LABORATORY #1
THE SCIENTIFIC METHOD & INTRODUCTION TO THE MICROSCOPE
Introduction
The scientific method is a way of solving problems. It is useful in the laboratory, in the shop,
in the home -- in the everyday affairs of life. Even though you do not plan to be a scientist, you will find the
scientific method useful in solving many problems in your life, be they
practical or philosophic. The best way
to appreciate and understand the scientific method is to apply it.
Application of the scientific method involves the following
basic steps:
1. Recognizing
and Stating the Problem - The problem at hand must be recognized and stated
as clearly and concisely as possible.
2.
Collecting Pertinent Information -
An attempt should be made to assemble all pertinent facts concerning the
problem. Much of this information may
be found in existing literature and can be accessed in a library.
3. Formulation
of an Hypothesis - On the basis of information assembled in step two above,
a tentative explanation or hypothesis is advanced. This is a trial idea, a possible solution to the problem.
4. Testing
of the Hypothesis - In this endeavor, the hypothesis is tested with further
observation and/or controlled experiments.
If the hypothesis is not confirmed by these tests, it must be rejected
or at least modified and subjected to further verification.
5. Publication
of Results - While your personal use of the scientific method does not
involve this step, it is vital to the scientist. This requires publication, in appropriate scientific literature,
of a detailed report of the problem, the hypothesis, all test results, and the
conclusions reached by the investigator.
This allows other scientists to repeat the investigation if they choose,
as a way of confirming the validity of the study.
Scientific Attitudes
To do science requires more than simply following a neat set
of steps in problem-solving. It also
requires a particular way of looking at problems. There are certain attitudes associated with the methods of
science that play a key role in this way of approaching a problem. Listed below are some of the attitudes that
scientists generally agree should be associated with scientific inquiry.
1. A sensitivity to problems - The habit
of an inquiring mind should be developed; that is, a mind that asks
questions. Every great scientist has
shown this attitude to a marked degree.
2. The attitude of intellectual honesty -
This involves the habit of divesting oneself of prejudice, and being honest
enough to admit error when facts indicate that to be the case. It requires that one seek out not only the
data that support one's hypothesis, but that one include all relevant facts and
consider them before drawing a conclusion.
3.
Open-mindedness - It means
substituting an open and inquiring mind for one that is biased and
intolerant. An open mind is one that
takes an objective approach rather than a subjective approach to problems. It is receptive to new ideas and
arguments. Open-mindedness involves a
willingness to consider all possible facts on a matter, even to the extent of
searching for opposing evidence. It
also involves an unwillingness to accept any answer to a problem as final. Science says, "This is true until
further notice," which is another way of saying that all truth is
relative.
4.
Accuracy in every phase of the
investigation - Obviously, habitual inaccuracy would be incompatible with
scientific thinking, since all efforts of science are directed toward the
discovery of facts. Whenever possible,
the scientist attempts to make objective quantita tive measurements, which are
of the greatest value in scientific studies.
5.
The habit of looking for true and natural
causes for observed phenomena, rather than accepting supernatural explanations
- In other words, it is the habit of trying to find the real causes for every
happening. This might be called a
cause-and-effect attitude. Science
assumes natural causes for natural phenomena and seeks in orderly fashion to
discover those real causes.
6.
The habit of suspended judgement -
This involves the patience to wait until all available facts on a question have
been collected, before drawing a conclusion.
If the data are inadequate, then the drawing of a conclusion should be
deferred.
7.
The habit of critical-mindedness -
This involves a constant lookout for possible flaws in all suggested
hypotheses, theories, evidences, conclusions and proposed remedies. This attitude also involves the ability to
criticize the thinking of others and especially the willingness to criticize
oneself.
8. The
review of findings by peers - Scientific investigations are written in a
concise but complete form and presented before groups of other scientists or
published in professional journals, as a way of making the study more generally
known. This exposes the methods and
conclusions of the investigation to other scientists who may find the information
useful in their own work, or who might suggest ways in which certain aspects of
the study could be improved.
ACTIVITIES
1. While viewing the videotape entitled "Scientific
Methods and Values," answer the following questions concerning it.
a. What are the two big ideas of science?
b.
Who discovered the theory of the pendulum clock?
c.
What philosopher/scientist claimed that diseases have natural causes, and
therefore must have natural cures?
d.
What are some stereotypes of scientists?
e.
What are the three ingredients of science?
f.
Who is quoted as saying, "Science is one percent inspiration and
ninety-nine percent perspiration."?
g.
To what was Isaac Newton referring when he said, "If I have seen further than
other men, it is because I have been able to stand on the shoulders of
giants."?
h.
Why is astrology not science?
i.
Why are scientists often looked upon as "absent-minded"?
j.
List five scientific values.
Introduction-
Until late in the sixteenth century, much of the body of
knowledge termed "biology" was unknown to science. This is due to the miniscule size of some
life forms, and the fact that a basic understanding of larger organisms
requires the ability to see objects not detectable to the unaided human
eye. The development of precision lense
grinding and the construction of an
instrument consisting of several lenses functioning together, revolutionized
biology. this instrument, the
microscope, has become a major tool of biologists and few laboratories are
without several. With the passing of
time and the development of new technologies, the microscope has proliferated
into a variety of models, each designed forparticular tasks. Among these designs are:
A. The
light microscope - The type we will use in our laboratory. These use
visible light which is magnified and concentrated by glass lenses. There are two models which we will use.
