Reading Assignment: Super-duper

detailed microscope usage instructions

Downloadable file: Detailed MScope Lab Exercise Sheet.docDownload Detailed MScope Lab
Exercise Sheet.doc
USING THE MICROSCOPE

The microscope is designed to make objects visible that are too difficult or too small to see
with the unaided eye. The microscopes in biology or anatomy labs are usually compound
binocular or monocular light microscopes. Compound means that the scopes have a minimum
of two magnifying lenses (the ocular and the objective lenses). Binocular microscopes have two
eyepieces and monocular microscopes have only one eyepiece. Light refers to the type of
illumination used, that is visible light from a lamp. The purpose of this laboratory exercise is to
become familiar with the microscopes we have in our lab and develop proper microscope
technique.

PARTS OF THE MICROSCOPE

1. Locate the parts of your microscope. Refer to the following description of a typical
microscope. In the spaces provided, answer the questions related to your microscope.

2. The head supports the two sets of magnifying lenses. One set is the ocular which is
the lens in the eyepiece. The ocular typically has a magnification of 10X (meaning it
increases the apparent size of an object 10 times). A pointer has been placed in the
eyepiece and is used to point to an object in the field of view, the circle of light that
one sees in the microscope. The pointer can be positioned by rotating the eyepiece.

Is your microscope monocular or binocular?

3. The other set of magnifying lenses supported by the head is the objectives.
The objectives are the three lenses on the revolving nosepiece. The shortest
objective is typically 4X and is called the scanning lens. The objective of next
longer length is10X and is called the low power lens. The next longer objective(high
 power lens) is usually 40X. The longest objective is the oil immersion lens (100X)
and is used with a drop of oil on the slide. We will not use the oil immersion lens.

In the following table, fill in the magnification column for the objectives of your
microscope (the magnification is on the side of each objective).

Objective Length Common Name Magnification

shortest scanning lens or objective

intermediate-short length low power lens or objective

intermediate-long length high power lens or objective

longest oil immersion lens

4. The arm supports the stage and condenser lens and iris diaphragm. The condenser
lens is located immediately beneath the stage and is used to focus the light from the
lamp on the specimen. The height of the condenser on some microscopes can be
adjusted by the adjustment knob. The iris diaphragm controls the width of the circle
of light and therefore the amount of light passing through the specimen. It is part of
the condenser apparatus and has a lever that controls the opening.

Is an adjustable condenser present on your microscope?

 5. The stage is the platform the slide rests on while being viewed. The stage always has
a hole in it to permit light to pass through both it and the specimen. There is a clip
apparatus that holds the slide. A mechanical stage can be moved right and left by one
of the stage adjustment knobs and back and forth with the other stage adjustment
knob. Practice moving the stage left and right and back and forth with these knobs.

6. The distance between the stage and the objective can be adjusted with
the coarse and fine focus adjustment knobs. The coarse adjustment knob is the
larger of the two and is located just outside the fine adjustment knob. These are the
knobs you will use to bring the slide you are viewing into focus. At this time,
practice moving first the coarse and then the fine adjustment knobs and observe how
they move the objectives.

7. The base acts as a stand for the microscope and houses the lamp. There is a switch to
turn the lamp on and off. In some microscopes, the intensity of the light that passes
through the specimen can be adjusted with the light intensity lever or knob.
Generally more light is needed when using high magnification than when using low
magnification.

Can you vary the light intensity on your microscope?

BASIC MICROSCOPE TECHNIQUES

1. CARE OF YOUR MICROSCOPE:

2. These microscopes are delicate, expensive instruments and have to be treated with
care.

1. Always carry the microscope with two hands; one hand supporting the base and the
other hand on the arm.
1. Only use lens paper to clean the lenses. The glass used in microscope lens is softer
than ordinary glass and it easily scratches. Do NOT use kleenex, paper towels or any
cloth to clean the lens. If you get water on the lens, wipe it off with lens paper. Your
fingers are oily; NEVER touch the lenses.

1. When you finish using the microscope, remove the slide from the stage and turn the
nosepiece so that the scanning objective is in position. Turn off the light switch and
pull out the plug. Wrap the cord around the base of the scope.

