BIO 111 Principles of Biology Fall 2021

Laboratory Session #3: Studying the Microscopic World

LAST NAME: FIRST NAME: LAB SECTION:

Lab Objectives for Lab Session #1:

Students will demonstrate their understanding of --
1. the different kinds of microscopes, how they are constructed, how they are used to study small
features: dissecting, compound, and electron microscopes.
2. how microscopes allow us to visualize living creatures too small to see without magnification.
3. how to prepare living organisms for microscopic examination using wet mounts.
4. how to make and cell preparations from mouth swabs.
5. how to use staining with biological dyes to make transparent cellular organelles visible.

DISSECTING MICROSCOPES ARE USED FOR STUDYING SMALL ORGANISMS

Dissecting microscopes are constructed to allow a researcher to visualize small features while
dissecting and to enable use of both hands in dissecting a specimen. These microscopes can also be
used to study organisms that only need a small amount of magnification to observe while they are
alive. Dissecting microscopes give good 3-dimensional view of the specimen being examined.
The photograph above shows an example of a dissecting microscope. There are many different
brands of these microscopes and so there are small differences but most are very similar in their
construction. Compare the microscope in the photograph to the labelled microscope in the laboratory
and learn the various terms for the parts of this microscope and what they do. You will be asked to
identify these features on lab exams.

LEARNING TO FOCUS THE DISSECTING MICROSCOPE

Take one of the dissecting microscopes out of the shelf at your lab desk. Note that the microscope
has an electric cord for the light source that should be plugged in below the desk.

Get one of the pennies available in the lab and place it on the stage of your dissecting microscope.
Try to get the penny into focus using the focus adjustment knob. Change the magnification using the
magnification adjustment knob. When using a microscope, it is usually a good idea to start with the
lowest magnification and then increase magnification after you have the microscope in focus. Return
the penny to the front desk when you are finished with this observation.

WHAT LIES BELOW? STUDYING A POND/MUSHROOM SAMPLE UNDER THE DISSECTING

SCOPE

Collect either a sample of pond water or a two pieces of mushroom from the front of the room. If you're
getting the pond water, only collect a few drops with some floating material in it. If you are taking the
mushrooms, be sure you get one of each kind so you can compare and contrast. Check the last page
for a simple sketch of some common organisms you may see.

In the space provided below, make a sketch of one/some of the organisms that you observed under
the dissecting scope or sketch both mushrooms and highlight any differences. Label the drawing
and any features you can identify.

How do you think this organism moves (can it move?)

Can this organism see you? Why or Why not?

COMPOUND MICROSCOPES USED FOR VIEWING SMALL FEATURES OF SPECIMENS
Compound microscopes are instruments for viewing biological specimens at a wide range of
magnifications, usually ranging from about 40X to over 1000X. Magnifications in this range are
capable of visualizing individual cells and, at the high end of the range, some of the larger internal
organelles of cells. Cells as small as bacterial cells can be studied with these microscopes.

Compound microscopes get their name from the fact that they have two sets of lenses, the ocular
lenses (eyepieces) and the objective lenses usually mounted on a revolving turret of three or four
lenses with different powers of magnification, e.g., 4X, 10X, and 40X.

To calculate the total magnification of a specimen, we have to multiply the power of the ocular lens
by the power of the objective lens in use. The table below lists the power of the lenses on the
microscopes used in this laboratory. Complete the table by calculating the Total Magnification for
each setting.

Ocular Lens Power Objective Lens Power Total Magnification

10X 4X
10X 10X
10X 40X
A typical compound microscope is shown in the picture below. The compound microscopes used in
this laboratory will be very similar to this.

Look at the demonstration microscope set up with labels in your laboratory. Make sure that you can
identify each of the labelled parts and state its purpose. The picture above will indicate the function of
each part.

Go to one of the wall cabinets and remove a compound microscope. Your laboratory instructor will
show you how to carry these back to your lab desk. Note the electric cord for the illuminator and
these must be plugged into an electrical outlet below your desk.

Viewing “Letter e” slide: Find one of the prepared slides labelled “Letter e.” Fit this slide into the
slide clip on the microscope stage. [IMPORTANT: The slide clip clamps a slide from the sides;
never try to put a slide under the prongs of the slide clip.] Be sure to let the instructor know if you
are having trouble finding the specimen in the microscope field.

Turn the objective lens turret so that the shortest lens (4X objective) is in place over the hole in the
stage. Center the letter e on the slide in the circle of light coming through the hole in the stage. Try
to focus the microscope using the coarse focus knob so, as you look through the microscope, the
letter e is seen in sharp focus. Is the “e” in normal reading position or upside down?

Use the stage control knobs to move the slide slightly to left and right. When you move the slide to
the left, which way does the letter e image move as you look through the microscope?
This upside down view of the object and backwards movement is a property of compound
microscopes. We say that the image is inverted. Remember this as you move the slide to study
future specimens.

Increase the magnification to 100X by turning the 10X objective (next longest length) into position
over the hole in the stage. The image should still be in focus or only require slight adjustment using
the fine focus knob.

