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Module 2 Module 18 Module 36

Module 3 Module 19 Module 37

Module 4 Module 21 Module 42

Module 5 Module 22 Module 43

Module 6 Module 23 Module 45

Module 7 Module 27 Module 46

Module 8 Module 28 Module 47

Module 9 Module 29 Module 49

Module 10 Module 30 Module 50

Module 11 Module 31 Module 51

Module 12 Module 32 Module 52

Module 13 Module 33 Module 53

Module 15 Module 34 Module 54

Module 16 Module 35 Module 55

Module 17 Module 38

Module 20 Module 39

PREVIEW

This module will be done primarily as an introduction to the microbiology lab and to the techniques that are used by our microbiology technician to prepare the media that will be provided for you as needed.

However, as many of you may someday need to do these techniques yourself, it is important that we go through the basic steps. Many of you may someday go over seas on short or long-term missions trips. Others may return to your home in these countries or in some small town in America where clinical labs are not available to do identification work for you. In each of these circumstances, you may very well find yourself having to perform these basic microbiological techniques on your own.

Media is the material we use to provide the microbes with most of the requirements they need to live out side their normal habitat. It is one of the most important thing that Robert Koch had to develop in his pioneering work on the Germ Theory of Disease. While the incubator provides the correct temperature and lighting conditions, it is the media that provides the nutrients, correct pH, moisture and other essential requirements. Therefore, there are many types of media available due to the varied requirements of the different microbes.

Regardless of the specific type of media, all media comes either as liquid, usually called broth, or semi-solid, called agar. The broth is provided in test tubes or flasks while the agar may come in test tubes, as slants or deeps, or in petri plates.

Preparing the media, regardless of the type, requires the same basic procedure. After you have determined the type of media to use you must gather the ingredients together. In most modern media, the ingredients are already mixed together and you may only need to add agar. The ingredients are then weighted out according to the directions which are usually provided for making up 1 liter. If more or less than that is required, the amounts must be increased or decreased accordingly. After weighing, the dry ingredients must be mixed into the water which is in a flask that is twice the volume of the final amount. This is then placed on a heating plate with a stir bar and allowed to hear just until boiling begins.

At this point, the next step depends on whether you are making plates or tubes. If you are making plates, the agar is autoclaved in the flask and the plates are poured afterwards. If you are making anything in a test tube, the tubes are poured first, then autoclaved and allowed to cool.

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None as this is only a demo

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PREVIEW

Whether you are making media or changing a patients bandages, it is imperative that proper sterility is maintained at all times. As you will see in the next exercise, bacteria, fungi, viruses and other microorganisms exist all around us, on us and in us. If you are not careful, some of these can get into your media or in your patient with serious effects.

Just as maintaining aseptic conditions in the clinical setting requires a certain set of procedures, keeping media and cultures contamination free requires its own set of methodologies. In the lab setting we are usually interested in keeping media and equipment complete sterile through the process of sterilization. This means that absolutely no organisms are present. When we are involved in transferring and culturing a single microbe type, we are then primarily interested in maintaining purity.

The most common way to do this in the lab is through the use of heat. The technique known as flaming uses very hot, dry temperatures to kill microbes on metal and glass pieces of equipment. The use of a dry oven is similar. Unfortunately, dry heat will not help when we are trying to sterilize liquids, large quantities of material or clothing. Wet heat can be used for several of these items and can be applied in a variety of ways. The simple process of boiling can be used to sterilize liquids but it takes a long time and results in the loss of liquid and change in its concentration. Steam avoids some of these problems but also takes a long time as it can only reach 1000 C. Therefore, the most useful sterilizing technique available today is the use of steam under pressure, which is commonly done in an autoclave. In most modern autoclaves, the pressure is allowed to reach 15 psi and the temperature can reach 1210C before boiling occurs. This allows much faster sterilization of material, usually requiring just 15 minutes, and can be used for solids, liquids and, in some cases, material.

Unfortunately, the reason heat is so effective is also the reason it cannot be used in some instances. In many cases, the proteins, amino acids and other chemical compounds in media are themselves destroyed by heat. In that case we can sterilize the media through filtration. In this technique, liquid media is filtered through filters that have pores so small that they stop even viruses. The filtrate is collected into a sterile container and is then poured.

In many cases, such as with large batches of clothing or bedding, neither of these techniques is practical. For these types of material, sterilizing gases may be used. These are toxic gases that can be released into large sealed chambers containing the material to be sterilized. The toxic gas kills the microbes as it penetrates through the clothing, etc.

Finally, sterilization is occasionally done using radiation of different types. This can be as mild as using ultraviolet light in a restaurant to gamma radiation in sterilizing fruit.

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None as this is only a demo

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PREVIEW

Since we assume that you have used microscopes in previous science classes, we will spend only a short time reviewing the structure and use of a microscope. However, since these each cost about $2,000.00 and even the smallest lens costs over $100.00, please ask for assistance if you do not understand or forget any of this material.

Due to the extreme size, by definition, of microorganism, the microscope will be one of our primary tools in this course. The ones we are using are most completely called "compound binocular light microscopes". "Compound" refers to the fact that two lenses are used to magnify the objects, "binocular" refers to the two oculars present, rather than the one in regular scopes, and "light" refers to the way the images are produced.

It is imperative that you study the diagrams in the manual and become familiar with the parts of the microscope and their functions. As you do this you will notice that there are several differences between this scope and those you have used in the past that make it much easier to use. First, there are the two oculars which allow you to use both eyes without having to close one eye. To properly use these, be sure to adjust them out/in so that you get one circle of light, not two. Then use the fine focus ring on one of the oculars, which one varies from scope to scope, to adjust for the differences between your left and right eyes.

Second is the degree of control you have over the light amount. Each of these have a rheostat switch that allows you to regulate the amount of light produced by the bulb. Usually, this should be set a maximum to get the whitest light. Then there is the diaphragm that opens and closes allowing more and less light, respectively, through it. Finally, the sub-stage condensor moves up and down beneath the stage focusing a cone of light into a single point. By raising and lowering it you can change where the pinpoint of light falls, changing the amount of light on the slide.

Finally, you will notice that the stage has a rather refined mechanism for moving the slide around on it. This stage manipulator will allow precise movement of the slide in a horizontal and vertical direction allowing you to carefully search the slide or to track an organism.

Another difference between these and the less expensive scopes you may have used is the addition of a fourth objective, the 100X oil immersion lens. This lens allows greater magnification which is necessary for seeing the bacteria that are our primary goal. It is able to do this because it is designed to be used with a thin layer of oil between the slide and the lens. This oil has the same index of refraction as the glass slide and the glass lens. This way the light will not refract, or bend, as it passes from one layer to the next. This allows more light to pass through into the lens to create a better image at that high magnification.

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As you prepare for the test, be sure that you know all the parts of the microscope and their function. Be sure that you understand the important terms and that you know how to properly use the instrument. One of the most important thing to keep in mind as you go through this course, is the relative size of the organisms you are looking at. While we will rarely be concerned with the exact size of an organism, it will be important to keep in perspective the size of a human blood cell versus the size of a paramecium versus the size of a bacteria. This will help you know exactly what to look for and when you have found the right thing.

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PREVIEW

While we will primarily be examining prepared slides in this class, we will frequently be making our own slides. When we view bacteria we will usually use a special set of procedures that will be explained during the staining labs. However, there are times when we will be using these techniques to view living bacteria, protozoans and fungi.

The first thing to keep in mind is the necessity for a clean slide. This is usually simplified in this class as we rarely reuse slides here due to the potentially pathogenic nature of the bacteria we use. However, it is always a good idea to examine the slides carefully and clean them completely if they appear dirty. Any dirt or grit may look like a microorganism under magnification and the oil the may remain from fingerprints may cause the stains and smears to not adhere.

The primary technique we will use to examine living organisms under the microscope will be the wet mount. This is a relatively easy technique that is done by simply placing a drop of the solution containing the organism on the slide. If you are working from an agar slant, for example, you may have to first place a drop of distilled water on the slide and then, using aseptic techniques, transfer a loopful of bacteria to the drop of water. Either way, the next step is to simply place the coverslip onto the drop in such a way that you to do not trap any air bubbles. The easiest way to do this is to first put one edge of the coverslip on the slide and slide it until it just hits the water drop. At this point, releasing the other edge of the coverslip so it falls down onto the water drop at an angle will force most of the air out to one side, reducing the number of air bubbles.

