Microscope Investigation
Introduction
The microscope is the biologists basic tool. It has been developed to help explore
the world of living things too small to be seen with the naked eye. Without the help of a
magnifying glass, your eye can only see only a limited amount of detail. For example,
two dots less than 0.1-mm (.004 in.) apart blur into a single fuzzy dot. When you
consider that many of your body cells are one-fourth the size of the smallest dot you can
see, the importance of the microscope.
Early microscopes, like the one Anton Van Leewenhoek (Dutch) made, had only
one lens and difficult to use. The magnifying glass is like early scopes, simple
microscopes. The biggest problem with these scopes was their magnification.
Magnification is the apparent increase in an object’s size. The more powerful the lensfor greater magnification- the closer we had to be to the lens. At very high magnification
the lens almost touched the eye.
A major advance in microscopes came with the invention of the compound
microscope. It has two simple lenses, which magnify objects much more a single lens.
Two Dutch eyeglass makers named Hans and Zacharus Janssen- are credited with
developing the first compound light microscope in 1590.
Light microscopes are important tools, but have one major drawback. These
microscopes can magnify an object any desired amount, but eventually the details of the
object become fuzzy. Why does this occur? The resolving power, or the ability of a
microscope to provide clear details depends on the objective lens. The best objective lens
can distinguish objects as close together as 0.2 micrometers (um). By combining this
objective with the right ocular, the viewer can get useful magnification up to 2,000 times.
Resolution is the ability to distinguish between two points.
QUESTIONS:
1. Who is credited with developing the first light compound microscope? Identify the
year this took place and the country they came from.
2. Describe the early simple microscopes.
3. Differentiate between resolution and magnification.
4. What is a major drawback of the light microscope?
5. What is the largest useful magnification that can be achieved with the light
microscope?
THE LENS SYSTEM:
One of the two sets of lenses on a compound microscope are the objective lenses.
They work much as did the lens of the early simple microscope. The objective lenses
make the initial or primary magnification. They are located on the nosepiece of the
microscope; you probably have three of them on your scope. Locate the objective lenses
on your microscope.
Inscribed on each objective is the magnification or power of that lens. This tells
the number of times the lens magnifies the image. For example if you are looking at a
strand of hair with 4X lens, the hair will appear four times the actual size.
Rotate the lenses in the nosepiece until they click into position. The objective
lenses are:
4X This is the scanning lens (not on all microscopes)
10X This is low power
40X This is the high power
The second kind of lens in the microscope is the ocular, sometimes called the
eyepiece. This lens is located on the top of the body tube. Locate this lens on your
microscope. The ocular lens serve as a small telescope, magnifying the image made by
the objective lens. This lens is involved in secondary magnification. The second lens
now forms the compound microscope. The most common magnification is the 10X
ocular. (which is what you have!)
The total magnification of the microscope is determined by multiplying the
primary magnification (objective lens) by the secondary magnification (ocular lens).
*CAUTION – Do not put your fingers on the lenses. If your lenses need cleaned use
LENS PAPER, Do not use anything but lens paper!!
QUESTIONS
1. How many objective lenses does your microscope have?_____________
2. Note the length of each lens. Which power of lens is the
longest?___________
3. List all the magnification of each of your objective lenses on your microscope.
4. Differentiate between secondary and primary magnification
5. What is the magnification of your ocular?______________
6. How is the total magnification determined?
7. What is the lowest total magnification of your microscope?_____________
8. What is the highest total magnification of your microscope?______________
9. What is the magnification of the low power objective?_____________
THE FOCUSING SYSTEM
In order to bring the image of the specimen into proper focus, it is necessary to
change the distance between the slide and the objective lenses. This change either takes
place by the nosepiece moving or by the stage moving.
Focus is controlled by two knobs. The coarse adjustment knob (largest knob) is
used first when the objective must be moved a significant distance. Locate this knob on
your microscope. The fine adjustment (smallest) is used when you wish to make only a
slight improvement in your focus. Depending upon the microscope the fine and coarse
knobs can be located in different positions.
PROCEDURE
1. Have your microscope positioned like the picture at the
left.
2. Turn the objective lens to low power. (10X)
3. Slowly turn the coarse wheel adjustment back and
forth. DO NOT force the wheel once it stops.
4. When it stops, turn it in the opposite direction. Note
which moves the, stage or the nosepiece.
5. Compare the distance and movement of the coarse and
fine wheel adjustment. First give one full turn of the
coarse adjustment, then do the same for the fine wheel
adjustment.
QUESTIONS
1. In which direction does the objective/stage move as you turn the coarse wheel
towards you?
2. In which direction does the objective/stage move when you turn the coarse
wheel away from you?
