Laboratory 1: Use and Care of the Microscope

USE OF THE MICROSCOPE

The compound microscope is a precision, highly specialized instrument that is
designed to allow the viewing of objects that are too small to be seen by the unaided eye.
The resolving power of your microscopes is approximately 0.2m (micrometers or
microns), which means it can discriminate objects down to a size of 0.2. Unfortunately,
many users fail to properly adjust the optical system resulting in unsatisfactory results or
resolving power. We will spend one lab period learning how to properly use a
microscope. For some of you this may be a review, for others, it may be the first time
you were properly instructed on the proper use of a microscope.
MAJOR PARTS AND FUNCTIONS
Optimum clarity of images produced by the microscope cannot be obtained if the
elements of the optical system are dirty. Before using the microscope, always clean the
lenses with lens paper provided for that purpose. Never substitute paper towels of facial
tissues as they contain grit and will scratch the lenses. Dust may be blown from the
lenses with a bulb. Do not touch the surfaces of the glass lenses with your fingers.
The compound microscope is basically a tube with magnifying lenses at each end;
the objective lens at the lower end, and the ocular lens at the upper end. The tube is
attached to a supporting frame that is equipped with coarse and fine focusing screws.
There is also usually a stage to support the specimen being examined. In the case of
binocular microscopes, there is an internal image splitting prism and mirror arrangement
to deliver the image to both oculars.
Most microscopes have more than one objective lens attached to a revolving
nosepiece, which allow the selection of objectives of different magnifications. These
objectives are constructed in such a way that they are parfocal; that is, they may be
rotated into position for viewing without danger of touching the specimen slide and
should require only minor adjustment to bring them into sharp focus. The light necessary
for viewing the specimen may be either natural or artificial, and may be directed into the
optical system by either a mirror or be beamed directly through the specimen from below.
When the mirror is used, the flat side is usually utilized with low magnification and the
concave side with higher magnifications. Modern microscopes also have a set of lenses
between the light source and the objective called the condenser. The condenser is
equipped with an adjustment screw so that the light can be focused to provide the most
effective illumination of the study specimen (always use the flat side of the mirror if your
microscope has a condenser). Immediately below the condenser is an iris diaphragm (a
lever on the condenser) which may be opened or closed to control the total light passing
through the optical system. On microscopes with built-in artificial light sources, there is

usually a rheostatic control which regulates the intensity of the light beam illuminating
the specimen.
ADJUSTMENT OF THE MICROSCOPE
The following procedures should be performed to adjust your microscope at the
start of each laboratory session:
1. Carefully carry a microscope back to your lab bench. Plug it in. Record the number
of your microscope here: # __________ . This will be your microscope for the rest of
the semester. You are responsible for the care and maintenance of this microscope, so
please be careful.
2. Clean all glass surfaced carefully with lens paper.
3. Place the 10X objective in position, turn the lamp on at approximately one-half
power. Obtain a prepared slide (letter e) and place it on the stage with the specimen
centered in the light path. Make sure the slide clips are snuggly against the slide
NOT ON TOP OF IT. While looking at the stage, bring the stage to a position near to
the objective lens.
4. Look through the oculars. Move the oculars farther apart or closer together until the
oculars match your inter-pupillary distance. DONT LEAN YOUR EYES ON THE
EYEPIECES AND MAKE SURE YOU ARE LOOKING THROUGH BOTH EYES!
What is your inter-pupillary distance? _____________
5. Lower the stage with the coarse adjustment slowly until the specimen comes into
sharp focus. If both oculars are not in sharp focus, follow these procedures. Close
your eye that is over the diopter adjusting ring (usually the left). Use your coarse and
fine focus to bring the image into sharp focus with your other eye (usually the right).
Now close the other eye, and use the diopter adjusting ring to bring the image into
sharp focus. You will probably have to do this every time you use these microscopes,
particularly if other people are using them in the meanwhile.
6. Gently close the iris diaphragm.
7. Raise or lower the substage condenser until the edges of the diaphragm are in sharp
focus when viewed through the oculars.
8. Slowly open the diaphragm until the edges disappear from the field of view.
9. Your microscope is now adjusted and ready to use.
10. Look critically at the letter e. Does the letter e appear the same when looked at with
the naked eye as it does when you look in the microcope? How is it different?