Occasionally, we may find ourselves using an alternative method called the hanging drop technique for viewing these objects. This is a way to prevent the squashed affect of having the coverslip resting right on top of the slide. Instead of following the directions in the manual we will either place the Vaseline right on the slide itself and then add the water in the middle of the ring of Vaseline or we will use a depression slide that has a thicker slide with a well that will take a drop of water. Placing the coverslip on top of this well will have the same effect as using the Vaseline.

However the most important aspect of this lab is learning and beginning to recognize the three primary shapes of bacteria cells as this is a critical aspect of bacterial identification. As shown in the lab manual, there are three basic shapes: cocci (spheres), bacilli (rods) and spirilla (spirals). However, there are modifications of each that may be seen and require additional study to differentiate.

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Notice that you can frequently know the shape of the organism by its name such as Bacillus and Micrococcus. Think back over the techniques you used and the success you had in not producing too many bubbles in the slide. More than one person in the class probably was embarrassed to find that the beautiful cell she or he was drawing was actually an air bubble. As you complete this lab, think over the various sizes of the organisms and place them in order from largest to smallest.

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This is a fun lab that is also designed to teach a very important lesson that is critical to your success both as a microbiologist in the lab and as a medical professional: microorganisms are all around you!! They are on you, in you, floating in the air, on the table top and EVERYWHERE!! Thus, if you are not careful, you may end up with an unwanted microorganism growing on your petri plate or, more importantly, on the catheter you just inserted into your patient.

This will also be your first exposure to what a group of microorganisms growing together on agar look like. This colony is an important concept in microbiology and is assumed to represent one original cell that, over the incubation time, reproduced numerous times until enough cells were present to become visible to the naked eye. Sometimes these colonies, however, run together as the result of numerous cells being clumped together or lying very closely together.

Another important concept that should come across in this lab is the effect that antimicrobial agents have on microorganisms. You will be doing before and after samplings at several points today. Be sure to remember and compare these together when you check them in a few days.

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As you examine these plates and tubes be sure to look at the lab exercise to refresh your memory of what you did to each plate or tube originally. Examine the soil tube to notice where the growth seems to occur. Most likely it will be throughout the entire tube with some on the bottom, some mixed throughout the broth and some floating on top. We will discuss what each of these growth patterns are called and what they represent in another exercise.

Be sure to look at all the other group's air plates as they will each have come from a different location. Notice that these plates probably have the most distinct colonies as the microorganisms were floating as single cells and landed in separate areas of the plate. There are also probably both fungal and bacterial colonies since both are present in the air. The fungal colonies are usually larger, fuzzier, and more likely to be colored than the bacterial colonies that are smaller, smoother or slimier, and tend to be whitish.

In two of these experiments, you made before and after cultures. Notice the difference between the number of organisms on the plate taken from the unwashed benchtop and the washed benchtop. Is there a difference in number? Does it appear that the material used to wash down the benchtop was effective against the microbes? Now think about the results from the handwashing experiment. Which had more organisms: the before or after washing? This might make you think that the soap used in the lab is ineffective. However, think about the kind of handwashing we do here in the lab versus the type of hospital or surgical scrub you use in that setting. In the lab, we are interested primarily in just getting rid of any microbes we might have picked up during lab, which would be just sitting on the skin surface. However, in the surgical setting, we are primarily concerned with anything that we might bring in as normal skin flora that might be lurking under the top layers of skin. Thus, a surgical scrub is designed to get down past those initial layers to fresh, uncontaminated skin. Our lab washing, however, just removes surface microbes and may actually expose more microbes existing as normal flora under our top layers.

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As we just learned in exercise 7, microorganisms are everywhere. In the lab, we need to be able to work with our material, primarily sterile media, in such a way as to prevent those microorganisms from getting into or onto our media. Sometimes this will mean trying to maintain sterility, such as when we pour a plate and do not want anything to get on it initially. Other times it will mean trying to keep anything from getting on the media except what we deliberately put there ourselves.

This is what we will be learning in this lab. We will be trying to take a specific bacteria and place it onto or into sterile media so that it, and only it, is transferred from the pure stock culture to the new media. Being able to do this allows you to insure that the cultures you work with only contain the one species of organism you want it to and no contaminants that might affect your studies.

There are a few helpful hints that you need to keep in mind as you do this:

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You will know if you have been successful with your transfers if each slant contains growth and if that growth is clearly from only one species of microorganism. The particular organism you are using in this experiment will help you see that more easily because of its obvious pigmentation.

On a slant there should be a nice straight line or zigzag line, depending on what you did, running along the slant. There will also probably be an accumulation of liquid at the bottom of the slant but that is normal. In a broth culture, you should see only the pigmentation of the transferred bacterium and it should all be either on the top, at the bottom or evenly dispersed throughout the broth.

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In a science lab such as this, we are always concerned with exactness, whatever it is that we are doing. This is particularly true when we are talking about measuring out amounts of material. One of the things we will be measuring out many times this semester is liquid, whether in measuring material to add to a broth culture or actually measuring out a given amount of that broth culture to put into something else.

For this type of measuring of liquids a serological pipette is usually the simplest, safest, and most accurate way to do this. A serological pipette is a devise that looks like a long tube with a narrowed tip and markings all along its length. At the top end will be a marking such as "1 in 1/100" or "10 in 1/10". The first number always tells you the total volume that the tube is capable of holding within the gradations while the second number tells you what the smallest markings indicate. Thus the first tube measures 1 ml and can measure it in 1/100ths of a ml while the second one holds a full 10 ml but each smallest marking only measures 1/10th of a ml.

In some cases, such as the 1 ml pipettes that we use in this lab, there may also be a few markings above the "0 ml" point. This will usually allow you to measure out an additional .1 ml so that, as we will be doing later in the course, you can measure out 1.1 ml so that you can do serial dilutions of 1 ml and .1 ml at the same time.

While the manual talks about glass pipettes that are reusable and have to be cleaned and sterilized, our pipettes are usually disposable and come already sterilized. However, remember that as soon as they are removed from the package, they are no longer considered to be sterilized and should be used immediately and then disposed of properly.

When using a serological pipette there are several things to keep in mind:

Following these simple rules will allow you to safely and successfully measure liquids accurately.

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None as this is primarily a demo.

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In most cases in this course we will provide you with petri dishes that already contain the agar in them. Occasionally, however, we will have you do pour plates, usually during serial dilutions. When this becomes necessary, you will need to pour the plates correctly to ensure success.

The first thing that will be needed is to melt the agar in the tubes. This also may be done for you if the tubes are already in a water bath. If not, it will be necessary for you to put the tubes in a beaker, fill it with water until it just covers the media and then place the beaker over the Bunsen burners. When the tubes are melted, which can be determined by gently shaking them using a test tube holder or by looking for a clearing of the media, the tubes can be removed from the water.

Before pouring the agar, be sure that the plate is right-side up so that you are pouring into the smaller diameter, deeper bottom of the plate. If you are pouring the agar into an empty plate, simply pull off the top of the test tube and quickly lift the edge of the plate just enough to allow the test tube lip to enter the plate. Then pour the agar into the plate in one swift motion and lower the top again. Gently swirl the plate on the table top to disperse the agar throughout the plate.

If you are pouring the agar into a plate containing some pre-added broth, such as in a serial dilution, you cannot pour the agar immediately after removing it from the boiling water as it will be too hot. Therefore, first cool the media slightly in a process called tempering. This can be a tricky process as it must be warm enough not to solidify and cool enough not to harm the microbes. The best way to do this is to simply place it in a water bath at 55o C for about 10 minutes. If such a water bath is not available, simply keep an eye on the test tube by gently touching the tube every minute or two. If you can hold the tube without burning yourself but it is still warm, that is the right time to quickly pour it.

