Practical 1_ Safety rules and the Light Microscope PDF

Microbiology Laboratory 0490-105 Faculty of Science Department of Biological Sciences Microbiology Program 1 Safety Rules 1. Lab coats must be worn during the lab session. 2. Long hair must be tied back in such a manner as to not be...

Microbiology Laboratory 0490-105 Faculty of Science Department of Biological Sciences Microbiology Program 1 Safety Rules 1. Lab coats must be worn during the lab session. 2. Long hair must be tied back in such a manner as to not be in danger of catching fire. 3. NO MOUTH PIPETTING. 4. Students must know where the safety equipment is located; they will be informed of this at the beginning of the semester by the instructor. 5. No unauthorized or unsupervised experiments are permitted. 6. No eating or drinking is permitted in the lab. 7. Students should wash their hands at the conclusion of every lab. 8. Fingers, pencils and other instruments are to be kept out of the mouth. 9. Students should avoid touching (rubbing) their eyes, mouth or skin before washing of hands. 10. Clean and sterilize your working area before and after working in the lab. 11. Use sterile tools and media. 12. All accidents are to be reported to the lab instructor. 13. Culture or reagent spills are to be reported to and cleaned up only by the instructor. The student responsible for the spill is required to inform the instructor & class members of any such accidents immediately. 14. Cultures, reagent and other materials can not be removed from the laboratory unless specific permission is granted. 15. Any accidents involving the release of BLOOD (e.g. glass cuts etc) must be reported to the lab Instructor immediately. 16. At the end of each lab session, make sure that you have placed all culture tubes and plates in their designated areas. 17. All used cultures, pipettes, tubes, and glass slides must be placed at designated places at the end of each lab session. 18. Make sure that you return the microscope to its proper place after cleaning it. 19. Turn off the gas burners when not in use. 2  Practical (1) The use of Light Microscopy In this Lab session, you will learn: 1. The parts of a compound light microscope, 2. The functions of those parts, and the proper use and care of the microscope. 3. You will also learn the technique of preparing wet-mount slides. 4. Use of oil immersion lens. 5. Use of the stereo microscope. Introduction The light microscope, so called because it employs visible light to illuminate small objects, is probably the commonly used research tool in biology. Most microbial specimens are too small to be seen by the eye alone, thus requires the use of an instrument that magnifies, or visually enlarges, the object. One such instrument, which is of great importance to microbiologists and other scientists, is the bright-field microscope. It is called a bright-field microscope because it forms a dark image against a bright background. Modern microscopes consist of two sets of lenses (objective lenses and ocular lenses, Figure 1), therefore also referred to as a compound light microscope. A compound light microscope consists of a light source or mirror that illuminates, an objective lens that magnifies the image of a specimen, and an eyepiece (ocular lens) that further magnifies the image of the object and projects it into the viewer’s eye (Figure 1.). Specimens, to be observed under a microscope are generally either temporary prepared on slides in the lab or are permanent commercial slides made to last a long time. Temporary slides are made to last only a short time—usually one laboratory period. 3  Compound Light Microscope 1. Care of the Compound Light Microscope The microscope is an expensive precision instrument that requires special care and handling. Always carry the microscope with both hands. Grasp the arm of the microscope with one hand and place your other hand under the base. Always hold the microscope in an upright position so that the eyepiece cannot fall out. Place a microscope on your worktable or desk at least 10 cm from the edge. Position the microscope with the arm facing you. Before you use the microscope, clean the lenses of the objectives and eyepiece with lens paper. Note: To avoid scratching the lenses, never clean or wipe them with anything other than lens paper. Use a new piece of lens paper on each lens you clean. Never touch a lens with your finger. The oils on your skin may attract dust or lint that could scratch the lens. 2. Parts of a Compound Light Microscope Referring to Figure 1. of the compound microscope at the end of this lab, locate the following features of the microscope available in your laboratory; it may have all or most of the features described: A. Ocular Lens The oculars are the lenses you look through. The oculars of most binocular microscopes can be adjusted to match the distance between the eyes of different observers (interpupillary adjustment). Oculars of different microscopes may have different magnifications, although most are 10X. You may have to remove the ocular from its holder to determine its magnification. B. Body Tube Light travels from the objectives through a series of magnifying lenses in the body tube to the ocular. In some microscopes, the body tube is straight; in others, the oculars are held at an angle and the body tube contains a prism that bends the light rays so that they will pass through the oculars. C. Objective Lens Attached to a rotating nosepiece, at the base of the body tube are a group of 3 or 4 Objective lenses or objectives. Rotate the nosepiece and notice that a click is heard as each objective comes into position. The objectives focus the light that comes 4  through the specimen, up the body tube, and through the oculars. Each objective has numbers stamped on it. One of these numbers identifies the magnification of the objective (e.g., 43X). Objectives are usually named according to their magnifying power, as follows: Scanning power - 4X; Low power - 10X; High power - 40X; Oil immersion - 100X. Note the magnification of each of the objectives on your