3. Which wheel moved the objective the farthest and the fastest?
4. Why is it necessary to have two different adjustment knobs?
The Stage
The specimen to be viewed through the microscope is mounted on a glass slide
and covered with a cover slip. The slide rests on the stage, the flat surface beneath the
body tube. Stage clips hold the slide in place if you are transporting it from one place to
another, otherwise don’t bother with them
Both the slide and the stage are extremely smooth. Water between them acts like
glue and causes the slide to stick to the stage. Try and keep both the stage and bottom
slide dry.
QUESTIONS
1. When should you use stage clips? Is it necessary to use stage clips when doing
normal viewing?
2. Why should you keep stage and slide dry?
The Lighting System
A camera, your eye, and a microscope all require light and all three also have a
means by which they can regulate the amount of light that enters the system. The lighting
system is located under the stage of the microscope. There are three different types of
lighting systems. We will be considering one.
Some scopes use a mirror as a light source, our scopes have a sub-stage light. When a
viewing a living specimen you need to be careful about leaving the light on too long. The
light gets hot and can easily destroy your organism. Some organisms even try to avoid
light which often makes finding them difficult.
Under the stage you will find a diaphragm. It is used to adjust the amount of light
that passes through the specimen. The diaphragm works likes the aperture on a camera
and like the pupil of your eye.
The type of diaphragm that can be found on your microscope is a disk diaphragm
This diaphragm has different size holes that can be rotated into place. On a camera the
wrong amount of light distorts a picture and makes the figures difficult to see. Too much
light on a thin or semitransparent specimen will make it very hard to see.
Plug in your microscope and adjust your diaphragm so that you can see when the
most and least amount of light is seen. Make sure your cord is positioned so that nobody
can trip over it. Turn off your light when finished.
Questions
1. What is the function of the diaphragm?
2. What precaution should you take when viewing a living thing with the light
on?
3. Which type of diaphragm does your scope have?
4. If you wanted to increase the amount of light coming into your scope, what
specific adjustment would you make to your diaphragm? Adjustment to
decrease the light?
5. What does the diaphragm compare to on the human body?
6. What light intensity would you use when a specimen is very thin or
semitransparent?
Part and Function Analysis
Identify the parts of the microscope below.
4. ________________
Body Tube
8.____________________
3._______________
_
10._____________
7. _________________
11. ________
12._______________________
5. Stage clips
5. stage
2. ________________
6. ____________
9.________
Match part with function
Function
1.______Scanning lens ( not shown)
2. ______Diaphragm
3. ______Fine adjustment knob
4. ______Ocular Lens (eye piece)
5. ______Stage and stage clips
6. ______Light
7. ______High power objective
8. ______Coarse adjustment knob
9. ______Base
10. ______Nose piece
11. ______Arm
12. ______Low power objective
A. Provide light to see object
B. Place to hold microscope when carrying
it (stand)
C. Place to hold microscope when carrying
it
D. Involved in secondary magnification
E. Hold the objective lenses
F. Primary magnification 10X
G. Primary magnification 40X
H. Brings objects into rapid focus
I. Brings objects slowly into focus
J. Holds specimen and keeps it in place
K. Controls amount of light entering scope
L. Primary magnification 4X
Techniques For Microscope Use
Position of Objects
We often make the statement that things are not as they seem. Well that certainly
is the case when viewing objects under the microscope.
PROCEDURE
1. Put the low power objective (10X) in place (not the scanning lens 4X)
2. Look through the ocular and adjust the light so that you can see
uniformly bright field of view. The field of view is that area you can
see through the lens.
3. If you see specks of debris in the field of view, clean both your ocular
and objective lens with lens paper.
4. Place the lowercase letter “e” in the center of the clean slide. The “e”
should be facing you in its normal position.
5. Place a drop of water on the letter. Next place the edge of a cover slip
over the “e”.
This type of slide is called a wet mount.
6. Place the wet mount on to your microscope stage. Position the slide on
the stage so the “e” faces you as it would on a magazine page.
7. Focusing always begins with the lowest power (4X). Click the proper
objective in
8. Turn the coarse adjustment wheel slowly until the letter “e” comes into
focus. Take it out of focus by turning the coarse adjustment wheel. DO
NOT USE ANY OTHER OBJECTIVE.
Questions
1. Draw the letter “e” as it appears when not looking through the microscope.
2. Draw the letter “e” as it appears when looking through the microscope.
3. While looking through the eyepiece, move the slide slowly from left to right.
In which direction does the letter appear to move when viewed when moved
through the scope?
4. Recenter the letter “e”. Push the slide away from you on the stage. Which
direction does the letter “e” move when viewed through the microscope?
5. Push the slide toward you. Which direction does the letter move?
Use Of The Diaphragm
This is one of the most important and most often forgotten tools on the
microscope. In order to see an object clearly you must adjust both the lens position and
the light intensity.
Prepare a wet mount of a few strands of absorbent cotton. Follow the same
procedure in the previous section.
All objects viewed under the microscope will require adjustment of light. Many
problems associated with microscope observation can be overcome by adjusting the
diaphragm for proper lighting. Don’t forget this later on when you have trouble locating
some objects.
