Name: ______________________________________________ Date Completed: ________________

Laboratory Activity No. 1:

Introduction to the Microscope Lab Activity

Introduction
"Micro" refers to tiny, "scope" refers to view or look at. Microscopes are tools used to enlarge images of
small objects so as they can be studied. The compound light microscope is an instrument containing two lenses,
which magnifies, and a variety of knobs to resolve (focus) the picture. Because it uses more than one lens, it is
sometimes called the compound microscope in addition to being referred to as being a light microscope. In this
lab, we will learn about the proper use and handling of the microscope.

Instructional Objectives
 Demonstrate the proper procedures used in correctly using the compound light microscope.
 Prepare and use a wet mount.
 Determine the total magnification of the microscope.
 Explain how to properly handle the microscope.
 Describe changes in the field of view and available light when going from low to high power using the
compound light microscope
 Explain why objects must be centered in the field of view before going from low to high power using the
compound light microscope.
 Explain how to increase the amount of light when going from low to high power using the compound light
microscope.
 Explain the proper procedure for focusing under low and high power using the compound light microscope.

Materials
 Compound microscope
 Glass slides
 Cover slips
 Eye dropper
 Beaker of water
 The letter "e" cut from newsprint
 Scissors

Pre-Lab

1. Each student is assigned a numbered microscope. That specific microscope must be used every time by
that student.
 a. True b. False

2. Each individual student must read and complete a Student Microscope Checklist each time a microscope
is used.
a. True b. False

3. Label the microscope parts below.

 Procedure
I. Microscope Parts and Function

1. Carry the microscope with both hands --- one on the arm and the other under the base of the microscope.
2. One person from each group will now go over to the microscope storage area and properly transport one
microscope to your working area.
3. The other person in the group will pick up a pair of scissors, newsprint, a slide, and a cover slip.
4. Remove the dust cover and store it properly. Plug in the scope. Do not turn it on until told to do so.
5. Examine the microscope and give the function of each of the parts listed on the right side of the diagram. Use a
separate sheet to list and define the function of each part of the microscope.

Examining microscope parts and functions

Parts of the light microscope

1. Stand: Arm and base.

All other parts of the microscope are attached to the arm or base.
2. Mechanical stage: The clamping device that secures and moves the slide while it rests on the stage.

3. X-Y mechanical stage control knobs: These knobs, under the left side of the stage, move the slide
horizontally (lower X knob) and vertically (upper Y knob). Never force any knobs.

4. Lamp.
The lamp on/off switch is located in the rear on the base. The light intensity is adjusted with the light
intensity regulator on the right side of the base. The lamp should be adjusted to a medium level at the
start of viewing.

5. Lens systems:
There are three lens systems: the eyepieces (ocular), the objectives (four), and the condenser.

a. Eyepieces (ocular): Magnification of 10X. The eyepieces are held rather loosely in the
eyepiece tubes. Never remove the eyepieces from the eyepiece tubes. A rubber eye
shield should be on the top of each eyepiece. The width between the eyepieces should be
moved until a full circle (the viewing field) is visible with both eyes simultaneously.
Students can record their proper setting by reading the scale between the eyepieces.

b. Objectives: There are four objectives: 4X, 10X, 40X, and the 100X (oil immersion
objective). The objectives are attached to a rotatable nosepiece.

The total magnification is calculated by multiplying the

ocular magnification and the magnification of the objective in use.

c. Condenser: The condenser is located directly beneath the stage. It gathers and conducts
the light to the specimen. The condenser height adjustment knob (on the right side of the
scope arm below the stage) should be moved clockwise until the condenser is in the
highest position. If the field of view is not uniform enough, it may be improved by
lowering the condenser slightly.

The iris diaphragm controls the amount of light from the condenser and is adjusted
with a thin lever under the stage.

6. Focusing knobs: located on the sides of the microscope.

The larger coarse adjustment knob moves the stage up and down much faster and farther than the smaller
fine adjustment knob. The coarse adjustment knob is used ONLY with the lower power (4X, 10X)
objectives. When focusing under the 40X or 100X objective, ONLY use the fine adjustment, never the
coarse adjustment.
Preparation for viewing- Refer to each of the following procedures every time the microscope is
used. Read FIRST, then perform the step.

1. If you have not done so, carefully remove a scope from the cabinet and set it on your lab bench.
Remove the dust cover. Check that no parts are loose or missing). They might be in the dust cover.
Immediately contact the instructor if parts are missing or anything is wrong with the scope. Write
down the number of your scope (found on the front of each scope) on the microscope checklist at the
end of the lab exercise instructions.

