Republic of the Philippines

Laguna State Polytechnic University

Province of Laguna

Exercise 1
THE COMPOUND MICROSCOPE

Most microorganisms like bacteria, fungi and parasites 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 a 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:
1. Bright-field, where the microscopic field is brightly lighted and the object to be observed
appears dark due to its ability to absorb or refract some of the incident light.
2. Dark-field, where the object appears luminous against a dark background since it reflects
some of the incident light in all directions.
3. 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 television screen.
4. 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.
5. 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.
 In this subject, the compound microscope will be used in many of the exercises so the students
should be able to use it proficiently and care for it properly.

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 iris diaphragm regulates the amount of light entering the condenser. Just below the iris
diaphragm is a slot to accommodate different types of light filters.

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

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 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.

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 and distinct. The greater the resolving power, the greater the
 definition of an object. This property is dependent on the wavelength of light used and an
optical property of the objective lens known as numerical aperture.
c. Numerical aperture is a measure of the resolving power of an objective. An objective with
0.25 NA allows the viewer to distinguish as separate 25 000 lines per inch. If a specimen is
known to be of order of 26 000 lines per inch, the observer can never see the lines as separate
no matter how much magnification is employed. Lenses with higher magnification usually
have higher NA but the medium through which the light passes also affects NA. NA is indicated
on the side of the lens.
d. Parfocal means that the objectives are optically and mechanically designed so that the
distance between the specimen and the aerial image is always constant. Slight refocusing with
the aid of the fine focus knob is sufficient to restore critical sharpness of the image after
changing from one object to another, thus, the coarse knob need not 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 ocular system through which the specimen is viewed.
The intermediate image projected by the objective is enlarged by the eyepiece. Hence, the term
compound microscope id 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.

The course adjustment knob is used to bring the object into approximate focus. For maximum
definition, the fine adjustment knob is used.

MATERIALS AND METHODS

 Microscope
 Slide
 Cover Slip
 Forceps
 Onion
 Lugol’s Iodine
 Toothpick
 Methylene Blue
 Dropper
For Plant Cells (Onion Skin Wet Mount)
1. Peel the delicate transparent tissue from the inner surface of a piece of onion using forceps (tweezers).
2. Make a wet mount by placing the tissue, unwrinkled, in a small drop of water on a glass slide.
3. Add one small drop of Lugol's iodine stain to the tissue and cover with a cover slip as directed. (*be
careful - the Lugol's can stain and burn the skin!)
4. Examine the onion cells at low power objectives, focus as necessary.
5. Next examine the cells at medium and high power objectives.
6. Prepare a diagram of onion skin tissue showing two to four cells. Label the structures you can identify
from the microscope. (e.g. - cell membrane, nucleus, etc.)

For Animal Cells ( Human Cheek Cell Wet Mount)

**Care must be taken when doing this part of the lab to handle and dispose of the cells with appropriate
concern. Using a prepared epithelial cell slide would also work.
1. Place a drop of water on a clean slide. Gently scrape the inside of your cheek with the blunt end of a
clean toothpick and stir the material on the toothpick in the drop of water on the slide. (properly dispose
the toothpick)
2. Add one small drop of methylene blue stain to the slide and then add a coverslip as directed.
3. Focus and examine the slide under low power objectives before moving to the higher magnifications.
4. Prepare a diagram showing two to four cells of the cheek and label structures you can identify.

For Bacterial Cells

**Prepared slide will be given to you**