Hello, I am Professor Sims and in this video I will discuss the ubiquity of microorganisms, culturing, and isolating bacterial colonies. This is the second in the series of ten lab sessions held as part of my Laboratory for the Fundamentals of Microbiology course. If you're a student currently enrolled in this course, please consult the syllabus, course Moodle site for assignments, quizzes, due dates, and other course information. The learning objectives for this unit include learning to perform proper aseptic technique, understanding the meaning and significance of microbes being ubiquitous in nature, learning about the types of media, liquid, broths, solid, aggers, slants, deeps, and plates, and becoming competent preparing streak plates and isolating bacterial colonies, being able to observe and describe bacterial colony morphologies. Of course, as always, understanding safety and disposal procedures relating to this lab.
Microorganisms are ubiquitous throughout the environment, which means that they are found everywhere. One characteristic of microorganisms that contributes to their ubiquity is the fact that they are very diverse. There are three major classes of microorganisms, which are prokaryotes, eukaryotes, and viruses. Prokaryotes are rather simple.
in their structure. They lack membrane-bound nuclei. They don't have organelles. Usually, they are unicellular.
They do have a cell membrane and ribosomes and chromosomes. Eukaryotes are much more complex. They have a membrane-bound nucleus. They have organelles. They can be unicellular or multicellular.
They have cell membranes and they have complex cell structures. And they're also usually much larger. than prokaryotes.
Viruses are really quite different from bacteria in that they have a super, super simple structure. It's basically just molecular material, DNA or RNA, surrounded by protein and lipid membranes, and they depend on an infected cell or a host. They have to have a host in order to multiply, in order to disperse.
Viruses are kind of a point of contention. Many scientists don't agree whether they're even living creatures or not. Another characteristic of microorganisms that contributes to their ubiquity is the fact that they are very old. They've had up to 4 billion years to evolve and adapt and to live in any and all environments on earth.
In fact, the fossil record shows that there have had some form of bacteria on earth for 3.5 billion years. So for experiment one, What we're going to do is we're trying to illustrate the ubiquity of microbes in our environment. And our environment is defined as the laboratory itself or the building surrounding the lab.
You can choose to sample things outside of the classroom itself, but you should not leave the building. So you choose two inanimate objects to test for bacterial contamination. Things like a lab bench or a sink or the bottom of your shoe or the toilet or door handle. So.
You make note of these choices, designate one, object A, object B. You get a nutrient agri-plate, divide it in half, and you swab on one side of swab culture taken from object A, one from object B, and that plate is incubated. And you want to form a hypothesis to see which one would have either more bacterial growth overall or which one would have more species or maybe one might have more growth and more species. So you want to have some kind of idea what you think is going to happen and some kind of rationale. And then the instructor is going to have, we'll do like an open air play just to see the ubiquity of microorganisms and contamination just from the air.
So that's pretty fun. Throughout this course, we will be culturing that is growing bacteria. And it may seem kind of counterintuitive to want to grow bacteria because you're used to killing it. But you can't study the bacteria without... growing it first.
So in order to grow it you need to know what kind of pH it means, what temperature it grows out, and its oxygen requirements. So here's some guidelines for that. Most bacteria are neutrophiles, meaning that they grow at a neutral pH of about 7. Microorganisms that grow optimally at a pH less than this are called acidophiles, and then at the other end of the spectrum are alkalophiles. These are microbes that grow at a pH between like 8 and 11. Organisms are mesophiles if they're adapted to modern... temperatures with an optimal growth temperature ranging from room temperature, which is about 20 degrees Celsius to all the way up to 45 degrees Celsius.
Your human pathogens are going to be mesophiles because they typically, well, they have to live at our body temperature. Our body temperature is about 37 degrees Celsius. So all of your human pathogens are classified as mesophiles. Organisms called psychotropes, these like cooler environments from about 25 degrees Celsius down to 4 degrees Celsius.
Cyclophiles are essentially cyclotrophs that can grow all the way down to zero degrees Celsius. So this is a freezing. They're found in permanently cold environments such as deep ocean water.
Thermophiles on the other end of the spectrum. Thermophiles are widely distributed in hot springs, geothermal soils. Miami environments such as compost piles where microbes break down kitchen scraps.
Hyperthermophiles, these grow in 80 degrees Celsius up to about 110 degrees Celsius. Note where the examples of these are Pyrobollus and Pyrodictum. These are archaea that grow in about 105 degrees Celsius.
