AP BIOLOGY
Cells
ACTIVITY #2
MICROSCOPE LAB
OBJECTIVES
1. Demonstrate proper care and use of a compound microscope.
2. Identify the parts of the microscope and describe the function of each part.
3. Compare magnification, resolving power, and contrast.
4. Demonstrate proper technique of preparing a wet mount slide.
5. Demonstrate inversion and depth of field.
6. Use the compound microscope as an instrument of measurement.
INTRODUCTION
The unaided human eye can detect objects as small as 0.1 mm in diameter. Most cells are between 0.01
mm and 0.1 mm in diameter and cannot be seen without a microscope. A microscope contains one or
more lenses and is used to view detail that cannot be seen with the unaided eye. The light microscope,
by virtue of its lens system, extends our vision a thousand times so that object as small as 0.1
micrometer (μm) in diameter can be seen. The electron microscope further extends our viewing
capability down to 1 nanometer (nm). At this magnification it is possible to see a virus and the outline of
individual protein or nucleic acid molecules. A lens functions by refracting (bending) light rays coming
from an object and focusing them to form an image of that object. Refraction of light is due to the angle
at which it passes from one transparent medium to another (for example, air to glass) and the difference
in density between the media. A magnifying glass is a simple light microscope. The microscope consists
of a set of lenses that focus an enlarged image of an object on the retina of the eye.
BASIC MICROSCOPY
The microscope is useful in making observations and collecting data in scientific experiments.
Microscopy involves three basic concepts:
Magnification
The degree to which the image of a specimen is enlarged.
Resolving Power
How well specimen detail is preserved during the magnifying process.
Contrast
The ability to see specimen detail against its background. Stains and dyes are added to sections
of biological specimens to increase contrast.
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BASIC MICROSCOPE LAYOUT
*Always keep the eyepiece facing away from the direction the stage faces.
*Never move the microscope to show your partner something. Instead- switch him or her
places.
*Never put away a microscope that is dirty or has a slide on the stage.
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Abbe Condenser:
A specially designed lens that mounts under the stage and is usually movable in the vertical direction. It has an iris type aperture to control
the diameter of the beam of light entering the lens system. By changing the size of the iris and moving the lens toward or away from the
stage, the diameter and focal point of the cone of light that goes through the specimen can be controlled. Abbe condensers really become
useful at magnifications above 400X.
Arm:
The part of the microscope that connects the tube to the base. When carrying a microscope, grab the arm with one hand and place your
other hand under the base.
Base:
The bottom support of the microscope (see arm above).
Coarse Focus:
This is the rough focus knob on the microscope. You use it to move the objective lenses toward or away from the specimen (see fine focus).
Do not use this if you are in HIGH POWER. You could crack the slide.
Condenser Lens:
A lens mounted in or below the stage whose purpose is to focus or condense the light onto the specimen. By using a condenser lens you will
increase the Illumination and resolution. Condenser lenses are not required on low power microscopes.
Cover Slip:
A very thin square piece of glass or plastic placed over the specimen on a microscope slide. When used with liquid samples, it flattens out
the liquid and assists with single plane focusing. Usually these are disposable.
Diaphragm:
Not present on these scopes.
Eyepiece Lens:
The lens at the top of the microscope that you look into. They are usually 10X but also are available in 5X, 15X and 20X.
Fine Focus:
This is the knob used to fine tune the focus on the specimen. It is also used to focus on various parts of the specimen. Generally one uses the
coarse focus first to get close then move to the fine focus knob for fine tuning.
If you turn this many many times you will really mess it up. It should not take many turns to find focus.
Field of View:
Sometimes abbreviated "FOV", it is the diameter of the circle of light that you see when looking into a microscope.
As the power gets greater, the field of view gets smaller. You can measure this by placing a clear metric ruler on the stage and counting the
millimeters from one side to the other.
Head:
The upper part of the microscope that contains the eyepiece tube and prisms. A monocular head has one eyepiece, a binocular has two (one
for each eye). We have monocular heads.
Illuminator:
A light source mounted under the stage. Our light source has a variable output instead of having a diaphragm under the stage and a fixed
lamp.
Mechanical Stage:
A mechanical way to move the slide around on your stage. It consists of a slide holder and two knobs. Turn one knob and the slide moves
toward or away from you. Turn the other knob and the slide moves left and right. Since everything is upside down on a (high power)
microscope it takes some getting used to but it is very convenient to have one especially when observing moving specimens like protozoans
or other pond water critters. Place the slide in the Mechanical Specimen Holder.
Nosepiece:
The part of the microscope that holds the objective lenses also called a revolving nosepiece or turret.