2. Plug in the microscope. Be sure the electrical cord is not dangling off of the lab bench and cannot
become entangled.

3. Use the coarse adjustment knob to move the stage to its lowest level.

4. Cleaning the microscope. Clean all objective lenses with swabs and liquid lens cleaner.

Place a small amount of liquid lens cleaner on the swab. Use a fair amount of pressure to clean the
lens in a circular pattern. Dry the lens with the dry, unused end different swab. Use different swabs
for each lens.

Do not use liquid lens cleaner on the eyepieces. Dust on the eyepieces, or elsewhere, may be
removed using the blower brush. NEVER remove any parts.

5. Before use, clean all slides, top and bottom, with Kimwipes. Place a coverslip over the specimen on
the slide. Do not use a coverslip if viewing prepared slides.

6. Rotate the nosepiece until the 4X (10X with bacteria) objective clicks into place.
Turn on the lamp. Adjust the on/off switch to a medium intensity.

7. Gently place the slide on the stage so that it is held within the mechanical stage clamping device. The
bow-shaped lever of the mechanical stage clamping device should be pulled outward and released gently
after the slide is placed on the stage within the clamping device.

The lever should not be released in the middle or the slide could be broken. The slide must lie flat on the
stage.
8. Using the mechanical stage X-Y movement control knobs, position the slide so that the specimen is in
the exact center of the light coming through the condenser. Do not use your hands to move the slide; only
use the X-Y control knobs.

Viewing and focusing the specimen under the 4X, 10X, and 40X objectives

1. Always start with a low power objective (4X or 10X when viewing bacteria) clicked into place.

The lower power objectives have the largest field of view (a larger portion of the slide can be seen),
making it easier to initially find the specimen.

2. Only now, with a lower power objective in place, move the stage to its highest point by rotating the
course adjustment know clockwise. In other words, the stage should be moved as close to the objective as
possible.

3. Optimal viewing by adjusting the eyepieces while focusing (steps

a. Inter-pupillary distance: While looking through the eyepieces, adjust the width between the eyepieces
(inter-pupillary distance) until a single image is seen simultaneously with both eyes. The left and right
fields of view should coincide completely. The position of the dot indicates the inter-pupillary value.
Note your inter-pupillary value.

b. Diopter adjustment: When you look through a microscope with two eyepiece lenses, you must be
able to change the focus on one eyepiece to compensate for the difference in vision between your two
eyes. The diopter adjustment does this.
 1) Rotate the right eyepiece to match your inter-pupillary distance value.

2) Close the eye over the eyepiece with the diopter adjustment (left eye), and normally focus
the microscope so that the open right eye sees the image in focus.

3) Next, switch eyes (close the right eye, open the left eye) and without changing the main
focus knobs, focus on the image by turning the diopter adjustment ring on the left eyepiece
only. Now with both eyes open, the image should be clear with both eyes.

c. Eye Shades

When wearing eyeglasses: Use the eye shades in the normal, folded-
down position. This will prevent the eyeglasses from being scratched.

When not wearing eyeglasses: Extend the folded eye shades outwards
(direction of the arrow) to prevent extraneous light from entering into
your line of vision.

4. Light control VERY IMPORTANT!!!!!

Light intensity is a essential aspect of microscopy. Perform the following steps to adjust the light. For
optimal viewing, the light must be adjusted several times while focusing. Always adjust the light while
looking through the eyepieces.

a. Adjust the light intensity switch that turns the lamp on. Begin at a medium level. Adjust
again while focusing.

b. The entire viewing area (field) must be filled with light. If this is not the case, contact the
instructor.

c. Locate the thin, iris diaphragm lever under the stage condenser. Adjust this lever to a
medium/low light level. The iris diaphragm will need to be adjusted as magnifications increase
and focusing continues.
5. Under low power, SLOWLY focus with the coarse adjustment knob counterclockwise, moving the
stage down, until the specimen comes into view. Adjust the light as instructed in step 3 above.

Many specimens, especially bacteria, are very small and may look like specks at this magnification. If the
stage moves too quickly, you may go past the specimen without seeing it. Look for ‘color’ since the
specimens are stained.
6. Refine the image with the fine adjustment knob and by adjusting the light.

7. Important! Before switching to the next objective, move the slide so that the desired specimen is
located in the center of the field (circular viewing area).