Once the hot agar hits the cool plate it will cause condensation to build up on the top of the plate. This is the reason we invert all petri dishes when we put them into the incubator as it prevents the water from falling down onto the colonies and dispersing them. This condensation can be reduced by stacking the cooling plates immediately on top of each other so that the heat from the top warm drives off some of the condensation from the plate below it.

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None as this is primarily a demo.

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In nature it is extremely rare to find microorganisms growing in perfect isolation from each other. If you remember back to module six when we tested the soil, the air, and your lips, among other things, the organisms were mixed together and not separated. However, we need to work with pure isolates of just one species so we somehow have to be able to separate or isolate out each species from a mixed group. This is particularly important in growing cultures from humans as, when you take a throat, urine, fecal, etc. sample from a patient, there will be both the pathogen and the normal body flora present. We must be able to isolate the possible pathogen from the normal microbes in order to identify it and know the exact nature of the problem.

There are basically three ways to do this. The first one is called a pour plate and is the techniques we will use today. The other two are the streak plate and the use of selective-differential media and we will use those techniques in the future. In a pour plate the idea is to place the mixed colonies in a liquid and mix them throughout the media well enough to separate the cells and dilute them throughout the media. The media is then poured into a plate and each separate cell is then allowed to grow into individual colonies. Frequently, there will be so many cells that you are still unable to separate and identify them from each other. In that case we usually do a serial dilution which uses dilution blanks of known amounts to further dilute the number of cells so that clear, separate colonies are formed.

In this experiment we will use two dilutions of the same bacteria. Instead of using a pipette to transfer an exact amount of culture we will simple use a loop as we are more concerned with technique than quantities. Notice the stress placed on thoroughly mixing the agar before pouring it. We will use a more efficient method of mixing than diagrammed here, however. Instead of moving it back and forth in our hands we will use a Vortex Mixer that cerates a much greater mixing action.

Remember also not to add the loopful of culture to agar that is too hot or the bacteria will be killed immediately. Be sure to temper the agar as discussed previously.

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Notice that both plates probably had way too many colonies to count. In fact, if you did not look closely, you probably thought there was nothing growing as there were so many that each grew only a very small amount. The very small size may also be due to the fact that so many of the colonies are growing in or under the agar and are thus suffering both from the large numbers of colonies, the effort of pushing against the agar itself, and, perhaps, the lower amounts of oxygen within the agar itself.

When we do this technique again, in conjunction with a serial dilution, there will be fewer cells in each plate resulting in fewer and larger colonies. Since we assume that the mixing action separates the cells and each colony represents groups of cells al arising from the same original cell, we could thus go in and take a sample of any one colony and transfer it to a fresh agar slant to create a pure colony. Thus, we would be isolating single species from a mixed group.

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In order to accurately and easily count and view colonies growing on agar plates, it is helpful to use a Quebec Colony Counter. This device has a number of features that make it easier to view and count colonies. First, it has a nicely backlite viewing screen. This screen is also etched with a grid that has the entire area covered in small squares and a number of those squares, forming an "X" across the screen, are further divided into smaller squares. Finally, there is a large magnifying lens that can be adjusted to enlarge the entire plate making it easier to view and count the colonies present.

The first decision that must be made in counting colonies is whether or not there are too many to count. Sometimes you have to realize that even with the Colony Counter there are simply too many colonies to keep straight which you have counted and which you have not. Then the decision has to be made as to whether or not there are few enough to count but too many to count the entire plate. In this case, only those colonies that fall into the "X" are counted. For our plates this means that you have counted about 20% of the entire plate so we multiply the number of colonies in the "X" by 5.5 to arrive at a whole plate count. Finally, if there are few enough colonies so that you can count them all, it is a simple process to start at one corner of the plate and, using the grid lines, move across the plate counting all the colonies that fall in that row. When the end of the plate is reached, simply move down one row and count colonies in the opposite direction. In either of these last two cases you must decide before hand what to do with colonies that fall on the line. It is best to simply say that you will count them in the upper row they fall into and not in the lower row they fall into.

When using this device it is best to center the plate on the screen, turn on the light, adjust the magnifying lens so that a large, clear image is seen. Sometimes it is best to count from the top of the plate, other times it is easier to count from the bottom of the plate, through the agar. This is true particularly if the condensation is thick on the cover. WHATEVER YOU DO, DO NOT REMOVE THE PETRI PLATE LID IN ORDER TO SEE THE COLONIES BETTER!! THIS WILL PROVIDE THEM WITH THE POSSIBILITY OF EITHER BEING CONTAMINATED OR CONTAMINATING THE LAB.

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This exercise is done with the dilution pour plates from exercise 10 which means you probably were not able to count the colonies as there were too many of them in either plate. If you were, however, you should have a pretty good feel for the unit and the process. However, if you were not able to really use it you will make use of it later on in the class.

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This lab teaches you the primary way to isolate pure colonies from mixed growths of microbes. The technique is called streaking and is usually used in conjunction with the third technique called selective-differential media. Today, however, we will simply concentrate on the technique of using an agar plate to dilute a loopful of culture over an entire plate so that, eventually, individual cells are dropped off the loop to grow into pure colonies that can be used to create pure cultures in new slants.

While there are many ways to do this, the method described in your manual is the most efficient and easiest to learn. The pattern of the four sections is actually drawn on the underside of the dish and labeled using roman numerals. Then, the original loopful of culture, or cotton swab of culture, is used to thoroughly inoculate the "0" section. Care must be taken to stay within the section but here you can go back and forth over your own line of inoculation. If you are working with a cotton swab, from a throat culture perhaps, you then toss the swab and continue with a flamed loop. If you are starting with a loop you simply flame it well to remove any excess bacteria. Remember the idea is to dilute the cells across the four sections so any bacteria still hanging on the loop will only add to the number of cells moved into the next section. Rotating the plate so the next section is across from you, open the plate very slightly and drag the loop once across the "0" quadrant and right into the "I" quadrant. Now you must be careful not to go outside section "I" and not to cross over the inoculating line you create in section "I". The process is then completed, while flaming the loop after each section, by dragging the loop across one section and into the next section.

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If, when you check these plates, you find massive growth without any clear distinctions on the first one, two or even three sections, that is fine. As long as in one of the quadrants there are small clearly distinct and separate colonies, you have been successful. Hopefully, this will occur in either sections two or three as this would be ideal.

If you had little or no growth on any section it may have been due to there being very few cells in the original stock. However, it may also have been due to the fact that you did not cross over the previous section's inoculating line or that the loop was too hot and the microbes were killed when you first touched them.

If you had too much growth in all sections it was probably because you did not flame after each section, you crossed over into previous sections and grabbed additional cells or, if you were really not following directions, you actually reinoculated the loop in-between every section!!

Take special note of the plates that did work and notice the technique you used in order to insure that you can repeat this important technique successfully each time you use it!

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Now that you have an idea of what a colony is, what it looks like and how to get nice separate, distinct ones from mixed cultures, we need to start working on learning the special characteristics that distinguish one species' colony from another species colonies. In Module 6 we already talked about the differences between most fungi colonies and most bacterial colonies. Now we will look more closely at the differences between different types of bacterial colonies.

Here, as throughout most of the rest of the class, we are not interested primarily in memorizing the characteristics of each species we use. Those kinds of things can be looked up in a text book should you ever need it someday. Rather, we want to concentrate on the various aspects of a colony that can be used to distinguish them from each other. That way, should you ever have to identity species based on colony characteristics, you can look up the species in Bergey's Manual of Determinative Bacteriology and understand what is meant by the description that is offered there. In this activity we will concentrate on the appearance of colonies on agar plates and in nutrient broth. However, additional details could also be discussed to describe streaks on slants.

On agar plates, colony size and shape are two of the primary aspects we are interested in . Be sure to learn the ones given in the book. In addition, the appearance of the edge of the colony, also called its margin, is also important. Finally, color is something that is also important as there are many variations of pigmentation that bacteria produce. However, the one aspect that the manual mentions that we will not worry about is the elevation as that is very difficulty to ascertain as this point in your microbiological career!

As you will see in this experiment, these characteristics may vary due to temperature, pH, or the presence of certain other environmental factors. In another module we will see that color changes with temperature as the proteins are affected by the heat. Here we will look at the difference between growth on different types of agar and the affect, for example, that salt has on some organisms.

