EXERCISE 2: THE MICROSCOPE

I. INTRODUCTION

The first microscope designed by Anton van Leeuwenhoek was very simple
but very useful. Today’s microscopes have much improved design, are easier to use,
and offer much greater magnification. The principles of microscopy basically remain
unchanged; that is, light passes through the object being viewed, and the image of
the object is enlarged by a series of lenses (objective lens and ocular lens).

With the help of the microscope, biologists are able to clarify the detailed
concepts of cell structure and function. The dissecting microscope, for example, is
used for the examination of gross specimens and for their dissection under low
power. Other types of microscopes, like the ultraviolet microscope and the electron
microscope, are very complicated and expensive but have much greater magnifying
power.

The type of microscope used in most Biology classes is the compound light
microscope. It contains a combination of lenses and can magnify objects normally
not seen with the naked eye. Under a microscope, the details of objects are very
sharp. The extent to which the microscope can distinguish two objects that are close
together is called resolution, or resolving power. High resolution is needed to see the
details of very small cell parts. Some microscopes have a special type of high-
magnification lens, called an oil immersion lens, which increases resolution.
Although microscopes can have the same magnifying power, they can differ in
resolving power.

In many cases, the details of an object may be difficult to see because the
object is colorless or transparent. The phase-contrast microscope makes it possible
to see different parts in cells without using stains. A phase-contrast microscope
changes the angle at which light waves pass through a specimen; hence, some parts
of the specimen appear lighter or darker than the others.

PARTS OF THE MICROSCOPE

There are three structural parts of the microscope i.e. head, arm, and base.
1. Head – The head is a cylindrical metallic tube that holds the eyepiece lens
at one end and connects to the nose piece at other end. It is also called a
body tube or eyepiece tube. It connects the eyepiece lens to the objective
lens. The light coming from objectives will bend inside this tube. In binocular
microscopes, they are adjustable so that the viewer can adjust the eyepiece
for maximum visualization.
2. Arm – This is the part connecting the base to the head and the eyepiece
tube to the base of the microscope. It supports the head of the microscope
and is also used when carrying the microscope. Some high-quality
microscopes have an articulated arm with more than one joint, allowing
more movement of the microscopic head for better viewing.
3. Base – The base is the lowermost part of the microscope that supports the
entire microscope structure. It provides stability for the microscope.

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 Illuminators, light switches, and electrical wiring systems are fitted in the
base.
Optical parts of a microscope and their functions

The optical parts of the microscope are used to view, magnify, and produce an
image from a specimen placed on a slide. These parts include:

1. Eyepiece – The eyepiece (ocular Lens) is closest to the viewer’s eye. They
are located at the top of the microscope. This part is used to look at the
specimen. These lenses come in different magnification powers from 5X to
30X, but the most common ocular lenses are of 10X or 15X magnification.
They magnify the image for the second time.
2. Eyepiece tube – It’s the eyepiece holder. It carries the eyepiece just above
the objective lens. In some microscopes, such as the binoculars, the
eyepiece tube is flexible and can be rotated for maximum visualization for
variance in distance. For monocular microscopes, they are none flexible.
3. Diopter Adjustment – Diopter Adjustment is a control knob present only in
the binocular microscope that is used to change focus on one eyepiece. It is
used to correct any difference in vision and compensate for the differences
in vision between the viewer’s two eyes.
4. Nose piece – A nose piece is a movable circular structure that houses all
the objective lenses. It is also called the revolving turret. It is connected to
the body tube and lies just above the stage. It can be rotated clockwise or
counterclockwise to increase or decrease the magnification. The change in
magnification results due to a change in the objective lens.
5. Objective lenses – The objective lens is the lens that is closest to the
specimen. They are fitted on the nosepiece. A standard microscope has 3 to
4 objective lenses of different magnifying powers: 4X, 10X, 40X, and 100X.
The objective lenses first receive the light transmitted from the specimen
and magnify the image for the first time. Objective lenses are color-coded
and are of different sizes. Size and color depend on the power of the lens.
The smallest lens is of the lowest power, and gradually, the longest will be
of the highest power. The high-power lenses i.e. 40X and 100X, are
retractable, i.e., their end can be pushed inward. In most optical
microscopes, objective lenses with 100X or more magnification are of oil
immersion type.
6. The Adjustment knobs – Adjustment Knobs are the control knobs used to
focus the microscope on the specimen. These knobs are of two types;
a. Fine Adjustment Knob: Fine Adjustment Knob is used for fine
adjustment. It is a smaller knob and is used to move the stage up or down
very slowly. The stage covers a very small distance on each rotation of the
fine adjustment knob. It is used to sharpen the image. It is mostly used while
viewing under high power.
b. Coarse Adjustment Knob: Coarse Adjustment Knob is used for focusing
the image under low power magnification. It is a larger knob and is used to
move the stage up or down very rapidly. The stage is raised or lowered
rapidly with the help of a coarse adjustment knob.
7. Stage – This is the section in which the specimen is placed for viewing.
They have stage clips that hold the specimen slides in place. The most
common stage is the mechanical stage, which allows the control of the

