Exercise 4 USE OF THE MICROSCOPE

Introduction
Many objects of biological interest are too small to be seen with the naked eye. Various
instruments, including simple lenses, dissecting microscopes, compound microscopes, and
electron microscopes, have been developed to provide enlarged images and enhanced resolution of
small objects.

meter
1 .1 .01 .001
millimeter
1 .01 .001
.1
naked eye micrometer
1 .01 .001
.1
dissecting light microscope nm
1
.1
compound light microscope

electron microscope

approximate  sizes  of  

biological  objects   cat house human bacterium protein
fly sperm molecule
mouse flea red blood virus amino  
cell acid
Figure 4.1 Visual range of microscopes as compared to the range of the unaided eye. molecule

A short description of two kinds of light microscopes and two kinds of electron microscopes
follow.
Light microscopes use light rays that are magnified and focused by means of lenses. The dissecting
microscope is designed to study entire objects in three dimensions at low magnification. The
compound microscope is used for very small or thinly sliced sections of objects under
magnification that is higher than that of the dissecting microscope. To improve contrast as light
passes through the specimen, stains or dyes are often used to bind to cellular structures and absorb
light.
Electron microscopes use a focused beam of electrons to examine objects at very high
magnification. The scanning electron microscope is somewhat analogous to the dissecting light
microscope in that it gives an image of the surface of an object. The transmission electron

Biology  171  Lab  Notebook   Exercise  4:  Use  of  the  Microscope     Page  36    
 microscope is analogous to the compound light microscope. The object is thinly sliced and treated
with heavy metal salts to improve the contrast.
In many of the exercises that you will perform in BIOL 171, you will be expected to use the
dissecting and/or compound microscopes. This exercise is intended to make you familiar with
their proper use.

The Compound Microscope

The microscope that you will use in this course is the Olympus model CHT, a good quality (and
expensive!) binocular model. Refer to the diagram of the microscope in Figure 4.2, and identify
the following components on your microscope:

       Eyepiece  or  Ocular  

                               Condenser  

                             Illuminator  

Voltage  control  
dial  

Figure 4.2 The parts of an Olympus compound light microscope

Photograph of microscope courtesy of  http://makezine.com/choosing-a-microscope/

Biology  171  Lab  Notebook   Exercise  4:  Use  of  the  Microscope     Page  37    
1) The microscope has a built–in ILLUMINATOR (light source) that requires standard line voltage.
Be sure the POWER CORD is properly inserted into the receptacle in the microscope base, then
insert the 3–prong plug into an outlet on the table.
2) The ON–OFF SWITCH for the illuminator is at the lower left on the front of the base. To turn
the light on, press I. To turn the light off, press O.
3) The VOLTAGE CONTROL DIAL is located on the right side of the base. The dial rotates from 1
(low intensity light) to 10 (high intensity light).
4) Important: Set the VOLTAGE CONTROL DIAL at 1 before turning the switch on or off. This
extends the life of the bulb. After turning the light on, turn the dial to 6.
5) Light from the illuminator passes through the CONDENSER. The condenser focuses a cone of
light onto the specimen mounted on the stage. The condenser contains a blue filter that
removes some of the yellow wavelengths of light from the microscope’s light source, giving the
image a more realistic color. The condenser also contains an IRIS DIAPHRAGM that adjusts the
amount of light reaching the specimen. Adjustment of the iris diaphragm can improve the
appearance of the specimen on the slide. This adjustment is made by moving the lever at the
front of the condenser to the left or right. For now, position the iris diaphragm lever so that
it points directly toward the front.
6) The MECHANICAL STAGE provides a platform to support the specimen and a device to move
the specimen from side to side and/or forward and backward. The use of the mechanical stage
will be described later.
7) The mechanical stage can be moved up and down by the COARSE and FINE FOCUS KNOBS.
Their use will be described later.
8) Four OBJECTIVE LENSES, of 4x, 10x, 40x, and 100x magnification, are mounted in the
“nosepiece”. The NOSEPIECE rotates to position any one of the lenses in the light path (i.e.,
directly above the condenser). The lenses can be distinguished by size; the shortest is the 4x
lens, and the longest is the 100x lens. The lenses also have color–coded markings: 4x (red),
10x (yellow), 40x (blue) and 100x (white).
9) Important: The microscope should always be stored with the shortest objective in place.
10) These microscopes are PARFOCAL. In other words, if you are focused on a specimen with one
objective, when you switch objectives, you will still be focused on your specimen (more or
less). You will have to fine focus your specimen.
11) WARNING:   The objective lenses are the most delicate and expensive components of the
microscope. Read the following instructions carefully.
a) When changing from one objective to another, always rotate by the nosepiece, never by
the objective. A “click” sound will tell you that the objective is in place.
b) When rotating the nosepiece, be careful not to hit an objective lens against the
mechanical stage or microscope slide. Leave plenty of clearance. If you use proper
technique for focusing the microscope (see below), this will not be an issue.

