Microscopy Exercises PDF

PART Microscopy 1 EXERCISES 1 Use and Care of the Microscope 2 Examination of Living Microorganisms Antoni van Leeuwenhoek is the first person known to have observed living microbes. His observations of the microbiota in tooth tartar and feces were the first descriptions of the human microbiome....

PART Microscopy 1 EXERCISES 1 Use and Care of the Microscope 2 Examination of Living Microorganisms Antoni van Leeuwenhoek is the first person known to have observed living microbes. His observations of the microbiota in tooth tartar and feces were the first descriptions of the human microbiome. Unfortunately, he was very protective of his homemade microscopes and left no description of how to make them (see the photograph on page 2). During his lifetime he kept “for himself alone” his microscopes and his method of observing “animalcules.” Directions for making a replica of van Leeuwenhoek’s microscope can be found in American Biology Teacher.* Fortunately, you will not have to make your own microscope. The microscope is a very important tool for a microbiologist. Microscopes and microscopy (microscope technique) are introduced in Exercise 1 and 2, which are designed to help you become familiar with the compound light microscope and proficient in using it. This knowledge will be valuable in later exercises. Beginning students frequently become impatient with the microscope and forgo this opportunity to practice and develop their observation skills. Simple observation is a critical part of any science. Making discoveries by observation requires curiosity and patience. We cannot provide procedures for observation, but we can offer this suggestion: Make careful sketches to enhance effective observation. You need not be an artist to draw what you see. In your drawings, pay special attention to: 1. Size relationships. For example, how big are bacteria relative to protozoa? 2. Spatial relationships. For example, where is one bacterium in relation to the others? Are they all together in chains? 3. Behavior. For example, are individual cells moving, or are they all f lowing in the liquid medium? Looking at objects through a microscope is not easy at first, but with a little practice, you, like Antoni van Leeuwenhoek, will make discoveries in the microcosms of peppercorn infusions and raindrops. In 1684, van Leeuwenhoek wrote the following: Tho my teeth are kept usually very clean, nevertheless when I view them in a Magnifying Glass, I find growing between them a little white matter as thick as wetted flour: In this substance tho I do not perceive any motion, I judged there might probably be living creatures. *W. G. Walter and H. Via. “Making a Leeuwenhoek Microscope Replica.” American Biology Teacher 30(6): 537–539, 1968. 9 I therefore took some of this flour and mixed it either with pure rain water wherein were no Animals; or else with some of my Spittle (having no air bubbles to cause a motion in it) and then to my great surprise perceived that the aforesaid matter contained very many small living Animals, which moved themselves very extravagantly. Their motion was strong and nimble, and they darted themselves thro the water or spittle, as a Jack or Pike does thro the water.* *Quoted in E. B. Fred. “Antoni van Leeuwenhoek.” Journal of Bacteriology 25: 1, 1933. 1 CENTIMETERS Lens 2 Location of specimen on pin 3 Specimen-positioning screw 4 Focusing control 5 6 Stage-positioning screw 7 8 9 A replica of the simple microscope made by Antoni van Leeuwenhoek to observe living organisms too small to be seen with the naked eye. The specimen was placed on the tip of the adjustable point and viewed from the other side through the tiny round lens. The highest magnification with his lenses was about 300 ×. CASE STUDY: Too Many Slides Your first microbiology field trip is to a large university hospital lab. Urinary tract infections are quite common, so it is not surprising that urine specimens make up a large proportion of the samples submitted for routine laboratory diagnosis. Nevertheless, you are surprised to learn that the lab technicians may examine 200 microscope slides of urine every day. You are given the opportunity to look at some of the slides and are asked to describe any microorganisms that you see. Use the following choices to indicate which type of microorganism the one described in each question is most likely to be. a. b. c. d. Alga Bacterium Fungus Protozoan Questions 1. In a wet mount of urine, you observe f lagellated, nucleated cells. Which type of microorganism is most likely? 2. In a fixed, stained slide, you don’t see any cells until you use the oil immersion objective. Which type of microorganism is most likely? 3. In a wet mount of the scrapings, you observe long filaments composed of many cells. Which type of microorganism is most likely? 10 Use and Care of the Microscope EXERCISE 1 The most important DISCOVERIES of the laws, methods and progress of nature have nearly always sprung from the EXAMINATION of the smallest objects which she contains. –JEAN BAPTISTE LAMARCK OBJECTIVES After completing this exercise, you should be able to: 1. 2. 3. 