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 slides by moving the slides using the mechanical knobs on the stage
instead of moving them manually.
8. Stage Control Knobs – Stage Control Knobs are the control knobs used to
move the stage mechanically. There are two knobs; one for moving left and
right and the other for moving forward and backward. This will move the
slide in the field of vision.
9. Aperture – This is a hole in the microscope stage through which the
transmitted light from the source reaches the stage.
10. Microscopic illuminator – A microscopic illuminator is a light source. In
some compound microscopes, a mirror, which reflects the light from an
external source to the sample, is used. In other optical microscopes,
different electric bulbs of low voltages are used as a constant light source.
Commonly used illuminators are tungsten-halogen lamps, 75-150W Xenon
lamps, tin-halide lamps, mercury vapor lamps, etc. The selection of types of
bulbs is based on the requirement of intensity and wavelength for
illumination.
11. Condenser – These are lenses that are used to collect and focus light from
the illuminator into the specimen. They are found under the stage next to
the diaphragm of the microscope. They play a major role in ensuring
clear, sharp images are produced with a high magnification of 400X and
above. The higher the magnification of the condenser, the clearer the
image. More sophisticated microscopes come with an Abbe condenser that
has a high magnification of about 1000X.
12. Diaphragm – It’s also known as the iris. It is found under the stage of the
microscope, and its primary role is to control the amount of light that
reaches the specimen. It’s an adjustable apparatus, hence controlling the
light intensity and the size of the beam of light that gets to the specimen. For
high-quality microscopes, the diaphragm comes attached with an Abbe
condenser, and combined, they are able to control the light focus and light
intensity that reaches the specimen.
13. Condenser focus knob – This is a knob that moves the condenser up or
down, thus controlling the focus of light on the specimen.
14. Abbe Condenser – This condenser specially designed for high-quality
microscopes makes the condenser movable and allows very high
magnification above 400X. High-quality microscopes normally have a higher
numerical aperture than objective lenses.
15. The rack stop – It controls how far the stages should go, preventing the
objective lens from getting too close to the specimen slide, which may
damage the specimen. It is responsible for preventing the specimen slide
from coming too far up and hitting the objective lens.
16. Light Switch – Light Switch is an electrical control device. Light switches
are used to on and off the illuminator.
17. Brightness Adjustment – The brightness adjustment system controls the
voltage supplied to the light bulb, controlling the intensity (brightness) of the
light bulb.

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Figure 1. shows the compound light microscope and its parts.

