Basic Microscopy Lab
Name_________________________
Purpose:
After completing this exercise, you should be able to do the following:
a. Name the parts of the compound microscope and the functions of each.
a. b. Describe how total magnification is calculated.
b. c. Know the correct procedure for using and caring for the compound
microscope.
c. d. Understand the concept of depth of focus.
PARTS OF THE MICROSCOPE
Read through this information carefully. Check off each step as you go. Be sure you
understand each item before proceeding to the next step.
1. Review how to carry a microscope: one hand under base, one hand holding arm
Obtain a microscope.
2. The major parts of the compound microscope are shown on your microscope
diagram that has previously been labeled. As you read this text, refer to this diagram and
label the parts that appear in bold print. (Your microscope may be slightly different
from that illustrated.)
3. The base rests on the table and, in most microscopes, contains a built-in light source
and a light switch. The arm rises from the base and supports the stage, lens system, and
control mechanisms. The stage is the flat surface on which microscope slides are placed
for viewing. A mechanical stage holds the slide in place with a clip and can be adjusted
side to side and up and down with control knobs.
4. Most microscopes have a condenser located below the stage. It concentrates the
light on the object and may be raised or lowered by the condenser control knob.
Usually the condenser should be raised to its highest position. Check to see that it is.
5. An iris diaphragm is built into the base of the condenser. The iris diaphragm
control lever varies the amount of light entering the condenser and lens system. Move
the lever back and forth to see how this works.
6. The body tube is supported by the arm and has a pair of ocular lenses at the upper
end and a revolving nosepiece with attached objective lenses at the lower end. The
nosepiece is rotated to bring different objectives into viewing position. The objectives
will click into place when they are in viewing position. Try this now.
7. The magnification of each objective is indicated on the objective itself. An objective
with a magnification of 4x is called a scanning objective. A low-power objective has a
magnification of 10x. A high-power objective has a magnification of 40x. Finally, the
objective with a magnification of 100x is an oil-immersion objective.
8. Look at your microscope and list the objective lenses it has here:
__________________________
_____________________________
__________________________
_____________________________
9. There are two focusing knobs. The coarse-focusing knob has the largest diameter
and is used to bring objects into rough focus when using the 4x and 10x objectives. The
fine-focusing knob has a smaller diameter and is used to bring objects into fine focus. It
is the ONLY focusing knob used with the high-power and oil-immersion objective
lenses.
MAGNIFICATION
1. The magnification of a microscope is determined by the power of the ocular and
objective lenses being used together. The ocular of your scope has a magnification of
10x. You have a list of the objectives above (in item 8). Total magnification is
determined by multiplying the power of the ocular by the power of the objective.
2. Enter the objective and ocular magnifications from you microscope in the table
below. Calculate the total magnification when each of the four objectives is being used.
OBJECTIVE
Lens
scanning
OBJECTIVE
MAGNIFICATION
OCULAR
MAGNIFICATION
TOTAL
MAGNIFICATION
low-power
high-power
oil-immersion
USING A MICROSCOPE
The following exercise is designed to help you develop skill using the compound
microscope. Follow the steps and check them off as you go. Write answers to all of the
questions.
1. Carry the microscope with two hands: one on the arm and one under the base.
2. Place the microscope on the lab bench with the arm toward you.
3. Use a piece of lens paper to clean the ocular and objectives of your microscope.
Only use specially prepared lens paper on the lenses. You could damage the optics using
anything other than lens paper. If the lens is particularly dirty, use specially prepared
lens-cleaning solution with the lens paper.
4. Plug the microscope cord into the outlet at your lab station.
5. Swing the scanning lens into place. You will hear the nosepiece click when the
objective is in position.
6. Looking from the side, NOT INTO THE SCOPE, rotate the coarse adjustment knob
one-half turn. Rotate it the other way. What happens to the stage?
______________________________________________________________________
7. In the same way, rotate the fine adjustment knob one-half turn. Rotate it the other
way. How far did the stage move this time compared with the coarse adjustment?
___________________________________________________________________
8. Turn the coarse adjustment so that the stage is as near to the scanning objective as
possible. Using a piece of paper, mark off the distance between the tip of the objective
and the stage. This is a rough estimate of how far the objective will be from the slide.
Record this distance by drawing it here
______________________.
9. Now swing the low power objective into place, making sure you hear the click. Mark
off the distance to the stage using the same piece of paper. Is it larger or smaller than that
for the scanning objective? ____________________________
10. Repeat the measuring procedure after swinging the high power objective into place.
Watch to see that it doesn’t hit the stage. What happens to the distance between objective
and slide as magnification increases?