And these guys are interesting because they survive. Hyperthermophiles can survive many of our regular sterilizing procedures like autoclaving. The growth of bacteria with varying oxygen requirements is illustrated in this figure over here to the right. In the first tube, all of the growth is seen at the top of the tube and these bacteria are obligate air rooms because they cannot grow without an abundant supply of oxygen.
So that's why they're all up here at the top close to where the oxygen is. In the second tube, the bacteria grow at the bottom. So these are the opposite.
These are obligate anaerobes. These are actually killed by oxygen. The third tube shows heavy growth at the top and growth throughout the tube.
So this is a typical result for a facultative anaerobe. Facultative anaerobes are organisms that thrive in the presence of oxygen, but they can also grow in its absence by relying on fermentation or anaerobic. respiration. The aerotolerant anaerobes in the third tube here, I'm sorry the fourth tube, these are indifferent to oxygen so they don't use oxygen because they usually ferment but they're not harmed by oxygen either.
Then the last tube on the right shows a Goldilocks culture. The oxygen level has to be just right for these bacteria to grow. They can't have too much.
It can't have too little. And these are called microaerophiles. They require a minimum level of oxygen at about 1% to 10%, but it has to be lower than the 21% that is found in the atmosphere.
So these are fun to grow in the lab. Microorganisms need a constant nutrient supply to survive, where they get their nutrients from. So if they're free living in nature, they usually will get their nutrients from the environment.
If they're parasitic... they're going to get their nutrients from their host. But in the lab, to grow the microbes in the lab, we use artificial culture media. So the media contains chemicals, nutritional requirements, and everything that the bacteria need to grow. And they can be of several different types.
So some of the physical types of media we're going to be using in lab today, we're going to use liquid broths, and use solid agar plates, solid agar deeps and slants. All of these different kinds of media are used for specific purposes in the lab. Liquid broth is very, it's very useful for culturing bacteria when you have to do it quickly.
That is the incubation lasts for less than 24 hours. Now that is a pro, that's an advantage of using a broth, but a con would be the bacteria depletes the nutrients in a broth very quickly. So it grows fast and it dies fast. You can tell that you have growth in your broth because it would be cloudy. This is known as turbidity.
Turbidity may be measured to provide an estimated number of microorganisms. But remember that broth are great for culturing, and especially if you need to culture something quickly. But they can't be used for isolating pure cultures or isolating colonies. Agar is a solidifying agent derived from seaweed. and has no nutritional value whatsoever.
Plated media are typically used for obtaining isolation of species. They're also used for different types of differential tests and for quantifying bacteria. material density so for counting the bacteria.
We have these other two types of solid media. One is a slant. It's got all of this surface area up top. So these are really good for growing things that have to have oxygen or at least can tolerate oxygen because they have a lot of surface area. And then deeps.
We saw the deeps when we were talking about oxygen requirements earlier. This is a semi-solid media and you actually inoculate these by stabbing them. So these are good for growing things that either don't need oxygen or can't tolerate oxygen and it's also good for testing to see if your specimen is modal.
In other words, if it has flagella or cilia and is able to move throughout this semi-solid medium. So for experiment two, we're going to be working with E. coli. It's already isolated on plates so it is a pure culture and we're going to use that to inoculate slants and deeps.
Okay, so students will inoculate one slant and one deep each with a sample of E. coli that's already isolated. You're going to sterilize your inoculating loop.
We'll go through how to use that in just a minute. I'm going to explain to you all how to use the back decelerator. We're going to demonstrate all of this in class, so don't worry. You do want to make sure that you allow your loop to cool after you sterilize it, or otherwise you're going to kill your bacteria instead of just transferring it. But you will obtain a sample of E.
coli from this streak. plate that will be provided to you and you're going to use it to inoculate your sterile slants and deeps. So to inoculate the slant you use a fishtail motion so you start where the slant actually starts at the bottom and use a wavy zigzag pattern to inoculate the surface and then you'll take another sample you collect from the plate and you use that to inoculate the deep. The deep you actually stab. So you put your lube right into the agar and you do that about halfway down the tube and pull it straight back out.
We'll demonstrate this in class. It's very important for students to learn early in this course how to use proper aseptic technique. So you already know about wearing your PPE, your lab coat, safety glasses, gloves.
But in addition to that and to avoid contamination of your sample and the lab range and yourself and other people, you want to make sure that you keep your agar plate covered as much as possible. Always sterilize your inoculation loop before and after you use it. Make sure you're keeping your specimen at an arm's length so it's not too close to your face or your body. Try not to sneeze or cough while working in the lab. If you can't help it, you want to sneeze into your elbow or you can wear a face mask.
