Microscope Intensive Course Lesson 3: Morphology of Bacteria

Transcription

1 Microscope Intensive Course Lesson 3: Morphology of Bacteria Now that you ve learned how do you basically use the microscope, the the mechanics of it, how to focus your eyes, focus the light, get it so that your both eyeballs are on the same plane of focus, shadow so you can see nice shadowing here, let s go through the different organism groups. First thing I want to show you is, we re at low magnification, so this is what you would normally see at a 4X objective lens or 40X total mag. So, you can see a piece of organic matter here. You can see lots of little dots. You can see some bigger dots, some nice, round, big guys, kind of oval. What all are these organisms? It s kind of hard to pick things out and be really sure of your identification at this magnification, certainly really difficult when you re first starting out. By the time you ve done, you know, a couple of hundred slides, you know that each one of these little dots are the bacteria, individual bacteria. Now, you re not going to be able to tell here whether it s a coccobacillus or an actual rod bacillus. Bacillus is Latin for rod, so when I say bacillus, I..I mean rod, so we can do interchangeable. Some of these are going to be really tiny cocci. Some of them are going to be bigger cocci and bigger and bigger. So, how are you going to really tell, is that a bacterium or is that a spore? So, we ve got to go up in magnification. Now, when you look at a big, round thing like this, you d want to focus in and out a little bit, make sure it s not an air bubble, but in fact, it s not, because as you focus that in and out, as you focus in and out on this, organism, it doesn t all go completely back and then as you focus back through, the walls become really thin. This stays, you know, round and clear in the middle. This is a ciliate cyst. Okay, so is that a ciliate cyst? Is that another one? Is that oh, probably. But, what about this? This is actually oblong. We got a round thing here and then we got some others. What is that? We re going to have to go up in magnification. We can see some, filaments. Are those fungi or are those actinobacteria? Interesting plant material. Are these fungi growing out of that plant material? So, we re going to have to go up in magnification. So, let s dial in our 10X objective, and you can see where things are starting to get a little bit clearer. we can definitely see these are not fungi because notice how they are narrower at one end and get larger and fatter. Yes, that s plant debris. You got another strand here but we can see more than one cell along that strand, and so, not a fungus. Lots of plants debris, this is an aggregate. We got lots and lots of bacteria. You want to count all those bacteria? Oh, you re going to drive yourself crazy, so, no. We re going to dilute this more in order to count the bacteria. Oh, what s this? See how sharp those edges are and how crystal clear the middle? As we focus in and out, this is very shiny, very refractive. This is a grain of sand or silt. We could measure the actual size and determine whether it was a sand grain or a silt grain but it s in that category. Lots of nutrients in there that can only be unlocked by the microorganisms, so that s why we need our bacteria and our fungi to make the enzymes to pull those nutrients from that piece of mineral material and convert it into organic matter. And then, the bacteria and fungi have to be eaten by protozoa or nematodes. So, all those bacteria, eou want to count them all? And, we re only a 100X total magnification. So, to really identify what we ve got, we ve got to we have to go up to the 400X, but certainly we can see bacteria here at this mag. Now, we ve got some strands. We have to go up in magnification to determine if that s one micrometer diameter or if it s two or five or whatever it might happen to be. And now, we can clearly see here that this is not a ciliate cyst. There is a ciliate cyst. There s another one, so, very different organism, not a ciliate, so wh at is it? So, we may have to go up in magnification to really figure that out, but by the time you get experienced, you ll recognize that that s an amoeba. It has pulled a plug of the plant material, so see the or the aggregate material. Lots of bacteria in there and oh, yes, the amoeba inside here likes to eat bacteria. So, to pull the plug of that material inside the shell, so this is a test that has been built by that amoeba to protect itself from predation. Once it gets inside its recreational vehicle, it doesn t matter what predators are out here, this guy is now safe. So, i t s a strategy on their part to build a mobile cave that they can get into and protect themselves. 1 M i c r o s c o p e I n t e n s i v e C o u r s e L e s s o n 3