Finally, increase the magnification to 400X by turning the 40X objective (longest lens) into position
over the hole in the stage. Refocus the image if needed.

Another thing you should notice is that the diameter of the field you see through the microscope
gets smaller as you increase the magnification. You see more structural detail but less of the
specimen as you go to higher magnifications.

USING THE COMPOUND MICROSCOPE TO STUDY VERY SMALL CELLS -- BACTERIA

The development of high quality compound microscope was very important for scientists trying to
understand the cause of human diseases. Many human diseases are caused by bacterial infection
and bacterial cells are very small. Bacteria can be found in diseased tissues and different species of
bacteria cause different pathology in tissue infected with these tiny cells. More powerful microscopes
have allowed scientists to correlate the presence of many kinds of bacteria with the diseases that
they cause. Bacterial cells belong to a type of cell referred to as prokaryotic; these cells do not have
a membrane-bound nucleus. They are probably similar to the earliest cell types that developed on
the Earth many millions of years ago.

Get one of the prepared slides labelled “bacteria types” from the front desk. Put this slide on your
microscope and focus it on 40X, then increase to 100X and 400X.

Make certain that you adjust the diaphragm lever below the stage so that the correct amount of light
is coming through the specimen. This adjustment is very important to improve the view of the
specimen.

How would you describe the bacterial cell specimens that you see on this slide?

PREPARING A WET MOUNT OF EPITHELIAL CELLS FROM YOUR CHEEK AND GUMS

Your compound microscopes are capable of allowing study of the cellular/tissue level of human body
construction. Some cells are rather loosely attached to your body. An example of this is the
squamous epithelium that lines your cheek and gum surfaces. The top layers of these cells are
constantly being shed and replaced. We will make a “wet mount” preparation of these cells to
examine with the compound microscope. Wet mounts are not permanent slides; they are made for
brief study and then discarded.
Follow this procedure to make your wet mount slide:
• Obtain a clean microscope slide and a toothpick from the front desk of your lab.
• Rub your gum surface near your back teeth with the toothpick (DON”T GOUGE!!) and smear
the material in the center of the microscope slide.
• Mix in a small drop of physiological saline.
• Touch the tip of the toothpick to methylene blue stain to obtain a small amount and mix the
stain with the saline on the slide.
• Carefully, place a clear plastic cover slip over the drop.
Now, clip the wet mount preparation into the slide clip of your microscope. Adjust your microscope to
the 40X total magnification (use 4X objective) and focus on the material under the cover slip. Try to
find the cells you scraped off your gum surface.
Squamous epithelial cells are roundish, somewhat irregularly shaped cells that are very flat. The
methylene blue stain that you used will stain the nuclei of the cells blue so that you can identify the
nucleus of each cell. Sometimes you can see small darkly stained dots on the cells’ surfaces. These
are bacteria found in the human mouth. Some people also have an ameba called Entameba
gingivalis living in their mouths. This may be seen in some preparations. This ameba is non-
pathogenic; it does have a pathogenic relative, Entameba histolytica, (that you will not find) that can
cause serious digestive system upsets.
When you have found the cells on the lowest magnification, carefully rotate the 10X objective into
place and refocus. Recenter the specimens in the field and carefully rotate the 40X objective into
place and refocus. This is the useful magnification for studying these cells.
Always be very careful when increasing to 400X. Using the long 40X objective carelessly may break
the slide if you get the lens too close to the slide. If you don’t see the specimen when you increase to
400X, go back to the 10X objective and refocus. Remember your instructor is here to help.
In the space provided below, make a pencil sketch of the squamous epithelial cells you find.

Label the drawing you made, note the magnification you were using, and label the nucleus of the
cells.

Why is it necessary to use the methylene blue stain?

USING THE COMPOUND MICROSCOPE TO STUDY SMALL ORGANISMS -- DAPHNIA

Daphnia is a small aquatic animal. It is a member of the group or phylum Arthropoda, a very large
group including many “joint-legged animals.” Creatures like Daphnia can be present in large numbers
in lakes, ponds and streams and can be very important to the aquatic food web. They are eaten by
fish and other larger aquatic animals. Note that, even though they are of microscopic size, their
bodies can have complex structures and function.

Take a prepared slide labeled, ”Daphnia w.m.” from the front desk. Use your compound microscope
to study this animal. Make a pencil sketch of one of the Daphnia specimens in the space provided
below. Use the pictures available in the laboratory to help you identify the structures you see. Label
all the structures, note the magnification you were using to view the specimen, and label the drawing.
POND SAMPLE UNDER THE COMPOUND MICROSCOPE

Find the pond sample container from earlier. Take a small drop from the sample container and
mount it on a slide like we have done today already. Be sure to revert back to the 4x scope before
you zoom in on the slide. Look at the slide and try to identify some organisms in the slide.

How does your perception of the life in this sample seen under the compound scope compare to the life
seen under the dissecting scope? Does it seem more or less active?

Ask your instructor to sign your lab before your leave for the day and take a copy of the Take-
home work.