Finally, we will frequently be growing cultures in broths and many species have a distinct type of growth when growing in liquids like that. Usually, this is due to their need for a certain type of oxygen requirements, which we will discuss further in future labs. If an organism ends up growing primarily on the top of the broth, in a thick layer, it is called pellicle growth. If it likes to grow throughout the broth, so the whole test tube is cloudy, it is said to exhibit turbid growth. Finally, many organisms will grow on the very bottom of the test tube. Your manual mentions only one such growth pattern, sediment. This is the type of bottom growth that simply rests on the bottom and is easily disturbed. If you are not careful, you may well make your sediment growth go to turbid simply by accidentally shaking it. However, there is a second type of bottom growth that you will encounter this semester. This type of growth is due to the presence of a slime layer around each cell that allows it to stick to other cells and the glass of the tube. Therefore, slime growth will lie on the bottom and be very hard to mix. Once you disperse it by mixing it, in fact, it may very well simply settle to the bottom in clumps.

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Obviously, a critical aspect of this exercise was getting successful single colonies in doing your streak culture. If you were unable to obtain good colonies you may have difficulty doing this analysis. In that case, do not just give up. Ask a colleague if you can share their results if they were able to get single colonies.

In activity 2 be sure to compare the two TSA plates with each other as well as the two NaCl plates with each other. In each case one species should grow "abnormally" compared to the other because of the difference the agar has on the species' growth pattern.

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For the next few labs we will be examining microorganisms other than bacteria. In each case, there are two labs for each group of organisms. Therefore, we will discuss the entire group in the first module and review it in the second module.

The Fungi Kingdom was frequently placed in other kingdoms for many years due to its weird characteristics. They are basically eukaryotic, non-photosynthetic cells that get their nutrition through absorption through the cell wall/membrane. While some of them are distinctly unicellular (Module 15) others are composed of many cells (Module 16). However, these are not really organized enough to be called multicellular organisms like plants or animals.

In terms of basic non-reproductive structure, the kingdom can be divided into those that are unicellular (yeasts) and those that are composed of hyphae (molds, etc.). The hyphae are actually chins of cells that grow in length through true mitosis. Sometimes the cell walls will remain between the cells in the hyphae creating septate hyphae. Other times the cell walls will disappear shortly after the cell is formed creating a single long aseptate strand. If a number of these hyphae are wrapped together a mycelium is formed and this is easily seen with the naked eye.

When it comes to reproductive structures, the unicellular fungi may frequently use a process known as budding. This process is unlike mitosis as both the mother cell and the new daughter cell, usually only one at a time, continue to exist after the process is over. Here, a small bud simply starts to form off of the mother cell and grows to a certain size. When it reaches that size it simply breaks off, allowing the mother cell to continue to live and going off to grow to its full size and repeat the process. Filamentous fungi have many more variations in reproduction, both asexual and sexual forms. In the asexual category are any type of spore that is formed from a single cell. These will frequently be found on complicated structures that are described in your manual. Be sure to know these terms and associate them correctly with each type of fungi you examine. Sexual spores are those that are reproduced when two strains of the same fungi come into contact and, from their union, arises a sexually produced spore. Due to their simplicity we do not use the term male and female. We usually refer to them as (+) and (-) strains. These types of spores are actually the basis by which we classify the fungi as discussed in the manual. Be sure to understand these divisions or classes (depending on how your manual classifies them) and which type of spores each group produces.

This is the first exercise where several things will be done. First, you will be looking at numerous prepared slides of different types of fungi. It is important that you devise some method for learning what each species looks like and how to distinguish it from the others you are responsible for. You may find that drawing elaborate diagrams works, or that making comments alongside the photographs and diagrams in your manual is best. Whatever you do be sure to allow yourself additional time before the test to examine the slides again. Do not rely simply on the single opportunity provided in this lab period.

Secondly, this is the first exercise were you will need to start connecting the specific names of the species with specific diseases or special characteristics of the species. This is very important in the medical profession as you will need to understand what pathogen causes what disease as well as any special things about it that might help you identify it, control it, or whatever. Again, how you go about learning this is up to you but you must be able to do so.

While we will usually be examining these organisms as potential pathogens, we will also be seeing that many microorganisms are beneficial to humans. Sometimes this will be because of things they do inside of us, other times, like today, it will be because of something they provide for us. In this case, we will be examining the production of alcoholic beverages. However, the same group of fungi also are involved in creating leavened or rising breads.

As we look at the filamentous fungi there are lots of terms that are important and that you will need to know. Page 144 lists many of these and you need to know all of them except for the following: aerial, coenocytic, diphasic, fruiting heads, rhizoids, and sterigma.

A final aspect of the module on the filamentous fungi will be the attempt to grow them on slides so that they can be viewed under a regular microscope while alive. As you will see, these types of fungi can get quite large, quite rapidily and will fill a petri dish in a day or two. As such they can be viewed under a dissecting scope but that does not allow you to see the details that you will see on the prepared slides. Unfortunately, the prepared slides contain dead fungi and some of the details are not visible. Therefore, we will grow them on small chunks of agar on top of a slide and then simply move the whole microculture to a microscope. Unfortunately, this small piece of agar will dry out very quickly in the incubator, before any significant growth can occur. Therefore, we will create a moist chamber in which to keep the microculture slide. This is simply a sterile petri dish with a piece of filter paper in it that we will keep moistened with sterile water. The water will maintain a high humidity level in the petri dish and prevent the agar from drying out.

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See next module unit.

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See previous module unit.

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As you examine the slides of the different types of fungi keep in mind that, except for the differences between the two yeasts that you examine, you will be required to identify the organisms on the basis of their physical characteristics. Be sure you can find distinguishing characteristics, besides color, that will allow you to separate them on an exam. Color is usually a poor indicator as it frequently is the result of artificially added stains that may vary from slide to slide depending on what was used.

In examining the grape juice tubes compare the differences between the two species of fungi used. Remember that one is a pathogenic species in humans while the other is the yeast used in commercial preparation of breads and alcoholic beverages. Obviously one should be a better grape fermenter than the other. In examining the effects of pasteurization on grapes, remember that the bloom found on grapes does not refer to the flowers but to the naturally occurring microorganisms that begin to break down the grapes. This grayish looking material may contain fungi capable of naturally fermenting the grapes. However, if you were successful in [pasteurizing your grapes, little fermentation should have occurred in this tube. Finally, the tube that had the bloom removed via pasteurization and was then inoculated with the commercial fermenter should have produced the most gas and alcohol.

Notice that as you look at the yeast colonies and the filamentous fungi colonies, there are significant differences. The former tend to look more like bacterial colonies while the latter are significantly different. You should also observe that the yeast grow much more slowly than the filamentous ones which will frequently fill the petri plate by the time you examine them. Regardless of which species you used in these two exercises, be sure to examine the results that other students got, using different species, as you are responsible for understanding what each looks like.

If your microculture was successful you should be able to see distinct hyphae and fruiting bodies under the low power of the compound scope. Hopefully, you should be able to see if your species has the cells walls in the hyphae or is aseptate. If your fungi is aseptate, see if you can see a process known as cytoplasmic streaming occurring where the cell cytoplasm moves along the hyphae.

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Modules 17 and 18 deal with the second group of non-bacterial organisms we will be looking at. These protozoans are in the Protista Kingdom and are true microorganisms being unicellular. They are eukaryotic and are usually heterotrophic with a few autotrophic species.

While many of these are free-living organisms, many are of medical significance, causing a wide variety of diseases. They usually exist in two stages, cysts and trophozoites. The cysts are the non-motile resting stages while the trophozoites are the adult form and are usually motile. In fact, the type of motility they exhibit is the primary basis by which they are classified. These modules contain excellent tables that list the important free-living and parasitic representatives that we will examine and you will be responsible for. The work will be exclusively on the microscope so, again, you will need to develop important skills in identifying these organisms on the basis of characteristic features. Much of the test on these organisms will simply be microscopes set-up with these slides. You will need to identify them, name them completely and tell what disease they cause if they are pathogenic.