_______________________________________________________________________
_______________________________________________________________________
11. Why should you NEVER USE THE COARSE ADJUSTMENT WHEN USING
THE HIGH POWER OBJECTIVE LENSES?
________________________________________________________________________
THE FOLLOWING PROCEDURE IS THE EXACT SEQUENCE OF STEPS THAT
YOU ALWAYS FOLLOW WHEN USING A MICROSCOPE! PAY ATTENTION!!!
1. Put the scanning objective into place.
2. Turn on the light of your microscope.
3. Raise the condenser to its highest position.
4. Looking through the eyepiece, sweep the iris diaphragm control all the way from
right to left. What happens?
____________________________________________________________________
5. Move the control about midway between the extreme left and right positions. You
should now have even illumination over the field of vision. The amount of light will
have to be adjusted for different specimens and different levels of magnification.
6. Take a slide of the letter “e”. Place the slide on the mechanical stage so that the clip
holds it securely in place.
7. Using the knobs on the mechanical stage, center the letter over the stage opening.
8. Show how the letter “e” is oriented on the slide as it appears WITHOUT
LOOKING THROUGH THE MICROSCOPE by drawing it in Figure 2.
Figure 2. Slide of the letter “e” as seen without using a microscope.
9. Turn the coarse adjustment knob to position the stage as close to the scanning
objective as possible.
10. Look through the ocular and adjust the light so that there is even illumination across
the whole field of view.
11. Slowly turn the coarse adjustment toward you until you have the letter in view. If
after a few turns it doesn’t appear, try centering the slide and adjusting the light again.
Repeat the focusing procedure. Now use the fine adjustment for getting sharp focus.
12. Compared to the picture that you drew in Figure 2, is the letter “e” right side up or
upside down?
________________________________
13. Does it face the same way or is it backwards? _____________________________
14. Using the knobs on the mechanical stage, move the slide to the right slightly. Which
way does the image move?
______________________________________________
15. Move the slide up. Which way does the image move? ____________________
16. In your own words, summarize what happens to the orientation and movement of
objects when viewed through the compound microscope.
_____________________________________________________________________
_____________________________________________________________________
17. Draw the letter “e” as you see it in Figure 3. Try to get it about the right size and in
the right orientation.
Figure 3. Image of the letter “e” at 40x total magnification
18. Before changing to a different objective, make sure that the object is in the center of
your field of view and in sharp focus. Looking at the SIDE OF THE MICROSCOPE,
swing the 10x objective into place.
19. Now use the fine adjustment knob to bring the image into sharp focus. If your scope
is parfocal, you should not have to adjust it more than ½ turn at any time. It may be
necessary to increase the light intensity when you use this objective.
20. How large is the letter “e” compared to what you saw at 40x total magnification
(using the scanning objective)?
___________________________________________________________________
21. At this point have your instructor check your work and initial your paper. ________
22. Draw the letter “e” in Figure 4. Again show the approximate size and orientation.
Figure 4. Image of the letter “e” at 100x total magnification
23. Looking at the SIDE OF THE MICROSCOPE, carefully swing the high power
objective into place. You should not have to touch the adjustment knobs if your
microscope is parfocal. Why?
________________________________________________________________________
________________________________________________________________________
24. Remember the coarse adjustment knob is NEVER used with high power. Focus with
the fine adjustment knob only.
25. What happened to the light when you changed to high power?
___________________________________________________________________
26. Draw what you see in Figure 5. Give a title to this figure.
Figure 5. __________________________________________
27. Swing the scanning objective back into place then remove the slide.
DEPTH OF FOCUS
1. Obtain a thread slide.
2. Using the procedures you learned in the last section, center the crossed threads on the
stage.
3. Focus first using the scanning objective, then the low power objective.
4. Center the threads so that the crossing point is in the middle of your field of view.
5. Now position the stage as close to the low power objective as possible using both the
coarse and fine adjustments.
6. Looking into the oculars, slowly move the stage down using the coarse and then fine
adjustment knobs until the first thread is in sharp focus. What color is the thread?
____________________________
7. This is the bottom thread on the slide! How do you know this is the bottom?
________________________________________________________________
________________________________________________________________
8. Now continue to move the stage down using the fine adjustment until a second thread
appears. What color is it?
_________________________________________
9. Does this thread lie over or under the thread you saw in number 6? How do you
know this?
____________________________________________________________________
____________________________________________________________________
10. Now continue to move the stage down using the fine adjustment until a third thread
appears. What color is it?
_______________________________________
11. When one thread is in focus, what is the appearance of the other two threads?
_______________________________________________________________
_______________________________________________________________
12. The importance of depth of field lies in the fact that all layers of a specimen on a
slide are seldom in focus at the same time. When the upper layer looks sharp, the middle
and lower layers are generally blurred. To examine those other layers, we must use the
fine adjustment to move the object downward. The vertical distance that remains in
focus at any one time is called the depth of focus.