microscope. D. Stage The surface or platform on which you place the microscope slide is the stage. Note the opening (stage aperture) in the center of the stage. On some microscopes, the stage is stationary and has clips to hold the slide in place. On other microscopes, the stage is movable and is called a mechanical stage. Movement is controlled by 2 knobs located on the top, side, or bottom of the stage. Note the horizontal and vertical scales on the mechanical stage. E. Substage The area under the stage, called the substage, may contain a diaphragm, a condenser, or both. i. Diaphragm: regulates the amount of light passing from the light source through the specimen and through the lens system of the microscope. By properly adjusting the diaphragm, you can provide better contrast between the surrounding medium and your specimen, thus greatly improving your image of the specimen. An iris diaphragm consists of a circle of overlapping thin metal plates. The lever that projects from the side of the diaphragm opens and closes these plates, thereby regulating the amount of light that enters the microscope. Most of you have a microscope with an iris diaphragm. ii. Condenser consists of a series of lenses that focus light onto the specimen. It is moved up and down by a knob at its side or by a lever projecting from the condenser housing. By properly adjusting the condenser, you can greatly improve the clarity of the specimen. A filter holder may be attached to the bottom of the condenser. It usually contains a blue filter. F. The Light Source Your microscope has an illuminator built into the base of the microscope and controlled by an on/off switch. You can control the amount of light entering the specimen by adjusting the diaphragm. You can also control the light intensity by adjusting the voltage of a transformer attached to the illuminator. Use low or medium voltage settings for most microscopic observations. You will need a higher setting when using the oil-immersion lens. 5 G. Coarse and fine adjustment knobs You can focus your microscope by using the coarse and fine adjustment knobs these raises or lower either the body tube or the stage, depending on the type of microscope you are using. Try this with the low-power objective in position. Rotate the coarse adjustment knob 1/2 turn clockwise while watching the low-power objective. Do the same with the fine adjustment knob. Figure 1. Parts of a microscope 6 3. The microscope field of view The microscope field of view (FOV) is the diameter of the area when looking through the eyepiece (Figure 2.). The field of view can be changed to observe different areas of the specimen on a slide by moving the stage controller. The FOV is largest at the lowest magnification (scanning objective lens), i.e., more of the specimen will be seen. When a specimen is viewed using a high magnification objective lens, a smaller area of the specimen will be seen, therefore the FOV might need to be changed. Figure 2. The microscope field of view (FOV). As the magnification increases the FOV decreases showing a smaller portion of the specimen. 7 4. Magnification at eye point Notice the numbers engraved on the objectives and on the eyepiece. Each number is followed by an “X” that means “times.” For example, the low-power objective may have the number “10X” on its side. That objective magnifies an object 10 times its normal size). The total magnification of a microscope (magnification at eye point) is calculated by multiplying the magnification of the objective by the magnification of the eyepiece. For example: Magnification X magnification = total of objective of eyepiece magnification 10X X 10X = 100X 5. Parfocality Parfocality refers to objective lenses adjusted so that when used interchangeably, the specimen in view will not be out of focus. Ideally, light microscopes should be parfocal so that it the image in view will remain in relative focus with other objective lenses on the microscope. 6. Image resolution An important feature of the microscope is to produce not only a magnified image but a clear one too. Therefore, the solution power of the objective lenses is important. Resolution power is the ability of an objective lens to distinguish between small objects that are close together. 7. The objective lens working distance The working distance is the distance between an objective lens and the surface of a specimen on a slide when it is in sharp focus. The working distance becomes small with the use of a high magnification objective lens and produces a higher resolving power. 8 8. Light Refraction & the Use of Immersion Oil The most powerful lens of the light microscope is the 100x oil immersion objective. Because light is refracted every time it passes through a medium with a different refractive index, (air to glass or vice versa) the quality of the image is reduced with each passage. Thus, by reducing the number of such passages to a minimum, the clarity, brilliance and resolving power is preserved. Immersion oil has been formulated so that it has a refractive index identical to that of glass. Thus, there is no refraction of light when it passes from glass to oil and vice versa. Thus, two changes in refractive index can be eliminated by placing a drop of immersion oil on the specimen, and immersing the 100x oil immersion objective directly into the drop. You should be struck by the clarity those results. Figure 3. The use of oil immersion. 9  Stereo Microscope Other than the compound microscope, a simpler instrument for low magnification use may also be found in the laboratory. The stereo microscope or dissecting microscope usually has a binocular eyepiece tube, a long working distance, and a range of magnifications typically from 5x to 100x (Figure 4). Some instruments supply lenses for higher magnifications, but there is no improvement in resolution. Such "false magnification" is rarely worth the expense. In a microbiology lab, the stereo microscope is used to examination microbial cultures in Petri dishes and macroscopic specimens. In contrast to a compound light microscope, a stereo microscope provides a 3-D image rather than a 2-D one. Figure 4. Parts of a stereo microscope. 