4. Demonstrate the correct use of a compound light microscope. Name the major parts of a compound microscope. Determine the relative sizes of different microbes. Identify the three basic morphologies of bacteria. BACKGROUND ASM: Demonstrate ability in using a brightfield light microscope to view and interpret slides, including (a) correctly setting up and focusing the microscope, (b) Proper handling, cleaning, and storage of the microscope, (c) correctly using all lenses, and (d) recording microscopic observations. Virtually all organisms studied in microbiology are invisible to the naked eye and require the use of optical systems for magnification. The microscope was invented shortly before 1660 by Zacharias Janssen of the Netherlands. The microscope was not used to examine microorganisms until the 1660s, when a clerk in a dry-goods store, Antoni van Leeuwenhoek, examined scrapings of his teeth, feces, and any other substances he could find. The early microscopes, called simple microscopes, consisted of biconvex lenses and were essentially magnifying glasses. (See the photograph on page 2.) To see microbes requires a compound microscope, which has two lenses between the eye and the object. This optical system magnifies the object, and an illumination system (sun and mirror or lamp) ensures that adequate light is available for viewing. A brightfield compound microscope, which shows dark objects in a bright field, is used most often. Play Lab Technique Video with Pre-Lab Quiz @MasteringMicrobiology Compound Microscope The Microscope You will be using a brightfield compound microscope similar to the one shown in FIGURE 1.1a. The basic frame of the microscope consists of a base, a stage to hold the slide, an arm for carrying the microscope, and a body tube for transmitting the magnified image. The stage may have two clips or a movable mechanical stage to hold the slide. The light source is in the base. Above the light source is a condenser, which consists of several lenses that concentrate light on the slide by focusing it into a cone, as shown in FIGURE 1.1b. The condenser has an iris diaphragm, which controls the angle and size of the cone of light. This ability to control the amount of light ensures that optimal light will reach the slide. Above the stage, on one end of the body tube, is a revolving nosepiece holding three or four objective lenses. At the upper end of the tube is an ocular or eyepiece lens (10* to 12.5* ). If a microscope has only one ocular lens, it is called a monocular microscope; a binocular microscope has two ocular lenses. Focusing the Microscope By moving the lens closer to the slide or the stage closer to the objective lens, using the coarse- or fineadjustment knob, one can focus the image. The larger knob, the coarse adjustment, is used for focusing with the low-power objectives (4* and 10* ), and the smaller knob, the fine adjustment, is used for focusing with the high-power and oil immersion lenses. The coarse-adjustment knob moves the lenses or the stage longer distances. The area seen through a microscope is called the field of vision. Depth of field is the thickness of the object that is in focus at one time. The magnification of a microscope depends on the type of objective lens used with the ocular. Compound microscopes have three or four objective lenses mounted on a nosepiece: scanning (4* ), low-power (10*), high-dry (40* to 45* ), and oil immersion (97* to 100* ). The magnification provided by each lens is stamped on the barrel. The total magnification of the object is calculated by multiplying the magnification of the ocular (usually 10* ) by the magnification of the objective lens. The most important lens in microbiology is the oil immersion lens; it has 11 12 EXERCISE 1: USE AND CARE Of ThE MICROSCOPE Ocular lens (eyepiece) Remagnifies the image formed by the objective lens Line of vision Ocular lens Path of light Body tube Transmits the image from the objective lens to the ocular lens Arm Prism Body tube Objective lenses Primary lenses that magnify the specimen Objective lenses Mechanical stage Holds the microscope slide Specimen Condenser Focuses light through specimen Condenser lenses Diaphragm Controls the amount of light entering the condenser Illuminator Illuminator Light source Base Coarse focusing knob Fine focusing knob Used for focusing the specimen; turning the knob changes the distance between the objective lens and the specimen Light intensity Adjusts current to lamp (a) Principal parts and functions Base with source of illumination (b) The path of light (bottom to top) FIGURE 1.1 The compound light microscope. (a) Its principal parts and their functions. (b) Blue lines from the light source through the ocular lens trace the path of light. the highest magnification (97* to 100* ) and must be used with immersion oil. Optical systems could be built to magnify much more than the 1000* magnification of your microscope, but the resolution would be poor. The Light Source Compound microscopes require a light source. The light may be ref lected to the condenser by a mirror under the stage. If your microscope has a mirror, the sun or a lamp may be used as the light source. Most compound microscopes have a built-in illuminator in the base. The intensity of the light can be adjusted with a wheel that regulates the amount of current to the bulb. Higher