MIC 461

GENERAL MICROBIOLOGY

PRACTICAL 1:
OPERATION OF MICROSCOPE
PRACTICAL 1: OPERATION OF THE MICROSCOPE

The Use and Care of the Microscope

The compound microscope is a precision instrument composed of a series of lenses designed and
arranged to provide, under optimal conditions, maximum magnifications ranging from
approximately 1 – 2000 times (x) the diameter of the specimen being observed.

The ordinary compound microscopic consists of a series of optical lenses, mechanical adjustment
parts and supportive structures from various components. The optical lenses include the ocular or
eyepiece, usually three objectives with different magnifying powers, and the sub stage
condenser. The coarse, fine and condenser adjustments knob together with the iris diaphragm
level comprise the major mechanical parts concerned with the operation of the instrument. The
various components of the scope are held in position by or contain within, supportive structures
such as the base, arm, pillar, body tube (barrel), and revolving nosepiece.

Successful operation of the microscope depends upon proper maintenance and correct use. Here
are some suggestions:

1. When carrying the instrument, grasp the arm with one hand, and place the other hand
under the base as a support. Keep the instrument in an upright position. In the event the
light source is not attached, take the scope to your laboratory table first, and return for the
light source. Do not carry both items at the same time.

2. Keep the microscope at least six inches from the edge of your laboratory table.

3. Do not handle the lenses with your fingers. Perspiration contains fatty acids and other
substances which can mar the lens glass. Always use the lens paper for the cleaning of
the optical system.

4. Wipe the lenses of the microscope before and after class use.

5. Do not temper with any components of the instrument. If the microscope does not seem
to be functioning, notify the instructor immediately. As other laboratory sections may use
the same instruments, any part previously damaged and subsequently reported may be
charged to you.

6. Do not allow chemicals to come in contact with any part of the instrument.

7. Remove immersion oil from the microscope with lens paper.

8. Always be certain that the low-power objective is in the working position, i.e. in line with
the body tube, before putting the microscope away.
Operation of the microscope.

A. Components of Microscope

Materials:

Compound microscope and appropriate light source

Procedure:
.

1. Remove the microscope assigned to you from the cabinet by taking the arm with one
hand and supporting the instrument at the base with the other.

2. Place the microscope approximately six inches from the edge of the laboratory table
with the microscope arm facing you.

3. Examine the microscope and locate the components listed below:

(a) Ocular (h) Condenser

(b) Body tube (barrel) (i) Iris diaphragm knob
(c) Revolving nosepiece (j) Course adjustment knob
(d) Low power objective lens (k) Fine adjustment knob
(e) High-dry objective lens (l) Mechanical stage
(f) Oil immersion (m) Pillar
(g) Stage

B. Use of the Microscope

Materials:

1. Glass slides and cover slips

2. Lens paper and xylene
3. Eye dropper
4. Newspaper
5. Scissors and inoculating needle

Procedure:

1. Clean a glass slide and cover slip as indicated by the instructor and place a drop of water
on the slide.

2. Next, cut a series of letters from the newspaper provided. Be certain that at least one of
the letters is a small “e”.
3. Place the string of letters into the drop of water. Cover the preparation with a cover slip.
In order to limit the number of air bubbles in this preparation observe the following:
(a) Place the edge of the cover slip on the drop of water on the slide so that the
fluid completely wets the edge.
(b) Then place the inoculating needle under the cover slip and lower the cover
slip onto the slide.

4. Examine the preparation under the low-power and high-power objectives.

Procedure for microscope operation:

1. Wipe all lenses with lens paper.

2. Move the low-power objective into position under the body tube. You will feel a click
when it is correctly in place.
3. Connect your light source and turn it on.
4. Place the slide preparation to be examined over the central opening in the stage.
5. Arrange the portion of the preparation to be examined over the central opening in the
stage.
6. Lower the low-power objective with the aid of the coarse adjustment knob to
approximately one quarter of an inch above the cover slip of your preparation.
7. Raise the condenser as far as it will go with the aid of the condenser adjustment knob.
8. Look through the ocular. Next, move the iris diaphragm lever so that the diaphragm is
open to its maximum limit. This is determined by looking through the eyepiece and
noting changes in the intensity of the light while the iris lever is being manipulated. If the
light is too bright, make the necessary adjustment.
9. Next, focus upward with the fine adjustment knob, until the specimen comes clearly into
view. With a microscope having the body tube in a fixed position, the respective
adjustment knob will control the stage. Focusing in this case will be downward until the
specimen comes into view. In other words, this is opposite to the procedure described in
6 and 9 because the objectives will remain fixed while the stage lowers the specimen.
10. In order to change objectives (switch from low-power to high-power) do not raise the
body tube, just simply swing the objective desired into place. The microscope should stay
in focus with any objective. If it does, it is said to posses the property of being par focal.
Questions
 1. (a) What is the total magnification obtainable with the low-power objective?
(b) What is the total magnification obtainable with the oil immersion lens?

2. What happened to the original orientation of the letter “e” when the slide was moved:
(a) side-to-side
(b) up and down

3. Complete the following table:

No. Microscope Parts Function (s)

1. Ocular Lens
2. Objective Lens
3. Condenser
4. Iris Diaphragm Lever
5. Coarse Adjustment Knob
6. Condenser Adjustment knob
7. Fine Adjustment Knob

C. Examination of Stained Cells

The numbers and types of organisms seen under the microscope are limited by the
magnification of the objectives used. Because of their extreme small size bacteria are not
generally studied with the low or high dry objectives but instead they are stained with the
oil immersion objective. This practical will give you the experience in the use of oil
immersion and also serves as a comparative study of the stained cells between bacterial
cells and typical plant and animal cells.

Materials:

1. Prepared slide of plant cell (dicot)

2. Prepared slide of animal cell (alveoli)

Procedure:

1. Examine all the slides under the oil immersion objectives.

2. Observe the size and shape and any visible structures of these organisms.

3. Make illustrations/drawings of all observations.

Questions

1. How would a stained preparation appear under the oil immersion objective without the
oil between the slide and the objective lens?

2. What are the structural differences between the animal and plant cell?
Could you observe them in this exercise?

3. Could you detect the presence or absence of the structures in Question 2 in the bacterial
cell?