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Microscopic Measurement
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Microscopic Measurement
Using a Light Microscope to Determine an Object’s Size
The microscope is a valuable tool used by biologists. It magnifies materials such as cells that the human
eye cannot distinguish. We cannot see objects much smaller than 0.1 mm in size. The most commonly
used compound microscope is the monocular (one eyepiece) light microscope. The microscopes found
in most high school laboratories magnify objects from 40X – 1000X. The resolving power is the
limiting factor that determines how much a microscope can magnify. Resolving power is the
measurement of how close two points can be and still be distinguished as separate. The resolving power
of the human eye is approximately 0.1 mm. This value is somewhat variable as some humans are
capable of making much finer distinctions than others and this ability can change with age.
A microscope is basically a tube with lenses at both ends. The lens that is closest to the eye is the ocular
lens. It is not unusual for this lens to magnify 10X by itself. The level of magnification is found on the
side of the ocular. Some oculars will include built-in pointers or measuring devices.
The lenses that are found on the revolving nosepiece are the objectives. Most microscopes will have two
or more objectives. The magnification of the objective lens is stamped on the side of the lens. The total
magnification of an object can be found by multiplying the magnification of the objective lens with that
of the ocular lens. For example, a microscope with a 10X ocular lens and a 10X low power objective
lens has a low power total magnification of 100X. The objectives are attached to a revolving nosepiece
so that turning the nosepiece can change the objectives. The advantage of using an objective with higher
magnification is an increase in detail than can be viewed. The disadvantage of viewing at a higher
magnification is that less of the slide can be viewed. For example, if there are 20 evenly distributed
yeast cells in the field of view at 50X, then when the microscope objective is increased to a
magnification to 100X, then, on the average, there should be only 10 cells visible. These yeast cells
however, will appear twice as large. The other disadvantage of using higher magnification is that the
depth of focus is much more difficult to control. For example, on 40X two crossed threads on a
microscope slide can both be brought into focus. However, when the magnification is increased to 100X
only one of the threads can be brought into focus. The other thread will be out of focus.
Underneath the stage is the condenser lens which has a diaphragm. This lens regulates the amount of
light passing through the specimen. Specimens to be viewed are usually thin enough that the light
passes right through them. Light reaches the eye after being passed through the objects to be examined.
There are times when the amount of light passing through the specimen is too much and the amount of
light needs to be reduced. The iris of the microscope removes any stray light that might cause the image
to be blurry.
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Light Microscope
Film or eye
Light beam
Ocular lens
Objective lens
Specimen on stage
Condenser lens
Iris diaphragm
Light source
PURPOSE
In this investigation, you will learn how to use and care for a microscope.
MATERIALS
cover slips
3 microscope slides
scissors
newspaper
dropping pipet
compound microscope
transparent metric ruler
Safety Alert
1. Caution: Scissors are sharp. Use care when handling.
PROCEDURE
PART I: CARE OF THE MICROSCOPE
1. It is important to take proper care of the microscope; it is probably one of the most expensive pieces
of equipment in the laboratory. When moving the microscope, always carry the microscope with
both hands, placing one hand on the arm while holding the base with the other hand.
2. Always set the microscope down gently and away from the edge of the table. If a lamp is attached to
the microscope, keep its wire out of any water. Remove all of your materials from the table except
those items needed for the lab.
3. If you wear mascara hold your eyes at a distance from the ocular lens. When your eye blinks, your
eyelashes can transfer some of the mascara to the ocular lens, making it difficult to see the specimen.
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4. Before using the microscope, clean the lenses carefully with lens paper. The lenses of the
microscope are the most expensive items on the microscope. Always clean lenses with the lens
paper designed for this task. Using filter paper, paper towel or any other substitute may scratch the
lenses.
5. To prevent damage to the high power objective, always return the revolving nosepiece to the low
power setting before putting away the microscope.
PART II: IDENTIFYING THE PARTS OF THE MICROSCOPE
1. Obtain a microscope and use the diagram above to guide you as you locate the various parts. Read
about the function of each part of the microscope below. Use this information to complete the prelab activity.
a. Ocular or eyepiece - Contains lenses to increase magnification, usually 10X.
b. Arm - Connects body tube to the base where stage and adjustment knobs are located.
c. Revolving nosepiece - Allows changing of various objectives.
d. Objectives - Contains lenses of different magnifications, usually 4X, 10X, and 40/43X.
e. Stage - Holds microscope slides and has an opening to allow light to pass through.
f. Stage clips - hold the slide in place.
g. Adjustment knobs - Can be found as one knob with two parts or as two separate knobs. The
outer knob or the larger knob is the coarse adjustment and is used to bring the slide into focus.