8. SIGNIFICANT POINTS WHEN FOCUSING UNDER THE 4X, 10X, AND 40X OBJECTIVES!

a. Always start with a low power objective (4X or 10X). After focusing and making
appropriate observations, rotate the nosepiece until the next objective clicks into place. Do
not skip objectives.

b. Do not move the stage up or down before rotating the nosepiece to the next objective. When
properly focused, there is no need to adjust the objective's distance from the stage before
increasing the magnification.

c. DO NOT USE THE COARSE ADJUSTMENT KNOB WHEN FOCUSING UNDER

THE 40X OBJECTIVE! ONLY USE THE FINE ADJUSTMENT KNOB!

d. Before increasing the magnification, always use the X-Y control knobs to move the slide so
that the desired specimen is located in the center of the field.

e. Remember to adjust the light each time the magnification is changed and while
focusing.
1. Be sure that the specimen has been optimally focused using the 40X objective and is in the EXACT
CENTER of the viewing field under the 40X objective.

2. NOTE! Again, do not move the stage up or down before rotating the nosepiece to the 100X
objective.

3. Rotate the 40X objective away from the slide but do not yet click the 100X objective into place.

4. Put a small drop of immersion oil on the slide/coverslip directly over the light.

5. Rotate the nosepiece until the 100X oil immersion objective is clicked into place. If there are oil
bubbles, revolve the nosepiece slightly to remove the bubbles.

6. DO NOT USE THE COARSE ADJUSTMENT KNOB WHEN FOCUSING UNDER THE 100X
OBJECTIVE! ONLY USE THE FINE ADJUSTMENT KNOB!

6. Adjust the light for optimal viewing.

7. If problems are encountered during viewing, re-clean the objectives and repeat the procedure. If
problems persist, review the Common Problems section at the end of this document.

9. In order to determine the size of your specimen, you will need to estimate utilizing the size of the field
(circular viewing area). When you observe your specimen, estimate its size by comparing it with the size
of the field.

Objective Total Magnification Size of field of view

40X 400X ~0.45 mm

100X 1000X ~0.18 mm

When finished viewing and drawing your specimens, complete the Student Microscope
Checklist. Turn it in with your lab report.
Common Problems

1. The field is dark.

Is the light on?

Is the objective securely clicked into place?
Is the diaphragm open?
Is the slide lying flat on the stage?

2. You are not sure if you are looking at dirt on the objective lens or the specimen.

Use the mechanical stage knobs to move the slide slightly while looking through the eyepieces. If what
you are looking at does not move, it is probably dust or dirt on the objective. If it does move, it is on the
slide.

Rotate the ocular gently between your fingers. If what you are looking at rotates, it is probably dirt on the
ocular.

3. You cannot find the specimen.

The stage may be too far from the objective. Did you start with the stage as close as possible to the low
power objective?
Is the specimen directly over the light?
Is the slide secure and flat in the mechanical stage?
Did you start with a low power objective and focus on the lower objectives first?
Have you adjusted the light?
Are you moving the adjustment knobs too quickly? Work slowly so you do not miss the specimen.
Remember, bacteria look like specks at low magnifications.

4. You are having trouble focusing.

Always start on a low power objective, and focus here first.

Focus SLOWLY! It is very easy to move past the specimen if the adjustment knobs are moved too
quickly.
Be sure to look in the ocular while you are focusing with the adjustment knobs or changing the light
intensity.
Adjust the light.
Press down on the stage with your fingertips. Does the stage move? If so, tighten using the knob next to
the coarse adjustment.

5. You lose the specimen when switching from the 40X objective to the oil immersion objective.

Was the specimen in the exact center of the field before switching to the 100X objective?
Is the 100X objective lens clean?
Have you adjusted the light?
Have you refined the image with the fine adjustment?

6. If double vision persists after adjusting the width of the eyepieces…

a. Open the iris diaphragm all the way.

b. Using your left eye only, use the 10X objective and focus on the specimen by adjusting the
coarse adjustment knob.

c. When the image is in view, refine the image to its sharpest focus by turning the fine adjustment
knob.

d. Using both eyes, rotate the right eyetube collar below the eyepiece (dioptric adjustment) until
the sharpest image appears.

e. Repeat several times to check.

8. One of the most common problems when switching objectives several times, especially after
using oil, is that the objectives need RE-CLEANING, and the student must refocus
beginning with the 10X objective again.

