Lab 1: The Microscope (10 points)

Transcription

1 Lab 1: The Microscope (10 points) Pierce College Student Outcome: Lab Outcome 1: Review basic microscopy techniques including focusing, illumination, contrast, measuring and review of parts of the microscope and other microscopy terminology. By completing and understanding this lab, you should be able to do the following: 1. Demonstrate the correct technique for the transportation, cleaning, use and storage of the compound microscope. 2. Identify the parts of a microscope. 3. Directly determine the FOV of a microscope using a micrometer at lowest power, then calculate the FOV at other magnifications from these data. 4. Estimate the size of objects under the compound microscope under different magnifications. 5. Demonstrate/explain how to prepare a wet mount. 6. Demonstrate/explain the appropriate technique for using human tissues. Exercise 1.1: Microscope Structure and Function Introduction The compound microscope works by magnifying light that has passed through an object placed on the stage of the microscope. The source of the light is a high-intensity light source built into the microscope that shines up through the light aperture (a hole in the stage), through Fig. 1.1: Structures of the Compound Microscope. Putman/Biol 241 Lab 1/ /Page 1

2 the specimen, then into the objective lens. Compound microscopes (and dissecting microscopes as well) employ two sets of magnifying lenses to do their job. The lenses that you immediately look into are called the ocular lenses. These magnify light that has been brought up through the body of the microscope from the objective lenses, the lenses closest to the stage; the objective lenses together sit on an area called the nosepiece. Dissecting microscopes are distinguished from standard compound microscopes by their use in observing whole specimens, oftentimes during dissections. The portion of the microscope that supports it is called the base, the bottom of the base being the foot. From the base emerges the neck of the microscope, which supports the body and the focus controls. In compound microscopes, there are two sets of focus controls, an outside knob for coarse focus and a smaller, inside knob for fine focus. Under the stage of a compound microscope and attached to the foot is the high intensity light source and switch. Light generated here proceeds up through an iris diaphragm, that regulates the amount of light, and a condenser lens, that focuses the light through the light aperture. Specimen-containing microscope slides are gently placed in (not on or under) and held by the stage clips of the mechanical stage. The controls of the mechanical stage then allow you to precisely control the movement of the slide, something you cannot do if you re just using your fingers to move the slide about! In compound microscopes, you can see the four objective lenses and their designation as 4x (shortest lens), 10x, 43x and 100x. These lenses, multiplied by the 10x ocular lenses, give you the magnification of the microscope. The 100x lens is also called the oil immersion lens because it necessitates the use of a special optical oil when we wish to use it. The purpose of this exercise is to introduce you to the parts, function of those parts, and care and maintenance of a compound microscope. Materials and Methods Read and follow these instructions carefully before using your microscope! 1. Using two hands, with one hand around the neck of the microscope and the other hand under its base, carry the microscope back to your lab station; DO NOT BUMP, JAR, OR SLIDE THE MICROSCOPE ONCE YOU HAVE SET IT DOWN! 2. Carefully and gently unwrap the electric cord from around the base and plug it in. 3. Obtain some lens tissue (NOT PAPER TOWELS!) and lens cleaning solution (or 95% ethanol); moisten the lens tissue with a small amount of the lens cleaning solution and gently wipe off the ocular and objective lenses; also use lens tissue if you need to clean the condenser or light source. Do NOT use paper towels on any glass areas of the microscope, Putman/Biol 241 Lab 1/ /Page 2