10  Lab activities 1. Cleaning the microscope 2. Preparation of a wet-mount slide of the letter ‘e’ Add a drop of Place the specimen Place the edge of a Slowly lower the water to a slide. in the water. cover slip on the cover slip to prevent slide so that it forming and trapping touches the edge of air bubble the water. 3. Using a Compound Light Microscope A. Focusing 1. Clean the oculars and objectives using lens paper. 2. Put the scanning (4X) objective in position. Hold your slide of the letter “e” so that the letter is in a normal reading position. Then place it on the stage in that position and examine it with the scanning. Also prepare a wet mount slide of unstained salt crystals. 3. Turn on the illuminator and open the diaphragm fully. If there is a condenser, position it as high as it will go, so that the top lens of the condenser unit is level with the stage aperture. Center the specimen over the stage aperture. 4. Position the scanning objective (4X) as close to the slide as possible; then, while looking through the oculars, use the coarse adjustment knob to back off slowly until the specimen comes into focus. 11  5. Use the diaphragm (and/or the transformer voltage regulator) to adjust the light intensity as necessary, and again center the specimen by moving the slide. 6. Switch from the scanning lens (4X) to the low-power objective (10X). Make certain the objective clicks into position. If the specimen stays in focus, your microscope is parfocal. You can sharpen the focus by small adjustments of the fine adjustment knob. If your microscope is not in focus after changing objectives, you may have to use the coarse adjustment knob and then the fine adjustment knob. But remember, do not do this with the high power or oil-immersion objectives in position. Ask your instructor for help if you have difficulty focusing your microscope. Re-center the specimen, adjust the diaphragm, and adjust the position of the condenser to increase the contrast of the specimen. 1. Switch to the high-power (40X) objective and adjust the focus using the fine adjustment knob. Note: These procedures are usually used when examining a wet mount or a commercially prepared micro-scope slide. Always use clean microscope slides. Always proceed from the lowest power to the highest power objectives, making minor focus and light corrections as necessary. Learn to fine-tune your microscope. 12  B. The Microscopic Image i. Orientation of the Image Move the image to the right, left, upward, and downward, and notice the direction in which you have to move the slide. When you want to point out something of interest in the field of vision to someone, you can describe its approximate location by referring to the field of vision as a clock. Thus, you could tell them to “look at 3 o’clock”, or “look just off-center toward 9 o'clock”, and so forth. Alternatively, in some microscopes, a thin black line appears to cut across the field. This is a pointer that has been added to the ocular of your microscope so that you can point out something by moving the object under observation to the end of the pointer. Record your observations in the report sheet. ii. Brightness of the Field of Vision and Working Distance Examine your unstained salt crystals slide, starting with the lowest power objective and progressing to the highest power objective (do NOT use the oil-immersion objective). Note any changes in the brightness of the field when you change objectives. When the object on your slide is in focus, for each objective, the distance between the slide and the objective lens, the working distance, decreases as the objective magnification increases. 4. The use of oil immersion lens 1. Obtain a commercially prepared slide from your lab instructor. 2. Focus your specimen as in section C. 3. Partially rotate the nosepiece so that the space between 40x and 100x objectives is directly over the center of your specimen. 4. Place a small drop of oil on the slide in the center of the lighted area. (Take care not to dribble on the stage.). Note the small drop of oil directly over the area of the specimen to be examined. 5. Rotate nosepiece so that the 100x oil immersion objective touches the oil and clicks into place. Focus only with fine focus. Hopefully, the specimen will come into focus easily. Do not change focus dramatically. If you still have trouble, move the slide slightly left and right, looking for movement in the visual field, and focus on the object which moved. 13  6. Never go back to the 10x or 40x objectives after you have applied oil to the specimen since oil can ruin the lower power objectives. [The 4x objective can be used because it is high enough to be above the oil.] 7. When you have finished for the day, wipe the 100x oil immersion objective carefully with lens paper to remove all oil. Wipe oil from the slide thoroughly. Clean the stage should any oil have spilled on it. 5. Using a Stereo Microscope 1. Place the provided bean in the center of the stage plate. 2. Adjust the eyepiece(s) so that you can look through the microscope comfortably without straining your eyes. 3. Bring your sample into focus. Note: with a stereo microscope you will often be viewing 3-D specimens that have many different levels. You will not be able to focus every feature clearly at the same time. Storage of a light Microscope 1. Clean the optical parts with lens paper and 95% alcohol. 2. Clean the non-optical parts with tissue paper. 3. Place the scanning objective lens into working position. 4. Fold the electrical cord then insert it under the stage. 5. ALWAYS cover the microscope with its dust cover before returning it to its designated place. 14  Lab Discussion 1. Why might it be a good idea to keep your microscope at least 10 cm from the edge of the table? 2. Why should a microscope slide and cover slip be held by their edges? 3. Why do scientists use microscopes? 4. Why should you use lens paper only once? 5. Why is it important to eliminate air bubbles from the slide? 15