PROPER USE AND CARE OF THE MICROSCOPE

1. Always carry the microscope with both hands. Hold the arm with one hand while
the other hand supports the base.

2. Always use the microscope with the tube perpendicular to your work surface,
especially when working with fresh mounts or with any slide being examined under
the oil-immersion objective. The oil and fluid mounting of the specimen tend to flow
onto the stage if the microscope is inclined.
3. Keep the microscope dust-free. Develop the habit of cleaning the microscope
before and after using it. Use clean soft cloth for cleaning the body, and lens paper
for cleaning the lenses and the mirror. Lens paper moistened with xylol is used to
remove the immersion oil.

4. The coarse adjustment screw is used to bring the object into focus, while the fine
adjustment screw is used to obtain a more sensitive and gradual adjustment for
focusing through the different planes and depths of the object.

5. A great deal of success in microscopic examination is dependent upon the

adjustment and control of illumination. This is done by reflecting light from the best
source, raising or lowering the condenser, and opening or closing the diaphragm.

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D. Focusing Under the Different Objectives

a. Low-Power Objective
Place the specimen at the middle of the stage above the aperture and secure
it with clips. Position the low-power objective until it is more or less 4-5 mm
from the specimen. Looking through the eyepiece, slowly move the LPO with
the coarse adjustment screw until the specimen appears more or less distinct.
To sharpen the image, use the fine adjustment screw. The LPO, particularly
the scanner, is usually the first to be used before any other objective. It shows
the larger part of the specimen and makes it easier to choose which parts of
the specimen to view under the high-power objective.
b. High-Power Objective After focusing under the LPO, click the high-power
objective into place. The objectives of the microscope are made to be parfocal
using one objective; that is, after focusing the image using one objective, the
image remains more or less in focus (requiring a bit of fine focusing) when
you switch to another objective. NOTE: If the objective is not parfocal, raise
the LPO slightly before placing the HPO into position. Then, lower the HPO
until it almost touches the specimen.

NOTE: if the objective is not parfocal, raise the LPO slightly before
placing the HPO into position. Then, lower the HPO until it almost
touches the specimen.

Manipulate the fine adjustment screw until you obtain a clear image of the
specimen. You may need to move the slide around to bring the desired parts
of the specimen into your field of vision.

The HPO is usually used before the oil-immersion objective.

c. Oil-Immersion Objective
This objective gives the highest magnification of all the objectives. It
has a very short working distance and, therefore, it must be used with
greatest care.

The specimen should first be focused under the high-power objective.

Move away the HPO from its position and put a drop of oil of cedar
(immersion oil) on the specimen. Any special oil having the same refractive
index as that of the glass may be used. Place the 010 into position and
manipulate the fine adjustment screw until the image is finely focused.

NOTE: If the objective is not parfocal, with the eyes at the level of the
stage, lower the OIO until it touches the oil and almost touches the
specimen. Looking through the eyepiece, manipulate the fine
adjustment screw until you obtain a clear image of the specimen.

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6. Develop the habit of having both eyes open when looking through the Wit
microscope. The left eye is used by the right-handed and the right eye is used by the
left-handed.

NOTE: If the objective is not parfocal, raise the LPO slightly before
placing the HPO into position. Then, lower the high-power objective
until it almost touches the specimen.

7. Report the loss of your microscope or any of its parts to your laboratory instructor.
If the loss occurs during use, report it immediately.

8. Any microscope which is not in good working condition should be reported to the
instructor. Do not attempt to fix the microscope yourself; a qualified technician will fix
it.

9. Use only the microscope assigned to your group.

10. Look after the microscope assigned to your group as if it were your own. Other
groups will use it and benefit from it as much as you did.

11. After every use, remove the slide. Clean the stage of the microscope with a clean
cloth, and the lenses with lens paper. Do not use other types of paper as these may
scratch the lenses.

12. Return the microscope with the parts in proper position: stage and revolving
nosepiece at the lowest position, objective with lowest magnification clicked in place,
mirror parallel to the stage, and iris diaphragm closed.

