Laboratory 2 Metric Measurement and Microscopy
(LM pages 9–22)
Time Estimate 1.5 to 2.0 hours
The actual time required to become familiar with each microscope is dependent on students’ amount of hands-on experience in previous classes.
Special Requirements
Living material. Euglena.
Fresh material. Onion.
Notes
Microscope supplies. Set aside an area in the laboratory for storage of clean microscope slides, coverslips, and lens paper. Post a notice in this area, outlining the established procedures for handling dirty slides. Possible procedures include:
1. Wash, rinse, and dry all slides, and return them to their boxes; discard plastic coverslips.
2. Wash and rinse all slides, and place them in the drying rack.
3. Place dirty slides in the detergent solution provided; discard plastic coverslips. Some laboratories prefer that the laboratory assistant wash all slides in an ultrasonic cleaner, rinse the slides in distilled water, and allow the slides to drain dry.
4. Discard plastic coverslips. Glass coverslips should be placed in detergent solution in a beaker.
MATERIALS AND PREPARATIONS1
2.1 The Metric System (LM pages 10–11)
_____ rulers, plastic millimeter
_____ graduated cylinders, 50 ml or 100 ml
_____ dropping bottles containing water
_____ index card, blank white (20 cm 3 30 cm)
2.3 Binocular Dissecting Microscope (Stereomicroscope) (LM pages 14–15)
_____ microscope, binocular dissecting with illuminator
_____ lens paper
_____ an assortment of objects for viewing (e.g., coins, plastomount)
______
[1]Instructions are grouped by exercise. Some materials may be used in more than one exercise.
2.4 Use of the Compound Light Microscope (LM pages 16–19)
_____ microscopes, compound light
_____ lens paper
_____ slide, prepared: letter e (Carolina 29-1406); or newspaper, scissors, slides, and coverslips
2.5 Microscopic Observations (LM pages 19–21)
_____ microscope slides (glass, Carolina 63-2950, -2956 or plastic, Carolina 63-2850)
_____ coverslips (Carolina 63-2898, -2900)
_____ lens paper (Carolina 63-2898, -2900)
_____ microscopes, compound light
_____ toothpicks, prepackaged flat
_____ ethyl alcohol (ethanol), 70% (Carolina 86-1261); or alcohol swabs (if toothpicks are not prepackaged)
_____ optional prepared slide: human stratified squamous epithelium, cheek (Carolina 31-2534)
_____ methylene blue solution, or iodine-potassium-iodide (IKI) solution (premade: Carolina 86-9051, -9053, -9055)
_____ biohazard waste container for toothpicks (Carolina 83-1660, -1665)
_____ container of 10% bleach solution for slides and coverslips (to be washed directly or autoclaved and washed at lab technician’s discretion)
_____ dropping bottles, or bottles with droppers
_____ onion, fresh
_____ scalpel
_____ cutting board
_____ Protoslo® (Carolina 88-5141) or methyl cellulose solution (Carolina 87-5181, -5183, -5185)
Methylene blue solution (LM page 20). Make up a 1.5% stock solution, using 1.5 g methylene blue stain (dye powder, Carolina 87-5684) in 100 ml of 95% ethyl alcohol (ethanol, Carolina 86-1281). Dilute one part stock solution with nine parts water for laboratory use. Methylene blue staining solution can also be purchased premade (Carolina 87-5911, -5913, -5915).
Iodine (IKI) solution (LM page 20). Iodine-potassium-iodide (IKI) solution can be purchased premade, or the ingredients can be purchased separately as potassium iodide (KI) (Carolina 88-3790, -3792) and iodine (I) (Carolina 86-8970, -8972). These dry ingredients have a long shelf life and can be mixed as needed, according to the following recipe:
To make a liter of stock solution, add 20 g of potassium iodide (KI) to 1 liter of distilled water, and stir to dissolve. Then add 4 g of iodine crystals, and stir on a stir plate; dissolution will take a few hours or more. Keep the stock reagent in dark, stoppered bottles. For student use, place in dropping bottles. Label as “iodine (IKI) solution.”
Iodine solution stored in clear bottles loses potency over time. If the solution lightens significantly, replace it. Small dropper bottles can be stored for about a month, and they are used in other exercises. A screw-capped, brown bottle of stock iodine can be stored for about six months. Dispose of it if the solution turns light in color.