The fine adjustment is the inner knob or the smaller knob and is used to sharpen the focus.
h. Diaphragm - Regulates the amount of light passing through the stage.
i. Light source - Directs light upward through the diaphragm.
j. Base - Supports the microscope.
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PART III: USING THE MICROSCOPE
1. Plug in the microscope. Turn on the lamp by using the lamp switch. This will allow light through
the stage. Most microscopes are equipped with a diaphragm for regulating light. Some materials are
best viewed in dim light, others in bright light.
2. Rotate the nosepiece to bring the low-power objective into place. When changing from one
objective to another you will hear a click as the objective snaps into position.
3. Using only lens paper, wipe the lenses to make sure that they are dry and free of fingerprints and
debris.
4. Obtain a prepared slide and look at it to locate the specimen. Place the slide on the stage so that the
specimen is over the opening on the stage.
5. When focusing, start with the objective with the lowest magnification. Make sure that both the
coarse and fine adjustment knobs are lowered down as far as possible. Do not allow the objectives
to touch the cover slip.
6. Adjust the light with the diaphragm so that an evenly distributed circle of light is visible.
7. Look through the ocular and slowly turn the coarse adjustment knob to raise the nosepiece until the
specimen comes into rough focus. Use the fine adjustment knob to sharply focus the specimen.
8. To increase the magnification, revolve the nosepiece to the higher objective. Be sure to snap this
lens in place. You should only have to sharpen the focus with the fine-adjustment knob. You should
not focus using the coarse-adjustment knob after changing to a higher magnification.
9. Sketch the specimen as it appears under high power magnification in the space provided on your
student answer page. Indicate the magnification power in the lower right of your sketch.
10. Remove the slide and return to the low power objective.
PART IV: VIEWING THE LETTER “E”
1. Cut a lowercase “e” from the classified section of the newspaper. The smaller the letter, the better.
Place it right side up on a clean slide.
2. This is a dry mount slide, so just place a cover slip over the newspaper.
3. Focus the letter “e” using steps 3 - 7 from Part III.
4. Notice how this letter appears in the field of view compared to how it appears on the slide. Draw
this letter as it appears in the field of view on your student answer page. In making microscope
drawings keep the following points in mind.
a. Microscope drawings are made in circles to represent the field of view.
b. Be sure to label the total magnification outside the circle to the lower right-hand side.
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c. Draw the field of view as it appears in the microscope and with the proper proportions. If an
object occupies half of the field of view when viewing it with the microscope, then when the
object is drawn, make sure that it occupies half the circle.
d. Include as much detail as possible.
e. When labeling, draw straight lines with a ruler, and print the label.
5. Slowly move the slide to the right. Observe which direction the “e” moves in the field of view.
Slowly move the slide away from you. Again, observe which direction the “e” moves in the field of
view.
6. Following step # 8 from Part III view the “e” on high power and draw the image as it appears in the
field of view.
PART V: MEASURING WITH THE MICROSCOPE
Because objects examined with a microscope are usually quite small, biologists measure objects viewed
under the microscope in units called micrometers, which is one thousandth of a millimeter. The symbol
for micrometer is µm, the Greek letter µ (called mu) followed by m.
1. You can estimate the size of a microscopic object by comparing it with the size of the field of view.
Determine the size of the field by placing a plastic mm ruler on the stage. Use the low power
objective to focus the divisions on the ruler.
2. Carefully move the ruler until its marked edge passes through the center of the field of view.
3. Count the number of divisions that you can see in the field of view. One mm or 1000 µm is the
distance from the center of one mark to the center of the next.
4. Record the diameter of the low power field of view for your microscope, in micrometers, on your
student answer page. This process can be used for objectives as great as 10X.
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ruler
1 cm
1 mm
Place a transparent metric
ruler over opening in stage
one
mm
The above field of view measures approximately 6750µm
The diameter of the high power (40X or 43X) cannot be determined using the process described above.
This is because the high power diameter is usually less than one millimeter. The high power diameter
can be determined indirectly. To determine the diameter on high power, you need to know the low
power field diameter and the magnifying power of both objectives. Since the magnification of the
objectives is inversely proportional to the field size, you can use the formula:
High Power
Field Diameter
=
Low Power
x Low Power
Magnification
Field Diameter
High Power Magnification
5. Use the formula above and determine the diameter of the field of view on high power. Record this
in the space provided on your student answer page.
6. Knowing the diameter of the field of view will allow you to estimate the sizes of objects viewed
under the microscope with both low and high power, by comparing them with the diameter of the
field of vision. For example, if a tiny organism takes up approximately one-half of a field of view
that is 1000 micrometers in diameter, then the size of the organism is one-half of 1000 micrometers,
or 500 micrometers.