ILLUMINATION
Increase or decrease the light intensity of the lamp by turning the variable rheostat
knob on the base of the microscope. Generally, you should avoid moving the substage
condenser to adjust the light intensity. However, for some purposes at lower powers
better contrast can be achieved by lowering the substage condenser.
OBJECTIVE LENSES
1. Magnification of your specimen is a determined by the magnification of your
objective lenses (i.e.4X, 10X, 40X, and 100X) times the magnification of your ocular
lens (ours are 10X). Therefore, fill in the following information:
Objective Lens
4X
10X
40X
100X

Ocular Lens
10X
10X
10X
10X

Total Magnification

2. Always start your study with the lowest power objective. Do not try to focus on a
fresh slide with an objective higher that the 10X. Start with the letter e at 4X. Make
sure it is focused and centered in the field of view (the illuminated circle you see
when you look in your microscope). What percentage of the total field of view does
the letter e take up? _______ Now, switch to the 10X objective and use the fine
focus to bring it into focus. What percentage of the total field of view NOW does the
letter e take up? ________. Do it again for the 40X and again notice the percentage
of the total field of view that the letter e takes up.
OIL IMMERSION OBJECTIVE
1. Obtain a prepared slide of bacteria. Start with the lower power objective and increase
the magnification stepwise until the specimen is in focus with the 40X objective.
2. Rotate the nosepiece to a position about halfway between 40X and the 100X oil
immersion objective.
3. Place a small drop of oil on the slide in the center of the illuminated area.
4. Carefully rotate the oil immersion objective into contact with the oil and into position.
5. Focus only with the fine adjustment knob.
6. Clean the slide and objective with lens paper after each use to remove the oil.

GENERAL CARE
1. The microscope is a precision instrument. Carry the microscope carefully, avoiding
any bumping or jostling that may knock it out of alignment.
2. Keep the microscope dusted and the optics clean.
3. At the completion of the laboratory or study session (not yet for this lab), turn the
lamp off, place the 4X objective in position, lower the stage with the coarse
adjustment knob, move the mechanical stage clamp to the extreme left position, and
replace the microscope dust cover.
4. Inform an instructor if your microscope is not working properly. Do not attempt
repairs yourself.
CALIBRATION OF THE MICROSCOPE AND SCALE DRAWINGS
CALIBRATING THE MICROSCOPE
The identification of many microscopic species of plant and animals requires that
you take careful measurements of morphologic characteristics. Such measurements may
require the use of a calibrated microscope measurement fitted with an ocular micrometer.
The unit of measurement is the micron (micrometer).
THE OCULAR MICROMETER
The ocular micrometer is a glass disc etched with lines or squares that are
numbered to facilitate counting of the divisions. The disc normally sits on a shelf in the
ocular, or eyepiece of the microscope. The goal in a calibration is to establish
measurement values for the divisions in the ocular micrometer. These values will vary
with the different lens combinations. Thus, the higher the magnification of a given lens
combination, the smaller will be the interval in microns per ocular division. Ocular
micrometers usually have five major divisions, numbered 1 through 5, with each major
division subdivided into 10 smaller divisions. We customarily count these smaller
divisions; therefore an organism that is as long as 14 of the smaller divisions is 14 ocular
divisions long, not 1.4 divisions.
To establish the ocular micrometer values, the divisions on the ocular micrometer
are compared with the scale divisions on a standard measurement slide known as a stage
micrometer. The scale on a stage micrometer is etched upon a glass microscope slide and
protected by a glass coverslip. Usually the scale is 2 millimeters (mm) in length with the
smallest divisions being 0.01 mm (10 microns ()). Stage micrometers may or may not
have scale numbers. Use great care in handling these micrometers.

