AP BIOLOGY
CELL UNIT
ACTIVITY #1
NAME____________________
DATE__________HOUR______
MICROSCOPE LAB
PART I: C OMPOUND M ICROSCOPE
OBJECTIVES: After completing this exercise you should be able to:
4 Demonstrate proper care and use of a compound microscope.
4 Identify the parts of the compound light microscope and describe the
4
4
4
4
function of each part.
Compare magnification, resolving power, and contrast.
Demonstrate proper technique of preparing a wet mount slide.
Demonstrate inversion and depth of field.
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. The
greater the area of the retina covered by the image of a specimen, the greater its
magnification.
A: PURPOSE OF THE MICROSCOPE
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.
Cell Unit Activity #1 page #1
The microscope is an expensive
precision instrument. When removing the
microscope from the storage area, always
grasp it with both hands. Place one had
around the arm and the other hand firmly
under the base. Hold it close to your body
for stability. Once you reach your work
area, set the microscope down gently on the
table with the arm toward you.
1.
Get a microscope from the microscope
cabinet and bring it back to your desk.
B: T HE COMPOUND M ICROSCOPE
This section covers the parts of a compound microscope. Make sure you can
identify each of the parts listed in this section on your microscope.
1.
Support Structures
Arm:
Supports the body tube and the stage of the microscope
Stage:
Platform where the slide is placed for viewing
Stage clip: Holds the slide firmly in place on the stage
Stage opening: The hole in the stage that allows light to pass from the
lamp, through the specimen, and into the body tube.
Base:
Lowermost part of the microscope; provides a firm and steady
support
Body tube: Holds the eyepiece lens and objective lens at the correct
distance for magnification.
Rotate the coarse focus knob. Does the stage or body tube move?
_____________________________________________________________
2.
Lighting
Lamp:
Provides the light needed to view the specimen.
Diaphragm: A disk located directly below the stage of the microscope;
regulates the amount of light passing through the stage
opening.
Condenser: Focuses the beam of light; located below the stage
Does your microscope have a condenser?____________________________
Describe the diaphragm on your microscope._________________________
_____________________________________________________________
Cell Unit Activity #1 page #2
What diaphragm setting (number) allows the most light to pass through the
specimen?
_____________________________________________________________
What diaphragm setting (number) allows the least amount of light to pass
through the specimen?
_____________________________________________________________
3.
Focus
Coarse focus knob:
Larger knob used to elevate or lower the body tube
or stage a large distance with each turn.
Fine focus knob: Smaller knob used to elevate or lower the body tube or
stage a small distance with each turn; used to make fine
adjustments when focusing on a specimen.
Where are the coarse focus knobs located on your microscope?
_____________________________________________________________
Where are the fine focus knobs located on your microscope?
_____________________________________________________________
4.
Optics
The compound microscope consists of
a set of lenses that gather light
transmitted through a specimen and
focus this light on the retina of the
eye. The diagram below shows the
path of light as it passes from the
lamp, through the microscope, and
into the eye.
The compound microscope has at least
two lens systems: an eyepiece that
you look into and an objective that
magnifies the specimen.
Eyepiece lens: Located in the upper end of the body tube and focuses light
on the retina of the eye. The power of the eyepiece is usually 10X.
How many eyepieces does your microscope have?______________________
Is it monocular or binocular?_______________________________________
Cell Unit Activity #1 page #3
Objective lenses: Attached to the revolving nosepiece. The number and
magnification of the objective lenses will vary with the type of microscope.
The objective lenses are housed in one end of several steel tubes that are
threaded into the revolving nosepiece. The desired objective lens is placed
in position by rotating the nosepiece until it clicks into place. The
microscopes used in this class have either three or four objective lenses. The
objectives include the scanning lens (4X), lower power lens (10X), high
power lens (40X), and the oil immersion lens (100X) in some microscopes.