Module 17 deals with the three phyla that have both free-living and parasitic representatives. These are all classified on the basis of the type of locomotion they display. Be familiar with the phyla, their method of movement and their representatives. You are also responsible for the material in the tables in these modules except for the material in columns 5 and 7. In addition, remember the more correct and specific disease names that will be given in class as the table occasionally names only the symptoms and not the specific medical disease associated with each.

With these first three phyla we will be looking at both living slides you will make and prepared slides that we will provide. It is a good idea to start with the prepared slides so you know what you are looking at as the living specimen material frequently contains other types of protozoans that are there by accident or to serve as food for the specimen we are after. While you must look at both the living and the prepared slides, when they are available, remember that we will be using only the prepared slides in the exam, so concentrate on those.

In module 18 we will deal with a phylum that goes by several names: Sporazoa, Haemosporina and Apicomplexa. You may learn whichever you like but the last two are more current. This group is different in several regards. First, there are no free-living representatives, all are parasitic. Second, they are not motile. Third, they all have spore forms and, finally, all have complex lifecycles. The primary one we will study will be the Plasmodium, the causal organisms of malaria. Study the lifecycle that, in this manual, is divided into two sections: one showing the portion in the human, the other illustrating what happens in the vector, the mosquito.

In the prepared slides you will be looking at, you may see any of the forms shown to be in the human but none of the forms that are found only in the vector. Therefore, it is important that you look carefully at the diagrams of the sporozoite, merozoite, gametocyte and the several forms that are found inside the erythrocyte. Each of these is fairly distinctive.

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See previous module unit.

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As you review the material from these two modules, be sure that you understand the phyla, their characteristic traits, and the representative organisms we discussed. Much of this material will also be discussed in lecture so that will give you additional exposure to it.

The information here on the specific diseases is not to be as detailed as what you will need to know for lecture exams. Here we are primarily interested in your being able to provide the causal organism, classify it and tell the disease that it causes. Most importantly, however, you must be able to identify the pathogen under the microscope. This is difficult, but not impossible. More than likely it will require that you spend additional time in the lab looking at the organisms again, drawing accurate pictures to refresh you mind and then, perhaps, teaming up with a partner and setting up slides for each other to see how well you really remember them.

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In Modules 19 and 20 we will look at a group of organisms that definitely do not meet the definition of a true microorganism; in fact, these organisms are actually in the Animal Kingdom which means they are true multicellular organisms. However, since they are frequently pathogenic and usually very small, especially in their infective stage, they are usually discussed in an introductory microbiology course such as this.

The first group is the Phylum Platyhelminthe which are also knows as the flatworms. This is further broken down into classes, of which two are important to medical microbiology. The first of these is the Class Trematoda which consists of the flukes. These organisms are flat but tend to be shorter and broader than the next group. They frequently have oral and ventral suckers which allow them to feed and attach to their host. These typically have fairly complex lifecycles with several hosts and various larval forms that infect the different hosts. Be sure, when you are examining the slides, that you realize what stage you are looking at or it can get very confusing. For some of these you will need to know the adult and the egg or ova form as both can be useful in diagnosing the problem your patient is having.

The second class is the Class Cestoda which is made up of the tapeworms. The tapeworms are flat and very thin and long. Some of them may get up to sixty-five feet in length but, because of their thinness, they can easily be held in one hand. While you will be examining one tapeworm which will fit entirely on one slide, Echinococcus, the majority of tapeworms are too large to fit on one slide. Therefore, you will be looking at separate scolex and proglottid slides in order to see the components of a typical tapeworm. The scolex is the head and the proglottids are the individual body segments that make up the majority of the tapeworm. Again, you will also be examining the ova and larval stages for several of these organisms so be sure which it is that you are looking at. Do not examine any stage that the instructor does not require you to know.

The table in this module summarizes the organisms you will need to know and the diseases they cause. In the case of the Taenia spps. you will not need to be able to distinguish between the T. saginata and T. solium on the basis of the slides. In fact, what you will probably be examining are the parts of T. pisiformis which is commonly known as the dog tapeworm.

Related to these, in terms of also being animals, are the roundworms in the Phylum Nematoda. These will be discussed separately in the next help module.

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Be sure that you are able to recognize the different pathogens discussed here in whatever stage you were required to look at. You will need to know the ova stage, larval stage and adult stage for several of these. you will also have to classify them and connect them to the disease they cause.

Rest assured, however, that the instructor will try to pick only those specimens that are truly representative of the species and the illustration in the manual.

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While the previous module dealt with the flatworms, this one will discuss the roundworms. These are also called nematodes and are placed in the Phylum Nemotoda or Nematyhelminth. While these at first look may look like common earthworms, they are different in several regards. The most significant of these is the fact that they do not have the segmented body that we associated with the earthworms. As you will see, there is also a tremendous amount of variation in length of this group of worms.

This variation of length is something you will need to be careful about. While it can be used as a useful technique for differentiating between some of the species, it can also prove problematic due to the variation that may occur within a species. Therefore, it is best if length is used only in certain cases and if other distinguishing features are identified. Be sure that when you examine Figure 20-1 you keep in mind Figure 20-3 and the measurements given in the first figure. This will help you understand the differences in length between the organisms you are responsible for.

Again, you will need to know both the adult, larval and ova forms of the organisms, depending on the species. However, it will not be necessary for you to be able to distinguish between the two sexes for any of these.

Be sure to study Table 20-1 and to be familiar with the organisms, their classification and the diseases they cause. The Discussion section of your lab manual will provide additional information of use to you as will your lecture component on these organisms.

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Be sure that you find distinguishing features for each of the nematodes you need to be able to recognize. Sometimes it will be the overall size of the organisms, other times it may be the changes in diameter along its length, or the shape of the mouth parts. With the eggs, you need to be able to identify them out of the numerous other objects that show up in the typical fecal smear that they are found in.

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Since the organisms we will be concentrating on in this class, the bacteria, are so incredibly small, being able to properly use a microscope is, as you have seen, very important. however, in many of our labs you will also need to become proficient in making your own slides and in staining the organisms so that they will appear. Module 21 is the first step in creating a good slide and you will put this technique into use each time you make a bacterial slide in this class.

While we will use a slightly modified version of the method discussed here, it is important to realize the proper way to perform this technique. Be sure you understand the need for a clean slide and the best way to obtain one. This will usually not be a problem in this class as we usually dispose of our slides and start with brand new slides each time to reduce the possibility of contamination. However, another problem may arise because of the newness of the slides. Frequently these slides will stick together and you may find yourself unable to properly focus because the two slides together make it too thick. Be sure that you just remove one slide at a time from the box.

The modifications that we will make to this technique start with the use of a wax pencil to draw a straight line about 1/3 of the way along the slide. This line will help you know which side is up, will give you something to focus on initially and will help you locate the bacteria you have added. The second variation occurs when you go to air dry the smear. Rather than waiting 30 minutes for true air-drying, we will cheat a bit by holding it high above the Bunsen burner so that it will dry in a shorter time period.

The critical portion of this technique really occurs during the "fixing" portion when the bacteria are killed and made to adhere to the slide. Since we will frequently be using potentially pathogenic bacteria and since we will not be using any artificial "glues" or coverslips on the slides, this technique is very important. Top do this correctly is as much science as it is art so do not be frustrated if it does not work well the first time.

After the drop of water has air dried, move the slide through the hottest part of the flame three times, moving at a moderate rate of speed, as demonstrated by the instructor. If you go too quickly it will not be heated enough and the bacteria may still be alive and will probably wash off during the staining procedure. If you go too slowly, they will probably be heated too much and the cell walls and cell membranes will be destroyed resulting in an appearance like melted crayons and improper staining.

Be sure that you do one slide at a time, then stain and examine each. This is different from the instructions in the manual but will allow you to check your technique immediately and help you avoid having made the same mistake on all the slides you do initially.

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One of the problems you most likely encountered in this module is making the smear either too thick or too thin. If you used an agar slant and transferred a loopful into a drop of water on the slide, you probably ended up with clumps or layers of bacteria that did not allow proper staining. It may even have been too thick to see through. However, if you went with a broth, you may have ended up having a hard time finding enough, or any, bacteria to examine. These are typical problems and do not reflect on your skills.

