THE MICROSCOPE
The microscope is a basic tool of biology that you will use
throughout the course. In this laboratory you will work with a Bausch &
Lomb compound microscope. In order to derive the maximum benefit
from this microscope, you should learn the names and functions of the
various parts.
Parts of the microscope
1. Base: The base supports the other parts.
2. Arm: The C- or L-shaped arm supports the stage and body-tube.
3. Stage: The stage is the horizontal platform which supports the
object, usually a slide, to be examined. The slide may be held in place
by the clips on the stage, or the clips may be removed.
4. Body-tube: The body-tube bears the lenses of the microscope - the
eyepiece or ocular lens at the top, and the objective lens just above the
stage.
5. Eyepiece: The eyepiece or ocular lens generally magnifies the
image 10 times. It may contain a pointer that can be moved by rotating
the eyepiece.
6. Nosepiece: The nosepiece bears the objective lenses. To position
an objective lens above a specimen, rotate the nosepiece until the lens
clicks into position.
7. Objective lenses: Most of the microscopes bear three objective
lenses: scanning (3.5x), low power (10x), and high power (43x). The
magnification is marked on the metal lens housing. Note: the higher the
magnification, the larger the objective lens.
In order to calculate the total magnification of an object viewed
through the microscope, multiply the eyepiece magnification times
the objective lens magnification. As an example, if a cell is viewed
through the low power (10x) objective, and the microscope has a
10x eyepiece, the total magnification of the cell is 10 x 10 = 100x.
8. Focus knobs: The image is focused by turning the large coarse
focus knob and/or the small fine focus knob. On the B & L microscope,
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the focus knobs raise or lower the stage to position the specimen at the
point where the objective lens is focused. On some scopes, the focus
knobs move the nosepiece up and down to change the position of the
objective lens with respect to a stationary stage.
9. Light source: The light is turned on and off by turning the switch
near the left side of the base. Don't forget to plug in the cord!
10. Condenser: The condenser collects light from the source to pass
through the specimen. The condenser should be positioned about 1/8”
below the stage.
11. Iris diaphragm: The condenser iris diaphragm adjusts the intensity
of the light passing through the sample. It is roughly equivalent to the
contrast control on a television. Opening the diaphragm allows more
light to pass through, which is critical in viewing dense objects or objects
at high magnification. Closing the diaphragm, in addition to protecting
your eyes from intense light, allows you to examine objects at low
magnification with high contrast.
Identify the parts of the microscope on the photo on the next page.
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Bausch & Lomb Compound Microscope
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Care of the microscope
Before you begin using the microscope, you should be familiar
with a few recommendations to extend the life of the instrument:
1. Carry the microscope carefully with one hand supporting the base
and the other supporting the arm. Hold it upright; the eyepiece can slip
out of the body-tube of a tilted microscope.
2. Beware of frayed cords and loose plugs. Inform your instructor.
3. Be careful when turning the nosepiece and when focusing that an
objective lens does not hit the slide or stage. This can break the slide
and damage the objective lens.
4. Clean the lenses with lens paper. Never use paper towels, tissues,
cloth, etc. They are abrasive and scratch the lenses.
5. Be sure to remove the slide from the microscope before returning it
to the microscope cabinet. Again, carry it carefully.
Orientation
1. Obtain an "e" slide from the supply table.
2. Using the coarse focus knob, move the stage downward so that you
have room to place the slide on the stage. Position the slide so that the
"e" is directly above the condenser, in the center of the hole in the stage
and facing you with the "e" right side up. Check that the condenser is
approximately 1/8" below the stage; use the condenser lens adjustment
if necessary.
3. Rotate the nosepiece to position the scanning (3.5x) objective above
the "e". Carefully raise the stage as high as possible without hitting the
objective - you should be looking directly at the lens and stage, not
through the eyepiece.
4. Now turn on the light source and look through the eyepiece. Adjust
the condenser iris diaphragm to give a comfortable light intensity.
5. Bring the "e" into focus by turning the coarse, then fine, focus knobs.
The stage will move away from the lens (if you raised the stage as
directed in step 3), so you don't have to worry about the lens hitting the
slide.
