Exercise No.

1: The
Compound Microscope
and Microscopic
Measurements
Introduction
Microbiology is the science concerned with the study of living organisms
made visible with the use of compound microscopes. Since the compound
microscope is a primary tool in microbiology, familiarity with and
understanding the functions of its parts developing the skill in its use and
manipulation, and learning how to calibrate it to be able to measure
microscopic objects, are prerequisites to any study in the science

Learning Outcomes
At the end of the exercise, the students should be able to:
1. familiarize with the parts of a compound microscope and their functions.
2. practice in the use and manipulation of the compound microscope; and
3. calibrate a microscope and measure microscopic objects.

Materials
• compound microscope, ocular micrometer, stage micrometer,
prepared slides of Paramecium, immersion oil, lens paper

Procedure

A. Review of Microscope Parts (see Figure 1) and Their Corresponding

Functions

The base keeps the microscope steady at any position of the stage.
The arm is used to hold the microscope together with the base when carrying
it.

The mirror, one side plane and the other concave, reflects the light into the
condenser.

To rid the microscopic field of objectionable window bar image in daylight

illumination, use the plane mirror in low power magnification and concave
mirror in higher magnification.

The diaphragm regulates the amount of light entering the condenser, of 2

types namely: a.) iris - mounted immediately beneath the stage apparatus. A
lever regulates its opening and closing; b) disc - mounted similarly as the iris
diaphragm, it is a circular plate with several openings of varied diameter.

The abbe condenser concentrates and sends light rays to the objective

The stage is a horizontal shelf upon which is placed the objects to be

examined. At the center of the stage is a circular opening to permit light to
pass through

The stage clips hold the slide in place,

The objective-magnifies initially the object and forms the real image.

The revolving nosepiece enables rapid. convenient exchange of one objective

for another

The body tube is a hollow tube through which light passes from the
objectives to the eyepiece. The upper portion of the tube is called draw tube,

The eyepiece or ocular further magnifies the image formed by the objective.
Hence the term compound microscope is derived from the fact that the
object is magnified twice, first by the objective and second by the eyepiece.
The final image formed is a virtual image

The magnification of the compound microscope is therefore, the product of

the magnifying power of the objective and the eyepiece.

The coarse adjustment knob is used for bringing the object into approximate
focus. For maximum definition, the fine adjustment knob is used.
B. Use and Manipulation of the Microscope

1. Low Power Objective

a. Place the microscope perpendicularly with the arm toward you on

your table, at least two inches from the edge

b. If the low power objective is not in position over the stage, raise the
stage one inch and revolve the objective into place.

c. Rotate the diaphragm to its largest opening (open it in the old

microscope by use of its lever)

d. With your eyes on the ocular, turn the concave side of the mirror (if
there is a plane and concave side) to get an even circle of light

e. Place a prepared slide of Paramecium right side up on the stage.

f. Looking at the objective from one side of the microscope, raise the
stage with specimen on the slide toward the objective using the
coarse adjustment until the lower lens of the objective is within 1/2
inch from the cover glass.

g. With eyes on the ocular (both eyes open), lower the stage slowly with
the coarse adjustment until the image comes into view.

h. Should this fail to occur, repeat procedures f then g.

i. Accurate focus can be obtained by one or two tums of the fine

adjustment in case coarse adjustment could not make a clear focus
of the specimen.

2. High Power Objective

a. To focus with the high-power objective, first find the image with the
low power. and then move the slide until the object is in the exact
center of the microscope field

b. Rotate the high-power objective and bring it over the object. Watch
from the side to make sure it does not hit the slide.
 c. if the two objectives are parfocal, that is, if they can be interchanged
without varying the focus, the high power will be approximately in
focus. If they are not partocal, the image is either blurred or totally
invisible. In either case, the fine adjustment screw should be used to
bring the image into focus Rotate it slowly in one direction. If no
image appears after these turns, reverse the direction of adjustment
doing it slowly until the image appears. Never use the coarse
adjustment with high magnification, The diaphragm should be opened
to admit lighter when changing to high power magnification.