Assignment (see attached Lab Report):

Each student must view and draw two prepared slides.

 When viewing bacteria, start with the 10X objective, and work your way up to the 100X objective.

Call the instructor after you have focused at 100X on each slide.

Label and draw the specimens on the Lab Report.

Remember to complete the Student Microscope Checklist. Be sure to write the microscope number of the
scope you used on the Checklist. The Checklist and the Lab Report will be returned to the instructor.

II. Preparing a wet mount of the letter "e”.

1. With your scissors cut out the letter "e" from the newspaper.
2. Place it on the glass slide so as to look like (e).
3. Cover it with a clean cover slip. See the figure below.

4. Using your eyedropper, place a drop of water on the edge of the cover slip where it touches the glass slide. The
water should be sucked under the slide if done properly.

Technique for Adding a Stain when making a Wet Mount

Water from dropper

Small piece of paper towel

Specimen under cover slip

 5. Turn on the microscope and place the slide on the stage; making sure the "e" is facing the normal
reading position (see the figure above). Using the course focus and low power, move the body tube
down until the "e" can be seen clearly. Draw what you see in the space below.

6. Describe the relationship between what you see through the eyepiece and what you see on the stage.
___________________________________________________________________________________________
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7. Looking through the eyepiece, move the slide to the upper right area of the stage. What direction does
the image move?
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__________________________________________________________________

8. Now, move it to the lower left side of the stage. What direction does the image move?
____________________________________________________________________________________
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9. Re-center the slide and change the scope to high power. You will notice the "e" is out of focus. DO
NOT touch the coarse focus knob, instead use the fine focus to resolve the picture. Draw the image you
see of the letter e (or part of it) on high power.

10. Locate the diaphragm under the stage. Move it and record the changes in light intensity as you do so.
____________________________________________________________________________________
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III. Measuring Field of View with a Microscope

1. Use a clear ruler to determine the width of the viewing field under the low power
objective. Position the ruler so that the millimeter marks are visible in your viewing
field. Remember that there are 1000 micrometers in a millimeter.

In the pictured field of view at the left, it can be observed

that there are approximately 3 1/2 divisions equal to a
length of 3.5 mm. Therefore this field of view is equal to
3.5 mm or 3,500 micrometers.

Estimate the length (diameter) of your viewing field in micrometers _______________

 Lab Report

Name:

Microscopy

1. Draw and label the letter "e" after focusing with 10X and 40X objectives.

2. Draw and label the crossed hairs after focusing with 10X and 40X objectives.

3.
Draw and label the cheek cells (squamous cells) after focusing with 10X and 40X objectives.
 4. If you had enough time to look at a prepared Human Blood Smear, what cells did you
identify?

Post- Lab Questions:

1. State TWO procedures that should be used to properly handle a light microscope.

2. Explain why the light microscope is also called the compound microscope.

3. Images observed under the light microscope are reversed and inverted. Explain what this means.

4. Explain why the specimen must be centered in the field of view on low power before going to high
power.

5. A microscope has a 20 X ocular (eyepiece) and two objectives of 10 X and 43 X respectively.

a) Calculate the low power magnification of this microscope. Show your formula and all work.

b) Calculate the high power magnification of this microscope. Show your formula and all work.

6. In three steps using complete sentences, describe how to make a proper wet mount of the letter e.

7. Describe the changes in the field of view and the amount of available light when going from low to high
power using the compound microscope.

8. Explain what the microscope user may have to do to combat the problems incurred in question # 7.

9. How does the procedure for using the microscope differ under high power as opposed to low power?
 Student Microscope Checklist
When finished using the microscope, complete the following.
Check off each step when completed.

Name: Lab Section:

Date: Microscope Number: HCC

Bring the stage to its lowest level.

Click the 4X objective into place.
Remove the slide and dispose of appropriately. Wipe prepared slides until ALL oil/dirt is removed (top
and bottom).
Clean objective lenses with swabs and liquid lens cleaner. Dry each objective after using the liquid
Clean eyepieces with dry swabs.

Clean condenser lens with dry swabs.

If necessary, clean the stage with a damp Kimwipe.

Turn the light switch OFF. Do not move the microscope for a few minutes before putting it
away,

Rotate the head so that the eyepieces are facing forward (away from the arm).
Replace the dust cover.
Return the microscope to the appropriate location in the cabinet in the appropriate postion.

Check to be sure all waste is removed from the sink and floor.