Microscope PDF
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This document covers microscope usage, safety procedures, and metric system conversions. It includes detailed procedure for preparing wet mounts, focusing specimens, and identifying microscope parts. Exercises on metric conversions are also included in the document.
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MICROSCOPE GOALS The idea behind conversion is a simple one. If the The...
MICROSCOPE GOALS The idea behind conversion is a simple one. If the The goals of this exercise are: units you have are larger than the units you want, to review metric system measurements you multiply by the factor difference between the to properly use and care for a sensitive scientific units (see table 1). For example, let’s convert 200 instrument meters into centimeters. You have just memorized to teach you the techniques required to prepare that 1 m = 100 cm. Since a meter is larger than a cells for viewing with a microscope centimeter, we should multiply. By how much? The to give you a sense of the size of cells difference between meters and centimeters is 100, so multiply the number of meters by 100. For to survey microscopic organisms from a few example, 200 m x 100 = 20,000 cm. kingdoms of life TABLE 1. Metric Measurements By the end of this lab you should: identify the parts of a microscope and functions “Saying” LENGTH MULTIPLE know how to properly carry, use and store a Kids kilometer (km) 1000.0 microscope Have hectometer 100.0 know how to prepare a wet mount slide Dropped decameter 10.0 view and focus specimens under a microscope Over meter (m) 1.0 compare the sizes of different kinds of cells Dead decimeter 0.1 estimate the size of a cell by using the size of the Converting centimeter (cm) 0.01 field of view Metric millimeter (mm) 0.001 0.0001 0.00001 A. METRIC SYSTEM REVIEW Micrometer (µm) 0.000001 Scientists (and most of the world) use the metric How would you convert 2 millimeters to system to communicate information concerning the centimeters? Since a millimeter is smaller than a temperature, length, weight and volume of a centimeter, so what you have is smaller than what material. When using the metric system, the you want, so you divide. The difference between measurements of temperature, length, weight, millimeters and centimeters is 10; so divide the volume are degrees Celsius (or centigrade), meters, number of millimeters by 10. grams and liters. 2 millimeters/10 = 0.2 centimeters. The metric system is a convenient one to use Double-‐check that you are moving the decimal in the because it is based on the decimal equivalents of the correct direction. Your conversion equation should measurement unit. This means that the always have the format of [big number] x [small unit] measurement is multiplied or divided by ten. The = [small number] x [big unit]. fractions or smaller parts of a measurement are named using the prefixes “centi-‐” (hundredth), Conversions to Memorize: “milli-‐” (thousandth) and “micro-‐” (millionth). The multiple is “kilo-‐” (thousand). Table 1 lists the 1 km = 1,000 m measurements for length in meters. But the same 1 m = 100 cm principle is used for volume (liters) and weight 1 cm = 10 mm (grams). The Greek letter “µ” stands for “micro-‐”. 1 mm = 1,000 µm Conversions. One metric unit is converted to another for reasons of comparison. If one Ø PRE-LAB: Go to the Data Sheet (page 5) measurement is in centimeters, and another is in and complete the problems for Metric millimeters, it is easier to compare the lengths if the System Conversions. units used are the same. Bio 101 Microscope - 1 B. COMPOUND LIGHT MICROSCOPE The compound light microscope is an useful tool for studying biology, to view small specimens that are not visible to the naked eye. The microscope uses bright light to illuminate through the specimen and provides an inverted image at high magnification and resolution. There are two lenses that magnify the image of the specimen -‐-‐ the objective lens on the nosepiece and the ocular lens (or eyepiece). To determine the total magnification of the specimen, multiply the objective lens magnification with the ocular lens magnification. Ø Calculate the three total magnifications possible with your microscope and fill in Table 1 on the Data Sheet. As the compound light microscope uses expensive glass lenses similar to professional camera lenses, the lenses can only be cleaned with special lens paper or cloth. Any other type of paper (tissue, paper towel, etc) can scratch the lenses. In addition, water and solvents can damage the various metal fittings. So be sure to follow these important rules and protocol about microscope use. Each person should get a compound light microscope from the storage cabinet at the back of the room. General Rules: Always START and END with the low power lens when putting on OR taking away a slide. Do not get any portion of the microscope wet -‐ especially the stage and objective lenses. Use only lens paper to clean microscope lenses. Carrying the Microscope: 1. Always use one hand around the microscope arm and one hand under the microscope base. 2. Carry it in a vertical position without swinging, tipping, dropping or bumping the microscope. 