EXERCISE 1

THE MICROSCOPE

Heidi C. Porquis, Marilou C. Marfil and Victoria T. Quimpang

I. INTRODUCTION

The microscope is one of the basic tools in biology for studying structures that could
not be seen by the naked eye. The first microscopes were light microscopes. This used the
principle of the refraction of light rays in producing enlarged images. The earliest ones were
only simple lenses made of glass globes filled with water and were probably used in
examining gemstones. Pliny gave the first report of its use in the first century although this
could have extended 500 years earlier. Leeuwenhoek later fabricated one of these simple
lenses so well that he was able to discover bacteria with it.

Many changes and developments have been done to improve the light microscope.
The first of these was the introduction of the compound microscope which is the type that
you will use and study in this laboratory course. Its design includes two sets of lenses, the
ocular, and the objectives on opposite ends of a closed tube. This was done to overcome
the problem of the early microscope which was not about magnification but about focal
distance which would decrease as magnification increases. This means that if magnification
is increased the viewer suffers the inconvenience of thrusting his eye into the surface of the
lens in order to view the object.

Other designs have been made to overcome other problems later encountered in
light microscopy. The phase contrast, the light interference, the dark field and the confocal
microscopes are a few of these. The electron microscope is also an important breakthrough
which uses electron beams instead of refracted light in producing highly magnified images.
The major problems however of all these new improvements on the microscope are cost
and size. For instance an electron microscope requires one big room which adds to its cost
of acquisition and maintenance. However recent developments in microfluidics and optics
produced a new concept on design of microscopes. This is known as Optofluidic microscopy
(OFM). In this new concept, the lens is replaced with a metal coated sensor called
Complementary Metal Oxide Semiconductor (CMOS) etched with an array of micrometer
sized apertures.

II. OBJECTIVES

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

1. Identify the parts of the microscope and their functions; and
2. Follow correct ways in the proper use and care of the instrument.
III. MATERIALS
Light Microscope Glass slide Cover slip
Tissue paper Piece of newspaper Medicine dropper
Scissors Clear plastic ruler Pencil

IV. PROCEDURE
A. Parts of microscope
(Note to the Student: the light microscope (LM) available to you in this
exercise is a compound microscope intended for student use. )

The parts of the microscope are classified either as optical or mechanical.

Locate these parts which are described as follows:

1. Mechanical Parts- These are the parts which are made of metal and function to
support and adjust the optical parts.
a. Base – the structure that supports the whole weight of the microscope;
may have horse-shoe shape or may just be a rectangular flatbed.
b. Pillar - a supporting piece which arises from the base and forms a short
column to join the arm.
c. Arm - short curved structure used in holding the instrument
d. Body tube- hollow cylinder which bears the two separate lens systems, the
objectives at the lower end and the eyepiece at the upper end
e. Draw tube - upper portion of the body tube which bears the eyepiece
f. Revolving nosepiece - structure at the lower end of the body tube which
can be rotated and bears the objectives
g. Dust shield - part above the revolving nosepiece which protects the
objectives from the dust
h. Stage - a square platform where the slide to be examined is placed (Note
the presence of an aperture at the center)
i. Stage clips - found on the stage to hold the slide
j. Diaphragm – found under the stage to regulate the amount of the light
admitted to the specimen. Your microscope may have either of these
two types:
1. Iris – immediately beneath the stage aperture whose opening is
regulated by a lever.
2. Disk- a plate with circular openings of varied diameters
k. Adjustment screw- maybe attached to the upper part of the arm or pillar.
This will move either the body tube or stage in order to get a clear view of
the specimen.
1. Coarse adjustment – produces greater movement (Be careful when
manipulating this screw.)
2. Fine adjustment – produces fine upward or downward movement.
2. Optical Parts - These consist chiefly of special type of glass and aligned on optical
axis.
 WARNING: DO NOT TOUCH THE LENSES OR GLASS WITH YOUR HANDS
NOR ALLOW THESE TO COME IN CONTACT WITH STAINS OR SHARP OR DIRTY
MATERIALS.

a. Mirror- found below the stage and is usually double-faced, one is flat
and the other is concave. For electric microscopes, a built-in light
system is instead present. Note: Electric microscopes should be
switched off when not in use to avoid overheating.
b. Condenser- if present, it is a secondary lens found immediately beneath
the stage to concentrate light rays and is provided with separate
adjustment screw. This maybe absent in the microscope.
c. Ocular or eyepiece- mounted in the draw tube where one peeps into
when viewing the microscope.
d. Objectives- attached to revolving nosepiece.
1. Very low power objective or scanner (may be absent) if present this
is the shortest tube which enables the viewer to see a larger
area of the object. It bears the number 3.5X or 4X.
2. Low power objective- gives a bigger image than the scanner and
more details of the specimens. Its magnification number is
10X.
3. High power objective- longer than the LPO; shows more details of
the specimen than LPO. Its magnification number is 40X or
45X.
4. Oil-immersion objective (may be present)- if present, this is the
longest objective and gives the highest magnification;
immersion oil is placed in mounting the slide to enable the
viewer to see the object. It magnifies 100X.

Your instructor will let you see few other models of light microscopes available. Identify
differences in these models.

B. Preparing your Microscope :

Follow these preparatory steps every time you use a microscope.

