Ex01_Microscopy_2023.pdf

Exercise 1 Microscopy LEARNING OBJECTIVES 1. Provide a definition of the term microorganism. 2. Compare and contrast the...

Exercise 1 Microscopy LEARNING OBJECTIVES 1. Provide a definition of the term microorganism. 2. Compare and contrast the three domains of life. 3. Cite the purpose for using a microscope. 4. Correctly identify the different parts of a bright light microscope. 5. Calculate the total magnification of a sample being viewed with each objective of a bright light microscope. 6. Distinguish between magnification and resolving power in microscopy. 7. Identify the appropriate type of microscopy to use when viewing a given specimen. 8. Describe how to properly use, care for, transport, and store a bright light microscope. 9. View stained smears using the 100X objective and identify both specimen color and morphology. I N T RO D U C T IO N Biology is the study of life and living organisms and the three domains of life include Eukarya, Archaea, and Bacteria. Microbiology is the study of living organisms that are too small to be observed using the naked eye; these are known as microorganisms. All members of domain Eukarya contain a distinct nucleus which is surrounded by a lipid membrane. Some Eukarya are macroscopic, while others are microscopic. Eukaryotic microorganisms consist of fungi (yeasts and molds), protozoa, algae, and certain forms of the helminths. The yeasts, some algae, and the protozoa are unicellular, while the molds, other types of algae, and the helminths are multicellular. Some eukaryotic microorganisms are responsible for causing some noteworthy human diseases, but others are significant contributors to the production of foods and beverages. On the other hand, members of domains Archaea and Bacteria do not contain a membrane-bound nucleus. Archaea and Bacteria are comprised of the archaeal and bacterial species, respectively. All organisms in these domains are unicellular and most are microscopic in nature. The Archaea are typically found in extreme environments, such as the hot springs of Yellowstone National Park and the deep sea hydrothermal vents, but rarely, if ever, cause clinical disease in humans. Many people Exercise 1 | Microscopy 1 believe that all bacteria cause disease and that they have no positive qualities. However, only a small percentage of the total number of bacterial species on Earth actually produce clinical disease. Bacteria benefit humans by participating in bioremediation (the use of microorganisms to degrade toxic compounds), the biogeochemical cycling of various nutrients required by humans for life, and the production of essential substances, such as insulin. The media collectively refer to microorganisms as “germs.” However, this terminology fails to account for differences in domain classification as well as individual differences between microorganisms. Microorganisms, like humans, demonstrate differences in appearance, size, movement, etc. Throughout the course of this semester, you will study representative members of domains Eukarya and Bacteria, and will learn to appreciate their differences, activities, and scope. In addition, you will also study viruses, which are nonliving entities that are not members of any of the above domains, but actually multiply within cells of the above domains. To begin this journey of discovery, you will need to master some basic concepts and techniques necessary for studying these organisms. Thus, we begin by learning how to observe microorganisms under the microscope. © bluedoor, LLC. Coccus Bacillus Spirillum Figure 1.1: Typical morphologies found in bacteria. You will use a microscope to observe three morphologies (shapes), coccus (circular), bacillus (rod), and spririllum (spiral), which are commonly found in bacteria. See Figure 1.1 for depictions of these bacterial shapes. In addition to having different shapes, microorganisms may display different arrangements of cells. For example, the pathogen Streptococcus pyogenes, the causative agent of strep throat, is a coccus-shaped organism whose cells are commonly arranged in chains resembling a pearl necklace. On the other hand, the coccus-shaped cells of Staphylococcus aureus, the causative agent of some types of foodborne infections, typically appear as grape-like clusters. Differences in both morphology and cellular arrangement are important features that aid in identification of species. It is vital to learn the proper operation of the microscope early so that visualizing bacteria and being able to distinguish these features become routine. Because microorganisms are so small, their study requires the use of a microscope. There are many different types of microscopes available, and each is specialized for viewing various preparations of organisms or their component structures. While in the laboratory, you will use a type of microscope known as a bright light microscope in which visible light rays pass through the specimen from a light source in the base of the microscope. Your microscope contains more than one magnifying lens – an objective lens located above the specimen and an ocular lens through which you view the specimen – and is thus also known as a compound light microscope. A monocular light microscope is a compound microscope with one eyepiece (ocular), while a binocular light microscope has two oculars. 