13. Check here with your instructor and have her initial you paper. _____________
ESTIMATING THE SIZE OF OBJECTS UNDER THE MICROSCOPE
1. The basic metric unit of length at the light-microscope level is the micrometer (m).
There are 1000m in 1mm. Knowing this, do the following conversions:
1m = _____________ mm
1m = _____________ m
1500m = _____________ mm
0.43mm = _____________ m
2. Obtain a clear plastic ruler. Swing the scanning objective into place and place the
ruler on the stage. Focus in on the millimeter side of the ruler. Estimate the diameter of
the field of view to the nearest tenth. Enter this number in Table 1.
3. Repeat step 2 using the low power objective. Enter the diameter of the field of view
in Table 1.
4. DO NOT REPEAT THESE STEPS WITH THE HIGH POWER OBJECTIVE.
WHY?
______________________________________________________________________
5. To estimate the diameter of the field of view of the high power objective you can use
the relationship: field 1 diameter (in m) multiplied by the total magnification of this
field equals field 2 diameter (in m) multiplied by the total magnification of field 2. You
know the diameter of the low power field, the total magnification of low power and the
total magnification of high power. Therefore, you can solve the following equation:
low power field diameter x total mag. at low power = high power field dia. x total
mag at high power
therefore: high power field dia = (low power field dia x low power total mag)
high power total mag
Use this relationship to figure the field diameter of both the high power field and the
oil immersion field.
Enter both of these field diameters into Table 1.
Ocular
magnification
Objective (___x)
Total
Magnification
Size of field of view
mm
m
scanning ____x
low power ___x
high power ____x
oil immersion ____x
Table 1. Diameter of the field of view
BEFORE PUTTING THE MICROSCOPE AWAY
1. Remove the microscope slide.
2. Turn off the light.
3. Clean all lenses using lens paper.
4. Move the mechanical stage so that there are no parts sticking out.
5. Place the cover on the microscope.
6. Carefully wrap the cord electrical cord around the microscope.
7. Put the microscope in the proper cubby of the microscope cabinet.
Conclusion: Summarize what you learned about using and observing objects using the
light microscope. Include a statement about sources of error. This conclusion should be
written in complete sentences.
Adapted 9/26/08 Bedinghaus
Advanced Techniques in Microscopy
OBJECTIVES
After completing this exercise, you should be able to do the following:
a. a. Understand how to properly use the condenser and iris diaphragm.
b. b. Understand how to use the oil-immersion lens.
c. c. Be able to pin point where the dirt is and remove it in order to obtain
crisp, clear images.
LOCATING DIRT PARTICLES ON THE LENS SYSTEM
1. 1. Turn the oculars. If the dirt turns with the ocular, then the dirt is on the ocular!
2. 2. Switch the objectives. If the dirt is present using one objective but not another,
then the dirt is on the objective.
3. 3. If neither 1 or 2 work, check the condenser and light source. Make sure both are
free from dirt.
FOCUSING THE CONDENSER
1. 1. Place a slide on the stage and focus on it using the low power objective (10x).
2. 2. Turn the light dimmer knob down to a low light setting.
3. 3. Close the iris diaphragm slightly.
4. 4. Raise or lower the condenser until a sharp hexagonal shape can be seen through
the ocular (this shape is the iris diaphragm). The condenser is now in focus for this
objective.
5. 5. You may open the iris diaphragm now until it is just outside the field of view.
6. 6. You will need to adjust the condenser each time you change objectives.
7. 7. In general, with increasing magnification you must increase the light (dimmer
and iris diaphragm) and move the condenser closer to the slide.
USING THE OIL IMMERSION LENS
1. 1. Place a slide on your stage with the specimen up.
2. 2. Using proper technique, focus on your specimen using the high power objective
(40x).
3. 3. Turn your nosepiece so that the high power and oil immersion objectives straddle
your specimen. (You are turning the objectives as if you are going to place the oil
immersion objective into place but you do not turn it all the way. Rather, you turn it
about half-way into position.)
4. 4. Place one drop of immersion oil on your slide directly where you see the light
coming through your slide.
5. 5. LOOKING FROM THE SIDE OF THE MICROSCOPE, rotate the oil
immersion lens into position. The lens should not hit your slide (if it looks like it
will, stop, you might have your slide upside down!). You should see the oil drop
make a continuous film between your slide and the objective.
6. 6. Now focus on your specimen using the fine adjustment knob.
7. 7. When you are done using the oil immersion lens, make sure that you clean both
the lens and the slide carefully