However, it will be necessary to overcome these problems in the future so that you do get good slides regardless of what you start with. If you are working from a slant it is important that you transfer only a small amount to the drop of water and that you spread it around as large as possible. If you are working from a broth, be sure to take your loopful from the most turbid portion of the broth, where ever that may be. Therefore, it is usually not a good idea to mix the broth before taking the sample.

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Now that the slide has been properly prepared, it is necessary to stain it so that it is more easily seen. Otherwise, the small size and the clear appearance will make it very difficult to see, let alone distinguish any parts of, the cell.

Stains that are typically used fall into one of two categories: acidic or basic dyes. Acidic dyes are those that have the colored ion on the negative portion of the molecule and, therefore, adhere to the positive components which are the basic portions of the cell. These are good for staining the cellular components inside the cell. However, what we are usually interested in seeing is the outside of the cell and its basic shape. Since these are negatively charged, we need a dye that is on the positive ion of the molecule. These dyes are in the Basic category of stains and are the most commonly used stains. The majority of the dyes that we will use in this class will fall into this category.

Each of the stains has a certain strength or affinity for the cell wall and can cause improper staining if left on for too long or too short. Therefore, be sure to leave the stain on for only the time noted and that you have the rinse water bottle handy when you add the stain as you do not want the time to expire and then have to look for the bottle to rinse it off.

Be sure to examine other students' slides as you will each be using different stains and bacteria species. Examining different slides will give you a better understanding of the appearance of the different stains and shapes of bacteria. This is a good time to refresh your memory regarding the three basic shapes of bacteria and their correct scientific terms.

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Basically, you will know that you have a successful slide if you see the bacterium that you added to the slide, if it is all stained one color and if there are at least some portions where the cells are far enough apart to distinguish their shape and growth patterns. If you do not achieve these goals then it is important to access what might have gone wrong so that you are able to improve your technique.

The most common problem, as stated in the previous module, will most likely be that the smear is either too thick or too thin. Again, if this is the case, reexamine the smear technique from Module 22 and rethink your technique. Then try another smear and stain to see if you can correct your technique.

If the smear is done correctly, the staining should go well as it is pretty straight forward. however, if the stain is too light or too dark it may be due either to improper timing or improper rinsing. If it is too light you may not have left the dye on long enough or you may have washed it too vigorously. Conversely, if it is too dark you may have done just the opposite. Again, rethink your results and try to avoid these mistakes with your next slide.

Be sure to examine other students' slides and compare them to yours. You should see differences in colors depending on the stain they used and in shape, depending on the species they used. You may very well find yourself using the stains and species they used today in the future so begin to learn what they look like.

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While the use of just one stain is called a simple stain, the use of multiple stains in the same techniques is usually refereed to as a differential stain as different cell types will stain differently. These are useful for distinguishing between different cell types which can frequently be useful in diagnosing a patient with a certain type of infection. In these techniques it thus becomes very important to get a smear of the correct consistency so that the entire smear stains one color or the other. This way you will know that any differences are due to the presence of two types of cells and not your poor technique.

This particular differential stain, the Gram Stain, was accidentally invented by C. Gram and it is still uncertain as to exactly why some cells stain one color and others stain the other color. The current theory is that it has to do with the differences between the cell wall in that Gram (+) cells have rigid protein walls while Gram (-) cells have a cell wall with a greater lipid concentration. This makes it difficult for stains to adhere to the Gram (-) cell walls so the dyes are easily removed.

The process begins with the preparation of a good smear as done in previous modules. Then a four reagent process is done that begins with an initial stain (crystal violet) which will stain both types of cells blue. A mordant (iodine) is added which will cause the initial stain to stick better to the gram (+) cells. However, when the decolorizer (acetone-alcohol) is added, it removes the dye from the gram (-) cells leaving them clear again. This means that when the counterstain (safranin) is added, the gram (-) cells are able to take it up and turn red. The gram (+) cells, however, are already stained blue so they are unable to take up the red stain and remain blue.

There are a number of things that can go wrong with this process and they are listed in the module discussion. Be sure to go through that list to avoid those problems and recheck it if you get a poor stain.

More importantly, from a clinical setting, there are also a number of important things that are usually different between the two types of cells besides the nature of their cells walls and the color they turn. The list in the module discussion is an important one because, after doing a simple, quick gram stain, you immediately know a variety of things that can help you grow the organism, kill the organism, know what problems it may cause or identify it.

Again, after you have successfully performed and examined your stain, be sure to look at your partner's slide so that you can see the opposite reaction.

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Hopefully, you were able to get a successful stain the first time around. If you were unable to, be sure to go over each of the steps and the list of possible errors in the manual. The most common problem usually involves the thickness of the smear as this greatly affects the ability of the various reagents to get into all areas of the smear. You, therefore, end up with patches of red and patches of blue. When this occurs you are not sure which color is correct or if you happen to have a mixed culture of gram (+) and gram (-) cells. Finally, too much decolorizer or leaving too much water on the slide after each rinse can also cause problems for beginning stainers.

Since our emphasis in this class is on the medically significant aspects of microbiology be sure to learn the physiological and cytological traits of each type of organism. Remember that the majority of cells are going to stain red as most are gram (-). However, as listed in your manual, some very important pathogenic bacteria are gram (+). This technique, along with recognizing the shape of the bacteria, is also critical to simply identifying organisms found in your patient, as we will see in later modules.

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This staining technique is also known as the Ziehl-Neelson stain after the men who refined the process first developed by Paul Ehrlich. These microbiologists were interested in finding a quick and easy method to identify the presence of Mycobacterium tuberculosis, the causal agent of TB, in patients. These cells, as well as a few others, have a very thick lipid layer in their cell wall and this accounts for their color after the process. Most cells, including human cells, do not have this lipid layer and stain the opposite color they retain. Therefore, if a doctor simply takes a specimen of the patient's sputum and creates an acid-fast stain from it, the non-acid-fast human cells will stain blue while any M. tuberculosis cells present will stain an acid-fast red.

As with the gram stain, four steps are used to create the differences seen between acid-fast and non-acid-fast cells. There is an initial stain (carbolfuchsin) that will stain all the cells red. Next, a mordant is used that is not chemical in nature but physical. In this case, heat is used to drive the initial stain into the lipid cell wall where it is permanently bound. We will use a different procedure than explained in the manual as their method is difficult and potentially dangerous. Instead of holding the Bunsen burner over the slide we will place the slide over a beaker of boiling water. This will perform the same function but in a better way. Just be careful in handling the slide over the steam as the steam can scald you. Use tweezers or slide holders to manipulate the slide over the heat.

The decolorizing technique is also more severe than in the gram stain procedure. Here a stronger acid-alcohol reagent is used and the decolorizer is allowed to sit on the smear and then added again until the color stops coming off the slide. This harsh treatment removes the initial stain from the non-acid-fast cells but not from those cells that have absorbed the stain into their lipid layers. These decolorized cells are then made clear again and will accept the counterstain (methylene blue) when it is added at the end.

Thus, acid-fast cells end of red while non-acid-fast cells will be blue. Again, be sure to examine the slides of your neighbors so that you can see both types of cells.

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Hopefully, by now you have gotten the smear technique down so that you are getting nice evenly stained slides without the patches of red and blue. If you are still having troubles with this aspect of the technique, talk to your instructor about possible remedies to your technique. You will be heavily dependent on a proper staining technique in your unknown procedure so you need to have this down pat.

If you did not get a correct stain with a good smear, examine the heating and decolorizing techniques as these are the most likely source of the problem. During heating the slide must be kept hot for at least five minutes and you must continually add the initial stain to prevent the slide from drying out. You also need to be sure that the decolorizing is done until any red color stops coming off but that no additional reagent is added beyond that time.

While we now have better techniques for determining if a patient is dealing with TB, this is still an important technique from a historical point of view and is still used in many countries. It is also a definitive test to supplement the other more modern diagnostic techniques in use today.

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The next series of exercises will deal with factors in the natural environment that affect the growth and survival of microorganisms, concentrating on bacteria. As we have seen in lecture, there are many things that affect the ability of an organism to grow and live. We will examine several of those in lab studies. In each case we must remember that if we want an organism to grow we would provide what they needed and if we wanted them to perish we would not provide them with what they needed.