1. Put the microscope away at its correct location. Match the microscope number (on
the arm) to its corresponding number within the cabinet.

2. HOW TO FOCUS:
3. Always start with the scanning or low power objective. Turn the nosepiece to rotate
this objective into position and listen for the “click”. If you do not hear the “click”,
the objective is not all the way in position.

1. Use the coarse adjustment knob to bring the scanning or low power objective as
close to the slide as possible. Watch the objective from the side when you are
lowering the objective to prevent breaking the slide or objective.

1. Look through the eyepiece and turn the coarse adjustment knob to slowly RAISE the
objective AWAY from the slide. STOP when the image comes into focus.

1. Refine your focus by using the fine adjustment knob. Note that when you manipulate
the fine adjustment you are actually focusing at different levels with the specimen.
This gives you depth perception of the material being viewed.

Move the stage so that the part of the slide you want to view is in the center of the
field. Experiment with the amount of light passing through the specimen by moving
the lever on the iris diaphragm. You will need to adjust the iris diaphragm for each
slide and each magnification.
 3. HOW TO CHANGE MAGNIFICATION:
4. Always start with the scanning or low power objective in position. Focus with this
objective and center your slide as described above.

1. Rotate the nosepiece to bring a higher power objective into position. Listen for the
“click”.

1. These microscopes are parfocal which means that if an object was in focus and
centered with low power, it should also be in focus or near focus with high
power. Use only the fine adjustment knob with the high power
objective. NEVER focus downward with the coarse adjustment knob when using
high power; a sure way to crack a slide.

1. Adjust the iris diaphragm so that the amount of light passing through your specimen
gives the maximum contrast. Usually the brightest light (maximum amount of light)
provides little contrast. You need to make this adjustment for every slide.

RULES FOR MICROSCOPE USE

1. 1. SOME IMPORTANT DO’S:

1. ALWAYS use the scanning or low power objective first. Get the slide in focus with
this objective, then switch to a higher power. Do this is for every slide you view, not
just the first slide of the lab period.

1. Only use lens paper on the lenses and slides.

 1. Treat your microscope gently.

1. Ask questions when it’s not clear how to do something.

2. 2. SOME IMPORTANT DON’TS:

1. NEVER use the coarse adjustment knob when using high power. Since this objective
is so long and the coarse adjustment moves it so fast, there is great danger of breaking
the slide or lens.

PROPER TECHNIQUE: On high power, use only the fine adjustment knob to focus.

1. NEVER focus downward with the coarse adjustment while looking through the
eyepiece. PROPER TECHNIQUE: You should lower the low power or scanning
objective first while viewing from the side(not the eyepiece) and then
focus upward while looking through the eyepiece.

1. DON’T try to find an object under high power that you cannot locate under low
power.

PROPER TECHNIQUE: First locate the object under low power and focus. Next move
the stage so that the object is in the center of the field of view and then switch to a higher power.

ORIENTATION OF IMAGES VIEWED THROUGH THE COMPOUND MICROSCOPE

Letter “e” slide
 1. Obtain a prepared letter “e” slide. Hold the slide up to the light and note the
appearance of the “e” to your naked eye. Place the slide on the stage and hold it in
position with the stage clips. Position the “e” over the opening in the stage.

2. Use the iris diaphragm to adjust the light. With the scanning lens in position, view the
microscope slide. Use the coarse adjustment knob to bring the “e” into focus. Be
certain to use the focusing technique described under “Basic Microscope
Techniques”. How does the “e” appear when viewed through the microscope
compared to the “e” seen with the naked eye?

3. While viewing the image, move the stage to the right. Does the image move to the
right or to the left?

4. When you move the stage away from you, which direction does the image move?

DEPTH OF FIELD

Thread Slide

1. The depth of field is the thickness of the specimen that may be seen in focus at one
time. Because the depth of focus is very short in the compound microscope, you must
focus up and down to clearly view all planes of the specimen. To demonstrate the
depth of field, we will use a prepared slide of three different colors of thread which
are layered one over the other.

2. Rotate the scanning objective into position and place the slide on the stage. Center the
slide so that the region where the three threads cross is in the center of the stage
opening.
 3. Use the correct focusing technique to get the region where the threads cross in focus.
Are all threads in focus at the same time?