2 Remember that we shook this sample before we put that drop on the microscope slide and spread it nice and thin so that we can see what s going on. But, it s pulled that plug because we shook that was a huge disturbance it was a massive earthquake, and oh, no, how do you survive an earthquake? Get back into your shelter, you get in a place where you know you re going to be safe. How long is it going to take for that amoeba to push that plug out and push its pseudopods out and start eating again? It could take hours. So, probably not going to happen before this drop of water dries up. How about right here? What s that? Well, it s another one, so there is another Testate amoeba. So, we start looking at these different materials and figure out what it is. Now, there is a silt grain right there. Some of these little tiny dots may be clays. So, when we see something that s slightly longer than it is wide, could be a clay colloid, and so, there is a mistake that we re often going to make here, where we re going to count clay as bacteria. We re just going to accept, using these methods, that there might be a little bit of an error. But, typically, we re not looking at just one sample, we re going to be looking at our soil over time. And, of course, over time, the clay is not going to change at all. So, the changes in what we count as bacteria has to be due to changes in the bacterial community, when we re all noting that we ve got more cocci now. We ve got more of the large cocci or fewer of the large we have more lactobacillus or we have, more little round coccobacillus, or we have, you know, differences, changes in the morphology. We know that that s due to the bacteria because the clay in the background is always going to stay the same. So, we can look at what s going on in our soils as we do timed courses But, of course, we re going to want to go up in magnification because we ve got a few things that we re going to have to look at closer. So, here we go, dialing in our 400X objective. Wow! Look at how much less of our sample we re actually going to be able to see using that 400X objective or 400 total magnification. Remember where this Testate amoeba was sitting in the last picture? Well, right in the center of the picture, so, this is the part of the picture that we re going to see when we dial in that 40X objective. But, look at how much less of your sample you re seeing as you go up in magnification. So, you know, we re much more likely to have a number of fields that actually have zero we re not seeing any fungi, we re not seeing any fungi, we re not seeing any protozoan, not seeing any nematodes, and then, whoop! We now got a couple and then we go on with lots of zeroes. So, it does become a challenge to get these organisms up to the higher concentrations so we can be really consistent in the data that we re getting. So, we re just going to have to live with some of that when our soil is in very low biology condition. So, go on up in that magnification, you can see that we ve got massive numbers of bacteria. We re going to have to take this sample or go back to our sample that s still in our test tube. Dilute that, shake that up a li ttle bit more, try to get these aggregates spread out. Take a look at this aggregate. How would you like to count all the bacteria in there? You have to be focusing in and out. Remember that we ve just got one out of the 15 micrometers depth of this sample. We ve only got one of those layers that were in focus here. We re going to have to focus down to our glass slide. We re going to have to focus up to the cover slip. How do you know when you ve gotten up to the cover slip? Well, there s nothing in focus. You re outside your sample, so turn around and go back down through your sample again, and when you get to the microscope slide, generally, you re going to find a nice layer of things sitting on the surface of that microscope slide, and as you go deeper than that, beyond that drop of water again, everything is out of focus, nothing, so, you know the limits of your sample. So, in this particular aggregate, you have to focus, is that a bacterium, is that one what s, just or more of a plant material and what are the actual bacteria in there, and then recognize that you re only going to see the number of bacteria on the surface. How about the underside of that aggregate? Oh, we re going to miss them altogether. So, if we re going to be accurate about counting our bacteria and then turning that into a biomass estimate, we re going to have to dilute so that we can actually not have layers of bacteria one on top of the other. So now, we ve got some strands here but notice that this is all kind of lumpy, it s bumpy. It s not in focus either, so you would have to focus on that, but it s not a fungus because all fungi are uniform in diameter. Well, is that a fungus? Well, probably not. Fungi don t break into those teeny tiny bits very often. So, not enough of it there anyway to count for much. So, is this a fungus? No, that s plant material because you can see the different cells of that plant material. You can see it s kind of tattered and torn and loosely falling apart. Fungi will never do that, so this is plant material here. And, we ve got some bacteria, so we should be counting all of those. Well, we re not going to actually this picture because way too high a concentration. But, let s say you wanted to look at diversity, so you re going to look 2 M i c r o s c o p e I n t e n s i v e C o u r s e L e s s o n 3