2 Microscopy | Exercise 1 The light source of the microscope is located in the base of the unit. The light passes through the condenser, which collects and converges the light rays. The specimen is placed in the slide holders on the mechanical stage of the microscope and the iris diaphragm controls the amount of light that passes through the specimen. Your microscope contains four objective lenses – the 4X, the 10X (low power), the 40X, (high dry), and the 100X (high power oil). These lenses magnify the image before it is transmitted through the body tube to the ocular lens located in the eyepiece. The ocular lens further magnifies the image. Ocular Lens © bluedoor, LLC. Objective Lenses Arm Specimen Holder Coarse Focus Knob Mechanical Stage Fine Focus Knob Condenser Stage Adjustment Condenser Height Knobs Adjustment Knob Diaphragm Power Base with Light Source Light Intensity Control Figure 1.2: A bright light microscope that is typically found in an introductory microbiology laboratory. Clearly observing your specimen is possible through the manipulation of several knobs on the microscope. The mechanical stage adjustment knobs allow you to move the stage for precise viewing of the specimen on a slide. When the 4X objective is rotated into place, turning the coarse adjustment knob allows for initial visualization of the specimen. The fine adjustment knob is used to bring the specimen into sharp focus once it has been located with the coarse adjustment knob. Your microscope is parfocal, which means that the objectives are mounted so that they can be interchanged without varying the focus greatly. Therefore, once you visualize your image on the 4X objective using the coarse adjustment knob, visualizing your image on the higher power objectives should require the use of the fine adjustment knob only. Refer to Figure 1.2 for a diagram of a microscope with the above items identified. The total magnification of a microscope is dependent upon the combination of the magnifications of the ocular and objective lenses. Each objective lens has the magnification it provides stamped on its side. The microscopes in your laboratory are equipped with 4X, 10X, 40X, and 100X objectives. The ocular lens magnification in your bright light microscope is 10X. Therefore, the total magnification is calculated by multiplying the objective magnification by the ocular magnification. The oil immersion Exercise 1 | Microscopy 3 objective, or 100X objective, is the most important lens in microbiology. However, if this objective is simply rotated into place, no clear image of the specimen would ever be obtained. This is because much of the light passing through the specimen would be refracted by its passage from air into glass and glass into air. Consequently, most of the light would not travel up through the objective lens. Thus, the 100X objective must be used somewhat differently than the other objectives. A drop of oil must be placed on the slide prior to rotating the 100X objective into place. Because oil has the same refractive index as glass, the loss of light passing through the lens is minimized. However, our ability to magnify an image is not the only factor that influences our ability to view a specimen. Resolution, or our ability to distinguish between two objects as separate entities, is also important. The wavelength of light used to illuminate the specimen and the numerical aperature (a measure of the light-gathering ability of a lens) dictate the resolution of the microscope. The shorter the wavelength of light and the higher the numerical aperature of the lens used, the better the resolution. When light is used to illuminate a specimen, the highest resolution that can be achieved is approximately 0.2 μm; therefore, objects closer than 0.2 μm apart cannot be resolved using a bright light microscope. Higher resolution can be achieved using electron microscopes, which are discussed below. The bright light microscope is useful for observing stained specimens and for enumeration of microorganisms. Because these microscopes are relatively inexpensive (the microscopes in the laboratory cost approximately $1300 each) and easy to use, bright light microscopes are found in a majority of introductory microbiology laboratories. In a dark-field microscope an object that blocks the light rays from being transmitted directly through the specimen is placed in the condenser so light is reflected off the specimen at an angle. Therefore, a light specimen is observed against a dark background. Dark-field microscopy is useful for viewing unstained, living specimens, as well as specimens that are difficult to stain and cannot be viewed by traditional bright light microscopy. A spiral shaped organism known as a spirochete is an example of an organism that can readily be viewed using dark-field microscopy. In order to examine the intracellular structures of a particular organism, another type of microscope must be used. Living organisms die when they are affixed to slides and stained, so intracellular structures are not visible. However, living specimens are examined using a phase-contrast microscope. In this type of microscopy, small differences in the refractive properties of the various cellular organelles are enhanced. Therefore, the microscopist detects variations in brightness between the organism, the background, and between organelles themselves. Additionally, one can visualize motility of various organisms using this type of microscope. Fluorescence microscopy is commonly used in medical diagnostics and scientific research. In this type of microscopy the specimen is coated with a fluorochrome dye. Upon exposure to ultraviolet light, molecules in the specimen are excited to emit light of a different wavelength. The different wavelengths emitted can be orange, yellow, or green, and are visualized against a dark background, as in dark-field microscopy. Many diagnostic procedures utilize fluorescent antibody staining to determine if a patient has been exposed to a particular infectious agent. For example, when the bacterium Mycobacterium tuberculosis is coated with the fluorochrome auramine O and exposed to ultraviolet light, the organisms glow yellow against a dark background. This allows researchers to quickly determine if these organisms are present in a sputum sample. This technique allows for diagnoses to be made much sooner than if other microscopic techniques are used. Electron microscopy utilizes a beam of electrons, rather than light waves, to increase the resolving power. Also, in much the same way that glass