6. Experiment with the condenser iris diaphragm. Open it to let fairly
intense light through, then slowly close it. What effect does closing the
diaphragm have on the quality of the image?
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Which of the following best illustrates the appearance of the
letter "e" as seen through the eyepiece?
7. Change magnification by simply turning the nosepiece to position the
low power (10x) objective over the condenser. It will click into place.
The objective should not hit the slide, but you should get in the habit of
watching the objective lens as you turn it - there will be times when it will
not clear the slide.
8. The B & L microscopes are parfocal, meaning that all of the objectives focus at the same point. Having just changed objectives, you can
check this feature. Is the image close to focus with the 10x objective?
If not, sharpen the focus with the fine focus knob. Adjust the iris
diaphragm to give proper contrast and light intensity.
Depth perception
Obtain a "Threads" slide from the supply table. On this slide you
will notice crossing red, yellow, and blue threads. The object of this
exercise is to examine the point of intersection of the threads at 100x
magnification and determine the order (bottom to top) of the three
threads.
How can this be done? Recall that the objective lens is focused at
a particular point, and that you focus by moving the object up or down to
reach that point. If you position the slide above that point (which is what
you ought to do to begin- see step 3 above), and then move the slide
downward, the first thread to reach the point of focus will be the bottom
one. Next, the middle thread will arrive at the focal point, and then the
top thread. Try it.
What's the order of the threads (bottom to top)?
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Size of the microscopic field
Obtain a "Graph paper" slide. On this slide you will see small
squares, with sides 1 millimeter (mm) in length. You can use the graph
paper as a ruler to measure the diameter of the microscopic field.
Simply observe the graph paper at 35x magnification, and count the
number of boxes which span the viewing area or field.
What's the diameter of the microscopic field at 35x magnification?
__________ mm
Now repeat the measurement, but at 100x magnification.
What's the diameter of the microscopic field at 100x magnification?
__________ mm
What's the lesson to learn from this exercise?
Here's a "thought experiment": Suppose you were looking through
the microscope at 35x magnification, and a microbe (a small worm
or something) came into view at one side of the field and swam
across to the other side in 5 seconds. Can you figure out how fast
the worm was swimming?
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Observing human cells
For this exercise, you will need to obtain a slide, coverglass, and a
toothpick. Gently scrape the inner lining of your cheek with the toothpick (as demonstrated by your instructor), then spread the scrapings on
the slide. Add a small drop of methylene blue solution, then place the
coverglass over the preparation. Examine under low, then higher
magnifications.
What is the prominent blue-staining structure within the cells?
Other cells
All organisms (except viruses) are cells or are made up of cells.
For the remainder of the session, observe and make drawings of the
cellular structure of the organisms provided:
Saccharomyces (yeast) - This is a single-celled organism that
reproduces by budding progeny cells from a larger mother cell. If the
culture is growing vigorously, lots of budding cells will be evident.
Tetrahymena - This is a ciliate, a single-celled organism that propels
itself through the water by using cilia, cellular projections, like flagella,
that are described in Chapter 3 of the text.
Elodea - This as a multicellular water plant. Tear a fresh leaf from a
plant, preferably near the tip. Lay the leaf down flat on a slide, add a
drop of water, then apply a coverglass. Notice the green, oval-shaped
bodies (chloroplasts) within the cells and the general shape of the cells.
Are there any specially shaped cells along the edges of the leaf?
Hay infusion or pond water - Examine the diversity of organismic and
cellular forms by placing a drop on the slide and applying a coverglass.
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Drawings
Saccharomyces
Tetrahymena
Elodea
Internet Resources
Many camera manufacturers also produce microscopes. Highquality microscopes from Olympus and a tutorial on microscopy can be
found at http://www.olympusmicro.com/primer/java/index.html.
To learn more about yeasts, including their use in brewing, see
http://genome-www.stanford.edu/Saccharomyces/VL-yeast.html.
Ciliates, the group that includes Tetrahymena, are featured at
http://www.microscopy-uk.org.uk/mag/wimsmall/cilidr.html.
Learn about Brazilian Elodea as a non-native species in American
waters at http://www.ecy.wa.gov/programs/wq/plants/weeds/egeria.html.
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