MAGNIFICATION

Magnification refers to the number of times an object is enlarged, or reduced, by lens

systems or in drawings.

With the use of a microscope, magnification is achieved by the system of curved

lenses of the eyepiece and the objectives. The objective magnifies the specimen to a
definite size. The eyepiece further magnifies the image formed by the objective so
that the image seen by the eye has a magnification equal to the product of the
magnification of the two lens systems. This is known as linear magnification.

The magnification of the eyepiece may be 6X, 10X, or 15X, while the objectives may
be 4X or 10X (low power), 40X or 45X (high power), and 100X (oil immersion). A
10X LPO means that it has a magnification power of 10 times. If the magnification of
the eyepiece is 10X and the objective used is 10X, then the linear magnification is
100X.

When illustrating a specimen viewed under the microscope, the magnification of the
drawing in relation to the actual specimen must always be indicated under the
drawing. This is done by writing a multiplication sign followed by a figure indicating
the number of times the drawing is bigger than the actual specimen (e.g., X2, X3,

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X50). If the drawing is a reduced image of the actual specimen, this is indicated by a
fraction (e.g., X½, X¼, X%).

To determine the magnification of microscopic objects, the formula is as follows:

Magnification (M) = Size of drawing (in mm)

Size of specimen (in mm)

II. OBJECTIVES

After the exercise, the students should be able to:

1. Identify the parts of the microscope.
2. Demonstrate proper use and care of the microscope.
3. Calculate the magnification of a specimen viewed under the microscope.

III. MATERIALS
Microscope seed (monggo, bell pepper, kalamansi)
pepper, squash)
Blue thread medium to large leaf
Red thread ruler
Hydrilla prepared slides of euglena, paramecium
Cover slip root & stem of corn, any other microscopic
Glass slide organisms
Scissors
Medicine dropper
Water

IV. PROCEDURES

A. Identifying the Parts of the Microscope

1. Identify the parts of the microscope
2. Observe the specimens in the microscope

B. Manipulating the Microscope

1. Cut a piece of blue thread about 1 cm long. Do the same with the red thread.
Place the threads on a clean glass slide, making sure that they cross each
other at right angles.
2. Place a drop of water on the threads using a medicine dropper. Then put a
cover slip over the threads.

Glass slide
Cover slip

3. Place the slide on the stage. Fasten with stage clips.

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 4. Position the low-power objective over the slide so that the objective is just
above the top of the slide. A click will indicate that the objective is positioned
properly.

CAUTION: Never allow the objective to touch the slide. It may scratch
the lens or break the glass slide.
5. Look through the eyepiece. Adjust the mirror and the diaphragm so that the
field is bright.
6. Slowly turn the coarse adjustment knob to raise the objective lens little by little
until the threads are clearly seen. Then use the fine adjustment knob to
sharpen the focus.
7. Rotate the high-power objective into place, making sure that the objective
does not touch the slide. Use the fine adjustment knob again to sharpen the
focus.
8. Move the slide to the left, to the right, up and down. Note the direction towards
where the threads appear to move each time you move the slide.

C. Measuring A specimen Under the Microscope (Microscopic Object)

1. Look through the eyepiece of your microscope using low power magnification.
The circle of light you see is called the field of vision.
2. Place a ruler (mm) directly on the stage. See to it that it covers half of the
stage opening.
3. Focus the ruler. The edge of the ruler should be exactly across the center of
the field of vision. Place the mm mark (vertical line) at the left of the field of
vision.

mm mark

NOTE: One milimeter is the distance from the middle of one mark to the middle of
the next mark.