magnification usually requires more light, which can be obtained by adjusting the iris diaphragm. Resolution Resolution, or resolving power, is the ability of a lens to reveal fine detail or two points distinctly separated. An example of resolution involves a car approaching you at night. At first only one light appears, but as the car nears, you can distinguish two headlights. The resolving power is a function of the wavelength of light used and a characteristic of the lens system called numerical aperture. Resolving power is best when two objects are seen as distinct even though they are very close together. Resolving power is expressed in units of length; the smaller the distance, the better the resolving power. Resolving power = Wavelength of light used 2 * numerical aperture Smaller wavelengths of light improve resolving power. The effect of decreasing the wavelength can be seen in electron microscopes, which use electrons as a source of “light.” The electrons have an extremely short wavelength and result in excellent resolving power. A light microscope has a resolving power of about 200 nanometers (nm), whereas an electron microscope has a resolving power of less than 0.2 nm. The numerical aperture is engraved on the side of each objective lens (usually abbreviated N.A.). Increasing the numerical aperture—for example, from 0.65 to 1.25—improves the resolving power. The numerical aperture depends on the maximum angle of the light entering the objective lens and on the refractive index (the amount the light bends) of the material (usually air) between the EXERCISE 1: USE AND CARE Of ThE MICROSCOPE Unrefracted light Oil immersion objective lens Without immersion oil, most light is refracted and lost Immersion oil Air Glass slide Condenser lenses Condenser Iris diaphragm Light source FIGURE 1.2 Refractive index. Because the glass microscope slide and immersion oil have the same refractive index, the oil keeps the light rays from refracting. objective lens and the slide. This relationship is defined by the formula: N.A. = N sin θ, where N = Refractive index of the medium between the objective lens and the slide θ = Angle between the most divergent light ray gathered by the lens and the center of the lens As shown in FIGURE 1.2, light is refracted when it emerges from the slide because of the change in medium as the light passes from glass to air. When immersion oil is placed between the slide and the oil immersion lens, the light ray continues without refraction because immersion oil has the same refractive index (N = 1.52) as glass (N = 1.52). This can be seen easily. When you look through a bottle of immersion oil, you cannot see the glass rod in it because of the identical N values of the glass and immersion oil. Using oil minimizes light loss, and the lens focuses very close to the slide. As light rays pass through a lens, they are bent to converge at the focal point, where an image is formed (FIGURE 1.3a). When you bring the center of a microscope field into focus, the periphery may be fuzzy because of the curvature of the lens, resulting in multiple focal points. This is called spherical aberration (FIGURE 1.3b). Spherical aberrations can be minimized by using the iris diaphragm, which eliminates light rays to the periphery of the lens, or by a series of lenses resulting in essentially a flat optical system. Sometimes a multitude of colors, or chromatic aberration, is seen in the field (FIGURE 1.3c). This is caused by the prism-like effect of the lens as various wavelengths of white light pass through to a different focal point for each wavelength. Chromatic aberrations can be minimized by using filters (usually blue); or by lens systems corrected for red and blue light, called achromatic lenses; or by lenses corrected for red, blue, and other wavelengths, called apochromatic lenses. The most logical, but most expensive, method of eliminating chromatic aberrations is to use a light source of one wavelength, or monochromatic light. GENERAL GUIDELINES The microscope is a very important tool in microbiology, and it must be used carefully and correctly. Follow these guidelines every time you use a microscope: 1. Carry the microscope with both hands: one hand beneath the base and one hand on the arm. 2. Do not tilt the microscope; instead, adjust your stool so you can comfortably use the instrument. 3. Observe the slide with both eyes open, to avoid eyestrain. Lens Lens Lens 13 Focal point (a) (b) (c) FIGURE 1.3 focal point. (a) An image is formed when light converges at one point, called the focal point. (b) Spherical aberration. Because the lenses are curved, light passing through one region of the lens has a different focal point from light passing through another part of the lens. (c) Chromatic aberration. The lens may give each wavelength of light a different focal point. 14 EXERCISE 1: USE AND CARE Of ThE MICROSCOPE 4. Always focus by moving the lens away from the slide. 5. Always focus slowly and carefully. 6. When using the low-power lens, keep the iris diaphragm barely open to achieve good contrast. Higher magnification requires more light. 7. Before using the oil immersion lens, have your slide in focus under high power. Always focus with low power first. 