Objective Lens:
The lens closest to the object. We have three (4X, 10X, and 40X)
Parfocal:
This is a focus issue. When changing from one objective to another, the new image should be either in focus or close enough so that you can
refocus with only minor adjustments. All of our microscopes are parfocaled.
Pointer:
When you look through the eyepiece lens, you may see a pointer. By turning the eyepiece, you can rotate the pointer around.
Slide:
A flat glass or plastic rectangular plate that the specimen is placed on. It may have a depression or well to hold a few drops of liquid.
Stage:
The flat plate where the slides are placed for observation.
X:
Times as in 200X or two hundred times magnification. The magnification of a microscope is determined by multiplying the power of the
eyepiece lens by the power of the corresponding objective lens
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Size of Objective
Magnification
Eyepiece Mag.
Smallest (Scanning)
10X
Medium
10X
Largest (High)
10X
Not Present (Oil
Immersion)
100X
Total Magnification
10X
Part A. General Microscopy Procedure
1. Obtain a prepared slide from the supply area.
2. Make sure the scanning power lens is in place. (4X) and turn on the power to the light source.
3. Turn up the light source intensity to about 75% or full range of dial.
4. Open up the iris fully to the left (toward the X&Y adjustment knobs)
5. Lower the stage with the course adjustment knob so you can place the slide on the stage
without hitting the objective lens.
6. Place the slide on the stage of your microscope and clip it into place. Turn the mechanical X & Y
stage position knobs until the center of the slide is right over the light source (circular lens
below the stage).
7. While looking at the microscope from the side, move the stage all the way up using the course
adjustment knob.
8. While looking through the eyepiece, turn the course adjustment knob away from you until the
specimen is in focus
9. Adjust the light intensity and iris until you have the best view of the specimen.
10. With the specimen in focus and positioned in the center of the field of view, rotate the
nosepiece lens to the medium power objective (10X). DO NOT move the coarse focus. Only fine
focus should be necessary to bring the specimen into sharp focus. Remember: The ability of the
microscope t remain in focus when switching from one objective lens to the next highest power
is called parfocal.
11. With the specimen in focus and positioned in the center of the field of view, rotate the
nosepiece lens to the high power objective (40X). DO NOT move the coarse focus. Only fine
focus should be necessary to bring the specimen into sharp focus.
12. Draw a pencil sketch of what you see in the front of your lab book. Be sure to note the name of
the slide, total magnification, topic (in this case microcopy), and date.
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13. Add color with colored pencils or crayons to make it look realistic. Consult your textbook, lab
manual, and/or - at last resort your teacher to see if you have the right details.
14. Label all visible structures.
15. Return microscope to scanning power, remove the slide and return it to the tray, and let your
partner do the next one! Both partners draw both specimens.
16. This sequence of events should be repeated every time you get a new specimen slide!
Part B. Specimen Orientation Procedure
1. Cut out a typed lowercase letter “e” from a scrap sheet of paper.
2. Place the e in the center of a microscope slide
3. Place the “e” slide on the stage so that the bottom of the “e” is facing the arm of the
microscope.
4. Make sure the scanning power lens is in place. (4X) and use the procedure (above) to find the
“e” in scanning power.
5. Sketch what you see in your lab notebook. Label it “e” and “40X”
What do you notice about the “e”? (What does the lens do to the image?)
When looking through the eyepiece, what happens when you move the slide forward?
When looking through the eyepiece, what happens when you move the slide back?
When looking through the eyepiece, what happens when you move the slide left?
When looking through the eyepiece, what happens when you move the slide right?
6. Repeat at medium power (including sketch).
7. Repeat at High power (including sketch). You may only be able to sketch PART of the image. Use
more detail under HIGH.
Part C. Field of View Measurement Procedure
Most of the objects you view under the compound microscope are smaller than two millimeters.
Obviously, measuring these microscopic objects could prove to be quite difficult and inexact if
millimeters are used as the unit of measure. To solve this problem scientists divide the millimeter into
1000 smaller units called micrometers (μm). Tiny objects can then be accurately measured in
micrometers. In this section you will learn how to estimate the size of the tiny organisms you view under
the compound microscope.
Obtain a transparent plastic ruler from the supply area.
1. Place the plastic ruler on the stage so that the ruler’s edge is centered in your field of view
under low power. Make sure you use the millimeter side of the ruler. Use the diagram for help.
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2. Look through the eyepiece… Position the ruler so one of the millimeter marks is
just visible to the left in your field of view. Use the diagram for help. Notice the
distance between the mark on the left and the next mark is one millimeter.
