MICROSCOPIC EXAMINATION
Objectives
The objectives of this exercise are to learn about the principles of microscopy and the
correct operation and maintenance of light microscopes. Practice using a microscope
by finding bacteria or protists and familiarizing yourselves with bacteria morphology.
Light Microscopes
The size of bacteria range typically from 1-10 microns (micrometers) and that of a
single celled eukaryote (like a protist) is typically 10-500 microns. Both are not visible
to the naked eye. However, they are just large enough to be seen with light waves
but require magnification using a light microscope.
Typically, a light microscope will have 4 objectives (lenses) with magnification as low
as 4X and as high as 100X. These lenses are called: scanning (4x), low (10x), high (40x)
and oil immersion (100x). The microscope also has an ocular (eyepiece) that you look
through when studying samples. The ocular also magnifies the image to 10X.
Total magnification of a sample being examined is equal to the ocular magnification
times the objective magnification.
Example: ocular (10X) × low objective (10X) = 100X total magnification.
Use of the Light Micsroscope
To use the light microscope, you will need to place a slide under the objectives and
rotate the objective lens to the scanning (4x) magnification. You will need to start
with the platform all of the way up and then slowly move it down using focusing knobs
, until the sample comes into focus. Please note, this is the only lens on which you
should use the coarse focus (the larger focus dial) to focus your specimen.
A condenser under the platform collects light from the lamp and directs it to the
slide being studied. There are two ways to control the light intensity directed toward
the sample 1) turn up or down the voltage to the lamp or 2) adjust the iris/diaphragm
found in the condenser. Too much light directed at the sample will wash out the
image. Too little light directed at the sample and the image will be too dark to see.
Once you have located your object using your scanning lens be sure it is centered in
your field of view (using the mechanical stage) and then increase your magnification
to 10X objective. You will likely need to focus the image slightly using the fine focus
(the small dial) only. If you are viewing a protist you might not need to increase your
magnification beyond the 10x lens, however if you are looking at bacteria due to their
exceedingly small size you will likely need to increase the magnification all the way to
the oil immersion lens to see each cell’s individual shape!
Using oil on the 100x lens
Looking at bacteria using the oil immersion lens (100X) is best when using immersion
oil. Immersion oil prevents the loss of light that would normally occur as light refracts
when passing from the specimen slide. The oil prevents refraction and more light
enters the objective lens, in other words the resolution of the lens is increased. This
increases the sharpness of the image by decreasing the dispersion of light away from
the lens. Figure 1 demonstrates the effect that immersion oil has.
Figure 1
Objectives
The objectives of this exercise are to learn about the principles of microscopy and the
correct operation and maintenance of light microscopes. Practice using a microscope
by finding bacteria or protists and familiarizing yourselves with bacteria morphology.
Light Microscopes
The size of bacteria range typically from 1-10 microns (micrometers) and that of a
single celled eukaryote (like a protist) is typically 10-500 microns. Both are not visible
to the naked eye. However, they are just large enough to be seen with light waves
but require magnification using a light microscope.
Typically, a light microscope will have 4 objectives (lenses) with magnification as low
as 4X and as high as 100X. These lenses are called: scanning (4x), low (10x), high (40x)
and oil immersion (100x). The microscope also has an ocular (eyepiece) that you look
through when studying samples. The ocular also magnifies the image to 10X.
Total magnification of a sample being examined is equal to the ocular magnification
times the objective magnification.
Example: ocular (10X) × low objective (10X) = 100X total magnification.
Use of the Light Micsroscope
To use the light microscope, you will need to place a slide under the objectives and
rotate the objective lens to the scanning (4x) magnification. You will need to start
with the platform all of the way up and then slowly move it down using focusing knobs
, until the sample comes into focus. Please note, this is the only lens on which you
should use the coarse focus (the larger focus dial) to focus your specimen.
A condenser under the platform collects light from the lamp and directs it to the
slide being studied. There are two ways to control the light intensity directed toward
the sample 1) turn up or down the voltage to the lamp or 2) adjust the iris/diaphragm
found in the condenser. Too much light directed at the sample will wash out the
image. Too little light directed at the sample and the image will be too dark to see.
Once you have located your object using your scanning lens be sure it is centered in
your field of view (using the mechanical stage) and then increase your magnification
to 10X objective. You will likely need to focus the image slightly using the fine focus
(the small dial) only. If you are viewing a protist you might not need to increase your
magnification beyond the 10x lens, however if you are looking at bacteria due to their
exceedingly small size you will likely need to increase the magnification all the way to
the oil immersion lens to see each cell’s individual shape!
Using oil on the 100x lens
Looking at bacteria using the oil immersion lens (100X) is best when using immersion
oil. Immersion oil prevents the loss of light that would normally occur as light refracts
when passing from the specimen slide. The oil prevents refraction and more light
enters the objective lens, in other words the resolution of the lens is increased. This
increases the sharpness of the image by decreasing the dispersion of light away from
the lens. Figure 1 demonstrates the effect that immersion oil has.
Figure 1