8. Keep the stage clean and free of oil. Keep all lenses except the oil immersion lens free of oil. 9. Keep all lenses clean. Use only lens paper to clean them. Wipe oil off the oil immersion lens before putting your microscope away. Do not touch the lenses with your hands. 10. Clean the ocular lens carefully with lens paper. If dust is present, it will rotate as you turn the lens. If needed, wet the lens paper with optical lens cleaner. 11. After use, remove the slide, wipe oil off it, put the dust cover on the microscope, and return it to the designated area. 12. When a problem does arise with the microscope, obtain help from the instructor. Do not use another microscope unless yours is declared “out of action.” Materials Compound light microscope Immersion oil Lens paper and optical lens cleaner Prepared slides of algae, fungi, protozoa, and bacteria PROCEDURE 1. Place the microscope on the bench squarely in front of you. 2. Obtain a slide of algae, fungi, or protozoa, and place it in the clips on the mechanical stage. 3. Adjust the eyepieces on a binocular microscope to your own personal measurements. a. Look through the eyepieces and, using the thumb wheel, adjust the distance between the eyepieces until one circle of light appears. b. With the low-power (10*) objective in place, cover the left eyepiece with a small card and focus the microscope on the slide. When the right eyepiece has been focused, remove your hand from the focusing knobs and cover the right eyepiece. Looking through the microscope with your left eye, focus the left eyepiece by turning the eyepiece adjustment. Make a note of the number at which you focused the left eyepiece so you can adjust any binocular microscope for your eyes. (a) (b) (c) FIGURE 1.4 focusing the condenser. (a) Using low power, lower the condenser until a distinct circle of light is visible. (b) Center the circle of light using the centering screws. (c) Open the iris diaphragm until the light just fills the field. 4. Raise the condenser up to the stage. On some microscopes, you can focus the condenser by the following procedure: a. Focus with the 10* objective. b. Close the iris diaphragm so only a minimum of light enters the objective lens. c. Lower the condenser until you see the light as a circle in the center of the field. On some microscopes, you can center the circle of light (FIGURE 1.4) by using the centering screws found on the condenser. d. Raise the condenser up to the slide, lower it, and stop when the color on the periphery changes from pink to blue (usually 1 or 2 mm below the stage). 5. Open the iris diaphragm until the light just fills the field. 6. Adjust the contrast by changing the diaphragm opening. Diagram some of the cells on the slide under low power. Use a minimum of light by adjusting the ____________________________. 7. When you have brought an image into focus with low power, rotate the nosepiece to the next lens, and the subject will remain almost in focus. All of the objectives (with the possible exception of the 4*) are parfocal; that is, when a subject is in focus with one lens, it will be in focus with all of the lenses. However, the working distance—that is, the distance between the objective lens and the specimen—will change. In general, the working distance decreases as magnification increases. When you have completed your observations under low power, swing the high-dry objective into position and focus. Use the fine adjustment. Only a slight adjustment should be required. Why? ____ ______________________________________________ _______________________________________________ _______________________________________________ More light is usually needed at a higher magnification. How can you increase the amount of light? ______________________________________________ ______________________________________________ Again, draw the general size and shape of some cells. EXERCISE 1: USE AND CARE Of ThE MICROSCOPE (a) Move the high-dry lens out of position. (b) Place a drop of immersion oil in the center of the slide. (c) Move the oil immersion lens into position. FIGURE 1.5 Using the oil immersion lens. 15 8. Move the high-dry lens out of position, and place a drop of immersion oil on the area of the slide you are observing. Carefully click the oil immersion lens into position. It should now be immersed in the oil (FIGURE 1.5). Careful use of the fineadjustment knob should bring the object into focus. Note the shape and size of the cells. Did the color of the cells change with the different lenses? ________ _______________________________________________ Did the size of the field change? ________________ 9. Record your observations, and note the magnifications. (Use the following figures as references: Algae: Figure 2.1a and b on page 14 and 34.3 on page 267; protozoa: Figure 2.1c on page 14 and 35.1 on page 273; fungi: Figure 33.2 on page 256.) 10. Repeat this procedure with all the available slides. When observing the bacteria, note the three different morphologies, or shapes, shown in FIGURE 1.6. When your observations are completed, move the nosepiece to bring a low-power objective into position. Do not rotate the high-dry (40*) objective through the immersion oil. Remove the slide. Clean the oil off the objective lens with lens paper, and clean off the slide with tissue paper or a paper towel. 