INTRODUCTION

A light microscope is a laboratory instrument that is used to detect and magnify

small objects or specimens. This microscope is used based on the system of lenses
and light to magnify the specimens. There are 4 major types of microscopes which are
dark field microscope, phase contrast microscope fluorescence microscope and bright
field microscope. Dark field microscope is simply designed for better contrast image of
the specimen. The light source for this microscope is indirect and it is from the bottom.
As for phase contrast microscope, it has intermediate between bright and dark field that
produces image that has darkness and brightness with differing degrees. Meanwhile,
the fluorescent microscope shows fluorescent-coloured images and it can either be
fluorescent in natural form or with staining caused by chemicals capable of fluorescing.
The bright field microscope is a most common type of light microscope and it has the
light source at the bottom which will directly focused to the microscope. This typical
bright field light microscope has major parts including ocular or eyepiece lens body
tube, arm, objective lenses that consists of 10X, 50X, 40X and 100X magnification
powers, rotating nosepiece, stage, slide holder, diaphragm, focusing knob, condenser,
light source and a base. In order to view an image of specimen, the microscope must
provide magnification. Magnification is the enlargement of the specimen to view or see
the organelles contain in the specimen. The total magnification of the specimen is the
product of magnification of objective lens and ocular lens. This can be increased by the
adding or increasing the magnification of lenses. The size of the magnified image of the
specimen can be measured using ocular and stage micrometers. Ocular micrometer is
the measuring instrument placed under the ocular lens to help to measure the images
under microscope while stage micrometer is a type of ruler which is used to measure
the size of specimen as well but it is mounted on the specimen slide. The stage
micrometer is can be 0.01mm or 0.1mm per space according to the manufacturer. This
is because the size of the ocular micrometer is remained unchanged even after different
types of objective lenses that have different magnifications were used.

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 Experimental Procedures

PART 2

Before viewing specimen:

1. Both hands were used to carry the microscope to move it. The arm of
microscope was held with one hand and other hand was placed under the base for a
support.
2. Equipment and materials were assembled. Microscope was set on a flat, sturdy
surface to have plenty of room to work.
3. Ocular and objective lenses was cleaned using lens paper.
4. Power supply and the plug connection was checked.
5. The microscope’s light source was switched on.
6. Light source was opened to maximum, allowing the greatest amount of light through.
7. The focusing knob was turned until the distance between stage and the nosepiece
unit is greatest. (This was to avoid knocking the objective lens to the stage).

The specimen was viewed at lowest-power objective lens (5X).

8. The objective lens was rotated to the lowest-power objective.
9. A microscope slide was placed on the stage and was secured with clips.
10. The condenser was positioned to almost touch the bottom of the slide (this was for
full focus of light intensity to the specimen).
11. The ocular lenses was looked through by the user to view the specimen on the
slide.
12. The large coarse focus knob was adjusted slowly until the specimen was in focus.
The small fine focus knob was then slowly adjusted to sharpen the focus of the
image. (IMPORTANT: When the objective no longer moves the turnings must be
stopped).
13. The ocular lenses position was adjusted to suit the user’s eyes (steps 13a–b).
a. Interpupillary distance was adjusted by adjusting distance between oculars so one
image is seen.
b. Dioptic adjustment was made by following 3 steps below:

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 • Coarse and fine adjustments was used to bring object into focus while
looking through the right ocular with right eye (and with left eye closed).
• The right eye was closed, the left eye was used to look into the left ocular,
and the knurled collar on the left ocular was used to bring the object into
sharp focus. (Coarse or fine adjustment must not be turned at this time.)
• Both eyes were used to look into the oculars to observe that object is in
clear focus. The procedure was repeated if it is not focused.