This module examines the relationship between bacteria and their oxygen needs. There are basically four types of bacteria based on how they make use of oxygen. The obligate aerobes are those that have to have oxygen to survive. The obligate anaerobes are those that must have an oxygen-free environment as oxygen is lethal to them. In between these two are the facultative anaerobes which do not require oxygen but can tolerate its presence. Finally, there are the microaerophiles which need oxygen but in lower than normal concentrations and usually prefer high carbon dioxide concentrations, also.

Since each of these categories contain pathogenic bacteria, as listed in your manual, we need to have ways to provide the correct environment to culture them in when we need to identify them from a patient sample. Conversely, once we have determined their identity we may need to provide a harmful environment to them in order to get rid of them or prevent their growth in the future.

When it comes to growing obligate aerobes and facultative anaerobes, things are pretty simple as the standard petri plates, broth tubes and agar slants that we use are all designed to allow oxygen to reach the cultures. However, when we want to provide an oxygen-free environment we need to make use of several special techniques. One of these is to use a special chemical in the media that is capable of removing oxygen. Thioglycollate is such a chemical so, if you use thioglycollate broth, it will all start as anaerobic but, overtime as the oxygen is diffused into the surface layer, it will eventually develop three distinct layers as shown in your manual.

Another technique for removing oxygen from an environment is to use a Brewer anaerobic jar. This device is made up of a tightly sealed jar and some kind of a packet that contains material that will absorb oxygen and release some type of inert gas to take its place.

Finally, a microaerophilic environment can very easily be created by using an old glass jar and a candle. Remember that a candle consumes oxygen and releases carbon dioxide. Therefore, if we place the cultures into a jar, place a lit candle inside it, and seal it up, the candle will consume most of the oxygen but, before it is all gone, it will go out and leave a low oxygen, high carbon dioxide environment, perfect for growing microaerophiles.

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As you examine the growth of each species in the thioglycollate broth, notice where the growth occurred in relation to the pink aerobic layer at the top of the broth. If the growth is in that area the bacterium is an obligate anaerobe. If it is at the interface of the pink area and the yellow area, it is probably a microaerophilic. The two types of anaerobes will be found throughout the broth if it is a facultative anaerobe and only near the bottom, below the oxygenated area, if it is an obligate anaerobe. Check with your instructor to see if each species grew where it should have.

The Brewer Anaerobic Jar is the best way to grow small amounts of facultative and obligate anaerobes. If they fall into this category, they should grow well here. However, if they are obligate aerobes, there should be little or no growth present.

The candle jar creates the best environment for the microaerophilics, of which Streptococcus pyogeness a perfect example. Therefore, it should grow better inside the candle jar than outside it. However, both plates should show some growth. This microorganism is the cause of strep throat so it is normally grown in a candle jar, or a similar device, after a culture is taken from a patient's throat.

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As has been explained in virtually every biology class you have had, temperature is a significant factor in the life of any organism. This is primarily due to the action it has on the proteins in the cells of the organism. Remember that proteins are important structural components, so too much heat will destroy those components, and that they also make up enzymes, which are necessary for all biochemical reactions occurring in the cell. Enzymes are designed to operate at certain temperature ranges and if they are placed in environments that are either too warm or too cold they will first operate less effectively and then operate not at all.

For this reason, bacteria can be grouped according to the temperature regimens they prefer. If they are adapted to colder temperatures they are called psychrophiles. If they grow best in the middle range of temperatures, such as the human body's temperature, they are called mesophiles. Finally, if they have special enzymes and proteins that allow them to function at higher than normal ranges, they are called thermophiles.

Understanding the temperature range for each bacterium will help you to provide the best environment if you wish to grow the organism or the worst type of environment if you wish to prevent its growth. Obviously, if you are dealing with a thermophile you will need to provide a much higher temperature to destroy it than if you were working with a psychrophile. Microbiologists try to determine the temperature limits under which a bacterium can operate in order to understand how to grow or control them.

There are several aspects that affect the action of heat on bacteria. Obviously, the species will be a factor as some are designed for different temperatures. Since some species produce resistant spores and others form clumps or clusters, this will all affect their susceptibility to heat. The amount of heat and the length of time they are exposed to heat are also important factors. Two terms are used in explaining the relationship between these two factors. Thermal death point (TDP) refers to the temperature an organism is killed by after a set time period, usually 10 minutes. This is the aspect we will examine today. Thermal death time (TDT) is used to refer to the time an organism dies after being exposed to a set temperature. Organisms will have different TDP's and TDT's depending on which temperature category they fall into.

We will be doing this experiment slightly differently from the manual's version in order to be more accurate and more efficient. Scattered around the lab will be five water baths set at specific temperatures. Instead of placing each tube into a beaker of water and chanting the water temperature, you will simply place each tube into the proper water bath before inoculating them onto the agar plates.

Besides survival and growth, temperature can also affect other aspects of a bacterium. We will examine the effect that temperature has on the production of pigments which are related to proteins either because they are composed of proteins or because they require certain enzymes for production.

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The first activity is rather straightforward as you should see either a pigmented culture or a non-pigmented one. Whichever temperature produced the pigmented culture is the temperature required for production of the pigment. The other temperature will probably not cause a significant change in growth but is sufficiently different to change the ability to produce the pigment.

As you examine the plates with the streaks from the different temperature water baths, remember that each streak represents a group of bacteria that were placed in a different temperature for 10 mins. If they were not affected by that temperature regime then you should see growth. If, however, the temperature was too hot for them, you should not see any growth. Whichever is the highest temperature at which you observe growth will be the TDP for that species, within the parameters of this experiment. You should then be able to place each species into one of the three temperature categories dependent on what temperature they were able to grow at.

POST-TEST

PREVIEW

Radiation may seem like an abnormal environmental factor but when you consider that, technically, all types of energy radiated from the sun is considered radiation it is a common factor indeed. The type of radiation we will concentrate on is the ultraviolet radiation that is in the ???nm range. These wavelengths are just the right size to enter the cell and disrupt the DNA in the cell. It does this by forming a dimer mutation that is characterized by the breaking of adenine to thymine bonds and the formation of thymine to thymine bonds. These types of mutations are destructive to the DNA and result in the death of the cell. incidentally, this is also the type of radiation that causes sunburns and tanning and it is the destruction of the skin cells' DNA that is causing this.

However, due to the nature of this type of radiation, several factors can affect how deadly this radiation is to the cell. Because radiation falls off rapidily as the distance between it and the organism increases, the cell has to fairly close to be affected by the radiation. Secondly, the longer you expose the cell to the radiation, the more effect it will have on it. Thirdly, this type of radiation is easily blocked by almost any type of material, including glass and plastic. Finally, some species are capable of repairing the damage to their DNA, to a certain extent, and so can survive the initial affects.

Because of the possibility of damage to all cells, it is very important that you do not expose yourself to the UV light while performing this experiment. This is particularly true of your eyes which are extremely sensitive to these wavelengths. Our set-up is designed to make this extremely difficult to do, however, if you really tried you could so don't!!

PRE-TEST

REVIEW

As you examine the results of the experiment, notice where growth occurred and where it either did not occur or was much reduced. Then go back to what you did for each plate and consciously make the connection between cause and effect. You should see that plate four, which was not exposed to the UV light, probably had great growth. The other plates should have varying degrees of growth and, for plates one and two, there should be a difference between the half of the plate that was covered and the half that was exposed.

POST-TEST

PREVIEW

While the previous factors discussed have all been naturally occurring environmental factors, humans have developed a number of artificial factors to affect the growth and survival of bacteria. In this module we will examine two groups of chemicals that humans have made in the laboratory that are used against microorganisms. These chemicals fall into one of two categories depending on their harshness and strength. The first group are the antiseptics that are mild enough to use on living tissue while the second group are the disinfectants that are too strong to use on anything but inanimate objects. In either case, these chemicals could be either bacteriostatic in that they simply inhibit the growth of microorganism or bacterocidal if they actually kill the cells.