3 at there s a rod there well, you might want to focus because what if this is one little small cocci and a second small cocci? So, you re going to want to make those calls. Is that a single cocci there, round? Cocci means round. So, we ve got another little bacterium, some slightly out of focus. We re going to have to focus on it, slightly rod-shaped there and a longer rod there, some individual cocci, so we ve got to be focusing on each of those and again, you can see why you want to dilute this out so you can see things individually. See how this kind of strand here, it s tattered, it s torn. Fungi never kind of fall apart like this. So, there are no fungi in this picture. Lots of bacteria, and yes, we have a protozoan. Here s a Testate amoeba. There s the individual bacterium. It s pulled the plug of this material inside to protect itself. So, the difference in size, how much of your sample are you going to see at a 4X objective compared to a 10X objective, as compared to a 40X objective, which is why we have to look at lots of fields when we re at that 40X objective quantifying what we re looking at. Going on. This is an air bubble. So, we ve got one air bubble here, we got another one there. It s slightly different layers within our drop of water under the microscope, drop of our sample under the microscope. As you focus, this black wall will take over and this thing will become will completely black, like this is almost. So, when you re at the top of the focus or the bottom of that air bubble, it s going to be completely black. But, as you focus through the air bubble, those black walls are going to get narrower and narrower and narrower until there s just kind of the clear bubble. So, don t mistake this for some wondrous new organism. What s causing the air bubble? Well, look at the oil drops? Someone didn t clean the slide very well. Notice in this picture too that all the other material in this picture, in this field of view seem very ghosty. There s not good contrast because somebody has also forgotten to shadow with their iris diaphragm. So, if you re seeing samples where everything seems just kind of wow, hard to focus on, hard to see it, get back down there and use that iris diaphragm and get things shadowed. Organisms, when we look at organisms, we re going to base identification on morphology. Now, that means we re going to miss some differences in species. It s really hard for us to see the subtle differences in morphology between different bacterial species. We are not going to be able to determine how many total species. We can say a lot of information about how communities of bacteria are changing, but we re not going to be able to identify each and every different species of bacteria. So, if that s what you want to know for some reason, is to identify all 75,000 species of bacteria that are present in a teaspoon or a gram of really good soil or compost, you better use DNA analysis. So, if you ve got to know that level of information, you re going to go with a different methodology. Here, we re just trying to get an idea of do we have lots of rods, do we have no rods, do we have lots of little tiny bacteria, cocci, more [indiscernible][0:15:54], so, those changes, we re going to be able to identify, and ce rtainly, we can pick out the bad guys, those things that should put us into a state of going, Whoa! I better wash my hands after having dealt with this soil or this compost. Well, it s not compost, it s not soil, it s dirt, you know, it s putrid organic material. If you re seeing these disease-causing organisms, if you re seeing the spiral cork screw-shaped bacteria, you know you ve got E. Coli, you know you have Salmonella. You know that there are all kinds of bad guys in there if you re seeing Spirilla. So, take appropriate care in dealing with that sample of material, going back to where it came from and dealing with the problem. Make sure you wash your hands as soon as you realize that you ve got some disease - causing organisms in any of these material. So, handwashing, very important. When you clean up the slide, when you clean up your test tube, make sure you get that really well-cleaned out and make sure it dries. Disease-causing organisms can t deal with absolutely no moisture. So, be aware, that s how we re going to deal with this problem. So, when we re dealing with bacterial morphology, well, here s just a reminder of what good guy bacteria do and bad guy bacteria, so, okay, and then, the morphology of all these different kinds of round-shaped bacteria. They can be tiny round, a little bit larger, a little bit larger. They can group up in groups of two. If you see a capsule around a group of two, wash your hands really carefully because that is a disease-causing organism that causes pneumonia. So, you see this, and I have never seen this in soil. I ve seen it in, manure. I ve seen it in all kinds of other animal waste materials but not in soil. Staphylococci form these clumps of grapes. Streptococci, the chains, so that s what Strepto means, chains. Staph means bunch, like this, when we re looking a t tetrahedrons in three dimensions. Now, bacillus, so the coccobacillus, round rods; straight rods; real stiff heavy rods are going to be bacillus species, genus, excuse me, genus; where the rounded, we would just call that one of the many different kinds of rods. Coryneforms that link up like this. Now, the bad guy bacteria, Vibrio, comma-shaped, we don t see those in soil. They will be in aquatic systems. Bacteria they re bacterial. You ll see the flagellum on the end but they re tiny. 3 M i c r o s c o p e I n t e n s i v e C o u r s e L e s s o n 3

