You will need
to record data and answer questions for part of this lab. All drawings must be labeled and
include the magnification used.
Students will
work with their lab partners. There will be 1 microscope for 2 people. It is assumed that you have read and
know the various parts of the microscope and their functions.
RULES
FOR MICROSCOPE USE:
Always observe the following rules for
microscope use:
- Always carry the microscope with two hands to your
lab desk.
- The lowest power objective should be in
position both at the beginning and end of microscope use.
- Use only lens paper for cleaning lenses.
- Do not tilt the microscope when viewing a
wet mount.
- Keep the stage clean and dry to prevent
rust and corrosion.
- Do not remove pats of the microscope.
- Report any malfunctioning.
Focusing the Microscope:
Lowest Power (Scanning) 4X
- Turn the nosepiece so that the lowest power
lens is in straight alignment over the stage.
- Always begin focusing with the lowest
power objective lens.
- With the coarse-adjustment knob, lower
the stage until it stops.
- Place a slide of the letter “e” on the stage, and stabilize it with the
clips or mechanical stage (instructor will demonstrate if necessary).
- Be sure that the lowest power objective
is in place. Then, as you look from the side, turn the coarse
adjustment knob until the distance
between the stage and the tip of the objective lens is no closer than 3 mm
above the slide.
- While looking into the eyepiece, rotate
the diaphragm (or diaphragm lever) to give the maximum amount of light.
- Slowly increase the distance between the
stage and the objective lens, using the coarse-adjustment knob, until the
object - in this case the letter “e” - comes into view, or focus.
- Once the object is seen, you may need to
adjust the amount of light. Increase or decrease the contrast by
rotatingthe diaphragm slightly.
- Use the fine-adjustment knob to sharpen the focus if
necessary. Practice having both eyes open when looking through the
eyepieces, as it greatly reduces eyestrain.
Higher Powers
Compound microscopes are parfocal; so
that once an object is in focus with lowest power, it should be almost in focus
with the higher power.
Bring the object into focus under the
lowest power by following the instructions in the previous section.
Make
sure that the letter “e” is centered in the field of the lowest
objective. Move to the next higher objective (low power, 10x) by turning the
nosepiece until you hear or feel it click into place. Do not change the
focus; parfocal microscope objectives will not hit normal slides when changing
the focus if the lowest objective is initially in focus. If any adjustment is
needed use only the fine-adjustment knob. Always use only the
fine-adjustment knob with higher powers.
Computing Total Magnification
When looking through the microscopes,
you are actually viewing the specimen first through the ocular lenses and then through
whatever objective is in place. In order to obtain the total magnification that
you are viewing the specimen with you must multiply the magnification of
the ocular lens by the magnification of
the objective.
Total magnification = Magnification of Objective
x Magnification of Ocular
For example, if the ocular is 5x
magnification and you are using the 10x objective, the total magnification
would be 50x, i.e. the specimen would appear 50x larger than its actual size.
For the compound, it is the objective
that is clicked into the vertical position above the light source.
Field of View (FOV)
The field of view (FOV) is the size of
the circular area that you can see when you look through the ocular and
objective lenses. Knowing the size of the FOV is important because you can use
it to determine the approximate size of a specimen. The FOV will change as you
change
the objective and therefore the
magnification.
How to view a specimen:
For all specimens, use the following
basic procedure.
1.) Clean the lenses of your
microscopes if you have not done so already.
2.) Plug in your microscope and turn on
the light source. You can experiment with how much light you allow using the
dimmer, iris and condenser lens with the compound microscope. It is generally
best
to start with a medium intensity of
light and adjust accordingly. Keep in mind that the highest intensity of light
is not necessarily the best for a given specimen.
Lab
Exercise 1 - Letter "e" Lab
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Supplies: |
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Standard
Equipment Needed: o
Microscope
o
Pencil
o
Prepared
letter "e" o
Paper
containing "e" s o
Scissors
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Lab Procedure: |
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1. Using microscope, slide, cover slip, water, scissors,
and newspaper make a wet-mount slide of a small-case letter "e". |
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The
letter"e" is a great way to learn how a microscope works. In this
lab you will set up your microscope and view
what a simple letter e cut out from a piece of paper looks
like. First let's examine the letter "e" with your naked eye. Draw
what you see in the circles below: |
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|
|
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Follow
these steps to use the microscope with a prepared slide: o
Place
your letter "e" slide, coverslip side up, on the stage. o
Place
the slide so that the letter is in its normal o
orientation
– in other words the way you would normally o
read
it o
Use
the low power objective. o
Secure
the slide with the stage clips or mechanical stage. o
Turn
on the light. o
Focus
on the letter "e" using the coarse focusing knob. o
Draw
what you see in the circle below. Try using the high o
power
objective. After focusing (fine focus only), draw what o
you
see. |
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Analysis Questions : What are some of the ways
the e you see with the microscope is different from the e you
see with the hand lens? If you are looking at the
"e" through the microscope and you push your slide to the left,
which way does the e in the microscope move? (Try this!) If you push the slide
away from you, which way does the e in the microscope move? (Try
this!) |
This
phenomenon is known as inversion.
Total
Magnification
Total
magnification is calculated by multiplying the magnification of the ocular lens
(eyepiece) by the magnification of the objective lens.
Exercise 2:
Calculate the
total magnification for each objective, and record your figures in a
table. To calculate the total magnification, multiply the power of the
ocular lens by the power of the objective lens. Your table should include
the powers of both lenses and the total magnification.
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Microscope |
Total Magnification |
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Value for
each ocular unit (10X) at 4X (Scanning) |
. |
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Value for
each ocular unit (10X) at 10X (Low Power) |
. |
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Value for
each ocular unit (10X) at 40X (High Power) |
. |
Diameter of Field
The
diameter of the field (the circle visible through the lens) is the length of
the field across the center.
Exercise 3:
SCANNING
(4x)
Place
a clear plastic ruler across the stage so that the edge of the ruler is visible
as a horizontal line along the diameter of the low-power field. Be sure
that you are looking at the millimeter side of the ruler. You will use
the ruler to measure the diameter of the field of view under various
magnifications.
Estimate
and record the number of millimeters (mm), to tenths, that you see along
the field: (Hint: Start with one of the millimeter markers at the edge of the
field.)
Convert the
figure to micrometers (mm) and record. This is your scanning
diameter of field. To convert from millimeters to micrometers, multiply the
millimeter value by 1,000.
|
Microscope |
Field of View |
|
Value for
each ocular unit at 4X (Scanning) |
. |
|
Value for
each ocular unit at 10X (Low Power) |
. |
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Value for
each ocular unit at 40X (High Power) |
. |
LOW POWER
(10x) Repeat the steps above to determine (LPD)
HIGH POWER
(40x) Repeat the steps above to determine (HPD)
Does
low power or high power have a larger field of view and allow you to see more
of the object?
Which
has a smaller field but magnifies to a greater extent?
Reminder:
To locate small objects that are seen on low
power, place these objects in the center of the field before rotating to
high power.
Depth
of Focus
The
vertical distance that remains in focus at one time is called the depth of
focus.
Constant use of the fine-adjustment knob when
viewing a slide with high power will give you an idea of the specimen's three
dimensional form.
There are a
number of other items that can be viewed depending on time. These include human
hair, human cheek cells, and red onion cells. Your instructor will demonstrate
how to set up slides of each of these
http://www.umanitoba.ca/Biology/lab1/biolab1_6.html#
http://shs.westport.k12.ct.us/mjvl/biology/microscope/microscope.htm#wetmount