Biology  171  Lab  Notebook   Exercise  4:  Use  of  the  Microscope     Page  38    
 c) Bring the mechanical stage up toward a slide only when looking at the lens from the side,
not through the lens. Again, this is to avoid hitting the lens against a slide. Then focus by
moving the stage down away from the slide.
d) Use only lens paper to clean microscope lenses. Never use Kimwipes, paper towels, tissues,
etc. to wipe lenses. These are coarse and can scratch the lenses.
e) Avoid getting the objective lenses wet. If a lens does get wet, immediately wipe it dry with
lens paper. (The 100x lens is designed for oil–immersion use; it should be cleaned
immediately after use.)
f) If you have difficulty viewing a specimen through an objective, clean it with lens paper. If
the specimen is still “fuzzy”, make your instructor aware of the problem.
g) NEVER REMOVE AN OBJECTIVE LENS FROM THE NOSEPIECE!
12) Light passing through the specimen and objective lens then passes into the OBSERVATION
TUBE to be reflected off of a mirror and toward the observer at a comfortable viewing angle.
13) The EYEPIECES (OCULARS) provide 10x magnification of the image. Two adjustments of the
eyepieces are possible: INTERPUPILLARY DISTANCE ADJUSTMENT and DIOPTER ADJUSTMENT.
Interpupillary distance adjustment matches the distance between the eyepieces to the distance
between your pupils. Diopter adjustment insures that both eyes focus on the same plane. The
use of these adjustments will be described later.

USING THE MICROSCOPE

1) While looking from the side, use the coarse focus knob to move the mechanical stage down,
away from the objective lenses.
2) Rotate the nosepiece to position the 4x objective above the condenser. It should “click” into
the correct position.
3) Obtain a microscope slide of the letter “e” and insert the slide into the mechanical stage as
demonstrated by the instructor. Using the mechanical stage controls (below the right side of
the stage), position the e in the light path, directly above the condenser. Notice that the lower
control governs side-to-side motion while the upper control moves the slide forward and
backward.
4) Now move the mechanical stage upward toward the objective lens by turning the coarse focus
adjustment. Remember to watch the stage from the side (not through the eyepieces) as you
move the stage upward so that you will not hit the objective lens with the stage or slide. With
the 4x objective in place, you should be able to move the stage upward to its highest position
without hitting the lens.
5) Look through the eyepieces. Turn the coarse focus knob to move the stage downward until
the e is in focus. Compare the orientation of the e on the slide to what you see through the
eyepieces.
You have made your first “discovery” regarding the operation of a compound microscope. Draw the letter
e as it appears in the microscope. What is your discovery?

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6) Before you proceed to make further observations, adjust the eyepieces as follows:
a) Look through both eyepieces. Adjust the interpupillary distance so that you can most
comfortably view the image. Note the number on the distance scale so that you can make
this adjustment quickly in the future.
b) Look at the e through the right eyepiece while keeping your left eye closed. Turn the fine
focus knob to bring the image into sharp focus.
c) Now, without changing the focus adjustment, look through the left eyepiece and close
your right eye. Bring the image into sharp focus by adjusting the diopter ring just below
the left eyepiece. Now the eyepieces should be focused for both of your eyes.
7) While looking through the eyepieces, move the e (using the mechanical stage controls) until it
is in the middle of the “field”. Then observe the position of the e above the condenser on the
stage. It should be in the center of the light path.
8) While looking through the eyepieces, move the e toward the right until it is at the “3–o'clock”
position in the field. Then observe the position of the e on the stage. Did the slide move to the
right as the e moved to the right in the microscopic field?
9) Center the e in the microscopic field. Observe the position of the e on the stage. Then move
the e in the field until it is positioned at “12–o'clock”. Did the slide move backward or forward on
the stage?
a) In addition to inverting the image, the microscope reverses left and right and forward and
backward. You should be aware of this as you move a slide around on the stage.
10) Light intensity is controlled by the VOLTAGE CONTROL DIAL. While viewing the e through
the eyepieces, adjust the voltage control dial on the right side of the base to give a comfortable
light intensity.
11) Contrast is controlled by the IRIS DIAPHRAGM. Reducing the iris diaphragm aperture (using
the lever below the microscope stage) reduces light intensity, but it also increases contrast
(details in the magnified specimen). While viewing the letter e through the eyepieces, move
the iris diaphragm lever to the right of center until you get maximum contrast (the sharpest
image of the letter e), while allowing sufficient light through to see the object. If you need
more light intensity, turn up the voltage control dial. Students often open the iris diaphragm
too much, causing the image to have less contrast and to appear washed out.
It may be necessary to readjust these controls each time you switch objective lenses; the lower
power objectives allow much more light to pass than the high power objectives allow. As you
increase the magnification, you usually must also increase the amount of light passing through the
specimen.

