Jan.

13, 2003 LAB EXERCISE :

Only objects 0.1mm and MICROSCOPY
larger can be visualized by
the human eye. Because
most microorganisms are much smaller than 0.1mm, a microscope must be utilized in
order to directly observe them. In general, the diameter of microorganisms ranges from
0.2 - 2.0 microns.
A light microscope, which uses light as a source of illumination, will be employed
in this lab. There are several types of light microscopes. The type used in this course is a
brightfield microscope, where the specimen appears darker against a bright background.

Resolving power

The resolving power (resolution) is a very important characteristic of a microscope.

Resolving power is the ability of the lenses to differentiate between two adjacent objects.
For example, if the resolving power of a microscope is 0.2 microns, objects closer than 0.2
microns will be seen as one object. Objects that are 0.2 microns (or farther) apart will be
seen as two separate objects. Therefore, the resolution determines the amount of detail
that can be visualized.

Care of the microscope Observe the following precautions when using the
microscope.

1. These microscopes are large and heavy. Always use both hands when carrying the
microscope. One hand holds the arm while the other hand supports the base. Always
carry the scope in an upright position. Do not bump the scope while removing it from the
cabinet.

2. Several parts are loosely connected to the scope. When the dust cover is removed or if
the scope is tipped at an angle, the following parts may detach from the scope:

1) Collector lens and blue filter (resting above the lamp),

2) Rubber eye shields (these attach to the top of the eyepieces, the viewer's
eyes rest on these shields),
3) The eyepieces, themselves, may be loose in the eyepiece tubes. Always take care
when removing the dust cover, and always carry the scope in an upright position.

3. Follow cleaning instructions carefully (p. 3, I. Preparation for viewing, 5.).

4. Do not let the electric cord dangle or become entangled.

5. Students must be assigned a specific microscope. The scopes are labeled by

number. Numbers 8793-8804 are in the main microscope cabinet at the front of the lab.
Numbers 8806-8809 are found in the far right cabinet under the lab bench at the back of
the lab. You must record the scope number each time a microscope is used on the
‘Student Microscope Checklist’.

1
2
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 platform on which the microscope slide rests and the clamping
device that secures the slide.

3. Mechanical stage control knobs: These knobs, under the stage, move the stage front
to back and the slide from side to side.

4. Lamp.
The lamp on/off switch is located on the base. The light intensity can be adjusted with this
on/off switch. The lamp should be adjusted to a medium level at the start of viewing. The
lamp, itself, has a blue filter that rests on the "collector housing" over the lamp apparatus.

5. Field diaphragm: adjusts the size of the lighted area in the field (circular viewing
area). The entire viewing area should be filled with light. As one focuses, the size of the
lighted area may need adjustment using the striated band (field diaphragm control)
encircling the collector housing over the lamp.

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

1) 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.

2) Objectives: There are four objectives: 4X (red), 10X (yellow), 40X (blue), and
100X (white, oil immersion objective). The objectives are attached to a
rotatable nosepiece (nose turret).

The total magnification is calculated by multiplying the

ocular magnification and the magnification of the objective in use.

3) Condenser: The condenser is located directly beneath the stage. It gathers and
conducts the light to the specimen. Although it can be raised and lowered with
the condenser adjustment knob, the condenser should remain at its highest
position. The iris diaphragm controls the amount of
light from the condenser and is adjusted with a thin, black lever under the stage.

7. Focusing knobs: located on both sides of the microscope.

The larger, inner coarse adjustment knob moves the stage up and down much faster
and farther than the smaller, outer fine adjustment knob. The coarse adjustment knob
is used ONLY with the low power (4X, 10X) objectives. When focusing under the 40X or
100X objective, ONLY use the fine adjustment, never the coarse adjustment.

3
Refer to each of the following procedures every time the
microscope is used.

I. Preparation for viewing

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 (see Care
of the microscope, #2). They might be in the dust cover. Immediately contact the
instructor if parts are missing or anything is wrong with the scope.

2. There are two ways to position the eyepieces: toward or away from the stage. The
‘head’ containing the eyepieces is loosened or tightened using the silver Head
Positioning Screw protruding below the microscope head. Do not loosen the screw
too much as the head may detach completely. To eliminate cord entanglement, rotate
the entire head containing the eyepieces so that the eyepieces are facing toward the
stage (this puts the cord attachment in back). Tighten the screw when it is in the
correct position. The instructor may loosen the screw to move the head for viewing
student work. In order to fit back in the microscope cabinet cubicle when the exercise
has been completed, the head must be rotated back so that the eyepieces are facing
away from the stage.