4 They re much smaller than protozoa. Clubs like this Corynebacteria, helical forms, bad guys again. So, when you re seeing these little they re not long cork screw-shaped, they are really short cork screw-shaped, be worried. Spirillum, 50% of almost all of the Spirilla are disease-causing fungi, so if you see these stiff cork screw shapes, time to wash your hands and fix the biology in there. The snakelike bacteria, usually much larger twists in there, waves in there, and they move like snakes, Spirochetes. About half of the Spirochetes are disease-causing organisms. The other half, perfectly fine, so a little less worry when you see something that moves likes a snake, but don t chance it, 50% chance it s something bad. Actinobacteria, they grow in filaments, but they re only one micrometer wide. And so, good under some conditions, bad under other conditions, and we ll go through that during the other courses. So, just a summary of the different shapes of bacteria, so we ve gone through that already. You know the bacillus or the rod shaped bacteria, long, short, fat, thin; the cocci; so just all of the different sizes and shape. The bad guy bacteria, in red here, so, you see Vibrio in soil or compost, get worried; see cork screw shapes, oh, bad news. Do something to aerate, get oxygen back in there, start growing the good guys instead of the bad guys. And so, yes, all of our materials can be fixed. You don t have to like go and incinerate or destroy the compost or whatever. So, you do have to get it aerobic, you do have to get the good guys growing again to get rid of the bad guys. So, just one more point, Lactobacillus, those long, slender rods, they re very stiff. They re not filaments, and so they don t bend and wind the way a fungus or Actinobacteria. Lactobacillus are very stiff. They can be very short, you know, 2 to 3 micrometers, five micrometers, ten micrometers. They can even be 50 micrometers long, but they are very stiff, very slender Lactobacillus. They do indicate reduced oxygen conditions. However, with Lactobacillus, you re not going to smell the bad smells because Lactobacillus, in those anaerobic conditions will prevent those bacteria that produce ammonia or hydrogen sulfide or phosphane gas. So, they prevent the bad guy bacteria from taking your nutrients and turning them into gas, your soluble nutrients and turning them into gas. So, in a way, Lactobacillus is quite useful if you ve got a situation where you re smelling stink and you want to shut that down and you want to shut it down right away. So, for example, EM has lots of Lactobacillus, got lots of yeast, the anaerobic form of fungi, yeasts. it s got some of the Actinobacteria in it, but all things that require reduced oxygen conditions. The positive part about EM is that it shuts down the bad smells from being made. Lactobacillus, yeast will typically outcocpete E. coli, Salmonella, Shigella, Pasteurella, the human pathogenic bacteria. So, you got a situation where you know the manure is coming from sick animals or sick people, get EM in there to start wiping out the diseasecausing fungi. So, not going to give you the benefits of the beneficial aerobic bacteria, but it s certainly going to prevent really bad things from happening. So, when we re looking at the picture, you can see that we ve got an aggregate of bacteria in the middle and then look at all the bacteria out in the solution. So, in this picture, we don t have a lot of aggregates going on. That means the structure in this soil or in this compost, whatever this material comes from is not going to be as good at holding water. It s not going to allow oxygen to move through this soil very rapidly. We just don t have the aggregates the structure built. Lots of bacteria, we got really good diversity. Now, we ve got, rods here. We d have to focus on them a little bit more to determine exactly how wide. Here is this rod is more in focus but notice that this rod is longer than those rods, and therefore, this is a different species than those. Now, we have this rod attached to in a chain with that rod, so this is yet a different species because that chaining ability is typically a whole different species than something that doesn t chain. You know, we could make a little bit of mistake there. Maybe these bacterium was just detached from the chain, whether through shaking or something else, but probably a whole different species. So, we ve got these smaller bacteria. You can see where ones are in focus, so cocci. These are round. You can see where we have really small cocci, a little bit larger cocci, another one, and then these slightly larger cocci, you know. So, where they re in focus, we can compare morphologies and come to conclusions of how much diversity. Notice that. It s not perfectly round. It s actually a little bit longer than it is wide. So, here is another one. This is actually a bacillus. It is longer than it is wide, so subtle differences in morphology. This is a round rod. That s a round rod. So, look at the diversity we have in this sample, it s massive, and that s very good. Now, would you want to count the bacteria in this field? No way! You couldn t even begin to estimate. So, this is one where we will dilute more. But, here at 1:5 magnification, we can look at diversity and be saying, Whoa! Great 4 M i c r o s c o p e I n t e n s i v e C o u r s e L e s s o n 3

