LAB: THE LIGHT MICROSCOPE AND CELL STRUCTURE 4) Always focus an image by moving the lens away from
image by moving the lens away from the slide. This way you
INTRODUCTION
The microscope is an instrument designed to observe objects that are too small to be
seen with the naked eye. In this class, you will use a compound light microscope
which functions by passing light through the object being viewed and focuses this
beam of photons into an image using optical lenses. Your microscope has the
capability of magnifying an object 1000 times its normal size and its resolving power
can distinguish two objects that are separated by as little as 0.5 um. You should be
able to identify and describe the function of the various components of a typical
compound light microscope as shown below.
Microscopes are expensive instruments; they can best be maintained by following a
few important guidelines:
1) Always carry the microscope to/from the storage cabinet with two hands, one on
the arm and one on the base. Set it gently on the counter (pretend it’s a baby’s
head) so that you don’t jar the lenses out of place.
2) Never tilt the microscope, the ocular may fall out.
3) To avoid scratching the lenses, never clean them with anything other than lens
tissue.
won’t accidentally grind the objective lens into the slide while looking through
the microscope.
5) When changing objective lenses, rotate the nose piece by turning it from its
circular base. Never rotate the nose piece by grasping an objective lens as this
will loosen the lens.
6) Never use the oil immersion objective lens (white ring at its base, labeled 100X)
unless directed to do so by the instructor. It requires that a special oil be placed
between the slide and the lens; without it, you may damage the objective lens.
7) When finished viewing, always store the microscope with the scanning objective
in place. This minimizes the chance of an objective lens grinding into the stage
during transport to/from the cabinet.
Learning to focus an image with the microscope may seem a bit tricky at first, but
the task will become second nature in a very short time. The diagram below
illustrates the distance between slide and objective lens for a properly focused image.
WORKING DISTANCE BETWEEN TIP OF OBJECTIVE
AND SLIDE WHEN AN OBJECT IS IN FOCUS
Scanning
Objective
Low Power
Objective High Power
4X
Objective
10 X
40 X
25 mm
8.3 mm 0.5 mm
microscope slide
The following procedure should enable you to focus an image with little difficulty.
1) Make sure the ocular and objective lenses are clean; use lens tissue if necessary.
2) Open the diaphragm to allow the maximum amount of light to pass from the
light source, through the lenses, to your eye.
3) Make sure the low power objective is in place. (If the object to be viewed is
very large, start with the scanning objective.)
4) Make a wet mount (see below) and place the slide on the stage, securing it with
the stage clips. Make sure the object to be viewed is in the center of the light
beam.
5) As you watch from the side, use the coarse focus knob to move the low power
objective all the way down (or until it is almost touching the slide).
6) Now, looking through the objective lens, use the coarse focus knob to raise the
objective lens until the object comes into view. SAMPLE EXERCISE: to be completed for homework on a clean sheet
7) Once the object is in focus, use the fine focus knob to sharpen the focus. of notebook paper and turned in at the beginning of the lab. Show all
8) Adjust the diaphragm to get the best lighting. work for credit.
9) If further magnification is desired, you will need to use the high power (40x)
Given the following information, estimate the size (in microns) of the
objective. As you watch from the side, rotate the nose piece so that the high organism in the diagram. Remember, there are 10 00 microns in a
power objective slides into position. The objective lens will be very close to the millimeter.
slide but it should not hit the slide. field of view (25X)
10) Now, looking through the objective lens, use the fine focus knob to sharpen the
focus. Never use the coarse focus knob when the high power objective is in
place because a small turn of the coarse focus knob in the wrong direction will
grind the objective into the slide.
11) Adjust the diaphragm to get the best lighting.
2 4 6 8 10 12 14 16 18
cm
Notice in the illustration below that there is an inverse relationship between the
magnification of the objective lens and the area of your specimen that is visible
through the microscope.
field of view (80X)
RELATIVE DIAMETER AND LUMINOSITY OF THE
organism
"FIELD OF VIEW" FOR EACH OBJECTIVE LENS

4X 10 X 40 X
This area is known as the “field of view.” As the power of the objective lens
increases, the opening at the tip of the lens decreases, allowing less light from the
illuminator to reach your eye. As a result, the image you observe will be too dark to
reveal important details. You will have to compensate for this by manually opening
the diaphragm, allowing more light to enter the system.
Therefore, the following summary equation can be used to find the diameter of the
field of view at any magnification, as long as the diameter of the field of view at a
previous magnification setting is known.
When viewing fresh, living material, you will need to make a “wet mount” as
outlined and illustrated below.
1) Place the specimen to be viewed on a clean slide.
2) With a dropper, add 2 drops of water onto the specimen.
3) Holding a coverslip at ~45 degree angle to the slide…touch the leading edge of
the coverslip to the slide where it contacts the water…then slowly lower the
coverslip down onto the slide. You should not have any air bubbles on the slide.
If you do, it is best to make a new wet mount. (Under the microscope, air
bubbles look like circles of varying size with very dark edges.)
PREPARING A WET M OUNT TO AVOID TRAP PING A IR BU BBLES
UNDER THE COVERS LIP