16 EXERCISE 1: USE AND CARE Of ThE MICROSCOPE (a) Bacillus (plural: bacilli) or rod FIGURE 1.6 Basic shapes of bacteria. (b) Coccus (plural: cocci) (c) Spiral Name: _____________________________________________ Date: _________________ Lab Section: ___________ EXERCISE LABORATORY REPORT Use and Care of the Microscope PURPOSE 1 ________________________________________________________________________________ ______________________________________________________________________________________________ ______________________________________________________________________________________________ EXPECTED RESULTS 1. The high-dry lens will be optimal for observing multicellular fungi or algae. Agree/disagree 2. The oil immersion objective is necessary to determine the morphology of prokaryotes. Agree/disagree RESULTS Microscope number: ___________________________ Monocular or binocular: ____________________________ Eyepiece adjustment notes: _______________________________________________________________________ Draw a few representative cells from each slide, and show how they appeared at each magnification. Note the differences in size at each magnification. Algae Slide of ______________________________________ Total magnification ____ * ____ * ____ * 17 18 EXERCISE 1: USE AND CARE Of ThE MICROSCOPE Fungi Slide of ______________________________________ Total magnification ____ * ____ * ____ * ____ * ____ * ____ * ____ * Protozoa Slide of ______________________________________ Total magnification ____ * Bacteria Slide of ______________________________________ Be sure to sketch all bacterial shapes observed. Total magnification ____ * EXERCISE 1: USE AND CARE Of ThE MICROSCOPE 19 CONCLUSIONS 1. Do your results agree with your expected results? _______________________________________________________ 2. What was the largest organism you observed? ___________________________________________________________ The smallest? ________________________________________________________________________________________ 3. What three bacterial shapes did you observe? ___________________________________________________________ 4. How does increased magnification affect the field of vision? ______________________________________________ 5. How does increased magnification affect the depth of field? ______________________________________________ QUESTIONS 1. Why is it desirable that microscope objectives be parfocal? _______________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ 2. Which objective focuses closest to the slide when it is in focus? ____________________________________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ 3. Which controls on the microscope affect the amount of light reaching the ocular lens? ______________________ _____________________________________________________________________________________________________ _____________________________________________________________________________________________________ CRITICAL THINKING 1. Assume the diameter of the field of vision in your microscope is 2 mm under low power. If one Bacillus cell measures 2 μm long, how many Bacillus cells could fit end-to-end across the field? How many 10@μm yeast cells could fit across the field? 20 EXERCISE 1: USE AND CARE Of ThE MICROSCOPE 2. Name two ways in which you can enhance the resolving power. 3. What are the advantages of the low-power objective over the oil immersion objective for viewing fungi or algae? 4. What would occur if water were accidentally used in place of immersion oil? CLINICAL APPLICATION Assume you are looking for microorganisms in a tissue sample from a lung biopsy. The microbes become apparent when you switch to 1000* . What type of microbe is most likely? Examination of Living Microorganisms EXERCISE 2 M MICROBIOME OBJECTIVES After completing this exercise, you should be able to: 1. 2. Prepare and observe wet-mount slides and hangingdrop slides. Distinguish motility from Brownian movement. BACKGROUND ASM: Demonstrate ability in properly preparing slides for microbiological examination including performing wet mount and/or hanging drop preparations and recording microscopic observations. In brightfield microscopy, objects are dark and the field is light. Brightfield microscopy can be used to observe unstained microorganisms in wet mounts and hanging drop slides. Observing Living Microbes In this exercise, you will examine, using wet-mount techniques, different environments to help you become aware of the numbers and varieties of microbes found in nature. The microbes will exhibit either Brownian movement or true motility. Brownian movement is not true motility but rather movement caused by the molecules in the liquid striking an object and causing the object to bounce. In Brownian movement, the particles and microorganisms all vibrate at about the same rate and maintain their relative positions. Motile microorganisms move from one position to another. Their movement appears more directed than Brownian movement, and occasionally the cells may spin or roll. Many kinds of microbes, such as protozoa, algae, fungi, and bacteria, can be found in pond water and in infusions of organic matter. The microbial