The drawing below shows the distance between the objective lens and the
slide. This distance decreases with higher magnification; therefore it is
important to use care when focusing with higher magnification. Only the fine
focus knob should be used. Notice the oil immersion lens requires a drop of
oil between the lens and the slide.
The Lens Table below will help you see the relationship between the unaided
eye and the magnification made possible by the light microscope. The
magnification is marked on the housing of each lens. The power of the
microscope is determined by multiplying the power of the eyepiece lens
times the power of the objective lens. The objective lenses have a color
coded ring around each lens which indicates the magnification of that lens.
5.
Complete the Table below.
Lens
Lens
Magnification
Ring Color
Total
Magnification
Eyepiece
Scanning
Low Power
High Power
C: FOCUSING THE M ICROSCOPE
6.
Obtain a prepared slide from the supply area.
7.
Make sure the scanning lens or the lower power lens is in place.
8.
Raise the body tube or lower the stage just enough to allow you to place the
slide on the stage without hitting the objective lens.
Cell Unit Activity #1 page #4
9.
Place the slide on the stage of your microscope and clip it into place. Move
the slide so the specimen is over the stage opening.
10.
While looking at the microscope from the side, move the body tube all the
way down or move the stage all the way up.
11.
While looking through the eyepiece, move the body tube up or move the
stage down until the specimen comes into focus.
12.
Adjust the diaphragm opening until you have the best view of the specimen.
13.
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. The ability of the microscope to remain in focus
when switching from one objective lens to the next highest power is called
parfocal.
14.
Adjust the diaphragm opening until you have the best view of the specimen.
12.
Have your partner repeat steps 6 – 12.
13.
Return the prepared slide to the supply area.
D: SPECIMEN ORIENTATION
14.
Prepare a wet mount slide of an R by first cutting a capital R out of a
newspaper. (Do not use one from a headline.)
15.
As shown in the illustration below, place a drop of water on the slide
(Diagram #1)
2
1
3
Cell Unit Activity #1 page #5
4
16.
Add the R to the drop (Diagram #2).
17.
Place one edge of a coverslip on the slide, in the water, next to the R. Use a
dissecting needle or pin to gently lower the coverslip onto the R (Diagram
#3).
18.
Get rid of any air bubbles by raising and lowering the coverslip until any
trapped air is released. Do not press directly down on the coverslip.
19.
Place the letter R slide right side up on the stage with the low power
objective lens in place. Center the letter in the field of view.
20.
Bring the R into focus under low power.
21.
Draw the R as you see it through the eyepiece with the low power lens in
place.
22.
Bring the R into focus under high power.
23.
Draw the R as you see it through the eyepiece with the high power lens in
place.
R viewed without
Microscope
24.
R viewed under
Low Power
R viewed Under
High Power
With the low power objective lens in place, move the slide to the right while
watching the image through the microscope.
In what direction does the image move?_____________________________
25.
Move the slide away from you. In what direction does the image move?
_____________________________________________________________
What is the relationship between the movement of the image and the
movement of the object?
_____________________________________________________________
Cell Unit Activity #1 page #6
E: M ICROSCOPE MEASUREMENT
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.
26.
Obtain a transparent plastic
ruler from the supply area.
27.
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
below for help.
28.
Position the ruler so one of the millimeter
marks is just visible to the left in your field
of view. Use the diagram at the right 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 low power?________
29.
What is the low field diameter in micrometers (µm)? (1 mm = 1000 µm)
_____________________________________________________________
30.
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:
High Power Magnification
=
Low Power Magnification
Low Power Field Diamter
High Power Field Diameter
What is the microscope’s calculated high power field diameter in µm?
_____________________________________________________________
Cell Unit Activity #1 page #7
31.
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.
32.
Obtain the prepared slides for this section from the supply area.
33.
Focus under low or high power to view each specimen and then estimate the
size of each. Record your observations in the table below.
Specimen
34.
Viewed
under Low
or High
power
Field
Diameter
# of
specimens
that fit
across field
Estimated
Specimen
Size
Return all slides to the supply area.