MAGNIFICATION AND FIELD DIMENSIONS

Total magnification of an object is determined by multiplying the objective magnification by the
eyepiece (ocular) magnification. The objective lenses are marked with their magnification: 4x, 10x,
40x, and 100x. To get the total magnification of an object, the objective magnification is
multiplied by the 10x magnification provided by the eyepieces. For example, total magnification
of an object when using the low power 4x is 40 times. This means that if the letter “e” on your
slide is 2 mm tall, under the 4x objective it will appear to be 80 mm (40 x 2 mm) tall.  
Biology  171  Lab  Notebook   Exercise  4:  Use  of  the  Microscope     Page  40    
1) Observe the letter “e” under the 4x objective to see how large it appears to be. Then, while
watching from the side, use the nosepiece to carefully rotate the 10x objective into position
over the letter “e”. What is the magnification of the letter “e” under the 10x objective?
2) Does the letter “e” appear larger or smaller as you increase magnification? Does the field of
view get larger or smaller as you increase magnification?

3) Fine focus the slide until the letter “e” is clearly in focus. Do not coarse focus while using
objectives higher than 4x power. In the space below, draw what the letter “e” looks like under
the 10x objective.  

 
 
 
 
 
DEPTH OF FIELD
As you focus on a field of view with the light microscope, some objects may go in focus while
others go out of focus. This is because the depth of field is limited on a light microscope. "Depth
of field" refers to the thickness of the plane in which objects are in sharp focus. It is important to
note that as magnification increases, the depth of field decreases. To illustrate the depth of field,
you are going to determine the relative vertical order (top, middle, bottom) of three overlapping
colored threads. With low magnification (such as 40X), there is a large (thick) depth of field, so
that all of the threads can be in focus at the same time. With higher magnification, there is a
smaller or thinner depth of field, so that only one thread can be focused at one time, and the
other threads will be out of focus.
1) Using the 10x objective, examine a prepared slide of three colored threads mounted on top of
each other.
2) Try to determine the order of the threads from top to bottom. If you focus properly, you
should see the bottom thread first, then the middle thread, and finally the top thread.
3) Re–examine the threads using the 40x objective.
4) Once you have determined the order of threads, ask your instructor to verify the correct order.
Depth Thread Color
Bottom ____________
Middle ____________
Top ____________

Biology  171  Lab  Notebook   Exercise  4:  Use  of  the  Microscope     Page  41    
OBSERVATION OF UNSTAINED AND STAINED HUMAN CELLS
Biologists don't examine letters under the microscope, they examine cells, tissues, and organisms.
Many of these subjects are nearly transparent, and as a result, difficult to view. The early
microscopists quickly realized that the application of natural or synthetic dyes might alleviate the
problem. What they discovered was that different dyes have different affinities for cellular
structures, and that these differences could be exploited to specifically stain nuclei, cytoplasm, etc.
In this exercise you will observe the difference between unstained and stained human cells.
1) Scrape the inside of your cheek with a toothpick, then quickly smear the material onto a
slide. Place a drop of alcohol on the smear, mix with a toothpick and cover it with a
coverglass. Toothpicks should be disposed of in the biohazardous waste containers
provided. Observe this preparation under the 10x objective (100x magnification). For
better contrast, adjust the iris diaphragm so that less light comes through the condenser. If
you have trouble finding cells, ask your instructor for assistance.
2) Repeat the procedure above, but instead of placing a drop of alcohol on the smear, place a
drop of the methylene blue (a dye dissolved in alcohol) on the smear.
3) Observe the stained cells under 100x magnification. What cellular structure is preferentially
stained by methylene blue?
4) Observe the cheek cells under 400x magnification. Carefully rotate the objective into
position without moving the mechanical stage. Fine focus the slide (never coarse focus on
the 40x objective). Draw a picture of a stained cheek cell in the box below. This drawing
should fill the box.