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PART VI: MEASURING THE AREA OF A PERIOD
1. Cut a period from the classified section of the newspaper. For the best results, use the smallest
period you can find. The smaller the period, the better the results will be. Place the period on the
microscope slide.
2. This is another dry mount slide, so just place a cover slip over the newspaper. Follow steps 3-13.
View this on high magnification and make a drawing of the field of view on the student answer
page.
3. Using the information and calculations determined in steps 5 and 6 in part V determine the diameter
of the period on high magnification. Next, calculate the area of the period in micrometers.
• Remember that the area of a circle is A = πr2 where r is the radius of the circle.
PART VII: MEASURING THE VOLUME OF A CYLINDER
1. Obtain two different-colored hairs, one light and one dark. Place both hairs on a clean glass slide.
Cross one hair over the other and place a cover slip over them. Put the slide on the stage so that
where the hairs cross is in the middle of the field of view.
2. Follow steps 3-13 in part III. View the hairs on high magnification and make a drawing of the field
of view on the student answer page. Notice that on low power both of the hairs can be brought into
focus. This is not true on high magnification.
3. Observe the hairs using high power. Draw these crossed hairs on your student answer page.
4. Focus on one of the hairs and imagine that what is viewed in the microscope is a cylinder.
Determine the volume of the cylinder, using the information in steps 5 and 6 in part V measure the
diameter of the of a hair and use the formula below to determine the volume of a cylinder.
• Remember the volume of a cylinder is V = Hπr2 where r is the radius of the circle and H is the
height of the cylinder.
• Record your volume calculations on your student answer page.
PART VIII: MEASURING THE VOLUME OF A SPHERE AND A BLOCK
1. Obtain a layer of an onion; tear the onion layer in half. Looking at the concave side, you should be
able to pull off a thin layer of epidermal cells. This layer of epidermal cells is usually one layer
thick and can be seen with the light microscope. Place this layer of epidermal cells on the slide.
Add a drop of iodine and place a cover slip on top of the epidermal cells. When placing the cover
slip on top of the slide, place one end of the cover slip on top of the slide and keep the cover slip at
about a 45o angle. Allow the cover slip to drop down. This should eliminate many air bubbles.
2. View this slide using steps 3-13. The cells may be too large on 400X to measure, if that is the case
continue using 100X. The cells have a block-like shape. The length and width can be measured by
using the microscope. The height or depth of the cell is approximately equal to the width of the cell
(or whichever dimension was smaller). Estimate the length and width of the onion plant cells. Draw
this field of view on your student answer page.
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3. Determine the volume of the block or cell, using the information in steps 5 and 6 in part V.
• Remember the volume of the block is V = L x W x H.
• Record this calculation on your student answer page.
4. The nucleus of the cell can be visualized because of the iodine stain. The nucleus is in the shape of a
sphere. Estimate the diameter of the nucleus and then determine the volume of the nucleus using the
information in steps 5 and 6 in part V.
• Remember the volume of a sphere is V = (4/3)πr3.
• Record this calculation on your student answer page.
5. Remove the slide from the stage, turn off the light with the switch, unplug and return your
microscope to its storage position. Clean your work area and return your lab supplies as directed by
your teacher.
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Name _____________________________________
Period ____________________________________
Microscopic Measurement
Using a Light Microscope to Determine an Object’s Size
PRE LAB QUESTIONS
1. Fill in the diagram of the microscope below.
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DATA AND OBSERVATIONS
PART III: USING THE MICROSCOPE
____X
PART IV: VIEWING THE LETTER “E”
____X
____X
List three things that the microscope did to the letter “e” when viewing under the microscope.
1.
2.
3.
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PART V: MEASURING WITH THE MICROSCOPE
For the field of view(s) that could not be measured with a ruler on the stage, determine the diameter
using the formula and the indirect method.
Magnification
Diameter of
field of view
(micrometers)
Can be measured
with a ruler
(yes / no)
40X
100X
400X
PART VI: MEASURING THE AREA OF A PERIOD
____X
Determine the area of a period from the classified section of the newspaper. Show your work.
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PART VII: MEASURING THE VOLUME OF A CYLINDER
____X
____X
Determine the volume of a cylinder from one of the hairs. Show your work.
PART VIII: MEASURING THE VOLUME OF A SPHERE AND BLOCK
____X
____X
Determine the volume of the onion cells or block. Show your work.
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Determine the volume of the nucleus or sphere. Show your work.
CONCLUSION QUESTIONS
1. What is the advantage of using high power?
2. What is the advantage of using low power?
3. If a slide has 28 yeast cells in a field of view on 100X, approximately how many yeast cells will be
seen when the magnification is increased to 400X?
4. What is the resolving power with regard to a microscope?
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