3 especially on the lenses, as the wood chips found in paper towels will scratch glass! 4. Make sure that the shortest objective lens (4x) is down before you start to use the microscope; to change lenses, rotate the nose piece until you see and feel it click into place. 5. When preparing your slide, make sure there is a cover slip over your specimen and that no fluid (preservative, water, etc) is on the bottom of the slide as this will make the slide stick to the stage of the microscope! If there is fluid on the bottom of the microscope slide or on the stage, remove it with a small piece of paper towel; it is okay to use paper towels moistened with DW or ETOH to clean microscope slides (not coverslips) or any part of the microscope not made of glass. 6. Your prepared slide should go gently into the stage clip, not on top of it or under it, so that you can move it around with the knobs of the mechanical stage. DO NOT FORCE ANYTHING! IF IN DOUBT, HAVE YOUR INSTRUCTOR HELP YOU! 7. Depress the light switch to turn the microscope on, then position your specimen over the light source, gently using the controls of the mechanical stage; REMEMBER TO NEVER FORCE ANYTHING! 8. Reduce the light intensity by sliding the iris diaphragm lever to just above minimum light, and initially focus using the coarse focus knob. Once you see something, use the fine focus knob to sharpen it. 9. Rotate the nose piece to the next lens (10x) and click it into position to increase the power. 10. When working with the 10x and 43x objectives, you should ONLY use the fine focus knob. 11. The fourth objective, 100x, if an oil immersion lens requiring the direct application of oil to the cover slip of your slide, making most wet mounts too unstable to effectively use under oil immersion. To use oil immersion, you should first make a permanent or at least stable mount of your slide (by putting petroleum jelly around the inside edges of the cover slip and gluing the cover slip down onto the slide), apply a drop of immersion oil directly to the cover slip, then carefully lower the 100x lens onto the slide. 12. When you are finished, return the microscope objectives to lowest power, lower the stage, make sure the light source is turned off, and clean the objectives and ocular with lens tissue moistened with 95% ETOH. If there is any fluid on the non-glass parts of your microscope, such as the stage, obtain a small piece of paper towel (no larger than 10 x 10 cm), dampen with DW, and wipe the microscope off; finish cleaning with 95% ETOH. Finally, wrap the cord loosely around one of the oculars. With both hands, return the microscope to its designated storage location. IF a microscope is put away incorrectly or is dirty or still has a slide on it, it will cost you 5 points. 13. Make sure you can identify parts of your microscope illustrated in Fig. 3.1, along with their correct use and function. Putman/Biol 241 Lab 1/ /Page 3

4 Exercise 1.2: Estimating the FOV and Using Size Rules. Introduction When you look into a microscope, you see a round circle of light with (hopefully) a specimen of some kind contained within it. FOV The distance from one side of the circle of light to the other side is called the field of view or FOV. The FOV is small, obviously, and is measured in units called micrometers, Fig. 1.2: The field of view, FOV. m. One millimeter equals 1000 m. The reason why the FOV is important is that it allows us to estimate the size of objects in the microscope. Clinically, this is very important as it helps us identify tissues, parasites, different types of blood cells, etc. You need to know how to do this. The purpose of this exercise is to determine the FOV of your microscope for each magnification; this is also called calibrating the microscope. We ll look at how to estimate sizes using FOV in the next exercise. Materials and Methods Record the name of your microscope in your lab report. In this way, you only have to determine the FOVs once for your microscope, if you use the same microscope throughout the course; you don t have to recalibrate it each time you use it! 1. Determine total magnification. a. To determine the FOV, first determine the total magnification corresponding to each of the objective lenses. The magnification (power) of the objective lenses should be 4x or 3.2 (scanning), 10x (low power), 40x (high dry power) and 100x (oil immersion). These, multiplied by the 10x magnification (power) of the oculars (eyepiece lenses) give you total magnification: 10 x 4x = 40x OR 10 x 3.2x = 32x total magnification 10 x 10x = 100x total magnification 10 x 40x = 400x total magnification 10 x 100x = 1000x total magnification Record the total magnifications in your lab report! b. Using a clear plastic ruler, measure the FOV directly under 40x (or 32x) total magnification (lowest power). You measure the FOV directly ONLY under lowest power; you CANNOT directly measure the FOV at higher magnifications! To measure the FOV directly, lay the clear plastic ruler on TOP of the stage clips, not between or under the stage clips. Using the mechanical stage controls, line up the leading edge of one of the millimeter increments of the plastic ruler with the edge of the field of view and focus clearly. Starting with the leading edge of the millimeter increments, count the number of millimeter increments across the FOV at its widest point. Determine the value Putman/Biol 241 Lab 1/ /Page 4