 
 
OBSERVATION OF POND WATER
1) Obtain a depression slide from the box on the side counter.
2) Place a drop of pond water in the depression slide well, add a drop of Protoslo to the water,
mix with a toothpick, and cover with a cover slide.
3) Observe the organisms under the lowest magnification (40x). Learn to track a single organism
by manipulating the mechanical stage.
4) Now switch to the 100x magnification and repeat the tracking.
5) Finally, view the organism under the 400x magnification. You may have to adjust the voltage
control dial and condenser.
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THE DISSECTING MICROSCOPE
1) If time permits, examine a Swift Stereo 80 dissecting microscope. Note the following:
A) Power cord
B) Light switch: 3 settings, for top, bottom, or top and bottom illumination.
C) Nosepiece: rotation of the nosepiece allows you to select either the 1x or 3x lens.
D) Focus control
E) Eyepieces, with diopter and interpupillary distance adjustments.
2) Place a specimen on the stage and examine it with top and/or bottom illumination. You
can also examine your skin, fingernails, jewelry, money, etc. under the dissecting
microscope.  

Study  Questions  
1) Which objective should always be in place, both when beginning to first view an object and
when putting the microscope away?

2) Describe the function of the major components of a compound light microscope.

a) illuminator:

b) voltage control dial:

c) condenser:

d) iris diaphragm:

e) mechanical stage:

f) mechanical stage controls:

g) coarse focus knobs:

h) fine focus knobs:

i) objectives:

j) nosepiece:

k) oculars/eyepieces:

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 l) diopter adjustment:

m) interpupillary distance adjustment:

3) Methylene blue preferentially stains: (a) cytoplasm, (b) the nucleus, (c) the plasma membrane,
(d) vacuoles.
4) Calculate the total magnification for the 4x, 10x, 40x, and 100x objectives.

5) You are observing pond water and see a protozoan located in the upper right side of the 40x
microscopic field. You want to magnify this cell more, so you increase magnification to 100x
and fine focus. Even after fine focusing, the protozoan is no longer visible. Why is the
protozoan no longer visible (other than “it swam away”)? What would you need to do to be
able to see the protozoan within the 100x field of view?

6) You focus on a cluster of plant cells from a leaf and notice that some of the cells are in focus
while other cells are out of focus. Explain why.

7) Compare dissecting vs. compound microscopes in terms of how they are used and the
advantages and disadvantages each has.

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8) How can you increase the contrast of an image under the compound light microscope without
using a stain or dye?

9) In which direction do you move the stage with the coarse focus knob when you are properly
focusing the microscope?

10) What is meant by the term “parfocal”?

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11) Organelles can best be seen and measured with an electron microscope. Below is a table
indicating the size (in micrometers) of most cellular organelles. Complete the rest of Table 4.1
as a study guide for Biol 171 lecture.

Table 4.1 Organelles Observed in the Electron Microscope

Organelle Size ( µ m) Function Where Present

Plant Animal
Plasma membrane 7–9x10–3 (thickness)

Cell wall Variable; a single fibril is as

thick as the plasma
membrane
Nucleus 4–10 (diameter)

Chloroplast 8 (length)

Mitochondrion 0.5–10 (diameter)

Vacuole Variable

Golgi bodies Variable

Microbodies 0.2–1.5 (diameter)

(peroxisomes)

Lysosomes 0.2–0.5 (diameter)

Endoplasmic 0.005–0.01 (tube diameter)

reticulum

Ribosomes 1.7–2.3x10–3 (diameter)

Flagella, cilia 0.2 (diameter);

2–150 (length)

Biology  171  Lab  Notebook   Exercise  4:  Use  of  the  Microscope     Page  46    
LAB OBJECTIVES - EXERCISE 4: USE OF THE MICROSCOPE
A. Identify the parts of the compound light microscope with the correct names and give
the functions of those parts.
B. Properly use the compound light microscope. This includes knowing how to:
• carry the microscope;
• clean the microscope lenses;
• focus the microscope
• change objectives using the nosepiece;
• adjust interpupillary distance and focus for both eyes using the diopter ring;
• adjust light intensity using the voltage control dial;
• and, adjust the iris diaphragm for best contrast and resolution.
C. Explain the rules of proper microscope use.
• Initially use the coarse focus with the 4X objective.
• When focusing, move the mechanical stage down, away from the objective
lens. Then change to a higher objective for observation.
• Microscopes are parfocal – after coarse focusing with the 4X objective, you
should only have to fine focus with the higher objectives (10X, 40X & 100X).
• Properly put away the microscope with it set at light intensity “1” on the
voltage control dial and with the shortest objective in place.
D. Calculate total magnification of an object.
E. Explain what biologists do to cells and tissues to highlight cellular structures when
using light micrscopy.
F. Compare and contrast compound and dissecting microscopes in terms of how they are
used and how objects appear when using them.

Biology  171  Lab  Notebook   Exercise  4:  Use  of  the  Microscope     Page  47