communities in environments are called microbiomes. Van Leeuwenhoek made some of his discoveries using a peppercorn infusion similar to the one you will see in this exercise. Direct examination of living microorganisms is very useful in determining size, shape, and movement. Using a wet mount is a fast way to observe bacteria, but motility is difficult to determine because of the movement of the water. Motility and larger microbes are more easily observed in the greater depth provided by a hanging drop. Using a petroleum jelly seal reduces evaporation of the suspended drop of f luid. In this exercise, you will examine living microorganisms by brightfield microscopy. Materials Slides Coverslips Hanging-drop (depression) slide Petroleum jelly Toothpick Pasteur pipettes Alcohol Gram’s iodine Inoculating loop Cultures Hay infusion, incubated 1 week in light Hay infusion, incubated 1 week in dark Peppercorn infusion 18- to 24-hour-old broth culture of Bacillus Techniques Required Compound light microscopy (Exercise 1) PROCEDURE Wet-Mount Technique 1. Suspend the infusions by stirring or shaking them carefully. Using a Pasteur pipette, transfer a very small drop of one hay infusion to a slide, or transfer a loopful using the inoculating loop, as demonstrated by your instructor. 2. Handle the coverslip carefully by its edges, and place it on the drop. 21 22 E X E R C I S E 2 : E X a M I n at I O n O f L I v I n g M I C R O O R g a n I S M S Euglena Diatoms (a) Algae Chlamydomonas Spirogyra Volvox Scenedesmus (b) Algae Paramecium Amoeba Stylonychia Vorticella Heteronema (c) Protozoa FIGURE 2.1 Eukaryotes found in pond water. Some common algae and protozoa that can be found in infusions. 3. Gently press on the coverslip with the end of a pencil or loop handle. 4. Place the slide on the microscope stage and observe it with low power (10*). Adjust the iris diaphragm to admit a small amount of light. Concentrate your observations on the larger, more rapidly moving organisms. At this magnification, bacteria are barely discernible as tiny dots. FIGURE 2.1 and Figure 34.3 on page 267 may help you to identify some of the microorganisms. 5. Examine the slide with the high-dry lens (40* to 45* ); increase the light and focus carefully. Some microorganisms are motile, whereas others exhibit Brownian movement. 6. After recording your observations, examine the slide with the oil immersion lens. Bacteria should now be magnified sufficiently to be seen. 7. If you want to observe the motile organisms further, place a drop of alcohol or Gram’s iodine at the edge of the coverslip, and allow it to run under and mix with the infusion. What does the alcohol or iodine do to these organisms? ____________________ ______________________________________________ You can now observe them more carefully. 8. Record your observations, noting the relative sizes and the shapes of the organisms (See Figure 1.6.). 9. Make a wet mount from the other hay infusion, and observe it. Record your observations. 10. Discard slides and coverslips in the disinfectant jar. Wipe the oil from the objective lens with lens paper. E X E R C I S E 2 : E X a M I n at I O n O f L I v I n g M I C R O O R g a n I S M S Hanging-Drop Procedure Petroleum jelly (a) Place a ring of petroleum jelly around the edge of a coverslip. Microbial suspension Petroleum jelly (b) Place a drop of an infusion in the center of the coverslip. Depression slide (face down) (c) Place the depression slide on the coverslip. Microscope objective lens Petroleum jelly Depression slide (face up) (d) Turn the slide over; place the slide, coverslip up, on the microscope stage; and observe it under the low and high-dry objectives. FIGURE 2.2 Hanging-drop preparation. 23 1. Obtain a clean hanging-drop (depression) slide. 2. Pick up a small amount of petroleum jelly on a toothpick. 3. Pick up a coverslip (by its edges), and carefully touch the petroleum jelly with an edge of the coverslip to get a small rim of petroleum jelly. Repeat with the other three edges (FIGURE 2.2a), keeping the petroleum jelly on the same side of the coverslip. 4. Place the coverslip on a paper towel, with the petroleum jelly–side up. 5. Transfer a small drop or loopful of the peppercorn infusion to the coverslip (FIGURE 2.2b). 6. Place a depression slide (well-side down) over the drop, and quickly invert it so the drop is suspended in the well (FIGURE 2.2c). Why should the drop be hanging? _____________________________________ ______________________________________________ 7. Examine the drop under low power (FIGURE 2.2d) by locating the edge of the drop and moving the slide so the edge of the drop crosses the center of the field. 8. Reduce the light with the iris diaphragm, and focus. Observe the different sizes, shapes, and types of movement. 9. Switch to high-dry and record your observations. Do not focus down. Why not?____________________ _______________________________________________ Do not attempt to use the oil immersion lens. Why not? _______________________________________________ 10. When finished, discard your slides and coverslips in disinfectant. Using a new coverslip, repeat the procedure with the culture of Bacillus. Record your observations. 11. Return your microscope to its proper location.