THE COMPOUND MICROSCOPE

Most microorganisms like protozoa, algae, fungi and bacteria are so small that they
cannot be seen by the naked eye. The smallest object that the eye can see at a distance of 250
mm is about 0.07 – 0.14 mm. The use of microscopes allows us to see these minute organisms,
since an object can be magnified a few hundred times to hundreds of thousand times.

There are two general categories of microscopes: the light microscope which uses light
waves and lenses that are associated with the light microscope, and the electron type which
employs electron beams and magnetic fields to produce the image.

Light microscopes can be classified as simple if they have short focal length, are held
close to the eye and magnify objects only up to 300X. On the other hand, the compound type
employs two separate lenses, an ocular and an objective, in order to achieve 2-5 times greater
magnification.

Other types of microscopes include:

 bright field, where the microscope field is brightly lighted and the object to be
observed appears dark die to its ability to absorb or refract some of the incident
light
 dark-field, where the object appears luminous against a dark background since it
reflects some of the incident light in all directions
 Ultraviolet (UV), which uses UV light, thereby allowing greater resolution and
magnification. It is used principally to detect or even measure substances in
specimens of living tissues that are known to absorb UV light at particular
wavelengths. UV is not visible to the eye, so the image formed is recorded with
the use of cameras or a television screen.
 Fluorescent, which makes use of the property of certain chemical substances that
release light at a different wavelength when exposed to UV rays. Such substances
convert UV light into visible waves of greater length. Bacteria and other
microorganisms are stained with fluorescent stain that can be detected in a
microscope illuminated with UV light.
 Phase-contrast, which utilizes the refraction that occurs when light passes from
one medium into another of different density. The special objectives and
condenser intensify slight differences in contrast produced by this bending of
light. It is useful in studying the internal structures of microorganisms because
structures differing in refractive index from the surrounding protoplasm become
visible, and their sizes and locations can be determined.

BIO103| Microbiology Laboratory Manual

 In this course, the compound microscope will be used in many of the exercises so the
student should be able to use it proficiently and care for it properly. Constant and proper use of
the microscope will increase one’s proficiency.

Objectives:

At the end of this exercise, the student should be able to:

a. Name and describe the parts of the compound microscope

b. Elucidate the function of each part
c. Handle and care for the microscope properly
d. Manipulate the microscope proficiently
e. Measure microbial specimens

Microscope Parts and their Corresponding Functions

The wide base keeps the microscope steady at any position of the stage.

The arm, fastened to the base through the inclination joint, permits the adjustment of the stage to
a desired angle.

The concave mirror reflects the light into the condenser.

The condenser concentrates the light rays received from the mirror and sends them to the
objective.

The stage is a horizontal platform upon which the specimen to be examined is placed. At the
center of the stage is a circular aperture.

The stage clips hold the slide in place on the stage.

The objective is that part of the optical system of the microscope which produces the specimen’s
initial magnified image (real) within the body tube.

The student microscope has three objectives: a dry low power, a dry high power, and an oil
immersion objective. The objectives are achromatic, that is, they are corrected for the spectral
colors of red and blue.

BIO103| Microbiology Laboratory Manual

Figure 1.1 Bausch and Lomb student microscope and its parts.

BIO103| Microbiology Laboratory Manual

Important features of the objectives:

a. Focal length (mm), an optical constant of the lens system, is the distance from the center
of the lens to the point where parallel rays entering the lens are brought to a focus.

b. Resolving power of an objective is that property to recognize features of a specimen that

are close to each other as separate or distinct. The greater the resolving power, the greater
the definition of an objective. This property is dependent on the wavelength of light used
and an optical property of the objective lens known as a numerical aperture.

c. Numerical aperture (N.A.) is a measure of the resolving power of an objective. An

objective with a 0.25 N.A. allows the viewer to distinguish as separate 25,000 lines per
inch. If the specimen is known to be of the order of 26,000 lines per inch, the observer
can never see the line as separate no matter how much magnification is employed. Lenses
with higher magnification usually have higher N.A. but the medium through which the
light passes also affects N.A. N.A. is indicated in the side of the lens.

d. Parfocal means that the objectives are optically ad mechanically designed so that the
distance between the specimen and the aerial image is always constant. Slight refocusing
with the aide of the fine focus knob is sufficient to restore critical sharpness of the image
after changing from one objective to another, thus, the coarse focus knob need not to be
operated.

The revolving nosepiece to which parfocal objectives are attached, allows convenient
shifting of the objectives.

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

The eyepiece or ocular is that part of the optical system through which the specimen is
viewed. The intermediate image projected by the objective is enlarged by the eyepiece. Hence,
the term compound microscope is derived from the fact that the specimen 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. If the magnifying power of the objective is 100X and
the eyepiece is 10X, the total magnification will be 1000X.

BIO103| Microbiology Laboratory Manual

The coarse focus knob is used to bring the objective into approximate focus. For maximum
definition, the fine focus knob is used.

Figure 1.2. Cross-sectional view of Bausch and Lomb achromatic objectives.

Figure 3 is a longitudinal section of the microscope showing the path of light. The condenser, the
objective and the eyepiece consist of systems of lenses. The mirror, the condenser, the objective
and the eyepiece comprise the optical system.

Figure 1.3. Path of light through the microscope.