3. Use of Oil Immersion Objective

a. After the specimen had been focused starting from the low power to
high power objective, with the image at the center of the field of vision
and without moving the side on the stage, move the high-power
objective out of the way Place a small drop of immersion oil on the
part of the specimen that is directly under the objective.

b. Carefully shift into place the oil immersion objective (looking at the
microscope from the side) until the front lens get immersed in oil and
almost touches the slide.

c. Focus again using the fine adjustment knob for more sensitive final
focusing.

d. If the front lens of the objective is no longer in contact with the oil,
repeat the procedure of raising the stage towards the objective until
the oil on the slide touches the objective looking into the eyepiece
until the image appears.

e. After using the oil immersion objective, blot out the oil from the
objective with lens paper only. After blotting out the oil from the oil
immersion objective and from the prepared slide, examine the slide
under the oil immersion objective without the oil. Compare
observation as when there is oil.

C. Calibration

1. Remove the eyepiece from the draw tube. Loosen the top lens of the
eyepiece and insert the ocular micrometer into the eyepiece. (Be sure
the disc is not inverted).

2. Screw back the top lens and return the eyepiece to the draw tube
 3. Place the stage micrometer on the stage and focus on the scale of the
stage micrometer

4. Arrange the stage micrometer so that one line on it coincides with the
line on the ocular micrometer scale

5. Look across the field for another line on the ocular micrometer
coinciding with a line on the stage micrometer.

6. Count the number of divisions on the ocular micrometer subtended by

the number of divisions on the stage micrometer.

7. Determine the value of one division on the ocular micrometer using

the formula:

ODV=SD X SDV
OD
Where:
ODV = value of each division of the ocular micrometer in microns
SD = number of divisions on the stage micrometer subtended by
the ocular micrometer (OD)
SDV = value of each division of the stage micrometer in microns (um)
OD =number of divisions on the ocular micrometer subtended by
the stage micrometer (SD)

Record the following:

Table 1. Value of Ocular Micrometer Division

Objective Ocular Stage Ocular Micrometer

Micrometer Micrometer Division Value
Division (OD) Division (SD) (ODV) (um/div)
Low power
High power
Oil immersion
D. Measurement of Microorganisms

1. Focus under the LP of the microscope the prepared slide of

Paramecium
2. Count the number of the divisions that will be covered by the length of
a Paramecium on the ocular micrometer at LPO. Record the result in
Table 2.
3. Place the measured Paramecium at the center of field of vision. Rotate
the revolving nosepiece until HP falls into place over the condenser.
Focus using the fine adjustment knob until the same Paramecium
viewed at PO appears. Count the number of divisions on OM covered
by the Paramecium length. Record results.
4. Adjust the slide so the measured Paramecium is at the center of field
of vision. Move the HPO out of the way (towards left side) then add a
drop of oil (immersion oil) over the slide (at the lighted area) and lower
the OIO in place.
5. Focus by adjusting the fine adjustment knob until the same
Paramecium is in view. Count the number of divisions on OM along the
Paramecium length. Record results,
6. Repeat step 1 to 5, measuring two more cells of Paramecium
7. Record all measurements in Table 2
8. Compute for average number of divisions of OM in each objective and
record results accordingly.
9. Multiply the average number of divisions on the OM with the ODV in
each corresponding objective to get the average length of the
Paramecium. Fill up the corresponding space in Table 2.
Fig 1. Parts of the Compound Microscope
Table 2. Average length of Paramecium.

Objective No. of Divisions ODV Average length

on OM along the
(um)
length of
Paramecium
1 2 3 Ave
LPO
HPO
OIO

Study Guide Questions:

1. When using the oil immersion objective, what does the oil serve as?

2. Why is it important to clean the oil immersion objective right away

after use?
3. Why is the ocular micrometer adjusted for each objective?