We have those if you need one. In order to reduce the risk of fire, we will use a back incinerator instead of a Bunsen burner in this class. You use these to sterilize your inoculating loops.
So in order to do that, you insert the loop toward the rear of the heating element, which is this part up top. You want to avoid scraping the sides. Your loop must remain within this heating element for five seconds, at least five seconds.
Then you want to allow your loop to cool, or I'll show you how to cool the loop on sterile media. But if your loop is too hot, then you're going to kill the bacteria that you're trying to culture. So the bacteria can't grow if it's dead.
When transferring bacteria hold the loop like a pencil. It helps to put your hand in a convenient position to hold two caps and other things with a pinky finger and we'll show you how to do that too. This is an important skill to learn since you can't put two caps on the lab bench or else they'll become contaminated.
When you isolate a bacteria you're going from a mixed culture to a pure culture. And then what you hopefully end up with are isolated colonies. A colony is actually derived from a single cell that has grown and reproduced such that all of the cells in that colony are genetically identical.
Colony morphology is what the colony is going to look like on a plate. This is a nice figure that gives a lot of examples for the terminology to use to describe the elevation, margin, shape, size, color, all of that stuff for your colonies. once they're isolated.
I will go over some of this in class with you guys and it does take some practice to observe and describe colony morphologies so you'll learn about that in your lab two especially when we're collecting the results from lab two. For experiment three you're going to try to get these isolated colonies from a mixed colony so you're streaking for isolation which consists of using a sterile loop to inoculate an agri-plate using a primary streak followed by a second and third and fourth streak and in between each one of these you're going to sterilize the loop. So during streaking your cell density decreases eventually leading to individual cells being deposited separately on the agar surface. So cells that have been sufficiently isolated will grow into these colonies which consists of one original cell type that has divided and grown and reproduced asexually.
After incubation single isolated colonies should be found on the plate. Usually in the third or fourth quadrant if the cells were sufficiently diluted. So this is the method here and again this will be demonstrated in class, but just quickly what you do is first you label the plate's base with four separate quadrants. You sterilize the loop and cool it by touching it on the sterile agar plate. You obtain your inoculum from the mixed broth and you only need one loop full of this.
Using a sterile loop, make sure your loop is sterile and cooled. You streak the mixed culture back and forth in the first quadrant and then you want to be really careful not to cut the agar. It's very important now between all four quadrants that you sterilize and cool the loop. It's also extremely important that you know that you're not going back into the broth. This here is the only specimen that you use.
You get a loop full of bacteria, you streak it in the first quadrant, then you sterilize the loop, cool it, pull a little bit out from the first quadrant and streak that out into the second. Sterilize the loop again, cool it, pull a little bit out from the second quadrant and streak it out into the third. Sterilize the loop again and cool it and pull a little bit out of the third quadrant and streak it into the fourth.
So your specimen should be getting more and more diluted as you go from each quadrant. And then after incubation your isolated colonies should appear in the third or fourth. quadrant. Go through your safety guidelines. This is the first time we're going to be using the back decenerator so make sure that you you unplug and turn off the back decenerator before you leave lab.
For your observations and interpretations, these are the things you're going to be looking for after your plates have incubated and you come back for the next lab session. So for experiment one you're going to be comparing your density of growth and the number of species from A to B and determining if your hypothesis was supported or rejected. For experiment two, you want to see if E. coli grew well on the slant and or the deep.
And you are going to have to look up some information about E. coli. You want to determine before you come to lab, google it or look it up in the book, what are the oxygen requirements for E. coli and is E.
coli a modal species? In other words, does it have flagella or cilia? And then for experiment three, You're going to see if you have isolation for one or both of the species that was in the mixed frog.
If you had any strange streaking patterns or cuts in the agar. If you had growth that was outside of the streaking pattern. So if you have growth that you didn't put there with the loop, think about what that could mean. And I just put up here some colony morphology slides for E. coli.
So when you're looking for E. coli, this is how you should look. And we've already seen in Micrococcus luteus's colony morphology so that should look pretty familiar to y'all. This is what we're looking for on the Experiment 3 plate to see if we isolated E.coli, if we isolated in luteus. Thank you guys for watching.
Don't forget to do the reading. Check the description for more videos related to these topics and leave your questions for me in the comments below.