3. Place the microscope on a table with the arm toward you. Cleaning the Microscope – only use LENS paper If needed, obtain a small square of lens paper (and ONLY lens paper) and gently wipe the microscope lenses directly across, in this order: 1. the lower surface of all the objective lenses 2. the ocular lens 3. the condenser lens and the light housing. Identifying the Microscope Parts £ Eyepiece (ocular lens) £ Nosepiece Ring (turret) £ Objective Lenses (low, medium, high power) £ Stage £ Stage Controls £ Iris Diaphragm £ Condenser Lens £ Light Source £ Coarse Focus Adjustment Knob £ Fine Focus Adjustment Knob Bio 101 Microscope - 2 C. OBSERVING SPECIMENS In order to observe specimens with the compound light microscope, always start with the low power objective lens and the nosepiece raised high above the stage, before placing the slide on the stage. Ensure that your specimen is in the circle of view (over the light source). Focus on the specimen at low power first, BEFORE attempting to look at specimen at higher magnification. As your microscope is PARFOCAL, it remains generally in focus at the higher magnifications, so that you should ONLY adjust with the Fine focus knob when using the medium and high power objective lenses. £ Obtain a slide with the letter “e” from the demo table. £ Follow the steps below to properly use the microscope. £ Focus, observe and draw the letter “e” with low power objective lens. Ø On the Data Sheet, draw what you see with the focused microscope, with the circle on paper representing the field of view (circle of light). Label the total magnification. £ Repeat with medium power objective lens and again at high power objective lens. Using the Microscope (protocol steps) 1. Before starting to place a slide onto the stage, check that the LOW power objective lens is in place (directly above the light source). 2. Rotate the large nosepiece disc that holds the objective lenses. Do NOT grab an objective lens to rotate the nosepiece! You may hear a soft click, that confirms the objective lens is in place. 3. Using the coarse adjustment knob, raise the nosepiece high. 4. Carefully, gently place the slide between the stage clips, with the brand name on the left side. 5. Center the “e” over the light with the stage control knobs. 6. Lower the nosepiece all the way down to the stage. 7. Look through the eyepiece and adjust the intensity of light with the diaphragm lever as needed. 8. Adjust with the fine adjustment knob until the letter “e” is in focus. 9. Try to have both eyes open to reduce eyestrain. 10. Your instructor will come to check your focus and help as needed. Focusing with Parfocal Medium and Higher Power Objective Lenses 11. While low power objective lens is in place, move stage control knobs so that the “e” is in the middle of the field of view. 12. Rotate the nosepiece disc to bring the medium power objective lens clicked into place. 13. You must NOT touch the coarse focus knob again. 14. Use the FINE focus knob to focus the specimen at this magnification, usually need less than a quarter of a turn. Draw as needed. 15. Again place the specimen in middle of the field of view. 16. Rotate the nosepiece disc to click the high power objective lens in place. 17. Use the FINE focus knob to focus the specimen, usually need less than a quarter of a turn. 18. When finished, rotate the low power objective lens in place. Raise the nosepiece away from the stage with coarse focus know. Remove the slide from the stage clips. Bio 101 Microscope - 3 WORK EFFICIENTLY in this long lab: Work with your lab partners. Each partner should prepare and focus a different specimen slide to the best magnification to see details of three whole cells. Then all lab partners can rotate around the table and observe and draw from each microscope. You must make your own drawings and measurements. Copying from someone else’s drawing is considered cheating. You will not be graded on artistic ability, just on your ability to see the details of each specimen. Your instructor will also explain the structures of each specimen on the video microscopy monitor! LAB GRADING: For each specimen, use the ONE magnification at which you can most clearly see whole cells with maximum detail. Draw and color at least three cells from each specimen as accurately as possible. Label the specimen name, total magnification used, arrow, and identifiable cell structures. DRY PREPARED SPECIMEN SLIDES WET MOUNTS THAT YOU PREPARE Spirogyra: cell wall, nucleus, chloroplasts Elodea leaf: chloroplasts Paramecium: cytoplasm, vacuoles, nucleus Human cheek: nucleus Human blood: nucleus, white & red blood Onion: cell wall, nucleus Human sickle cell anemia: sickle cells Pond water Amphibian blood: white, red blood Preparing Wet Mounts Onion epidermis Obtain a slide from the container of alcohol. Unless {Note: Iodine can stain skin & clothes.} Place a drop otherwise indicated, use a flat slide that contains no of iodine on a dry microscope slide. Peel the thin, depressions. Alcohol kills cells! DRY it off with a white, transparent membrane from the inner concave quarter strip of paper towel, not expensive lens side of an onion section and quickly place it paper! Place it on the lab bench. Place a small drop smoothly into the iodine drop. Count slowly to ten. of the medium (water or stain usually) on the slide Gently cover with a dry coverslip. Place the slide first! Obtain your specimen and place it in the inside a folded towel and gently dab out excess medium. Obtain a coverslip (very thin, small square iodine. Look under medium power to find the cell glass) from the container of alcohol. Be very careful wall, cytoplasm and nucleus of the rectangular cells. when drying -‐ they break easily. (Where do you Human Cheek cells dispose of broken glass?) ________ {Note: Methylene blue stains skin & clothes.