CALIBRATION PROCEDURES
The steps described here can be applied to the calibration of both compound and
dissecting microscopes. If no stage micrometer is available, the markings on a high
quality millimeter ruler can be used to obtain an approximated calibration of the low
power objectives of the microscope, and all lens combinations of the dissecting
microscope. You should prepare a calibration chart for all of the lens combinations to be
used.
The procedure for calibration is:
1. Place the stage micrometer on the stage of the microscope.
2. Focus on the stage micrometer scale with the low power objective of the
microscope.
3. Rotate the ocular micrometer so that its scale is parallel to that of the stage
micrometer. Move the stage micrometer to superimpose the two scales so that
the left etched line of the ocular micrometer exactly coincides with the left
etched line of the stage micrometer. If you are unsure which scale is the
ocular micrometer and which is the stage micrometer, turn slightly the
eyepiece containing the ocular micrometer or slightly move the stage
micrometer.
4. With the left-hand lines exactly superimposed, scan with you eye to the right
of these two coinciding lines until you find a point where the lines of the two
micrometers again exactly coincide. Greater accuracy is obtained if these two
points are as far apart as possible. Count and record the number of scale
divisions between these two points. With high magnifications, the scale
etchings on the stage micrometer will be broad, therefore measure from center
to center of the etchings.
The following example illustrates the calibration procedure.
Ocular micrometer divisions
=4
Stage micrometer divisions
= 5 of the 0.01 mm divisions or 0.05mm
therefore, 4 ocular divisions = 0.05mm (4O.D.= 0.05 mm)
or, 1 ocular division = 0.0125 mm,
(divide each side of the equation by 4)
or 12.5 (there are 1000/mm), so we multiplied by 1000.
from this information, youd know that 2 ocular divisions = 12.5 x 2 = 25
5. the final step in calibration is to calculate the ocular micrometer values for
each lens combination and prepare a calibration chart. These are especially
useful when many measurements are necessary.

LAB EXERCISE:
Prepare a table of calibration for your compound microscope and fill in the
following table.
Microscope # ______
Ocular Divisions
1
2
3
4
5
6
7
8
9
10

4X

Micron equivalents for the indicated lenses

10X
40X

SCALE DRAWINGS
Accurate measurements of microscopic specimens are very useful for proper
identification and description. In addition, they are very useful in the preparation of
scientific illustrations, where images may be greatly enlarged. Thus, illustrations may be
designated as X200 or 200X, indicating that the image of the specimen has been
enlarged 200 times.
To prepare such scale illustrations, you must first measure your specimen to
determine its dimensions in microns, millimeters or some other standard unit of measure.
then multiply these figures times the magnification desired and prepare the illustration
with the calculated dimensions. Be sure that your units of measure are the same when
making your calculations, for example, do not multiply microns times millimeters.
Consider these examples:
1. A protozoan is 20 long and 12 wide. To make a drawing to scale at a
magnifications of X3000 your figure will be of the following dimensions:
20 (= 0.020 mm) X 3000 = 60 mm long
12 (=0.012 mm) X 3000 = 36 mm wide

100X

Larger specimens can be measured with a millimeter ruler to determine the

dimensions of a scale drawing.
2. The actual size of a specimen can be calculated from its illustration as
follows:
An illustration of a specimen is given as X2000. The length of the illustration
is measured and found to be 48 mm. The actual length of the specimen from
which the illustration was made can be calculated to be 24 microns long.
The actual size of the specimen is 1/2000 of the size of the illustrations;
therefore:
1/2000 of 48 mm= 48mm/2000 = 0.024mm = 24
3. The magnification of an illustration can be calculated if the dimension of the
original specimen is known.
In a publication the author states that the specimen was 20 long, but the
magnification of the illustration is not given. You measure the length of the
illustration at 50 mm. The magnification can be calculated to be X2500 by the
following method.
The magnification can be calculated by discovering how many 20 lengths
will be needed to equal 50 mm.
50 mm divided by 0.020mm = 2500X
4. Line bars in the illustration can be used to indicate the actual size of the
specimen. This is commonly done by two different methods.
a. In the first method a line equal to the actual length of the specimen is
drawn beside the illustration along the corresponding axis.
b. In the second method, appropriate for microscopic specimens, a line is
drawn whose length is the equivalent of 1 in relation to the size of the
illustration, and labeled as 1 micron (kind of like a scale of miles on a
road map).
5. Sometimes you will see a photograph of a macroscopic specimen which
includes a ruler or even a coin as an indicator of scale.

LAB EXERCISE
A. Use the calibration data below and calculate the length in microns of an
organism that is 15 ocular micrometer divisions long. Show your calculations
in the space below.
5 ocular divisions equal 8 stage micrometer divisions. Each stage micrometer
division equals 0.01 mm.

B. Calculate the length of a drawing for an animal whose actual length is 2 mm

and the magnification is X100. Show calculations in the space below.

C. Calculate the actual length in microns of an animal when the drawing of the
animal is 24 cm long and the magnification is X3000. Show calculations in
the space below.

D. Make a wet mount of the specimens provided for you to study by

coverslipping a drop of each suspension. Examine these under your
microscope. Draw to scale and label one of these protozoans, indicating the
scale beside your drawing.