BIO103| Microbiology Laboratory Manual

METHODOLOGY

Before performing the exercise, note down the manufacturer’s name, serial number and
department number of the microscope (if these are available) assigned to you and fill in the
characteristics of the objectives in the table provided on the answer sheet. Check and
immediately report to your instructor any defect or damage since you will be held responsible for
any damage after checking.

A. Operation of the Microscope

1. Place the microscope close to the edge of the table. Select a suitable stool so that when
looking into the eyepiece, your back is straight and your neck is bent at the nape.

2. Lower the body tube by turning the coarse focus knob until the 10X or 16mm objective
reaches the downward stop.

3. Look through the eyepiece and adjust the mirror to the position which provides the
brightest and most evenly illuminated field of vision (the circular area seen in tht
eyepiece).

4. Place the slide on the stage and fasten it using the stage clips.

5. Position the specimen area of the slide over the center of the stage aperture.

6. Looking through the eyepiece, raise the coarse focus knob until the image appears. Focus
as sharply as possible. The low power objective has a much greater depth of focus and is
generally used for initial focusing and viewing.

The free space between the specimen surface and the objective is the working distance
(mm) the higher the magnification of the objective, the smaller the working distance.

7. Adjust the fine adjustment knob to sharpen the image in the center of the field of vision.

8. When a feature of the specimen is to be examined at a higher magnification, move the

slide so that the feature is centered in the field of vision. The higher the power of the
objective, the lesser is the area of the specimen surface included in the field of vision.
Shift the high power objective into place and adjust with the fine focus knob.

BIO103| Microbiology Laboratory Manual

9. Keep your eye at a certain distance from the eyepiece. Be relaxed when looking into the
eyepiece and keep both eyes open.

If a sharp image is not obtained despite the application of the above procedure, it is
possible that:

 The fine focus knob has reached a stop. On the right side of the microscope at the
rack and pinion area are three lines. Center the line (located on the fine focus
knob side) between the two lines (located below the coarse focus knob) by turning
the fine focus knob.

 Focusing attempts are too rapid. Use the fine focus knob and adjust slowly.

 The objective is covered with dried oil from previous use. Under the supervision
of your instructor, remove the dried oil with xylene or 95% ethanol and dry the
lens right away.

 The cover slip is too thick. The optimum thickness is 0.17 mm.

 The slide is inverted.

10. Use of the 1.8 mm or Oil Immersion Objective

a. Examine the specimen under the 16 mm objective. Locate first the smear. Focus
and select an area where the yeast or bacteria are clearly seen, and move this to
the center of the field.

b. Shift to the 4 mm objective and focus again using the fine adjustment knob to get
a clearer image of the specimen.

c. Shift into place the 1.8 mm or oil immersion objective. Use the fine focus knob
for final focusing. Write down your observation and draw the stained smear as
seen under the 1.8 mm objective without oil.

d. Slightly rotate the revolving nosepiece to allow application of small drop of oil on
that part of the specimen at the center of the stage aperture.

e. Return the 1.8 mm objective into place. The front lens is now immersed in oil and
almost touches the slide.

BIO103| Microbiology Laboratory Manual

 f. Look into the eyepiece and use the fine focus knob for sensitive focusing. Write
down your observations and draw the stained specimen as seen under the 1.8 mm
objective with oil.

After using the oil immersion objective:

 Blot out the oil with lens paper

 Clean the lens with lens paper wet with xylol (do this under the
instructor’s supervision)
 Blot-dry the lens with fresh lens paper

B. Calibration of the Microscope

Microscopic objects can be measured by means of an ocular or a Filar

micrometer. Both must be calibrated first with the stage micrometer. The unit of linear
measurement in microbiology is the micrometer (µm), which is equivalent to 1/1000 mm
or 1/25,400 inch.

The ocular micrometer (OM, Fig. 1.4) is a glass disc with mounted scale. It may
or may not have numbers on it. It is inserted into the eyepiece and must be calibrated for
the particular objective, eyepiece and tube length before measurements are made. The
student microscope has a fixed tube length.

Figure 1.4. Ocular micrometer (5X)

A stage micrometer (SM, Figure 1.5) is a glass slide with graduations of known
intervals. Some micrometers have numbers to indicate the length of the divisions. One
small division is 0.01 mm or 10 µm, where one big division is 0.1 mm or 100 µm.

Figure 1.5. Stage Micrometer (60X)

BIO103| Microbiology Laboratory Manual

1. Calibration of the Ocular Micrometer

a. Remove the eyepiece from the draw tube.

b. Unscrew the lens cove of the eyepiece.

c. Insert the ocular micrometer.

d. Replace the lens cover and return the eyepiece to the draw tube.

e. Look into the eyepiece to check the disc is not inverted.

f. Place the micrometer in the stage and focus on the scale. Arrange the stage
micrometer so that one line on the far left portion corresponds in the line of the
ocular scale. The lines are properly positioned if the line of the ocular micrometer
coincides with the center of the stage micrometer line.

g. Look across the field (to the right) for another line on the ocular micrometer
properly coinciding with another line on the stage micrometer scale.

h. Within the area of proper line coincidence, count the number of divisions on the
ocular micrometer subtended by the number of divisions in the stage micrometer
and vice-versa.

i. Record the number of divisions (OM and the value of the ocular and the stage
micrometer division used).

j. Determine the value of one division of the ocular micrometer using the formula
below.

BIO103| Microbiology Laboratory Manual