} Place Hold the cover glass at an angle on the slide and a drop of methylene blue stain on a slide. Rub a clean allow it to fall over the specimen. This gets all of the toothpick on the inside of your cheek very gently only air out from under the cover glass and minimizes the one or two times. DO NOT gouge the inside of your number of air bubbles in with your specimen! cheek! DO NOT draw blood! Rub toothpick onto slide. Cover and gently dab out excess stain with cover glass cover glass paper towel strip. Throw away your slimy toothpick, specimen specimen as no one else wants to clean it up for you! slide Figure 1. Proper placement of a cover glass. slide Pond water or culture DO NOT GET OBJECTIVE LENSES WET!!! Wipe Go to the "Single Depression Microscope Slide" jar to immediately with lens paper if needed. dry off a thick glass depression slide. The depression slide has a curved indent in the middle of the Elodea leaf slide, which allows the living creatures to move Put a drop of water onto a dry slide. With around and not get squashed. Put a drop of methyl forceps, place a very small leaf (or use half of a cellulose and a drop from the pond water or cultures. larger leaf) into the water and coverslip. Carefully put on a cover slip. If you have too much Examine the rectangular cells at high power to liquid on the slide, then gently use a corner of a paper find the cell walls, nucleus, cytoplasm, and green towel to absorb the excess liquid. Use the low chloroplasts. As you focus through the leaf, you and medium power objectives only! will be able to distinguish different cell layers These creatures are very difficult to find and move very through the leaf. Cytoplasmic streaming occurs quickly! The organisms are swimming in the liquid, often, which is evident by chloroplasts moving so just take a drop of it and place both water and inside the cells. organisms on a slide. Bio 101 Microscope - 4 Partners: Name: Bio 101 Lab: MICROSCOPE DATA SHEET PRE-‐LAB: METRIC CONVERSIONS 1. 1 cm = _________ mm = _______________ µm 6. 0.025 m = __________ cm = ____________ mm 2. 1000 µm = _________ mm = ____________ cm 7. 45 mm = _________ cm = _______________ m 3. 100 mm = ________ cm = _______________ m 8. 185 µm = __________ mm = ____________ cm 4. 1 km = ___________ m = _______________ cm 9. 0.04 km = __________ m = ______________ cm 5. 0.123 mm = ________ cm = ____________ µm 10. 54,321 µm = __________ mm =__________ cm TABLE 1. TOTAL MAGNIFICATION CALCULATION Low Power Medium Power High Power Ocular Lens Magnification 10 x Objective Lens Magnification Total Specimen Magnification Drawings of the Letter “E” Slide Specimen _____________ Specimen _____________ Specimen _____________ Total Mag ____________ Total Mag ____________ Total Mag ____________ Ø How does the orientation of the letter “e” when observing under a light microscope DIFFER from observing with your naked eye? Name two differences. ____________________________________________________ Ø While looking at the stage, move the slide VERY SLIGHTLY to the right. Which way did the letter move in the field of view? _______________________________ Ø While looking at the stage, move the slide VERY SLIGHTLY to the left, which way did the letter move in the field of view? ________________________________ Bio 101 Microscope - 5 Microscope Lab Data Sheet (page 2) Name: Specimen Observations – color and label Specimen _____________ Specimen _____________ Specimen _____________ Total Mag ____________ Total Mag ____________ Total Mag ____________ Specimen _____________ Specimen _____________ Specimen _____________ Total Mag ____________ Total Mag ____________ Total Mag ____________ Specimen _____________ Specimen _____________ Specimen _____________ Total Mag ____________ Total Mag ____________ Total Mag ____________ Bio 101 Microscope - 6 Microscope Lab Data Sheet (page 3) Name: LAB CHECK-‐OUT Cleaning Wet Mounts: To put the microscope away, follow these directions: q Rinse slide into beaker of water, place cover slip and slide into proper jars q low power objective in position q Put Prepared Slides back onto the correct tray on q raise nosepiece to the top position the demo table. q remove slide from stage, turn off light q Tighten all reagent bottle caps. Clean up demo table. q be sure black bar from stage clips does not stick out q Wipe off all tables with wet sponge. q unplug, wrap cord according to your instructor’s instructions q carry microscope properly to cabinet and place it Instructor signature_________________ on the correct shelf space STUDY QUESTIONS 1. Name the parts of the microscope. 2. How is a microscope properly carried? 3. How is the microscope properly put away? 4. What is the magnification of a) high power objective lens? b) medium power objective lens? c) low power objective lens? d) ocular lens? 5. How is the total magnification of a specimen determined? 6. When the magnification increases, how does the size of the field of view change? 7. Why is it important to place the medium on a slide before selecting the specimen to be mounted? 8. Name one way to be sure the specimen can be found in the field of view when you change magnification. 9. What are the distinguishing characteristics of a plant cell versus an animal cell? Bio 101 Microscope - 7