4. Calculate the length of the remaining segment of the milimeter.

5. Record the measurement of the Lower Power Field (LPF) diameter in mm.
Calculate the the LPF now in micrometers (m).
1 mm = 1000 m

6. Determine the High Power Field (HPF) diameter using formula:

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 H= Lower−Power Objective ( LPO ) Magnification x LPF
High-Power Objective (HPO) Magnification

7. Record your data in the data sheet.

8. Obtain prepared slides of three different organisms. Label them as specimen
1, specimen 2, specimen 3. Focus under LPO.
9. Position the slide such that same-sized cells span across the diameter of the
field of vision, and estimate the number of cells that fit the diameter.
10. Determine the size of a cell by dividing the LPF diameter by the number you
obtained.
11. Repeat steps 8-10 using HPO, this time using the HPF diameter in step 10.
12. Draw your observations and record all your calculations in the Report Sheet.

D. Magnifying Macroscopic Objects

1. Get seed (monggo, bell pepper, kalamansi, pepper, squash)
2. Draw your observation in the Report sheet.
3. Get a medium-to-large leaf. Draw it smaller than the actual size. Compute its
magnification using the formula.

References:

V. RESULTS AND OBSERVATIONS: (Accomplish the data sheet)

VI. CONCLUSIONS

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Name: ___________________________ Rating: ____________________
Laboratory Schedule: __________________ Date Performed: ______________

EXERCISE 1
THE MICROSCOPE
A. Parts of the Microscope
1. __________________ 11. __________________
2. __________________ 12. __________________
3. __________________ 13. __________________
4. __________________ 14. __________________
5. __________________ 15. __________________
6. __________________ 16. __________________
7. __________________ 17. __________________
8. __________________ 18. __________________
9. __________________ 19. __________________
10. __________________ 20. __________________

B. Manipulating/Adjusting the Microscope

Observations
1. What happened to the brightness of the field of vision when the magnification
was changed from low power to high power?

2. When you used the fine adjustment knob, how many threads were in focus at
a time? Would you able to see the threads better if they were larger or
thicker? Explain.

3. When you moved the slide in four different directions using adjustment knob,
how would you describe the threads?

a. up or away from you - ___________________________________________

b. down or towards you - ___________________________________________
c. to the right - ___________________________________________________
d. to the left - ____________________________________________________

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 C. Measuring Specimens under the microscope. (It should be drawn)

Diagram of specimen 1 Diagram of specimen 2

Organism: ____________________ Organism: ____________________
Magnification: _________________ Magnification: _________________

Diagram of specimen 2
Organism: ____________________
Magnification: _________________

Data Specimen 1 Specimen 2 Specimen 3

LPO HPO LPO HPO LPO HPO
# of cells that fit
side by side
Diameter of field of
vision
Estimated cell size

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in micrometers
(m)

D. Magnification of Macroscopic Objects

1. Seed:
Actual size: ________________ mm long
Size of drawing: ____________ mm long
Computation:

2. Leaf:
Actual size: ________________ mm long
Size of drawing: ____________ mm long
Computation:

GUIDE QUESTIONS

1. Why are you advised never to lower the HPO with the coarse adjustment
knob?

2. Why is it always necessary to put a cover slip over the specimen?

3. Why is it necessary to add a drop of water between the slide and the cover
slip? Explain your answer in terms of the difference in refractive index of water
compared to that of glass.

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 4. Suppose you are to observe a specimen under HPO, what are the steps that
you should follow?

5. Why is it necessary to wipe off the immersion oil after using the oil immersion
objective?

6. How would you define the following?

a. Parfocal-

b. total magnification

c. resolving power

d. field of vision

7. how would you convert the following measurements?

a. 8.5 mm = __________m
b. 10.5 mm = _________m
c. 700 m = __________mm
d. 61 m = ___________mm

8. Does the field of vision increase or decrease as you move from low-power
magnification to high power magnification?

9. If approximately 400 cells of a certain type of bacteria can fit across the LPF,
what is the approximate size of a single bacterium in micrometers? Show your
computation.

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 10. If a microscope has a low-power magnification of 60X, a high-power
magnification of 600X, and LPF of 1200 m, what is the HPF in m? Show
your computation.

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