Laboratory
2
Metric Measurement and Microscopy
(LM pages 9–28)
Time Estimate for Entire Lab: 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. The length of time spent on pond water organism location, identification, and/or drawings will also affect lab length.
Special Requirements
1. Living material (order in advance for timely delivery): Paramecium
2. Fresh material (obtain locally, close to time of use): onion, pond water
Seventh Edition Changes
This was Laboratory 1 Use of the Light Microscope in the previous edition.
New or revised figure: Figure 2.3 Temperature scale
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
1
The Metric System (LM pages 9–13)
_____
rulers, plastic millimeter
_____
meterstick, metric and English
_____
long bones from disarticulated human skeleton
_____
cardboard (10 cm 30 cm), two pieces
_____
thermometer, Celsius
_____
beaker, 50 ml
_____
water, cold tap
_____
water, hot tap
_____
water, ice
_____
balance scale
_____
wooden block, small enough to hold in the hand
_____
object, such as a penny, a piece of granite, or a trilobite fossil, small enough to fit
through the opening of a 50 ml or 100 ml graduated cylinder
_____
graduated cylinders, 50 ml or 100 ml
_____
test tubes (large enough to hold 20 ml of water)
_____
dropper bottles containing water
_____
index card, blank white (20 cm 30 cm)
_____
pipette, graduated
Note: “Materials and Preparations” instructions are grouped by exercise. Some materials may be used in more than one exercise.
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2.3
Use of the Compound Light Microscope (LM pages 16–21)
_____
microscopes, compound light
_____
lens paper
_____
slide, prepared: letter e (Carolina 29-1406); or newspaper, scissors, slides, and coverslips
_____
rulers, plastic millimeter
_____
slide, prepared: colored threads (Carolina 29-1418); or to prepare your own, you
will need slides and coverslips, three or four colors of sewing thread (or hairs),
scissors, and a dropping bottle of water
2.4
Microscopic Observations (LM pages 22–25)
_____
slides and coverslips
_____
microscopes, compound light
_____
lens paper
_____
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
_____
pond water, or live Paramecium culture (Carolina 13-1540 to -1554)
_____
Protoslo® (Carolina 88-5141) or methyl cellulose solution (Carolina 87-5181 to -5185)
_____
pictorial guide, such as: Needham, J. G., and P. R. Needham, A Guide to the Study of
Freshwater Biology: With Special Reference to Aquatic Insects and Other Invertebrate
Animals, 5th ed. Springfield, Ill.: Charles C. Thomas. ISBN: 0070461376; or
Jahn, T. L., et al. 1979. How to Know the Protozoa, 2d ed. Dubuque, Iowa: Wm. C. Brown
Publishers, ISBN: 0697047598 (Carolina 45-4100).
Methylene blue solution. Make up a 1.5% stock solution, using 1.5 g methylene blue stain (dye powder,
Carolina 87-5684, -5690) in 100 ml of 95% ethyl alcohol (ethanol, Carolina 86-1281). Dilute one part stock
solution with nine parts water for laboratory use, or use iodine (IKI) solution. Methylene blue staining solution can also be purchased premade (Carolina 87-5911, -5913, -5915).
Iodine (IKI) solution. 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 868970, -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 dropper 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. Because of increased awareness of hazards connected with human tissue
samples and body fluids, you should take special precautions if students are preparing their own epithelium slide. Use a biohazardous waste bag for toothpick disposal, and wash slides and coverslips in a 10%
bleach solution.
7
Dropping bottles. Various styles of dropping bottles are available—for example, dropper vials, glass screwcap (Carolina 71-6438, -6434) with attached droppers; Barnes dropping bottles (Carolina 71-6525); and plastic dropping bottles(Carolina 71-6550). See also Carolina’s Apparatus: Labware section.
Pond water. Collect pond water during an active growing season from any local pond or stream. Include
some algae and a small amount of organic debris and living aquatic (aquarium) plants, such as Elodea. For
best results, pond water should be aerated using an air stone and pump if it is to be maintained for any
length of time. Keep the culture in diffuse window light or under artificial illumination in a container with
a transparent cover and a large amount of surface area, such as:
1. A large culture dish (Carolina 74-1006) covered with a second large culture dish
2. A small aquarium and aquarium cover (1.5 gal. plastic, Carolina 67-0388)
3. A small glass aquarium with a lid
This culture can grow and provide material for future labs, even if the labs occur during midwinter. If live
cultures of pond water organisms are purchased, add them and Planaria to the culture, once they are no
longer needed for a particular laboratory.