F: DEPTH OF FIELD
35.
Obtain a microscope slide of silk fibers from the supply area.
36.
Look at the slide under low power where the threads cross. Adjust the
diaphragm to give the sharpest view. Are all three thread colors equally
visible under low power?
_____________________________________________________________
Cell Unit Activity #1 page #8
37.
Look at the slide under high power where the threads cross. Adjust the
diaphragm to give the sharpest view and fine focus. Are all three thread
colors equally visible under high power?
_____________________________________________________________
38.
Slowly fine focus up and down to determine the order of the thread colors.
Which color is on top?___________________________________________
Which color is on the bottom?_____________________________________
How did you determine the order of the thread colors?__________________
_____________________________________________________________
39.
Return the slide to the supply area.
PART II: STEREOSCOPIC DISSECTING M ICROSCOPE
OBJECTIVES: After completing this exercise, you should be able to:
4 Explain how a dissecting scope differs from the compound microscope.
4 Identify the parts of the dissecting scope and give the function of each
part.
4 Describe how to locate and focus on a specimen using the dissecting
scope.
4 Identify situations in which a dissecting scope would be more useful than
a compound light microscope.
INTRODUCTION:
In order to be viewed with the compound light microscope, specimens must be thin
enough for light to pass through them. They must be mounted on glass slides;
usually they are covered by a coverslip, so little manipulation of the specimen is
possible while it is being viewed. In addition, the minimum magnification is
typically 4OX.
The stereoscopic dissecting microscope, usually referred to as a dissecting scope,
provides a lower range of magnifications, usually between 5X and 50X, and a large
working distance for manipulation of specimens. This type of microscope is also a
compound microscope, since there are two lens systems, but in this case the
objective lenses are housed inside the scope and are changed using a knob or dial.
The number and magnifications of objective lenses vary considerably from one
model of microscope to another. Some have only two settings, low power and high
power, while others can be varied continuously like a zoom lens.
Cell Unit Activity #1 page #9
All dissecting scopes have two eyepieces. In contrast to a binocular compound
microscope, in which you see the same image with both eyes, in the dissecting
scope each eye sees through an independent system – there are actually two
objectives. The result is that you see depth the way you do with unaided eyes.
A. P ARTS OF A DISSECTING M ICROSCOPE
40.
Locate the following parts on your dissecting scope.
Eyepieces
These lenses magnify the specimen. Notice that the distance between the
eyepieces can be adjusted to fit the distance between your eyes. Often one
eyepiece has a diopter ring on it, which allows you to adjust the focus of the
two eyepieces independently.
What is the eyepiece magnification on your dissecting scope? ____________
Does your microscope have a diopter ring on one of the eyepieces?________
Stage
Usually the specimen may be placed directly on the stage to be examined. A
slide is required only for some types of specimens (for instance, if the
specimen must be viewed in a drop of water). A hole in the center of the
stage allows light to be transmitted from beneath the stage.
Magnification Changer (Zoom Lens)
This dial or knob changes the objective lenses inside the microscope. It is
marked to indicate the magnifications possible for the objectives. There may
be a setting that is less than 1 (for example, 0.7). If the eyepiece is 10X,
this objective would give a total magnification of 7X.
What are the range of total magnifications for your dissecting scope?
_____________________________________________________________
Focus Knob
Note that there is only one type of focus knob on this kind of microscope.
Light Sources
Two types of lighting are used with dissecting scopes: transmitted light,
which comes from beneath the stage, and reflected light, which shines down
on the specimen from above. Transmitted light is used when the specimen is
thin and transparent. Reflected light is used for specimens that are opaque.
It enhances the three-dimensional quality of the image.
Describe the illumination system in your microscope.
_____________________________________________________________
Cell Unit Activity #1 page #10
B. USING THE D ISSECTING SCOPE
41.
Cut out a section of newsprint with an “R”.
42.