5 diversity! Or, we could be looking at something, and we will in the next couple of pictures that says, Not so good. We don t have as much diversity. Now, go up here in this corner. Isn t that something interesting? What is that? Well, sometimes, when you re looking at kind of weird alien invader organisms, you know, [indiscernible][0:26:04] Pacman or something, whoo, what you re going to want to do is tap the surface of your cover slip. So, get a needle, get the end of your ink pen or the end of your pencil, and just lightly tap the sample, but be looking through the microscope when you tap. All of this material that s actually in your sample is going to move around and zoom and go all kinds of different ways, but this thing doesn t move at all, which tells you that it s not actually in your sample. When you re looking at this, see these really straight lines coming off, you know, where you can see that probably there was a sand grai n here the last time this slide was cleaned, there was a sand grain, and as we wiped that sample clean, it left behind these long streaks, very straight, very, gouge-like. Okay, so that s what that is. It s just, when we cleaned the slide, we gouged it. It wasn t sand, and so, it s not anything more than that. When you sometimes see these lighter splotches like this, it just has to do with the way the light is refracting within your sample. There is a mineral in here that s reflecting and that s what causes these, especially when they re, you know, kind of six-sided or eight-sided patterns like that with a dark area around it. It s just reflection from something in your sample. Moving on. How many bacteria in this field. Okay, we ve got a whole lot less but you got to remember to count the little tiny guys. A little bit out of focus, so we d focus down in order to count that. You can see that this is quite a bi t out of focus but going the other way, so we got to focus up. This one s pretty much in focus, so pretty much in focus. You can see little tiny guys, slightly larger tiny guys. Now, there s a lot of bacteria in that aggregate. How are you going to count that? So, we re going to dilute. Notice this particular sand grain. You can see the mineral outline of that sand grain but it s completely covered with a layer of organic matter and billions of bacteria, sticking and holding that organic matter to the surface of that sand grain. There is no way you re going to count that. So, we re going to dilute this, shake a little bit more, try to get most of those bacteria off these surfaces, try to get these aggregate split up so we can do a better job of counting and not have to try to keep track of hundreds, thousands of bacteria in a picture like this. But again, this is the 1:5 dilution, so take a look at diversity. You can see that there is nowhere near the diversity in this sample as we had in the last sample. Do remember, if you want to focus to whatever that is, some of these things in the background, you want to focus all the way to the slide, all the way up to the cover slip. Make sure you count everything, or if you re looking for diversity, then you see all the different sizes and shapes of the bacteria. Jot it down, how much diversity you had, and ultimately, you re going to make better compost, you re going to see if your soil is going in the right direction, your compost [indiscernible][0:29:25] whatever. Start recording some of these things. Now, do we have any protozoan in this picture, and is it a good guy protozoa or a bad guy protozoa? Well, there we are. We ve got our Testate amoeba. You can see the opening on the test. There is the test, the shell that the amoeba built. There is the amoeba down at the bottom and he is protecting himself by having pulled in some of this aggregate material, protecting himself, and giving himself some food to eat while he s hunkered down waiting for the earthquake to stop. Not all Testate amoeba have openings here at the top. Sometimes, the opening will be at the side. Sometimes the Testate amoeba will actually, have a very ornate, opening where it will almost look like a vase. Lots of different variations on test structure, but you re still going to be looking for the amoeba living dow n in here or maybe the amoeba completely occupying all of this, and with the pseudopods sticking out, eating the bacteria in that region, moving along, oozing along, dragging his shell behind him. So, no fungi in this picture. There were no protozoa other than the Testate amoeba, so we start to get an understanding of what s the structure of the food web in this soil. Going on. This is a very different kind of soil, because of that deep red, orange color. This is telling you something about the minerals in that soil. This is an oxidized iron that you see typically in clay soils that have gone anaerobic in the past. You re going to see this intense orangish-red color, and when you see this, that s all that it means. Sometime in the past, was that yesterday or ten years ago or 500 years ago or a thousand years ago, we don t know. But, Georgia red clay, for example, that s actually what this sample is. It s from some soils in Georgia. We can see a nice aggregate here. This is probably a worm poop or a microarthropod poop. So, aggregates that are just kind of that big and pushed together, and really they do have a layer of carbohydrates, slima material holding that whole aggregate together, and bacteria love to grow, on the surface. So, we ve stained this sample with fluorescein 5 M i c r o s c o p e I n t e n s i v e C o u r s e L e s s o n 3