1.
The dissecting microscope (Figure 2b
in your Photo Atlas) is designed to
study thick or opaque objects at fairly low magnification.
2.
The compound microscope (Figures 1a,
1b, and 2a) is used to observe small and/or thin objects at higher
magnification. Specimens must be thin enough
for light to pass through from below.
This is the "workhorse" of biology and the one you will most
often use.
B.
The electron microscope uses a beam
of electrons rather than light. The
electron beam is focused onto a photographic plate by means of electromagnetic
"lenses". There are two types
of electron microscopes, both of which are large and expensive and consequently
confined to research labs.
1.
The transmission electron microscope
is used for specially-prepared transparent specimens. It is capable of magnifying by thousands of diameters with great
resolution. Most of the photographs on
pages 5 - 9 in the Photo Atlas were
made using a microscope of this type.
2.
The scanning electron microscope is
not capable of extreme magnification, but is can be used on opaque objects and
shows remarkable surface details. The
photograph of Didinium on page 285 in your text book is an outstanding example
of scanning electron microscopy.
C.
The dark-field microscope has the
light entering from the side, where it is reflected off the object being
observed. This shows a brightly illuminated object on a dark background. See
Figure 20c in Perry and Morton.
D. Phase-contrast microscopes are useful
for examining live, transparent objects.
Differences in thickness and composition are converted into visible
differences which would be less apparent on another instrument.
PRELIMINARY INFORMATION - Please
read before proceeding!
Microscopes are very expensive to
replace; therefore, be particularly careful when handling and using them. Always carry them with both hands, holding
the instrument upright, so that the eyepiece does not slip out. Use only clean dry lense paper to clean the
lenses. Do NOT use Kleenex, paper towels, sleeves, nor the front of yout
T-shirt, as these can scratch the soft optical glass used in these lenses. If there is a problem with your microscope,
report this to your instructor immediately. You will not be penalized in
any way.
ACTIVITY 1.
The Microscope and its Parts - Select one
of the Wolfe compound microscopes from the cabinet at the front of the room,
carry it to your lab station and plug the power cord into a convenient
outlet. Using Figure 1b, in Perry and
Morton, a microscope similar to yours, locate all the parts shown.
a.
The ocular (Figure 3d) is the lens
through which you view the object. This
lens magnifies the image passing through it by 10 times. It also has a pointer which may be used to indicate objects being viewed.
b.
The revolving nosepiece holds four objective
lenses. Grasp the silver ring and
note that it may be rotated, bringing each objective lens into place. Feel the distinctive "click" as
each objective is in proper position for viewing.
c.
The objective lens performs the
initial magnification. In general, the lower the magnification, the shorter the
objective lens and the farther it is positioned from the specimen. Your scope has four of these, located on the
revolving nosepiece. They are as
follows:
- The scanning lens is a
short lens which magnifies by 4 times. It as the broadest field of all your
lenses and is used for initial location of objects.
- The low-power objective (Figure
3e) has yellow color bands and
magnifies by 10 diameters. You will
probably find this the objective most often used.
- The high-power objective, with its blue color bands and 40X
magnification, is much longer than the first two. It is the most powerful lens used in this class.
- The longest objective is the
one with black bands and 100X magnification.
This is the oil-immersion lens
(Figure 3f) and will not be used in this course. PLEASE DO NOT ATTEMPT TO USE
THIS LENS.
d.
The stage is the flat surface with
the round hole or aperture. The slide to be viewed is positioned on the
stage over the aperture. On your
microscopes, the stage is equipped with a mechanical device into which the
slide is clipped. This so-called mechanical stage (Figure 3a) allows
the slide to be manipulated by means of the two knobs located underneath the
stage.
e.
The substage condenser (Figure 3c)
is a package of lenses located under the stage and controlled by a knob on the
side opposite the mechanical stage
control knobs. Its function is to
condense the light and focus it on the specimen. You need not be concerned with this device. SIMPLY
RAISE IT TO ITS MAXIMUM HEIGHT AND LEAVE IT THERE.
f. The
iris diaphragm is located on the condenser and functions to control the
amount of light passing through the specimen.
A tiny lever projects from the front of this device and is moved from
side to side to affect a change in adjustment.
g.
The substage illuminator is the
silver-colored device on the microscope base beneath the stage. It is the source of light which illuminates
the object being viewed.
h.
The illuminator dial, located on the
side of the base just below the mechanical stage adjustment knobs, controls the
light output of the illuminator. A value of from five to seven is usually
sufficient for our purposes.
i. The adjustment
knobs are located on both sides of the microscope, just above the
base. There are two of these, one
located outside the other.
- The coarse
adjustment knob is the largest of these and surrounds the fine adjustment knob. Its function is
to focus the scanning and low-power objectives. In our microscopes, the coarse adjust ment is very tight and may
require the use of both hands.