1. Align the scanner with the aperture by turning the nosepiece. You will then hear
a “click” which means the objective is already set.
2. Align the large opening of the diaphragm with the stage aperture.
3. Look through the eyepiece. Do this with both eyes open (but this you have to
practice). You will then see a circular area called the microscopic field. Take note
also of the tiny rod seen through the eyepiece. This is called the pointer.
4. Look again through the eyepiece at the same time adjust the mirror until the
microscopic field is lighted. (Do not let the mirror face direct sunlight).
Note: No need to do the step if your microscope has built-in light source.
 5. Adjust diaphragm (with your eye on eyepiece) until the microscope field is bright
enough but not glaring.

C. Preparing a Wet Mount

Always follow this procedure every time you make a temporary mount of a specimen.

1. With the use of medicine dropper place a drop of water on the slide. The water
here is a mounting medium which helps produce a clear image.
2. Place the newsprint letter “e” in the drop of water.
3. Cover your mount with the cover slip. To do this, hold the cover slip by the
edge. Let one edge touch the glass slide at an angle of about 45°. Then lower the
opposite upper edge carefully so as not to trap bubbles as they will interfere
with your viewing. Air bubbles appear as circular objects with thick dark
outlines. Be sure you do not confuse these with your specimen.
Note: If your mount begins to dry, you can add a drop of water at the edge of
the cover slip.
4. Place the wet mount on the stage aperture and clip the slide to hold it in place.

D. Focusing: Getting a clear view of the specimen.

1. Bring down the scanning objective by turning the coarse adjustment screw while
watching from one side of the stage.
2. Look into the ocular. Slowly raise the objective until the letter comes into focus.
The letter you see is called the image. Take note of its position. Move the slide
until the image comes into the center of the microscopic field while still looking
into the eyepiece and without removing the stage clips.
3. Using the fine adjustment screw bring the image into its sharpest focus. Try to
adjust the opening of the diaphragm to get proper illumination. This increases
clearness of the image.
4. Align the LPO (Follow what you did with scanner in Procedure B-1).
5. Examine under the LPO. Follows steps 1-3 focusing (Procedure D). Note the
appearance of the image and also if the objectives are parfocal.
6. Watch from one side of the stage while you turn the nosepiece to align the HPO.
See to it that the HPO does not touch the cover slip. (at least 1mm apart). This
prevents breakage of the slide and cover slip and damage to the lens. To do this,
turn the coarse adjustment screw while still looking from one side.
7. You are now ready to examine with HPO. Get the sharpest focus using the fine
adjustment screw. NEVER USE THE COARSE ADJUSTMENT SCREW IN FOCUSING
WITH HPO. The reason should be obvious to you. Note the appearance of the
image. DO NOT DISCARD THIS SET UP. YOU WILL USE THIS IN Procedure E. Align
scanner back to the aperture.
E. Magnification

1. Magnification of Microscopic Specimens. Theoretically it is a product of the

magnification power of ocular and magnification power of objective used. This is called
LINEAR OR THEORETICAL MAGNIFICATION.

2. Magnification of Macroscopic Specimens. Make three proportional drawings

of an object (reduced, same size and enlarged) in your illustration sheet. Compute the
magnification index as follows:

Size of drawing
X = --------------------------------
Size of object

Show your computation beside your drawings.

V. RESULTS AND OBSERVATIONS. Answer completely the answer/data sheet.

REFERENCES:

ChanghueiYang. 2008. Optofluidic Microscopes shrinks to fit on a chip

http://images.iop.org/objects/optics/analysis/13/9/4/pdf.pdf
Gray, P. 1958. Handbook of Basic Microtechnique. McGraw-Hill Book Co. USA
Samaniego, L.A., E.P. Leaño, H. C. Porquis and L.D. Obsioma. 2003. Basic Microscopy and
Photomicrography. In: A Manual of Basic Techniques in Teaching Biology.
Unpublished. Department of Biology, Central Mindanao University.
Cameron, A.M., R. G. Birch and J. A. Fuerst.1990 BL 131 Cell Biology Laboratory Manual.
Name: _______________________________________ Rating: ______________
Laboratory Schedule: ____________________________ Date Performed: _______
Instructor: _____________________________________

EXERCISE 1
THE MICROSCOPE

V. RESULTS AND OBSERVATIONS:

A. Parts of the Microscope

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.
 Fig. 1. The Compound Microscope
B. Magnification

1. Microscopic Specimen

X_____ X_____ X_____

Under scanner Under LPO Under HPO

2. Macroscopic Specimen

X_____ X_____ X_____

Smaller than the object Same size as the object Bigger than the object

A. Give the magnification index of the:

a) Scanner______ b) LPO_______ c) HPO_______ d) Ocular______

B. What happens if the mirror reflects direct sunlight to the stage aperture?

C.
a. Describe an air bubble.

b. Give reasons why the cover slip is very important in microscopic examinations.

D.
1. Which adjustment knob produces greater movement?

2. What is the position of the image under the microscope?

3. What is the direction of movement of the image as you move the specimen to the
right?

4. Which objective showed the biggest area of the specimen? The smallest area? The
clearest detail of parts?

E.
1. If the magnification index of the drawing is less than one, what does this tell you of
the object?

2. What characteristic of the specimen does the magnification index indicate?

VI. CONCLUSION:

VII. GUIDE QUESTIONS:

1. Why do you think the microscope is considered an indispensable instrument in the

study of living things?
2. Using short sentences, list down at least 5 habits/guidelines which you should develop/
observe in the care and use of the microscope.
a.

b.

c.

d.

e.

3. What are the distinguishing features of the following:

a. light microscope b. Electron microscope

c. confocal microscope d. optofluidic microscope