14. The specimen was examined using the stage controls to move the slide in a left and
right or backward and forward pattern while looking through the oculars. (The
specimen structure was recorded) The view at high-power objective lens
was been proceeded after viewing at low-power objective lens.
The specimen then proceeded to view at high-power objective lens (40X)

15. High-power objective was changed. (IMPORTANT: The objective lens must not be
moved when looking through the ocular lenses to avoid breaking up the slide with
objective lens. The stage must be lowered down a little bit to give space to objective
lens.)
16. The view the object was looked through oculars on the slide.
17. The small fine focus knob was slowly adjusted to sharpen the focus of the image.
(IMPORTANT: When the objective no longer moves, it should be stopped turning).
The specimen was examined using the stage controls to move the slide in a left and
right or backward and forward pattern while looking through the oculars. (The
specimen morphology/structure was recorded) The highest-power objective lens
was proceeded to view after viewing at high-power objective lens.

When the specimen viewed at the highest-power objective lens (100X):

18. The 100X objective lens was rotated slightly to the side of slide.
19. One drop of immersion oil was placed on the slide directly over the condenser.
20. Then, the 100X objective lens was rotated into its position. (IMPORTANT: The
objective lens must not be in contact with the immersion oil).
21. The view of specimen was looked through the oculars. The fine adjustment was
adjusted slowly until the image is clear. (IMPORTANT: The fine adjustment was
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 used only to focus the oil-immersion objective to avoid knocking the objective lens to
the stage or specimen). The specimen was examined using the stage controls to
move the slide in a left and right or backward and forward pattern while looking
through the oculars. (Specimen morphology or structure must be recorded etc.)
(Note: After viewing at 100X objective lens, the lens must be cleaned and slided with
lens paper before other lower power objective lenses repeated for next use. (Other
objective lens can be avoided to touch the immersion oil).

After viewing specimen:

22. After viewing the specimen, the lowest-power objective lens was rotated back into
its position. (IMPORTANT: The other objective lenses must not be in contact to
immersion oil).
23. The slide from the microscope stage was removed and the oil from the slide was
blotted gently with lens paper.
24. The oculars, low-power objective and high-power objective were cleaned with clean
lens paper.
25. Lastly, the oil-immersion objective was cleaned with lens paper to remove all oil
(NOTE: The oil-immersion objective was cleaned lastly to avoid spreading the
immersion oil to other objective lens with the same lens paper). Then, all oil from the
microscope stage and condenser were cleaned.
26. The microscope light was turned off and the microscope power supply was
unplugged.
27. The stage was centered so that the either side of the microscope does not project.
28. The body tube was turned back to position.
29. The microscope was covered and returned to the storage.

PART 3

1. Low power objective lens was been used to start the experiment. (5X).
2. Ocular micrometer was placed on the eyepiece lens.
3. The stage micrometer was put on stage and was secured with clips.
4. The stage micrometer was lined up with the ocular micrometer. When in place, the
two micrometers were appeared.

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5. The lines at the left edges of the two micrometers were matched by turning the ocular
micrometer in the microscope tube or by moving the stage. It was continued until the
lines of the ocular micrometer are parallel with those of the stage micrometer.

6. After the stage micrometer scales were matched with the ocular micrometer scales,
the numbers of spaces on ocular micrometer that match defined spaces on stage
micrometer were started to count.

7. Calculation method:
Eg: For objective lens 5X, 10 spaces on the ocular micrometer = 7 spaces on the stage
micrometer.

8. The calculated data was recorded.

9. Objective lenses was changed to the other ones and steps 2-8 were repeated.
A chart of size for each magnification lens was created.

10. Then, the stage micrometer from the microscope stage was been removed and
specimen slide was put on the microscope stage.

11. The size of specimen at any objective lens was been proceeded to measure using
the ocular micrometer as the calibrated the ocular micrometer was done.

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 Conclusion

At the end of this report, the locations and labels of different major parts,
functions and also the locations of a light microscope were able to understand. More
than that, the ways to use a light microscope to examine the microscope using different
magnification powers using objective lenses such as 4X, 10X, 40X, and 100X were also
been identified. The steps to use both the micrometers which are the stage and ocular
micrometers and to measure the size of a specimen or the size of microorganisms were
also been identified. As an overall aspect, the uses, ways and more information of a
typical microscope especially a light microscope in a science laboratory was been
known and learnt through this practical session and report.

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