3. Plug in microscope. Be sure the electrical cord is not dangling off of the lab bench or is
not entangled.

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

5. Cleaning the microscope. Clean all objective lenses with swabs and, if necessary, liquid
lens cleaner. Place a small amount of liquid lens cleaner on the swab. Use a fair amount
of pressure to clean the lens. Dry the lens with a 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.

6. 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.

7. Adjust the condenser to its highest level. Turn on the lamp. Adjust the on/off switch to
a low/medium light level.

8. Rotate the nosepiece until the 4X objective clicks into place (10X if viewing bacteria).

9. Place the slide on the stage so that it is held within the slide holder clamping device.
The slide must lie flat on the stage. Using the mechanical stage knobs, position the slide so
that the specimen is in the exact center of the light coming through the condenser.

10. While looking through the eyepieces, adjust the width between the eyepieces until a
single, circular field is seen simultaneously with both eyes (see Parts of the light
microscope, #6 Eyepieces).

11. If problems are encountered during viewing, repeat the procedure. If problems persist,
review the Common Problems section at the end of this document.

4
II. Viewing the specimen under the 4X, 10X, and 40X objectives

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

The lowest 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. When viewing bacteria, start with
the 10X objective.

2. Be sure to bring the stage to its lowest point with the coarse adjustment knob. In other
words, the stage should be as far away from the objective as possible.

3. Light control VERY IMPORTANT!!!!!

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

a. Adjust the light intensity switch that turns the lamp on. Begin at a low/medium
level.

b. The entire viewing area (field) must be filled with light. The lighted area may
become smaller while focusing or changing magnifications. As needed, adjust the
field diaphragm using the striated band encircling the collector housing over the
lamp (see Parts of the light microscope #5).

c. Locate the thin, black iris diaphragm lever under the stage. Adjust this lever to
a medium/low light level. The iris diaphragm will need to be adjusted as
magnifications increase.

4. Under low power, SLOWLY focus with the coarse adjustment knob 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.

5. Refine the image with the fine adjustment knob and by adjusting the light.

6. 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).

5
7. SIGNIFICANT POINTS WHEN FOCUSING UNDER THE 4X, 10X, AND 40X
OBJECTIVES!

a. Always start with a low power objective (4X or 10X). After 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 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.

6
III. 100X Oil Immersion Procedure
Immersion oil is used with the 100X objective because it increases the resolution. The oil
should come in contact with both the lens of the 100X objective and the slide/coverslip.
This prevents light rays from escaping.

1. Be sure that the specimen 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.
Rotate the 40X objective away from the slide but do not yet click the 100X
objective into place.

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

4. Rotate the nosepiece until the 100X oil immersion objective is clicked into place.

5. 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. 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 Size of field of view

40X mm

100X mm

IV. When finished viewing, complete the Student Microscope Checklist.

Turn it in with your lab report.

7
V. 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?

8
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.

Assignment (see attached Lab Report):

Each student must view 2 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.

9
 Name:

Section:

Introductory Microbiology
Lab Report
Microscop
y

1. (1pt.) Draw and label the two specimens after focusing with the oil immersion objective.

2. (1pt.) Define resolution.

3. (1pt.) What is the total magnification of the following?

Ocular magnification Objective magnification Total magnification

10
10X 40X

10X 100X

11
4. (0.5pt.) What is the function of the iris diaphragm?

5. (0.5pt.) What is the function of the field diaphragm?

6. (1pt.) List two instances when the coarse adjustment knob is never used.

7. (1pt.) When should the lenses be cleaned? What is the correct way to clean them?

8. (1pt.) List the 3 lens systems on the light microscope.

9. (1pt.) Why is immersion oil used with the 100X objective?

10. (2pt.) List two common problems associated with using the microscope and how you
would go about solving it.

12
 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, if using oil, liquid lens cleaner.

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.

Do NOT wrap the cord around the base. Fold the cord and secure with
velcro.

In order to fit back in the microscope cabinet cubicle, the head must be rotated so that the
eyepieces are facing away from the stage.

Replace the dust cover.

Return the microscope to the appropriate location in the cabinet.

13