6 isothiocyanate so the active bacteria will take up that stain and fluoresce that green color. You can see where we ve got another bacterium here but out of focus. We ve got quite a few bacteria but out of focus. We ve got to remember to focus up and down. So, lots of active bacteria. Well, even now, here, there s a fair amount of active bacteria that aren t quite in focus, so that s how we can instantly tell activity. Using our small shadowing microscopes, we re going to look at how much those bacterial numbers change as we go through time. So, clearly so many bacteria in here, we re going to have to dilute, but no fungi, no protozoa we re seeing zooming around, and for protozoa, we are looking for the motion, the oozing motion of amoebae. We re looking for the fast movement of the flagellates gallumping or bumbling a little bit as they go for one or two flagella. When we re looking for the ciliates, we re looking for, little, hairs over parts or all of the body. So, look at the colors of things and start coming to conclusions. This is just a colony. So, we went up in higher magnification. This is 1000X magnification, and you can see how much bigger those little tiny, rod-shaped bacteria. So, all of these are actually little coccobacilli. All of them are active stained with the fluorescein isothiocyanate. Excuse me. Diacetate. So, when we re looking at all of these bacteria to determine biomass, we don t have to be extremely precise, but we want to get in the ballpark. So, a countable number of bacteria per field is going to be around 30 to 50 in a quarter of the field or in the whole field. Most of the time, the density of the bacteria in our initial dilutions of 1:5 or 1:10 is too high to easily count, so we re going to dilute more. So, we re going to dilute up to that point where it s a countable number, so we may be diluting 1:500, 1:1000. That s a typical normal number where we re counting about 30 to 50 at 1:500 or 1:1000. If you start going over the 1:1000, oh, you oh, yes, the suspicion would be that you re going anaerobic. So, let s go through a set of dilutions. Here is the 1:10 dilution, and this is the 400X magnification, and, oh, good luck counting that. And, remember, this is just one layer. You ve got to go through all of the layers in that drop that you ve got under your cover slip. And, how could you tell whether you ve actually counted this bacterium already or not? Was it in focus in the last layer or is this a new bacterium? You ll just drive yourself insane. So, think about trying to count all those little tiny bacteria in there and, you know, just in this little area, we ve probably got close to a hundred bacteria. So, no, we just go, we re going to dilute some more. So, here is our 1:100 dilution, and you can see how we ve spread all of those bacteria out. You can start to see more space, but still, good luck. Even if you did a quarter of that field, good luck counting all of those bacteria in a quarter or even an eighth or even a sixteenth of the of the just and again, layers, as you go through this, still just too much. So, we re going to dilute some more. Here is our 1:500. Are the bacteria spread out enough? So, now, what s actually a bacterium in here and what s organic matter. So, there is a clump of organic matter, there are some organic matter, but you re going to have to focus on these and be moving that iris diaphragm to shadow, because on the surfaces of that organic matter, we could well have a couple of bacteri a and you want to be able to see that. So, you know, you start counting, there s one I suspect as a bacterium, and you know, a little out of focus, a little out of focus. And so, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 probably a bacterium right there, and that s a little organic matter 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 did I count that one already? Oh, yes, you re going to drive yourself crazy. It would take you a good, you know, 3 or 4 minutes to count this, and that s not what we want to be spending our time doing. Let s spend our time diluting. So, here is our 1:1000 dilution, so when we re counting this, we ve got some organic material on here, we d have to focus on these and come to a decision that whether there s actually bacteria on these surfaces or not. So, counting the bacteria, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, probably not 21, 22, 23, 24, 25, 26. So, going through here and 135 bacteria per field, you know, comparing counts with other people, shadowing a little bit better when the picture is taken from a computer, it s quite a bit more difficult to really show the detail. A lot of this stuff clearly is just debris that we didn t count, and so you re focusing through your microscope, a lot better count is going to be taken when you re actually using your microscope resolution, it s a lot better. So, diluting it out until you can see that this is, in that right range. Maybe you would want to dilute this 1:5000 because then you really would get just, you know 30 to 50 bacteria per field. Scanning electron microscope shot. What you do here is take a little bit of your material. This is a compost, for example. So, you take a little bit of compost, a little chunk of the compost, glutted to a stub, and then, critical point dried to remove all the excess moisture and then coated this with about a one nanometer layer of gold Palladium. 6 M i c r o s c o p e I n t e n s i v e C o u r s e L e s s o n 3