Human epithelium cheek slide (LM page 20). To eliminate the possibility of contact with pathogens, this exercise can be done as a demonstration using a flexscope or videoscope for students to view from their seats. Otherwise, because of the hazards connected with human tissue samples and body fluids, you should take special clean-up precautions if students prepared their own epithelium slides. Use a biohazardous waste container for toothpick disposal, and wash slides and coverslips in a 10% bleach solution. Microscopes should also be wiped with a disinfecting solution.
Dropping bottles. Various styles of dropping bottles are available—for example, dropper vials, glass screw-cap (Carolina 71-6438, -6434) with attached droppers; Barnes dropping bottles (Carolina 71-6525); and plastic dropping bottles (Carolina 71-6550). See also Carolina’s Laboratory Equipment and Supplies section.
Protoslo® (or methyl cellulose solution) (LM page 21). You can also use glycerol (Carolina 86-5530) and water as a substitute for Protoslo. Note: Thickened Protoslo can be reconstituted with distilled water.
EXERCISE QUESTIONS
2.1The Metric System (LM pages 10–11)
Length (LM page 10)
Experimental Procedure: Length (LM page 10)
1. How many centimeters are represented? usually 15
One centimeter equals how many millimeters? 10
1 µm=0.001 mm
1 nm=0.000001 mm
1 mm=1,000 µm=1,000,000 nm
2. Measure the diameter of the circle shown to the nearest millimeter. This circle is 38 mm = 38,000 µm = 38,000,000 nm.
Volume (LM page 10–11)
Experimental Procedure: Volume (LM pages 10–11)
1. For example, use a millimeter ruler to measure a wooden block to get its length, width, and depth. Answers will vary according to the size of the block used. Computations of volume will also vary.
3. What procedure would allow you to determine the total volume of the test tube? Fill the test tube with water, and pour the water into the graduated cylinder. Read the volume in milliliters. What is the test tube’s total volume? Answers will vary.
4. How could you use this setup to calculate the volume of an object? Fill the cylinder with water to the 20 ml mark. Drop the object into the cylinder, and read the new elevated volume. The difference between the two readings is the volume of the object alone.
5. How could you determine how many drops from the pipette of the dropper bottle equal 1 ml? Using a 10 ml graduated cylinder, count the number of drops it takes to get to 1 ml.
How many drops from the pipette of the dropper bottle equal 1 ml? approximately 10 (Answers will vary with student’s technique and with the type of pipette.) Are pipettes customarily used to measure large or small volumes? Small
2.2Microscopy (LM page 12–13)
Electron Microscopes
Conclusions (LM page 13)
• Which two types of microscopes view the surface of an object? (1) binocular dissecting microscope; (2) scanning electron microscope
• Which two types of microscopes view objects that have been sliced and treated to improve contrast? compound light microscope and transmission electron microscope
• Of the microscopes just mentioned, which one resolves the greater amount of detail? transmission electron microscope
2.3Binocular Dissecting Microscope (Stereomicroscope) (LM pages 14–15)
Identifying the Parts (LM pages 14–15)
2. What is the magnification of your eyepieces? 10X or 20X
Locate each of these parts on your binocular dissecting microscope, and label them on Figure 2.4.
Figure 2.4: (Left, top to bottom): eyepiece lenses, binocular head; (Right, top to bottom): magnification changing knob, illuminator, focusing knob
Focusing the Binocular Dissecting Microscope (LM page 15)
4. Does your microscope have an independent focusing eyepiece? yes (most likely) Is the image inverted? no
5. What kind of mechanism is on your microscope? Answers will vary.
2.4 Use of the Compound Light Microscope (LM pages 16–19)
Identifying the Parts (LM pages16–17)
Identify the following parts on your microscope, and label them in Figure 2.5.
Figure 2.5 Left side, top to bottom: eyepiece(s) (ocular lens or lenses), nosepiece, objective lens or lenses, stage or mechanical stage (optional), diaphragm/diaphragm control lever, condenser
Figure 2.5 Right side, top to bottom: arm, stage clips, coarse-adjustment knob, fine-adjustment knob, mechanical stage control knobs (optional), light source, base
1. What is the magnifying power of the ocular lenses on your microscope? The magnifying power of the ocular lens is marked on the lens barrel (usually 10X).