5 to the nearest tenth; values such as 3.1, 4.5, mm are reasonable. Do not attempt to go to the hundredths place as this would not be significant! An example is given in Fig. 3.3 below Figure 1.3: Example measurement of the FOV at 40x total magnification. FOV in the figure to the right is approximately 3.6 to 3.7 mm, or about 3,600 to 3,700 m. Below is how you estimate FOV. 4 mm 2/3 = 0.6 or mm 2 mm 1 mm 0 mm c. Record the FOV for 40x (or 32x) total magnification in micrometers ( m, 1000 m = mm). For instance, 4.1 mm would be reported as 4100 m and 4.5 mm would be reported as 4500 m. (See Fig. 1.3 above.) Have your instructor check your value to make sure it s correct and sign your lab report. (No credit without instructor s signature!) d. For 100x, 400x and 1000x you CALCULATE each FOV from the directly-measured 40x (or 32x) FOV. You do this using the following formulas. Below are worked examples, based on a hypothetical 40x FOV of 3500 m (your scanning FOV will probably not be 3500 m): FOV 40x = directly measured FOV 100x = (FOV at 40x)(40)/(100) FOV 400x = (FOV at 40x)(40)/(400) FOV 1000x = (FOV at 40x)(40)/(1000) 3500 m (3500 m)(40) = 1400 m 100 (3500 m)(40) = 350 m 400 (3500 m)(40) = 140 m 1000 e. Record the results of your FOV calculations in your lab report; ALSO, make a copy of the results of your FOV calculations and keep it safe in your lab notebook for future reference when you re using your microscope! Putman/Biol 241 Lab 1/ /Page 5

6 Exercise 1.3: Vital Staining of Human Tissue Introduction Vital stains are those which color specific structures of a cell without killing it. Such stains include Bismark brown, methylene blue, neutral red, toluidine blue, Janus green B, brilliant cresyl blue, Nile blue sulfate, and other compounds. Exactly how much to use of each often is determined using trial and error, beginning with weak concentrations. Of course, for many commonly-studied laboratory organisms, the optimal concentrations are known. Of these stains, Janus green, which stains for mitochondria, and methylene blue, which stains proteins, coloring the nucleus dark blue, are quite useful. Animal cells useful for practicing staining techniques are easily available; we re animals and we re loaded with cells that can be painlessly isolated for such a purpose. One of the convenient tissues to sample is stratified squamous epithelium, the cells that cover the outside of your body and extend into your oral cavity, lining the inside of your mouth. Sampling these cells is easy and interesting because of all the bacteria in your mouth! The purpose of this exercise is to teach you how to use differential vital staining to observe nuclei in human stratified squamous epithelia and how to work with human tissue. Materials and Methods Before you proceed, read and understand the lab safety advisory below. Lab safety advisory: Biohazard! We will be working with human tissues that may carry the HIV virus, as well as other pathogens. If you come in contact with the squamous epithelia or saliva of another student, immediately wash the area in question with soap and water. Make sure you throw all contaminated waste (toothpicks) into the biohazard container provided by your instructor. 1. Obtain a microscope slide, coverslip and toothpick. Take a sample of squamous epithelium by firmly scraping the inside lining of your cheek with the side of a flat-tipped toothpick. Lay the flat of the toothpick with your sample down in the middle of a standard microscope slide and spread the sample out so that you have covered about 1/3 of the surface of the slide. Throw the toothpick away in the indicated biohazard container. Allow your tissue sample to air dry for about 3 minutes. 2. Once your tissue sample is dry, place the slide on a staining tray over the sink. Add a drop or two of methylene blue and leave it there for about three minutes. After about three minutes, dribble approximately 20 drops of water over the sample to remove excess stain into the sink. The goal here is to wash away the excess stain without washing away your specimen. Putman/Biol 241 Lab 1/ /Page 6