The first of these was carboxylic acid, also known as phenol, and it was used by Joseph Lister in the first attempt to control infections in the hospital setting. Since then, numerous other artificial chemicals have been discovered and your text book lists many of them. Your lab manual, however, lists only four groups with representatives that you need to be familiar with. Phenol is still used today both as a disinfectant and as a way of measuring the relative strength of other antimicrobial agents. The Phenol Coefficient is done by comparing the effectiveness of other agents to phenol, which is given the coefficient of 1. Anything with a number higher than 1 is stronger than phenol, while anything less than 1 is weaker than phenol.

These chemicals are tested in a fairly standard way that allows developers to test the effectiveness of new chemicals against bacteria and as compared to one another in a relatively simple and efficient manner. The technique is usually called the agar diffusion method and makes use of the ability of the chemical to diffuse out from a central area into the agar. As it does so it comes into contact with the test bacteria and either kills them, inhibits them, or does nothing to them. In either case a distinct appearance is seen surrounding the disk that originally contained the chemical and measurements can be made to compare the effect of one chemical to another. Obviously, the more successful antimicrobial agents are those that will leave the largest diameter kill zone around the disk.

However, if you remember anything about the process of diffusion from intro biology classes you may recall that there are several factors that affect the rate of diffusion. Several of these will not be of import to this experiment as each of the chemicals will be kept at the same temperature, which can speed up or slow down diffusion, and the experiment will be run for the same length of time, removing the effect of time on diffusion as a factor. However, the concentration of some of these chemicals will vary and this may affect the diffusion rate and some of these are larger molecules than others and this will slow down their diffusion rate. However, these factors are not sufficient to reduce the effectiveness of this test as an introductory test.

To make this test effective you need to be sure that you do a very complete job of streaking for confluent growth. This is a very different type of streaking than you have done before as the object here is create a dense, complete covering of the plate with the test organisms. Secondly, you need to be sure to use the same test chemicals against both organisms so that you can see any differences between the species and how they react to the different chemicals. Remember back to the module on gram staining and the differences between gram (+) and gram (-) cells in the way they are affected by antiseptics and disinfectants.

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Again, it is important that you examine the two plates together and compare the effect that each chemical had against each species of bacteria. If you were able to get a complete coverage of the plate with the bacteria there should be distinct zones of inhibition around at least some of the antimicrobial agents. By measuring the diameter of the zones you can determine which agent was most affective and how effective it was against each species. At this point you need to determine the gram staining group of each species used and see if your results confirm what was reported in the gram staining module.

POST-TEST

PREVIEW

While the previous module dealt with chemicals that were originally created artificially by humans for use against many types of microorganisms, this module deals with chemicals that originally were created naturally by fungi and bacteria and are effective primarily against bacteria. Humans have then modified these and learned to make them artificially to produce the numerous types of antibiotics we now have available to us. Examples of fungi that make antibiotics include the Penicillium genus while Bacillus and Streptomycetes are examples of bacteria that make antibiotics.

The test used to test the effectiveness of antibiotics is a modification of the technique employed in the previous module. When it is used to test antibiotics, however, it is usually called the Kirby-Bauer Method. This test makes use of disks that are preimpregnated with the antibiotic, at carefully controlled concentrations, and a special agar called Mueller-Hinton Agar. Other than that, the technique is virtually identical.

However, when the petri plates are read following incubation, special care must be taken in examining the zones of inhibition that form around the disks. Table 32-1 in your manual lists the more common antibiotics along with a series of zone diameters. Notice that in most cases even a fairly significant clear zone around the disk may not indicate effectiveness of the agent against the bacteria. These variations are due to such factors as the species used, the concentration of the agent, the amount of inoculum, etc.

Another aspect of this test is the fact that many species of bacteria are developing resistance to many types of antibiotics. This is occurring for a number of reasons including overuse of antibiotics, improper use by patients, and mutations occurring in the bacteria. This is becoming an area of increasing concern for both microbiologists and medical professionals and will impact your career in the future.

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Again, as with the antiseptic/disinfectant test, be sure to examine the two plates next to one another and compare the results. Some of the antibiotics may be effective against one species and ineffective against another. Several antibiotics may be effective against one species but not equally so. Be sure to compare your measurements to the table in the module and record whether the organism was resistant, intermediately susceptible or susceptible to the antibiotics.

If you see any zones that are fairly clear but still have a few bacteria in the zone, you may be seeing examples of resistance against that antibiotic.

POST-TEST

PREVIEW

The next series of tests, from module 33 to module 39, are frequently used as tests in identifying an unknown microorganism. You will see many of these again as you use the BacterioIdent CD-ROM program to work on the unknown bacterium. They are used, in various forms, by clinical labs to diagnose the pathogens that patients are dealing with when a doctor sends in a specimen. They are therefore, very important to all aspects of microbiology.

The first module deals with three of the basic food sources for any organism: starch and proteins. AS with most of these exercises, the tests are dealing with the organisms ability to produce the enzymes necessary to metabolize the various organic compounds. These are frequently called exoenzymes since they are released outside of the cell. This allows the bacterium to break down compounds that are too large to be brought across the cell membrane and create smaller compounds that can make it through the membrane. Since it is the basic genes of the organism that give it the capability to produce these enzymes, these are terrific tools for helping to identify the bacteria.

In the first activity we will look at whether or not organisms can produce amylase which is the enzyme needed to digest starch. If they do produce this enzyme the starch will be broken down into maltose and eventually glucose. This is a type of chemical reaction called a hydrolysis reaction because, along with the enzyme, water must be added to lyze or breakdown the starch. We will be able to determine if this happens by adding iodine to the starch agar plate after incubation. Iodine reacts with starch to create a black/purple complex that will color the starch plate. However, if the bacterium has digested the starch immediately around its colony, there will not be any for the iodine to react with and a clear zone will be seen around the colony. This would indicate a positive starch hydrolysis test.

Activity two and three involve the metabolism of two kinds of proteins. In the first of these, the protein casein will be present in the Milk Agar. This protein is what gives milk its characteristic white color. If your organism produces caseinase, the enzyme required to break milk down, there should be a clearer area around the colony as the casein is hydrolyzed.

Finally, in activity three we will look for the ability to hydrolyze the protein gelatin by producing the enzyme gelatinase. This protein is what makes things like gelatin a solid at room temperature after it has cooled from being a liquid. If it is hydrolyzed the gelatin substance will not stay firm and you will have a liquid again. Therefore, a positive gelatin hydrolysis test will show as an agar that will not harden even after being cooled in ice. The technique used in this test is different from any other you have done as this requires a stab inoculation. The technique here is to take an inoculating needle, not a loop, and coat the bottom portion in the organism. The needle is then stabbed straight down into a gelatin deep until either the handle of the loop come close to the top of the deep or the needle itself comes close to the bottom of the test tube. It is important to check this test at 48 hrs. and at 7 days to see if your organism is a fast liquifier, a slow liquifier or a negative test. After pulling the tube out of the incubator, it may very well be liquefied simply due to the temperature in the incubator. You then need to place it in a beaker of ice, not just in the refrigerator as the manual says, for about 10 minutes. If a significant portion of the agar remains liquid you have a positive test. However if it all hardens you have a negative test. After the 48 hr. test, put it back into the incubator and repeat the test after 7 days.

We are not doing the fat hydrolysis test in this class.

PRE-TEST

REVIEW

In examining the results of these activities the most important thing is remembering what a positive test looks like and what you have to do to test for it. The supplemental lab books available for you in the lab will help as they have photos showing positive and negative test results. With the starch plate, you first have to add iodine which should turn all areas containing starch a blue/black color. If no starch hydrolysis was done by the bacterium, everything should be that color up to and around the colony. If, however, your organism produced amylase, there should be an area that is not that color right adjacent to the colony. It will look like a halo around the entire colony.

The casein hydrolysis test is even easier to see as you do not have to add anything. Simply hold the plate against the light or a dark background and see if there is any change in appearance immediately around the colony. Sometimes this will be a very clear and spectacular clearing. Other times it may be just a thinning of the white in the plate as the caseinase has not had time to completely remove the white casein.

Finally, the gelatinase production requires immersion in an ice bath, being sure that it goes all the way to the top of the a