4. Rotate the low power (10X) objective into position and focus on the cross. Are all the
threads in focus at the same time?

Does the scanning objective (4X) or the low power (10X) objective have a shorter depth of
field?

5. Still using the low power (10X) objective, adjust the focus so that the slide is slightly
out of focus BELOW the three threads. You are to determine which thread is on the
bottom, which is in the middle and which thread is on top. Using the fine adjustment
knob, SLOWLY RAISE the objective lens until the bottom thread comes into clear
focus, then the middle thread and finally the top thread. Repeat this procedure at
least one more time to be certain you are correct.

6. There is a code letter on the label of the slide. Record the code letter and the colors of
the bottom, middle and top threads.
 Color of bottom thread
Color of middle thread
Color of top thread

Magnification and Field of View

1. The microscope is used to magnify objects. There are two lenses which increase the
size of the image viewed, the ocular lens (in the eyepiece) and the objective lens. The
ocular lens makes the object appear 10X larger. The objective lenses increase the
size of the object 4X, 10X or 40X depending upon the objective used. The total
magnification is the product of the powers of the two lenses (ocular x objective).
Therefore, if the scanning lens is in position, the total magnification is 40 times (10
x 4).

2. The field of view is the portion of the slide that you are actually seeing. The size of
the microscope field of view decreases proportionally with increased magnification.
That is, the higher the magnification, the smaller the area of the slide that is seen
through the microscope. To demonstrate, we will measure the field of view with a
clear plastic ruler. Place the ruler on the stage with the metric portion over the
opening in the stage. The small distance between two lines is one millimeter (1
mm). Record the diameter of the field of view for each of the objectives. (If less
than one millimeter is visible, you will only see a line or part of a space. The width
of each line is approximately 0.2 mm.)

3. Determine the total magnification of an image for each objective and calculate the
diameter of the field of view. 1 millimeter (1mm) = 1000 micrometers (1000 µm).
 Field of View Field of View
Objective Lens Total Magnification
(in mm) (in µm)

4X

10X

40X

ESTIMATE OF SIZE OF CELLS

We can use the information on the Diameter of Field of View determined in the exercise
above to help us estimate the size of cells viewed under the microscope. We will use a slide of
your cheek cells to do the determination. The size of an individual cell can be estimated from
the following formula:

Diameter of Field of View ¸ by number of cells that can fit across the Diameter of Field of
View = size of an individual cell

Preparation of a Cheek Cell Smear Slide

1. The materials you need to prepare a cheek cell smear are on the cart in the front of the
room. Obtain a plain, clean microscope slide and place one drop of 0.9% saline
(NaCl) in the center of the slide. (If the slide has water spots on it, rinse it with water
and dry with lens paper.)
 2. Use a clean toothpick to gently scrape the lining of your mouth. Stir the cheek cells
into the drop of saline. Add a small drop of methylene blue to stain the cells and stir
again. Carefully place a coverslip over the fluid and return to your microscope.

3. Place the slide on the microscope stage. If there is any excess fluid that will come in
contact with any part of the microscope, clean it up with lens paper. The saline (salt)
solution can damage the microscope.

4. Locate the cells under low power, using the proper focusing technique. Then switch
to high power and use the fine adjustment knob to focus.

5. Sketch a cheek cell as it appears under the microscope. The dot in the center is the
nucleus of the cell.

6. Estimate how many cells you could line up across the diameter of the field of view.
(If you have cells filling the whole field of view, count the number stretching across
the diameter of the field of view. If you have only a few cells in the field of view,
then imagine the total number of cells needed to stretch across the diameter of the
field of view. ASK if these directions are not clear.

7. Fill in the following table and calculate the size of an individual cell using the
formula at the beginning of this exercise

Diameter of Field of # of Cells that Can Fit across the

Size of Individual Cell
View Diameter

8. Place your slide and coverslip in the pan labeled “used cheek slides”. Make certain
that the microscope stage and lenses are dry (use lens paper to dry the lenses). Put
away your microscope according to the procedure described under Basic Microscope
Techniques.