Protoslo® (or methyl cellulose solution). You can also use glycerol (Carolina 86-5530) and water as a substitute for Protoslo. Note: Thickened Protoslo can be reconstituted with distilled water.
2.5
Binocular Dissecting Microscope (Stereomicroscope) (LM pages 26–27)
_____
microscope, binocular dissecting with illuminator
_____
lens paper
_____
an assortment of objects for viewing (e.g., coins)
EXERCISE QUESTIONS
2.1 The Metric System (LM page 9)
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 =
0.001 µm
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.
3. How many centimeters are in a meter? 100
How many millimeters are in a meter? 1,000
The prefix milli means a thousandth.
4. For example, if the bone measures from the 22 cm mark to the 50 cm mark, the length of the bone
is 28 cm. If the bone measures from the 22 cm mark to midway between the 50 cm and 51 cm
marks, its length is 285 mm or 28.5 cm.
5. Record the length of two bones. Recorded lengths will vary.
Weight (LM page 11)
A paper clip weighs about 1 g, which equals 1,000 mg.
2 g = 2,000 mg; 0.2 g = 200 mg; and 2 mg = 0.002 g
Experimental Procedure: Weight (LM page 11)
2. Measure the weight of the block to the tenth of a gram. Answers will vary.
3. Measure the weight of an item that is small enough to fit inside the opening of a 50 ml graduated
cylinder. Answers will vary.
8
Volume (LM page 11)
Experimental Procedure: Volume (LM pages 11–12)
1. For example, use a millimeter ruler to measure the wooden block used in the previous
Experimental Procedure 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. Hypothesize how could you find 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 the item you weighed previously? 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
Temperature (LM page 13)
Experimental Procedure: Temperature (LM page 13)
1a. Water freezes at either 32°F or 0°C.
1b. Water boils at either 212°F or 100°C.
2. Human body temperature of 98°F is what temperature on the Celsius scale? 37°C
3. Record any two of the following temperatures in your lab environment. Answers will vary.
2.2 Microscopy (LM page 14)
Electron Microscopes (LM page 15)
Conclusions (LM page 15)
• 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
2.3 Use of the Compound Light Microscope (LM page 16)
Identifying the Parts (LM pages 16–17)
Identify the following parts on your microscope, and label them in Figure 2.5. Figure 2.5: 1. eyepiece(s)
(ocular lens or lenses); 2. body tube; 3. arm; 4. nosepiece; 5. objectives (objective lens or lenses); 6. coarseadjustment knob; 7. fine-adjustment knob; 8. condensor; 9. diaphragm/diaphragm control lever; 10. light
source; 11. base; 12. stage; 13. stage clips; 14. mechanical stage (optional); 15. mechanical stage control knobs
(optional)
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 10).
5a. What is the magnifying power of the scanning lens on your microscope? (usually 4).
5b. What is the magnifying power of the low-power objective lens on your microscope? The magnifying power of the low-power objective is marked on the lens barrel (usually 10).
5c. What is the magnifying power of the high-power objective lens on your microscope? The
magnifying power of the high-power objective is marked on the lens barrel (usually 40).
5d. Does your microscope have an oil immersion objective? depends on microscope
14. Does your microscope have a mechanical stage? depends on microscope
9
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. (The letter should be in 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? 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 2.3 Total Magnification*
Objective
Ocular
Lens
×
Objective
Lens
Scanning Power (if present)
10
×
4
=
40
Low power
10
×
10
=
100
High power
10
×
40
=
400
Oil immersion (if present)
10
×
100
=
1,000
=
Total
Magnification
*Answers may vary with equipment.