5a. What is the magnifying power of the scanning objective lens on your microscope? (usually 4X).
5b.What is the magnifying power of the low-power objective lens on your microscope? The magnifying power of the low-power objective lens is marked on the lens barrel (usually 10X).
5c. What is the magnifying power of the high-power objective lens on your microscope? The magnifying power of the high-power objective lens is marked on the lens barrel (usually 40X).
5d. Does your microscope have an oil immersion objective? depends on microscope
14. Does your microscope have a mechanical stage? depends on microscope
Inversion (LM page 18)
Observation: Inversion (LM page 18)
1. In space 1 provided here, draw the letter e as it appears on the slide (with the unaided eye, not looking through the eyepiece). The letter should be in the normal position.
2. In space 2, draw the letter e as it appears when you look through the eyepiece. The letter should be upside down and reversed.
3. What differences do you notice? The letter is inverted—that is, it appears to be upside down and backward compared to its appearance when viewed by the unaided eye.
4. Which way does the image appear to move? Explain. When moved to the right, the slide appears to move to the left.
Total Magnification (LM page 19)
Observation: Total Magnification (LM page 19)
Table 2.3Total Magnification*
Objective / Ocular Lens / Objective Lens / Total MagnificationScanning power (if present) / 10X / 4X / 40X
Low power / 10X / 10X / 100X
High power / 10X / 40X / 400X
Oil immersion (if present) / 10X / 100X / 1,000X
*Answers may vary with equipment.
2.5Microscopic Observations (LM pages 19–21)
Observation: Human Epithelial Cells (LM page 20)
3. Label Figure 2.7. 1. plasma membrane; 2. nucleus; 3. cytoplasm
Observation: Onion Epidermal Cells (LM pages 20–21)
4. Label Figure 2.8. 1. nucleus; 2. cell wall
Table 2.4Differences Between Human Epithelial and Onion Epidermal Cells
Differences / Human Epithelial Cells (Cheek) / Onion Epidermal CellsShape (sketch) / Flattened, rounded, random orientation / Square or rectangular, oriented end to end and in lines/rows
Euglena (LM page 21)
Observation: Euglena (LM page 21)
5. How do your specimens compare to Figure 2.9? Both have long flagellum, eyespot, contractile vacuole, nucleus, chloroplast, same shape.
LABORATORY REVIEW 2 (LM page 22)
1. Make the following conversions:
a. 1 mm = 1,000 µm = 0.1 cm
b. 15 mm = 1.5 cm = 15,000 µm
c. 50 ml = 0.05 liter
d. 5 g = 5,000 mg
2. Explain the designation “compound light” microscope:
a. compoundThere are two sets of lenses—objective and ocular.
b. lightLight is used to view the object.
3. What function is performed by the diaphragm of a microscope?The diaphragm regulates the amount of light coming through the lenses.
4. Briefly describe the necessary steps for observing a slide at low-power under the compound light microscope.Center the slide on the stage. Looking from the side, decrease the distance between the slide and the objective lens until the lens comes to a stop. Looking through the ocular lens(es), use the coarse-adjustment knob to increase the distance between the slide and the lens until the object comes into view. Adjust the light, and fine-adjust the focus.
5. Why is it helpful for a microscope to be parfocal?Little, if any, adjustment is needed when switching from low to high power.
6. Why is locating an object more difficult if you start with the high-power objective than with the low-power objective.The diameter of field is smaller in high power than in low power.
7. How much larger than normal does an object appear with a low-power objective lens?100X
8. A virus is 50 nm in size.
a. Would you recommend using a binocular dissecting microscope, compound light microscope, or an electron microscope to see it? electron microscope Why?Only an electron microscope has the capability of observing an object this small because it magnifies more and has greater resolving power.
b. How many micrometers is the virus?0.05 µm
9. What type of microscope, aside from the compound light microscope, might you use to observe the organisms found in pond water?binocular dissecting microscope
10. State two differences between onion epidermal cells and human epithelial cells. Human epithelial cells are flat, round, and have a random orientation. Onion cells are square and oriented end to end in rows.
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