- The
fine adjustment knob is surrounded by and projects from the coarse adjustment knob. Its function is to focus the high-power and oil
immersion objectives. DO NOT USE WITH LOW-POWER OR SCANNING
OBJECTIVES.
ACTIVITY 2.
Magnification - The compound microscope combines the
magnifying power of the eyepiece with that of the objective lens. The magnifying power is marked on the housing
of each lens. Simply multiply the
magnification value of the objective times that marked on the eyepiece to see
how many times the specimen is enlarged.
Calculate the magnification of your microscope with each of the
objectives in place.
ACTIVITY 3.
Resolving Power - This is a measure of lens quality. Quality lenses have high resolving power,
the ability to deliver a clear image in fine detail. If a lens has high magnification but low resolution, it is of
little value. Although such an image
would be large, it would not be clear enough to show detail. Resolution is also affected by the
cleanliness of the lens. It is a good
idea to clean your lenses every lab.
ACTIVITY 4.
Field of View - This is the size of the area that the lens
views. The higher the magnifying power
of an objective lens, the smaller the area viewed. When you switch to a higher-powered lens, you are actually
looking at the central portion of what was visible under low power. It is important to center the specimen
before increasing magnification.
ACTIVITY 5.
Using the Microscope - With your microscope sitting before
you, grasp the monocular head and carefully rotate it. Try turning the microscope so that the arm
is away from you. The rotating head allows
you to position the instrument either way.
Which is most comfortable to you?
Now rotate the illuminator dial to the 7 position. Carefully lean the microscope back in the
direction of the arm. Look under the
stage and locate the iris diaphragm.
Move the control arm of the diaphragm and see how it affects the light
passing through the condenser.
a.
From the slide tray on the counter at the side of the room, select a slide
marked "Letter e”. Open the spring arm of the mechanical stage as
shown by your instructor, place the slide in the proper position on the stage
and carefully return the spring arm to its closed position. This will hold the slide in place. Using the control knobs, maneuver the slide
about on the stage and finally position the tiny "e" over the stage
aperture.
b.
Turn the scanning objective so that it snaps into place directly over the
aperture. Now raise the stage so that
the slide is as close to the objective as possible. While viewing through the eyepiece, use the coarse adjustment
knob to move the slide away from the objective and bring the letter
"e" into focus. Using the
mechanical stage, move the "e" around. If you move the slide to the right, what is the apparent
direction of movement of the "e"?
Look at the tiny "e" with your naked eye and sketch a picture
of it. Now look at it through the
microscope and sketch that image. Do
you see a difference? Carefully center
the "e" in the field (the circle of light seen through the
microscope) and proceed to the next activity.
c.
Without making any other adjustment to the microscope, turn the low-power
objective into place. Focus the image
(using the coarse adjustment knobs) and compare it to the previous one. Is it still in the center of the field? If not, center it once more.
d.
Again without making other adjustments, rotate the high-power objective into
place. Viewing the objective from the
side, note how close it is to the slide.
Look through the eyepiece and, using the fine adjustment knob, bring the
"e" into focus. If all is
dark, move the slide slightly. Now use
the iris diaphragm to change the amount of light. Did it make a difference?
A
feature of a good microscope (of which your's is one) is its parfocal
capability. This means that when a
specimen is in focus under low-power magnification, you can switch to
high-power magnification and have the specimen remain in fairly good
focus. Usually a slight adjustment with
the fine focus knob is all that is required for a sharp image.
e.
When you have finished, turn the scanning objective back into place and remove
the slide.
f.
Select a couple of other slides from the tray indicated by your instructor and
practice bringing them into focus.
Always use the same procedure shown above. DO NOT TRY SHORTCUTS!
ACTIVITY 6.
Making a Wet Mount - While most
of the slides you will use in this course are prepared slides; fixed, stained
and mounted by professionals, from time to time you will be asked to prepare a
temporary slide of some material. These
are termed wet mounts. You will now prepare a wet mount as directed below:
a.
Select a glass slide and a coverslip from those provided. Carefully clean the slide as you would
eye-glasses, using one of the KEMWIPE tissues provided. The coverslips are unused and should be
clean. Always handle both slides and
coverslips by the edges.
b.
Using a clean toothpick, carefully scrape some cheek cells from the inside of
your mouth, as demonstrated by your instructor. Touch the toothpick to the center of your slide, depositing a
milky mixture of saliva and cheek cells.
c.
Place a drop of iodine stain on the cheek cells and carefully mix with the
toothpick. This will render the cells
more visible.
d.
Now take a coverslip and carefully drop it into place on the drop of stained
cells. Set down one edge first, then
slowly lower the coverslip into place.
Ideally, any air bubbles will be displaced by this maneuver. Do not be distressed, however, if you get a
few bubbles in this initial preparation.
If the fluid does not come to the edge of the coverslip, add a bit of
water at one edge.
e.
Place the slide on your microscope and view the cheek cells. Use all three objectives. These are flat, tile-like cells with a
prominent nucleus which should be stained golden brown by the iodine. As there will probably be clumps, move the
slide around until you find some isolated cells to view.