Estimate the remaining distance in decimal fractions of a millimeter across the
diameter of the field of view. What is your total field of view size in millimeters
under scanning power?
____________________ mm
3. What is the scanning power field diameter in micrometers (μm)? (1 mm = 1000
μm)
____________________ μm
4. What is your total field of view size in millimeters under medium
power?
____________________ mm
5. What is the medium power field diameter in micrometers (μm)? (1
mm = 1000 μm)
____________________ μm
6. Switch to high power. Look at the marks on the ruler. You will find
that the high power field of view is less than 1mm or 1000μm. For that reason, it is difficult to
estimate the diameter of the field of view using the same technique used for low power.
However, you can determine the field of view under high power by using the formula below:
7. Using the simple algebra formula above… What is the high power field diameter in micrometers
(μm)? (1 mm = 1000 μm)
____________________ μm
8. Now that you know the diameter of your field size under both high and low
power, you can use that information to estimate the size of objects you
observe under the microscope. For example, in the diagram at the right, 10
circular objects fit across the field of view. The field of view is 2000μm in
diameter. Since each object takes up 1/10 of the 2000μm field diameter, the
size of each object is 200μm. You can use this method to estimate size of
objects you view under your microscope once you know your microscope’s
field diameter.
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9. Obtain three prepared slides from the tray and use the methods (above) to estimate the size or
thickness of the objects. Record your findings in the data table.
Number of
Estimated
Magnification?
Field
Specimen Name
Specimens
that
fit
specimen
size or
Whichever allows you to est.
Diameter
size best.
across
thickness
Part D. Depth of Field Procedure
1. Obtain a slide of colored threads from the supply area.
2. Look at the slide under low power where the threads cross. Adjust the knobs to give the
sharpest view. Are all three thread colors equally visible under low power?
3. Look at the slide under high power where the threads cross. Adjust the knobs to give the
sharpest view. Are all three thread colors equally visible under low power?
4. Slowly fine focus up and down to determine the order of the thread colors.
a) Top
b) Middle
c) Bottom
*no drawings necessary
The “Wiggle” Technique
This will come in handy in the future!
1. Sometimes a slide will be a very thin section of a tissue. In this case you must not let the light
“wash out” the specimen. Put the light very low and close the iris.
2. Once you place the specimen right in the middle, wiggle the slide back and forth slightly while
focusing with the course adjustment knob. When you get close you will start to see the slide
moving back and forth in your field of view. If you see no “back and forth” then you are not
close to being in focus!
*no drawings necessary
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QUESTIONS
1. Identify the parts of the
microscope from memory.
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
l.
m.
2. Describe how a compound microscope should be held and carried.
3. How is the total magnification of a microscope determined?
4. If the eyepiece on a microscope has a magnification of 10X, what is the total magnification with
a 15X objective?
5. If the eyepiece on a microscope has a magnification of 15X, what is the total magnification with
a 45X objective?
6. An image is located in the lower right hand corner of the field of view. How would you move the
slide to center the image?
7. Objects viewed under a compound microscope are frequently lost when switching from low to
high power. Give one reason why this happens.
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8. How did the light intensity change when you switched from low power to high power objective?
9. Do you observe more or less area in your field of view when under high power compared to low
power?
10. If a microscope has a low power magnification of 100X and a high power magnification of 500X,
and a low power field of 1500μm, what is the high power field in μm? (SHOW YOUR WORK)
11. If 20 objects fit across the diameter of a low power field of view whose field diameter is
4000μm, what would be the approximate size of each object?
12. Why is it more difficult to measure the diameter of the high power
field of view than the low power field of view?
13. The circle at the right represents a microscope’s field of view with a
black dot under 10X magnification. Draw how large the dot would
appear under 40X magnification. Also, draw a circle to indicate the
size of the field of view under 40X
14. Sketch the number 4 as it appears through the lenses of the
compound microscope.
15. How has the lens system of the compound microscope changed the orientation of the numeral?
16. A student focuses on a specimen at low power and carefully centers it before changing to high
power. At high power, however, he doesn’t see the part of the specimen he was interested in.
What might be the problem?
17. Inspired by her biology lab, a student decides to make a closer study of the food she eats. She
uses a razor blade to make a very thin section from a raw potato and mounts it in a drop of
water on a slide. To her disappointment, she can barely make out the cells under the
microscope. What might she do to improve her results?
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18. How is magnification different from resolving power?
19. What are the advantages and limitations of studying cells using light microscopy?
20. Compare and contrast transmission electron microscopy (TEM) and scanning electron
microscopy (SEM).
21. What are the advantages and limitations of studying cells using electron microscopy?
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