Observation: Diameter of Field (LM page 20)
Low Power (10) Diameter of Field (LM page 20)
2. Estimate the number of millimeters, to tenths, that you see along the field.
approximately 1.6 mm
Convert the figure to micrometers. approximately 1,600 µm
High Power (40) Diameter of Field (LM page 20)
1. To compute the high-power diameter of field (HPD), substitute these data into the formula given.
(Students record the data in a., b., and c. for their specific microscope—answers may vary with equipment):
a. LPD = low-power diameter of field (in micrometers) =1,600 µm
b. LPM = low-power total magnification 100
c. HPM = high-power magnification 400
HPD = (1,600 µm) (100) = 400 µm
(400)
Conclusions (LM page 20)
•
Does low power or high power have a larger field of view (one that allows you to see more of the
object?) low power
•
Which has a smaller field but magnifies to a greater extent? high power
10
Depth of Focus (LM page 20)
Observation: Depth of Focus (LM page 20)
Table 2.4 Order of Threads (or Hairs)*
Depth
Thread (or Hair) Color
Top
Red
Middle
Blue
Bottom
Yellow
*The order of threads given is that of Carolina Biological Supply Company slide 29-1418. The order of threads in other slides may
be different.
2.4 Microscopic Observations (LM page 22)
Observation: Human Epithelial Cells (LM page 22)
3. Label Figure 2.8. 1. plasma membrane; 2. nucleus; 3. cytoplasm
Observation: Onion Epidermal Cells (LM page 23)
4. Label Figure 2.9. 1. nucleus; 2. cell wall
5. Count the number of onion cells that line up end to end in a single line across the diameter of the
high-power (40) field. for example, five cells
What is your high-power diameter of field (HPD) in micrometers? 400 µm
Calculate the length of each onion cell (HPD number of cells). for example, 80 µm
Table 2.5 Differences Between Human Epithelial and Onion Epidermal Cells
Differences
Human Epithelial Cells (Cheek)
Onion Epidermal Cells
Shape (sketch)
Flattened, rounded, random orientation
Square or rectangular, oriented end to end
and in lines/rows
Cell wall (present
or absent)
Absent
Present
Euglena (LM page 25)
Observation: Euglena (LM page 25)
5. List the labeled features that you can actually see. long flagellum, eyespot, contractile vacuole, nucleus,
nucleolus, chloroplast, pellicle. See also Fig. 2.10 in the lab manual for other structures that may be visible,
depending on slide.
2.5 Binocular Dissecting Microscope (Stereomicroscope) (LM page 26)
Identifying the Parts (LM page 26–27)
2. What is the magnification of your eyepieces? 10
Locate each of these parts on your binocular dissecting microscope, and label them on Figure 2.11.
Figure 2.11: 1. binocular head; 2. eyepiece lenses; 3. focusing knob; 4. magnification changing knob;
5. illuminator.
Focusing the Binocular Dissecting Microscope (LM page 27)
4. Does your microscope have an independent focusing eyepiece? yes (most likely)
Is the image inverted? no
5. What kind of mechanism (zoom or rotating lens) is on your microscope? Answers will vary.
11
LABORATORY REVIEW 2 (LM page 28)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
11 mm equals how many cm? 1.1 cm
950 mm equals how many m? .95 m
2.1 liters equals how many ml? 2,100 ml
122°F equals how many degrees Celsius? 50°C
4,100 mg equals how many grams? 4.1 g
Which type of microscope would you use to view a Euglena swimming in pond water? compound
light microscope
What are the ocular lenses? the lenses located near the eye
Which objective always should be in place, both when beginning to use the microscope and
when putting it away? lower power objective
A total magnification of 100 requires the use of the 10 ocular lens with which objective? lower
power objective
What part of the microscope regulates the amount of light? diaphragm
What word is used to indicate that if the object is in focus at low power, it will also be in focus at
high power? parfocal
If the thread layers are red, brown, green, from top to bottom, which layer will come into focus
first if you are using the microscope properly? green
What adjustment knob is used with high power? fine adjustment knob
If a Euglena is swimming to the left, which way should you move your slide to keep it in view?
right
What is the final item placed on a wet mount before viewing with a light microscope? coverslip
What type of object do you study with a binocular dissecting microscope? whole, opaque
Why is a binocular dissecting microscope also called a stereomicroscope? It produces a
three-dimensional image.
Thought Questions
18. A virus is 50 nm in size. Which type of microscope should be used to see it? Why? For this size
object, use an electron microscope which magnifies more and has greater resolving power.
19. Why is locating an object more difficult if you start with the high-power objective rather than
with the low-power objective? The diameter of field is smaller with high power than with low power.