7 3. With a square of paper towel (~ 5 x 5 cm is adequate), imbibe up the excess water from the ends of your slide without touching the specimen; also, make sure there is no water on the bottom of your slide as this will cause it to stick to the mechanical stage, making it impossible to move. 4. Add a coverslip and examine your preparation. You should be able to see faint patches of blue-stained cells with your naked eye. 5. Gently put your slide between (NOT UNDER) the stage clips of the mechanical stage of your compound microscope. (Review Exercise 1.1 above, if necessary.) Beginning with scanning magnification (lowest power, shortest lens), focus on your specimens. Use your diaphragm and other lighting controls to optimize your light intensity. Use the coarse focus knob to lower the scanning objective lens down as close as it can get to the specimen while you re looking in the ocular. Use the mechanical stage to move the slide around until you spot a field of blue-stained squamous epithelium cells. If you can t do this, have your instructor help you! 6. Once you have located some cells, you are ready for Exercise 1.4. Exercise 1.4 Estimating the FOV and Using Size Rules. Estimating Size 1. Rotate the nosepiece to change magnification. Once you have found squamous epithelial cells, increase magnification to 100x and focus. Then increase magnification to 400x and focus. Finally, increase magnification to 1000x and focus. The 1000x is also called the oil immersion lens, meaning that optimal resolution is obtained by adding a drop of immersion oil directly to the coverslip of your slide. Generally speaking, if you can adequately observe a specimen under 1000x without oil, do so; using oil requires you to triple-clean the 100x objective and the slide, which is a lot of work if you don t need to use oil! We won t use oil in this lab. 2. Notice that stratified squamous epithelial cells imbricate, that is, they overlap. Try to find one or two cells that are not imbricating. Draw them as accurately as you can in the space provided in your lab report; note that you do not need to draw your cells in a circle! Label the cell membrane, nucleus and bacteria, which are on the outside of the cell. Also enter the magnification and the FOV in the box to the right of your drawing. 3. Draw a size rule the size of your cell next to your drawing of the cell (Fig. 1.4); the size rule indicates how big the cell is. While examining your cell under the microscope, estimate how many cells would fit across the FOV at the widest point, side-by-side. Divide the FOV by this number. This is the size of your size rule, if you made your size rule the same size as the cell! Enter this value next to your cell drawing in your lab report. Putman/Biol 241 Lab 1/ /Page 7

8 Figure 1.4. Estimating the size of an object using a compound microscope. FOV If your magnification is 40x, the field of view would be 3,900 m. About 3.5 of the organisms to the left would fit across the field of view. So the length of the organism would be 3,900 m 1,100 m 3.5 What you then draw, with your size rule: Size rule = m This thing is a size rule! 4. When you re finished with your drawing, have your instructor check and initial it. (No credit without instructor s signature!) 5. When finished with your slide, put it in the biohazard boxes provided. Please make sure the lab is in order before you leave! Putman/Biol 241 Lab 1/ /Page 8

9 Biol 241 Name: Lab 1 Report: Microscopy (10 points) Date: Lab Section: Exercise 1.1: Microscope Structure and Functions 1. Identify the parts of a compound microscope: a) b) f) g) c) h) i) d) e) j) k) l) m) 2. Based on your reading of the introduction to this lab, identify the part of a compound microscope based on its function! a. The foot upon which the microscope sits. b. Generally a 10x lens, it s where you look into microscope. c. Supports upper part of microscope; along with one arm under the base, allows you to carry the microscope. d. Provides illumination. e. Turns light source on. Putman/Biol 241 Lab 1/ /Page 9

10 f. Controls amount of light that shines up through specimen. g. Focuses light up into microscope slide. h. Holds microscope slide. i. Allows you to move microscope slide! j. You turn this to change objective lenses. k. Lenses closest to microscope slide; usually 4x, 10x, 40x and 100x. l. Use this to initially move stage and slide up to objective lenses and to bring the image of specimen roughly into view. m. Use this to bring specimen clearly into view, especially when using higher magnifications. 3. Should you put a microscope slide on, between or securely under the stage clips of a mechanical stage? (This is an important question; be sure of your answer!) 4. Briefly, and in your own words, what is a FOV? Exercise 1.2: Estimating the FOV and Using Size Rules. Microscope Name: Total magnification and reported/calculated FOVs for your microscope; ALSO copy this information somewhere in your lab notebook for future reference! Total Magnification FOV ( m) Instructor s signature for scanning magnification FOV Putman/Biol 241 Lab 1/ /Page 10

11 Discussion Questions: 1. A cell under the microscope has a diameter of about ¼ the FOV at 1000x total magnification. If the FOV is 140 m, what is the diameter of the cell? 2. Why can t we use the plastic ruler to directly measure the FOV under 100x, 400x and 1000x total magnifications? Exercise 1.3: Vital Staining of Human Tissue Discussion Questions: 1. Why is working with human tissue potentially dangerous? 2. What organelle did the methylene blue stain? 3. Why do we call methylene blue a vital stain? Putman/Biol 241 Lab 1/ /Page 11

12 Exercise 1.4: Using Size Rules Drawing of squamous epithelium cells under 1000x total magnification, with a size rule and labeled with the following terms: cell membrane nucleus bacteria Magnification: Field of View: Rule = m Identification of Drawing: Instructor s signature: Discussion Questions 1. What is the purpose of including a size rule in drawings of what you observe under the microscope? Putman/Biol 241 Lab 1/ /Page 12