concentration of the original stock solution of ethanol and only knew what the new flask concentration had? This would be like having only the plate, and we only know how many CFUs are in the new plate. We don't know how much was in the original tube.
Could we solve for how much was in flask A? Okay, so first let's figure out what we do know. How much ethanol was added to flask B? Well, 5 mL of this ethanol solution. How much of the water was added to flask B?
5 mL again. Intuitively, what do you think the concentration of ethanol is now in flask B? Well, it should be 25%. It's half and half.
The original ethanol flask was 50%. But what if we had to solve in reverse? So let's go ahead and do that. So we used to know this. Now we're trying to figure out what percent is the original ethanol solution.
So we're pretending we didn't know this. Now, again, we'll just put together our numbers here to figure out our dilution. How much did we add a flask A?
Well, it says we added 5 mils. How much did we add a flask C? It said we also added 5 mils.
Therefore, what is the total volume in flask A? b. The total volume is 10 mils.
So what is the dilution of the original solution? Hint, it's the volume added divided by the total volume. See if you can figure it out.
Well, the volume added was 5 mils and the total volume is 10. So it's going to be a dilution of one half, one over two. And as you know, looking at our formula at top here, the original concentration is equal to the final concentration in this solution times the dilution factor. And so now we need to figure out the dilution factor. What's the dilution factor?
And the hint is you take the reciprocal of your dilution. Should have said 2. So now plug it into the formula above. New concentration is 25%.
Our dilution factor is 2. And so 25% times 2 equals 50%. We know the the original ethanol solution was a 50% solution. Now let's put this to work.
Here's our serial dilutions. Okay. One mil, we put it in nine mils. So what's our dilution there?
Well, we added one mil to nine mils. So the total volume is 10 mils. So it's a one to 10 dilution. We take another mil of this broth and put it into a new tube of nine milliliters.
It's another one to 10 dilution, but we've done two dilutions. You can figure out the new total dilution by timesing each dilution against each other. So 1 to 10 times a 1 to 10 dilution. means we made a 1 to 100 dilution. So this is 1 100th the strength of the original inoculum.
Now we did it one more time. I did 1 mil to 9 mils. So what's our new total dilution?
It's 1 to 100 times 1 to 10 for 1 1000th. If we dilute that by 1 10th again, it becomes a 1 10,000th dilution. and we do it one final time one to one hundred thousandth dilution we now have our dilution we can figure out our dilution factor so we take the inverse of this dilution factor which is 100 000 or this dilution which is 100 000 multiply it by 3 it gives us 300 000 bacterium per milliliter in this solution Let's take an example. One mil of culture was diluted into a nine mil broth tube. One mil of this culture was diluted in another nine mil broth tube.
One milliliter of this final tube was plated and 25 CFUs were counted. What was the concentration of the original tube? Okay, so we're going to plug in our formula.
the final concentration of cells was 25 so we'll put in 25 here now we diluted it twice we did one one to ten dilution here and then we did another one to ten dilution here so we did uh two one to ten dilutions so we're going to find the dilution factor of each which is 10 times 10 or it's 100 right same thing So 25 times 100 equals 2,500 cells per milliliter in that original tube. This is also going to be, there'll be other examples of this in that math practice PowerPoint, so please take a look at that. The plate to the right represents a 1 to 10,000 dilution of the original culture. How many bacteria are...
estimated to be in each milliliter of original culture. So in this checkpoint, you need to count up the CFUs, times it by the dilution factor, and that'll tell you how many cells are in the original culture per milliliter. We've covered how to do plate counts.
Plate counts essentially take a culture, you do serial dilutions, you plate those dilutions, and then you count the colony-forming units. Colony-forming units represent the number of live bacterium in that culture. And though not all bacteria may be alive, and so it can be flawed because only the bacterium that are living are going to be counted.
And if these bacterium land very closely to each other, they may be, or they might even be stuck to each other. and land on the same spot in the plate, it might grow as a single colony. So it doesn't perfectly represent the exact number of bacterial cells in that culture.
If you want to count the exact cell numbers in a culture, whether they're alive or dead or sticking together, you can use something called a hemocytometer. Hemocytometers were invented for counting red blood cells. In fact, heme for red, cyto for cell, and meter.
So it measures their number of red blood cells. But microbiologists use these for measuring a variety of different types of cells. And what it allows you to do is count the number of cells under a microscope per a given unit of volume that you've added to it.
In this example here, we can see that there's the bottom of the slide piece, this right here. And then you put a little glass slide over the top and you add a solution here. then it gets absorbed and wicked over under the cover slide, and it'll hold about 0.01 milliliters of bacteria over all of these grids. You put it underneath the microscope, and you can count all of the bacteria here, or you can count just a number of squares, and using a little math, assume how many more bacteria you would count if you counted all of them together.
But essentially you know how many bacteria exist in... 0.01 milliliters of sample. And so then you would times that by 100. because there's 100 0.01 milliliters in one milliliter and that'll tell you how many bacteria are in a milliliter there are also disadvantages for the direct microscopic count as well one of these is uh the micro if microbes are motile so we've talked about how uh microbes can be modioles, they have flagella, uracilia, etc.
It makes it more difficult to be counted because they're moving around and you can't keep track of each one of them as they move from one square to the next. Additionally, sometimes you don't want to count the dead bacteria. So if you're doing direct microscopic counts, you're going to be counting the dead bacteria with the live ones. Also, it's going to be very vulnerable to sampling error. And that's because if you imagine with the plates, these...
With the plates, those colony forming units aren't going anywhere. And they're very dispersed out if you've done your dilution factors or your serial dilutions. They're like little cities. They're in place there.
You can take your time and count them. It's really hard to count all the cells in a liquid sample because they're kind of drifting around and moving around. And so from user to user, you might get much more different results than you would with a plate count. 6.6.
You view several 0.01 milliliter samples of bacteria using the direct microscopic count method and find an average of 126 bacteria per sample. How many bacteria are there per milliliter in the original culture? Alright, thank you so much for sticking through this one.
I know it was a tough one. Hopefully the math practice slides make it a little bit easier for you to get the hang of the math problems there. I hope you all have good luck with working through those math problems.
Go ahead and please review them, and they'll be posted under the lecture unit for this unit here. And when you're done, please email me if you have any specific questions about the math, or if you want to meet during office hours, I'd be more than happy to. Thank you very much for your time, and we'll see you during the next one.