Download use of compound microscope

USE OF COMPOUND MICROSCOPE
© Institute of Lifelong Learning, University of Delhi
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USE OF COMPOUND MICROSCOPE
LIFE SCIENCES (LAB MANUAL)
© Institute of Lifelong Learning, University of Delhi
2
LIFE SCIENCES (LAB MANUAL)
USE OF COMPOUND MICROSCOPE
Aim
To learn the use of a light microscope in the laboratory.
Learning Objectives
Life Sciences laboratories are usually concerned with organisms so small that they cannot be
seen distinctly with the naked eyes. In such laboratories, Microscope is of crucial importance
and is commonly used in the lab practical.
By this exercise we will be able to know various parts of this instrument and also to learn the
working of the compound microscope with best possible results.
Materials / Requirements
Equipment: A Light Microscope
Material: Any simple permanent slide may be of animal/plant tissue.
Theoretical aspects of the experiment
The microscope is employed to see fine details which are not visible to the naked eye. The
primary function of the common lab microscope is not to obtain a very high magnification but
to yield an image in which small details are optically resolved.
The most common
type of microscope found in laboratories is the LIGHT MICROSCOPE also called as
COMPOUND MICROSCOPE which is an optical microscope capable of magnifying the
apparent size of an object.
Visible light is projected through the specimen such as a
squamous epithelium mounted on a slide and is kept on the stage of the microscope.
Glass lenses, the eye piece and the objective then enlarges the image and project it to
a human eye.
The two important factors in microscopy are the magnification and the resolving
power. Magnification means an increase in an apparent size of the object as compared to
its actual size. Resolving power or resolution is the smallest distance between two
point-like objects which can just be recognized as separate. It is achieved if the visual
angle between the object details themselves, or in their magnified image, is large enough so
that light rays from either point will impinge upon different receptor elements in the
retina. Each optical instrument such as microscope, telescope has a limit to its resolving
power. For example, human eye can resolve points that are as close together as 1/10
millimeter (mm).
The resolving power of light microscope is about 0.2 micrometer
(µm).
Thus, the principle of working of a compound microscope is that when a ray of light passes
from one medium to another, REFRACTION occurs. This means that the ray is bent
at the interface. The REFRACTIVE INDEX is a measure of how greatly a substance
slows the velocity of light, and the direction and magnitude of bending is determined by
the refractive index of the two media forming the interface.
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LIFE SCIENCES (LAB MANUAL)
USE OF COMPOUND MICROSCOPE
The most important part of the microscope is the objective, which produces a clear image,
not just magnifies one. The resolution and magnification are extremely important in
viewing an object in the microscope.
Resolution is the ability of a lens to separate or
distinguish between small objects that are close together. The minimum distance (d)
between two objects that reveals them as separate entities is given by the following
equation:
d
 0.5
sin 
Lambda () is wavelength of light used to illuminate the specimens nSin is the numerical
operator
As d becomes smaller the resolution increases. To achieve higher resolution, increase
refractor index using immersion oil which is a colorless liquid with same refractive index
as glass. If air is replaced with immersion oil, many light rays that did not enter the
objective due to reflection and refraction at the surface of the objective lens and slide will
now do so.
Preparation for the experiment
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A compound microscope with its component parts
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A permanent slide/temporary mount of a animal/plant tissue
© Institute of Lifelong Learning, University of Delhi
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LIFE SCIENCES (LAB MANUAL)
USE OF COMPOUND MICROSCOPE
The microscope consists of the following parts as shown in the Figure:
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Study metal body.
Stand composed of BASE and an ARM to which remaining parts are attached.
A light source, either a mirror or an electric illuminator located at the base.
Two focusing knobs the fine and coarse adjustment knobs. These are located on the arm
and can move either stage or the nosepiece to focus the image.
5. The stage is positioned a out halfway up the arm.
6. Microscope slides are held by simple slide clips. These clips are fixed on the stage.
7. A mechanical stage allows the operator to move a slide around smoothly during viewing
by use of stage control knobs.
8. The sub stage condenser is mounted within or beneath the stage and focuses a cone of
light on the slide.
9. The curved upper part of the arm holds the body assembly, to which a nosepiece and
one or more eyepieces or oculars are attached.
10. The nose piece hold three to four objectives with lenses of differing magnifying power
and can be rotated to position any objective beneath the body assembly.
© Institute of Lifelong Learning, University of Delhi
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USE OF COMPOUND MICROSCOPE
LIFE SCIENCES (LAB MANUAL)
Procedure of using a microscope
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Place the microscope on a stable place on a laboratory bench. Place a stool behind the
laboratory table for sitting to look into the microscope. The stool should have a
convenient height to let you see through eye piece without bending.
Place microscope near the window if daylight is used for illumination. Provide
illumination from any of the three sources – build in lamp, external lamp or sunlight.
Use the concern side of the mirror. Use the plane side of the mirror if there is
condenser.
Direct the path of light to pass through the hole of the stage with maximum intensity
while setting the mirror.
Put the slide between the clips provided on the stage.
Revolve the nosepiece and align the low power objective (10X) to examine the object
on the slide.
Adjust illumination to improve the clarity of the object on the slide.
Put one hand on the focusing knob (coarse or fine) and another on the screw to move
the stage. The low power objective is commonly used to screen the field of view. Bring
the objective of interest in the centre.
Switch to high power (40X) and increase illumination as needed. Repeat the process of
focusing.
After screening under the low power and examining under high power, if desired and
required, oil-immersion objective is used for obtaining greater details of the object.
Sources of error and precautions
Care is required at each stage in handling the microscope and also during focusing of slide.
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Always sit straight.
The stool used for sitting to look into the microscope should have a convenient height to
let you see through eye piece without bending.
Always focus the slide first under low power.
Use lens paper/tissue paper to clean the mirror, eyepiece and objectives. Never use
your fingers to clean them.
Carefully use the fine adjustment while focusing the slide under high power.
Clean the stage, lenses etc. after finishing the work with the microscope, never leave it
dirty.
Always hold the microscope with both hands, putting one hand below the base to
prevent falling of mirror while carrying it from one place to another.
Observations
Magnification is the magnifying capability of a compound microscope. It is the product of the
individual magnifying powers of the OCULAR Lens (the lens nearest to eye) and objective
lens (lens nearest to specimen). A typical microscope used in an undergraduate laboratory
has objective lens of 10s, 40s and 100s and an ocular lens of 10x, thus capable of
magnifying the image of specimens 100, 400 and 1,000 times respectively.
One has to observe the specimen present on the slide at various magnifications.
© Institute of Lifelong Learning, University of Delhi
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LIFE SCIENCES (LAB MANUAL)
USE OF COMPOUND MICROSCOPE
Results
The structure of a tissue is seen clearly under low and high powers.
Discussion/Interpretation
The working of the light microscope is to be shown with the help of a ray diagram.
Questions
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What are compound microscopes?
What are the different microscope magnifications and what do they mean?
Is it superior to have one eyepiece or two?
What are the several problems associated with light microscopy?
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