7 Gold Palladium all over the surface of everything here. The gold Palladium will reflect ele ctrons when you, put that sample into an electron beam, electron microscope, it will reflect those electrons and the speed at which those electrons come back to the collector allow us to do the surface area. So, it s a really good way to be looking at surfaces of things. We re not looking at the inside structure. We re looking at the surfaces. So, what are all these organisms that we re looking at? It gets a little scary when you think about, all of this is organisms or the glues made by organisms. The only organic matter here that s not covered with microorganisms is a little bit right here. Well, there are bacteria, there s some short, tiny little rods, some cocci, but there is some not colonized organic matter right there, and again, right here. Oh, there s a couple of cocci, there s a rod, there s another cocci, so not completely sterile. But, out of all of that massive organic matter that you can see or the surface the organic matter on which these organisms are growing, the only bare surface is this little tiny bit. So, when you pick up a handful of compost, where do you actually touch them? It s all organisms. So, think about what you re doing next time you pick up soil or you pick up good compost. It should be really good sets of organisms. So, who are these organisms here? Well, we ve got some yeasts. Yeasts are the anaerobic forms of fungi, so, gosh, this is not really good news. Let s say you did 20 fields of this, scanning electron microscope bit of compost and you were seeing this many yeasts in every field of view. You would get really worried that you re dealing with anaerobic conditions here and not beneficial conditions. Now, also, look right here. Can you see the cork screw shape bacterium right there. So, cork screw shape, what does that mean? We ve got a Spirilllum in here, another indicator that we re anaerobic. So, you ve got to be going through a number of these fields. How often are you running across the Spirillum? If you re seeing Spirilla in each and every field, this is not compost. It s not soil. It s dirt. It s putrid, you re going to have to fix it, got to do something to get rid of the anaerobic conditions in that compost so that this is beneficial for our plants. Now, looking at a cocci, a rod, so you can see it s a coccobacillus, another coccobacillus, some more cocci, a slightly smaller cocci, so a different species, really small cocci here, another one there. So, you can see a lot of well, how many species in this picture? Any of these places where we ve got the slime that the bacteria are producing, these are the glues that these bacteria are producing to hold themselves to these surfaces. These are bacterial glues. So, when you want to really think about what makes glue in soil, it s the bacteria. For a long time we ve known that these bacteria produce massive amounts of glues. It s what holds them to the surfaces of all of the organic material, of the roots, of a sand [indiscernible][0:43:02] and clay grains. Bacteria are what make the glues that make the, sticky material in soil. Now, you can also see fungi. You can see some fatter fungi, really big wide diameter fungi, some narrower fungi. So, there is a fair amount of fungi in this picture, all this little thready things. Unfortunately, with the scanning electron microscope shot, we can t use this picture to determine diameter because when we critical point dried what happens to microorganisms when you dry them out? Microorganisms are just like human beings. We re like what, 80%, 85% water, so you dry them down, they re going to shrink. Think about these glue layers, those slime layers. You see how spiky that is? That s because the glue layer has dried down, leaving those glycoproteins, leaving the carbohydrates, leaving that glue material, dehydrated. So, what would this have really looked like if it hadn t been dried? So, all of these glues produced by aerobic bacteria are going to be alkaline. If these are anaerobic bacteria growing in here, and we ve got some indicators that they might be anaerobic bacteria, then these glues are going to be acidic, very acidic. So, bacteria, you kind of have two choices, alkaline, ph above seven if it s aerobic; if it s anaerobic, very much below a ph of 5.5. The only way to have a ph below 5.5 anywhere is if you re dropping into those anaerobic conditions and these anaerobic bacteria are growing, producing these very acidic compounds. But, we do have fungi in here, so we ve got some really good fungi and quite a bit of it. So, but are those fungi, nice strands of filamentous fungi? Isn t that indicating that this has to be aerobic? Well, mixed signals here. Which set of organisms is actually now winning the contest? We can t tell with a one-time look at what s going on in this sample. Now, it takes a long time to prepare a scanning electron microscope shot, and remember, you re coating with gold Palladium, so this is not inexpensive to produce. So, this is not a way that you re going to do time course studies. Our small microscope is going to be the way that we do time course studies. 7 M i c r o s c o p e I n t e n s i v e C o u r s e L e s s o n 3