Position the newsprint with the “R: on the stage so that the R is directly over
the hole in the center of the stage.
43.
Set the objective magnification at its lowest power.
44.
Turn on the transmitted (substage) light.
45.
Focus on the R. (You may need to complete step 46 to get the best view).
46.
While looking through the eyepieces, move them together or apart until you
see a single image.
47.
How is the image of the R oriented compared to the image of the R on the
slide itself?
____________________________________________________________
48.
Look through the eyepiece and move the slide to your right. What direction
does the R move?
____________________________________________________________
49.
What happens when you move to the slide toward you?
____________________________________________________________
50.
Turn the magnification knob so that you see the R through the entire range
of magnification available.
51.
Turn off the transmitted light and turn on the reflected light. How does this
change the image you see?
_____________________________________________________________
_____________________________________________________________
52.
What light source is better for this slide? ____________________________
53.
Next, get a photograph that has been cut out of a newspaper and place it on
the stage.
54.
Using reflected light, focus on the image. When you magnify the picture, you
can see that it is composed of dots. The individual dots are not resolved by
the unaided eye.
Cell Unit Activity #1 page #11
55.
Turn off the reflected light and turn on the transmitted light. How does this
change the image you see?
____________________________________________________________
____________________________________________________________
PART III: Q UESTIONS
56.
Describe how a compound microscope should be held and carried.
_____________________________________________________________
_____________________________________________________________
57.
How is the total magnification of a microscope determined?
_____________________________________________________________
58.
If the eyepiece on a microscope has a magnification of 10X, what is the total
magnification with a 15X objective?
_____________________________________________________________
59.
If the eyepiece on a microscope has a magnification of 15X, what is the total
magnif ication with a 45X objective?
_____________________________________________________________
60.
A microscope gives a total magnification of 1500X, but the image is too
blurry to be useful. What might be the problem with the microscope?
_____________________________________________________________
_____________________________________________________________
61.
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?
_____________________________________________________________
_____________________________________________________________
Cell Unit Activity #1 page #12
62.
Objects viewed under a compound microscope are frequently lost when
switching from low to high power. Give one reason why this happens.
_____________________________________________________________
_____________________________________________________________
63.
How did the light intensity change when you switched from low power to high
power objective?
_____________________________________________________________
64.
In general, how would you have to adjust the diaphragm after switching from
low to high power?
_____________________________________________________________
65.
Do you observe more or less area in your field of view when under high
power compared to low power?
_____________________________________________________________
66.
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?
_____________________________________________________________
67.
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?
_____________________________________________________________
68.
Why is it more difficult to measure the diameter of the high power field of
view than the low power field of view?
_____________________________________________________________
_____________________________________________________________
69.
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 magnification.
Cell Unit Activity #1 page #13
70.
Sketch the number 4 as it appears through
the lenses of the compound microscope.
How has the lens system of the compound
microscope changed the orientation of the
numeral?
____________________________________
71.
Sketch the number 4 as it appears through
the lenses of a dissecting scope.
How has the lens system of the dissecting scope
changed the orientation of the numeral?
____________________________________
72.
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?
_____________________________________________________________
_____________________________________________________________
73.
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?
_____________________________________________________________
_____________________________________________________________
74.
What are two methods used to study cells?
___________________________________________________________
___________________________________________________________
Cell Unit Activity #1 page #14
75.
How is magnification different from resolving power?
Magnification
76.
Resolving Power
What are the advantages and limitations of studying cells using light
microscopy?
Advantages
77.
Limitations
Compare and contrast transmission electron microscopy (TEM) and scanning
electron microscopy (SEM).
TEM
SEM
Similarities
Differences
78.
What are the advantages and limitations of studying cells using electron
microscopy?
Advantages
Cell Unit Activity #1 page #15
Limitations
79.
List the steps in cell fractio nation.
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
80.
What are the advantages and disadvantages of studying cells using cell
fractionation?
Advantages
Cell Unit Activity #1 page #16
Disadvantages