lenses are used to focus light waves, electron magnets 4 Microscopy | Exercise 1 are used to focus the beam of electrons. Because the wavelength of an electron beam is 1,000 times shorter than that of visible light waves, the resolving power and degree of magnification of an electron microscope are greatly increased. There are two different types of electron microscopes – transmission electron microscopes (TEM) and scanning electron microscopes (SEM). In TEM, a beam of electrons is directed through the specimen. This allows for visualization of the internal structures of the specimen. On the other hand, SEM allows for the visualization of the surface details of the specimen because the beam of electrons is scattered back and forth across the surface of the specimen. Although microscopes are a common piece of equipment of the laboratory and will be used quite often, they should always be treated with care. The microscopes are housed in special cabinets at your workstation and must be transported to the workspace by gripping the microscope arm with one hand and by supporting the base of the microscope with the other hand. The microscope should be carried close to the body so that collisions with other students and furniture are minimized. Never attempt to carry more than one microscope at a time. The microscope should be gently placed on the laboratory bench. Adjust the height of your stool so that you can comfortably look through the oculars without tilting the microscope. The electric wire of the microscope should be uncoiled and plugged into a nearby electric outlet. The electric cord should not be allowed to dangle over the side of the workspace. Small pieces of dust, lint, oil, or eyelashes will reduce the efficiency of the microscope, so each objective lens and the ocular lenses should be cleaned before the microscope is used. Clean the lenses with lens paper (found in the drawer at your workstation) moistened with a small amount of 70% ethanol. Paper towels and Kimwipes should never be used to clean a lens. The procedures for focusing the microscope are outlined in the procedure below. When looking through the oculars, be sure to observe the slide with both eyes open to avoid eyestrain. If you have a problem with a microscope, always obtain help from the graduate teaching assistant. Once the laboratory exercise is completed, the microscope must be cleaned and returned to its original position in the cabinet. Any slides should first be removed from the stage. All lenses should be wiped clean with lens paper moistened with 70% ethanol. The 4X objective should be rotated into position and the stage should be moved away from the objective lenses using the coarse objective knob. The stage should be centered and the electrical cord should be wrapped neatly around the bottom of the stage. The microscope should be carefully transported to its designated slot in the appropriate cabinet. The microscope should be stored with the arm towards the front of the microscope cabinet. Exercise 1 | Microscopy 5 Today’s Procedure 1. Each student should collect one each of the three slides provided. Each slide is a stained smear of bacteria displaying rod, cocci, or spiral morphology. 2. Begin with the 4X objective in the down position. Place the slide (specimen side up), on the stage and move the stage to center the specimen directly over the light source. 3. Using the coarse adjustment knob, raise the stage to its highest position while watching this procedure from the side. Do not do this procedure while looking through the ocular lenses. 4. While looking into the ocular lenses, slowly lower the body tube using the coarse adjustment knob until the specimen comes into focus. You may use the fine adjustment knob to bring the specimen into sharp focus. 5. Open and close the iris diaphragm and raise and lower the condenser. Notice the effects each has on the appearance of the specimen. Adjust both the diaphragm and condenser to achieve optimum illumination. 6. When rotating a higher power objective (10X and 40X) into place, recall that a turn of the fine adjustment knob will bring the specimen into focus. 7. To view the specimen with the 100X objective, place a small drop of oil directly over the area of the slide illuminated by the light source. The oil can be found in the drawer at your bench space. Rotate the 100X objective into the oil so that the lens contacts the oil. Look through the oculars and focus the specimen using the fine adjustment knob. You may move the mechanical stage back and forth to look for the specimen. 8. If you cannot locate your specimen using the 100X objective, do not simply rotate the 40X objective (or any lower power objective) back into place. This will allow oil to get into the 40X lens, which will ruin the lens. You must use a Kimwipe to remove the oil from the slide before rotating the 40X objective into place. 9. Each student should view each of the three slides and be able to identify which slide contains stained rods, cocci, and spirilla. It is important that students become familiar with the use of the microscope at this point as it will be useful in future exercises. 10. Record your results. 11. When you are finished, do not return your slides to the slide boxes. Dispose of the slides by placing them in the pan of quat 50 disinfectant located in your laboratory. 12. Clean the microscope as described earlier and return the microscope to the proper microscope cabinet. 6 Microscopy | Exercise 1 Results Slide 1 Slide Color: ______________________ Morphology: _____________________ Color of Organisms: _______________ Magnification: ____________________ Slide 2 Slide Color: ______________________ Morphology: _____________________ Color of Organisms: _______________ Magnification: ____________________ Slide 3 Slide Color: ______________________ Morphology: _____________________ Color of Organisms: _______________ Magnification: ____________________ Exercise 1 | Microscopy 7