8 So, lots of organisms. Look at all the bacteria. How would you like to count each and every one of those? Oh, scary! Good guys as well as bad guys, so, really the information that s most useful is that we can look at diversity, we can see that if we ve got bad guys, good guys, strands. So, which way is this transitioning though is going to take another sample, in a day, two days, five days, to see what s happening in the sample. We re looking at a sample that s 1:5 dilution of the sample but a slightly lower magnification than the 400X because we wanted to catch more of the different kinds of organisms in this. We are looking at a fungal hypha here. This thing looks a little narrow in diameter because it s out of focus, but the rest of that fungal strand is lying pretty flat, sitting on the surface of the microscope. You ll notice that it s got this kind of barred-looking, you know, empty holes and a little bit of cytoplasm, another big empty hole, because it hasn t been too long ago that the anaerobic bacteria were kind of bellied up to the bar [indiscernible][0:47:03] sucking out all of the cytoplasm. So, this is a dead fungus in this anaerobic conditions. Well, how do I know that this is anaerobic? because all the massive number of these, Lactobacillus only grow to these high numbers, and you can just see there is lots and lots of these Lactobacillus throughout this sample. And, of course, as you focus in and you focus out, you would see many, many more. Now, before I get going on the motion here, I want to point out a couple of subtle things that you want to pay attention to. See this wave-shaped bacterium here? It s actually a stiff cork screw shape, and as it moves through the, medium it spins and it sets up a kind of a wave motion. Here is another one right there where you can see that cork screw shape. These Spirilla, and so, that s what you call these cork screw-shaped bacteria, are almost invariably pathogens. So, you see these bad guys in your sample, and you know right away that this is anaerobic, that you ve got pathogens in the system. If we re seeing these Spirilla almost invariable again, we re going to have E. coli or Salmonella or Shigella growing, some really bad critters. So, I would want you to be very careful about this sample. Wash your hands. If any part of you came in contact with this sample, if it splashed on your table, go get some alcohol or go get something that s, you know, chlorox and bleach, something that s going to kill the pathogens that are in this sample. So, whatever container you have this sample in, when you clean it up, make certain that you use some bleach or some alcohol or peroxide to clean that test tube out, any [indiscernible][ 0:49:09] that you used, because you don t want to end up sick, because, you know, you had your fingers in contact with this sample and you thought you cleaned your hands. Maybe you didn t, and then you go eat lunch or something, and you eat a French fry and you lick your fingers, and whoops! So, be very careful when you see these organisms. Well, what if this was from a soil? Well, you got some work to do, to get good aerobic organisms back into the soil, build the structure, get the oxygen in there. What if this was a compost? you re going to want to re-compost this material because clearly, it didn t get composted correctly. What if this was from the early stages in a compost? Okay, you got your work cut out for you to get this aerobic again and get the benefici al organisms growing. Now, as I turn the movie on, you ll see that a lot of these organisms are going to be zipping around and and moving. And, what you ve got to decide is can you see cilia on the outsides of this, and of course, if we went up to the 400X, you d be able to see that a lot easier; or can you see the flagella? So, you want to be looking at these model organisms and try to decide. Because we are at 200X magnification, you might have to decide just based on how they re moving. Are they bumbling as they move along or do they have that sideward movement, or are they moving pretty smoothly, in which case they re probably ciliates, not flagellates. So, we do have flagellates in here, we do have ciliates in here, so pay attention. You might suspect that some of these are flagellate cysts especially if they don t move. We have some good aggregates, looking at all the bacteria in there. I mean, these are massive numbers of bacteria. So, ooh, there s another Spirillum here. I haven t picked him out before. So, have fun watching this as it, starts to move, and just look at all the action in here. The sample is drying, and so you ll notice that a lot of the movement things are all in the same direction because it s drying, and so the water is moving. That s not motility to the organisms. They re just floating in the water, so things that are moving in that direction, you can t count as motile. Things that are moving against the current, okay, then they are motile, so they are clearly underway. And, you can see a lot of these Spirilla kind of organisms in here, lots of Lactobacillus, some of which are motile because they re moving upstream. Here we are at a 400X, and so you can see the cilia on those ciliates. Now, there is a higher mag, so we re about, 800 X magnification. Very clearly, you can see the cilia only on a portion of the body, not the whole body covered by cilia. But, when we ve got these tufts of cilia, we definitely know what it is. So, seeing that guy, when you re seeing the cilia on the end... But, like that was a flagellate, there is a 8 M i c r o s c o p e I n t e n s i v e C o u r s e L e s s o n 3

9 flagellate. Here is an insect larva in the same sample. Well, insect larvae tell you the same thing. Anaerobic bad guy organisms are growing, and of course, we re selecting for the growth of things like insect larvae, the pathogens, the problem organisms. Just look at all the Lactobacillus in there. And, you ll pick up a Spirillum or two as you re observing in this area. So, massive numbers of bacteria, lots of indicators of bad things going on. 9 M i c r o s c o p e I n t e n s i v e C o u r s e L e s s o n 3