So you ask, what is the point of this exercise? You will routinely estimate the size of
objects that you view with the microscope and one way to do this is to compare the
longest length of your specimen to the known diameter of the field of view. For
example, if the diameter of the field of view is known to be 1200 microns, and if the
length of a cellular organelle extends a third of the way across this field of view, then
the organelle’s size can be estimated at around 400 microns.
The objectives of this lab are four-fold. First, note the optical distortion that the
lenses of a light microscope have on the image of an object being viewed. Second,
learn and apply a simple technique for estimating the size of a microscopic structure.
Third, identify those organelles that are commonly seen in a typical plant or animal
cell with the aid of the light microscope. Fourth, compare the structural similarities
and differences between prokaryotic and eukaryotic cells.
You will be asked to draw an accurate depiction of several different kinds of cells in
this exercise. For each drawing…
1) Choose the most appropriate total magnification for viewing the cell. All
drawings must be labeled with the magnification at which they were observed.
Total Magnification = ( power of ocular lens ) ( power of objective lens )
2) Each drawing should include 2-3 representative cells. Out of this group, only
one cell needs to be labeled.
3) Estimate the approximate size of the cells in your drawing.
4) Make your drawings of reasonable size (not so small that you have difficulty
drawing in small details). It is important that the detail of your drawings show
proper proportion.
5) Label all designated organelles; work only in pencil, no pens.
6) Answer any specific questions directed to you in the handout.
PROCEDURE
Eukaryotic Animal Cells
Human Cheek Cells
1) With a wooden toothpick, gently scrape the inside of your cheek to obtain a
cluster of epithelial cells.
2) Gently smear the saliva-laden cluster onto a clean microscope slide
3) Add a drop of Methylene Blue stain (instead of water) and a cover slip; let stand
for two minutes. (Methylene Blue adheres to DNA and thus identifies the
location of this macromolecule in a cell.)
4) Draw a 2-3 cheek cells; label the plasma membrane, nucleus, and cytoplasm.
5) You may notice some very small, darkly stained particles “within” or “on top
of” your cheek cells. What might these particles be? (You should be able to
come up with two possibilities.)
Eukaryotic Plant Cells
Elodea Cells: part 1
1) Secure a leaf from near the tip of an Elodea stem. Place the leaf (concave side
down) on a clean microscope slide and make a wet mount. Examine at 400X
total magnification.
2) The Elodea leaf is several cell layers thick; playing with the fine focus knob will
allow you to focus up/down through these layers. Play with the focus until you
have a good representation of an Elodea leaf cell. Draw 2-3 Elodea cells; label
the cell wall, cytoplasm, chloroplasts. You may or may not observe the nucleus.
Elodea Cells: part 2
1) Drop two drops of salt solution from the NaCl dropper bottle onto the slide at
the edge of the coverslip. Touch a small piece of paper towel to the opposite
side of the coverslip, allowing capillary action to “draw” the salt solution under
the coverslip and make contact with the Elodea cells.
2) Examine the Elodea cells near the edge of the leaf where the salt solution was
added. The cells should look distinctly different. (If they do not, remove the
cover slip, gently blot the excess water from the leaf surface, drop two drops of
salt solution directly onto the leaf, replace the cover slip.) Draw and label a few
of these cells; include the new organelle that is now visible.
3) Explain what happened to these cells when you added the salt solution.
Prokaryotic Cells
Bacterial Cells
1) Obtain prepared (preserved) slides of bacterial cells from the back counter.
Examine these cells at 400X total magnification.
2) You should observe several different shapes (cocci, bacillus, spirilla) and
groupings (solitary, chains, clumps) of bacteria. For each, draw a few
representative cells and label any organelles/structures that are visible.
3) Make a quantitative comparison (ratio calculation) of the size of a prokaryotic
cell vs. a eukaryotic cell.
This is a picture of the microscopes present in m104.

The distance between the binocular eyepieces must be adjusted to match the distance
separating your eyes. While looking through the oculars, slide the left & right
eyepieces laterally until you see one large circular image (field of view).