Observe the cotton fibers with low power. While looking through the microscope,
change the amount of light entering the microscope by adjusting the diaphragm.
Questions
1. Under what diaphragm setting (maximum, medium, or little light) are the
cotton fibers sharpest?
2. What should you do if you are having difficulty locating a very transparent
specimen?
Depth Of Field
When viewing living moving samples we often difficulty seeing objects because
they move to different depths within their small aquatic environments.
PROCEDURE
1. Make a wet mount using two different colors of thread cross
the two different strands and form an X.
2.
3.
Locate the stands under low power.
Center the slide so you are looking at the point where the two
strands cross.
4. Adjust your diaphragm so that you see the different light
intensities. Use the amount of light that you feel is correct. Not
what you see.
5. Now locate the center of the threads under high power
The lens system of your microscope allows you to see clearly only one depth at a
time under high power. In order to see objects at different depths, do the
following:
 Turn the fine wheel adjustment back and forth by a quarter of a turn while
looking through the microscope. This movement will give a three
dimensional view of the object. Try this technique while looking at the
crossed threads.
Questions
1. Can both strands be seen clearly at the same time under low power?
2. Can both strands be seen clearly at the same time under high power?
3. Make two drawings of the threads on high power, first with the top thread in
focus, and next with the bottom thread in focus
Stains as an Aid to Microscope Work
Many objectives observed with a microscope are colorless. Thus, they appear
almost transparent and are difficult to see. Stains often are used in microscope work to
color objects for easier and more detailed observations. Remember the reason they are
called stains is because they stain!
PROCEDURE
1. Add a drop of water on a glass slide.
2. With a scalpel or razor blade, gently scrape the end of a peeled
potato. You will then have an accumulation of potato juice.
DO NOT cut a solid piece of potato.
3. Mix the potato scrapings with the water and add a cover slip
4. View the wet mount with low power. You are looking at starch
grains, they are colorless.
5. Remove the slide from the microscope and add a drop of iodine
solution to your slide along one edge of the cover slip as shown
in figure A.
6. Place a piece of paper towel along the edge of the cover slip
opposite the iodine solution.
7. Allow the tissue paper to touch the water of the wet mount as
shown in figure B. This will soak water into the tissue paper,
drawing the iodine stain under the coverslip and into contact
with the starch grains.
8. Observe under low power (10X). Find an area where you have
an even distribution of grains. You don’t want an area that has
hundreds of grains. Draw and color what you observe. Call you
teacher over to approve your slide. ___________ Teacher
initials.
DO NOT MOVE YOUR SLIDE! KEEP THIS SLIDE FOR THE NEXT SECTION!
Questions
1. What color are the stained starch grains?
2. How many starch grains are visible under low power?_________ If you have
too many to count you should do the following. Divide your field of view into
four even sections. Make sure you have an even distribution of grains. Next,
count the number of grains in one section and multiply that by 4. This will
give you the total number under low power.
NOTE- If you where unsuccessful in staining your starch grains try again. Except this
time try adding your iodine directly on the potato
Comparison of Fields of View
Field of view is the area seen through a microscope. Is the field of view greater
with high power or low power, or are they the same? This exercise will help you answer
that question.
PROCEDURE
1. Switch to high power and count the total number of starch grains. __________
2. Draw a picture below of what you observe under high power.
Starch grains
high power
Questions
1. Do the number of starch grains that are visible under high power decrease or
increase.
2. What happens to the size of the individual starch grains under high power
3. To calculate how much wider the area under low power is than high power do
the following:
high power magnification = # of times low power width is greater
low power magnification than high power width
4. How much larger is your low power area than the high power objective?
4. As total magnification increases the total area observed________________
5. Imagine that you located a specimen under the low power and a specimen is
not centered but instead is off to one side of your field. What do you think you
will then see under high power? If you can’t figure this out try it with the
letter “e” for confirmation.
Comparing the Stereoscope to the Compound Light Microscope
The stereoscope is also referred to as the dissecting scope. Its light source is
different than that of the compound light microscope. Also, the eyepieces are
significantly different, as well. One of the scopes has a greater magnification than
the other.
1. The stereoscope can view objects that are opaque (light can’t pass through). How
does the difference in the light source make this
possible?____________________________________________________________
2. The stereoscope can produce images that have depth to them like a 3D image. What
about the eyepieces makes this possible?
_____________________________________________________________________
3. How does the greatest magnification of the two scopes compare?
_________________________________
Now for fun. View something exciting like an insect or a $5 bill on the stereoscope.
Get it in focus with both your eyepieces. Move the object while watching through
the microscope.
4. When you move the object to the left, what direction does it appear to move through
the scope?__________
5. Draw a picture of what you see.
How is the image you see different from the image you saw through the compound light
microscope?
________________________________________________________________________
______________
3. View another object you are interested in (the cuticle of your finger, a hair, or a
scab might be really cool). ***Extra Credit***
Draw what you see.