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Experiment 4: Microscopy: Compound Light Microscope

This document describes an experiment involving microscopy. It introduces different types of microscopes like light microscopes, binocular dissecting microscopes, and compound light microscopes. The procedures describe identifying parts of these microscopes and how to focus objects under different magnifications. The objectives of the experiment are to learn microscope parts, focusing steps, and calculating total magnification and field diameter under different objectives.

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91 views

Experiment 4: Microscopy: Compound Light Microscope

This document describes an experiment involving microscopy. It introduces different types of microscopes like light microscopes, binocular dissecting microscopes, and compound light microscopes. The procedures describe identifying parts of these microscopes and how to focus objects under different magnifications. The objectives of the experiment are to learn microscope parts, focusing steps, and calculating total magnification and field diameter under different objectives.

Uploaded by

Ehaz
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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EXPERIMENT 4:

MICROSCOPY
INTRODUCTION
Because biological object can be very small, a microscope is often used to view them. Many kinds of
instruments, ranging from the hand lens to the electron microscope, are effective magnifying devices.

Light microscope uses light rays that are magnified and focused by means of lenses. The
binocular dissecting microscope is designed to study entire object s in three dimensions at low
magnification. The compound light microscope is used for examining small or thinly sliced section of
object s under magnification that is higher than that of the dissecting light microscope. Illumination is
from below, and the light passes through clear sections but now the opaque section. To improve
contrast, microbiologists use stains or dyes that bind to cellular structures and absorb light.

OBJECTIVES
1. To name and give the function of the basic parts of the compound light microscope and
dissecting microscope.
2. To list in proper order of steps for bringing an object into focus with the compound
microscope.
3. To calculate the diameter of the field and the total magnification for both low and high power
microscope.

COMPOUND LIGHT MICROSCOPE


MATERIALS
 Compound light microscope
 Slides
 Marker pen
 Letter ‘e’ from newspaper

PROCEDURE
Identification of parts
After the instructor has explained how to carry a microscope, one microscope is taken and
placed securely on the table. The following parts is then identified from the microscope.

1. Eyepiece (ocular lens): Topmost series of lenses through which an object is viewed.
2. Body tube:
Holds nose piece at one end and eyepiece at the other end; conducts light rays.
3. Arm:
Supports upper parts and provides carrying handle.
4. Nosepiece:
Revolving device that holds objectives.
5. Objectives (objective lenses): Four common types of objectives as follows:
(a) Scanning power objective: Holds 4x lens used to view the whole slide.
(b) Low power objective: Holds 10x lens used to view the object in greater detail.
(c) High-power objective: Holds 40x lens used to view the object in even greater detail.
(d) Oil immersion objective: Holds 100x lens and is used in conjunction with immersion oil
to view the object with greatest magnification.
6. Course adjustment knob: Knob used to bring object into approximate focus; used only with
low power objective.
7. Fine-adjustment knob: Knob used to bring object into final focus.
8. Diaphragm or diaphragm control lever: Controls amount of illumination used to view the
object.
9. Light source: An attached lamp that directs a beam of light to view the object.
10. Base: The flat surface of the microscope that rests on the table.
11. Stage: Holds and supports microscope slides.
12. Stage clips: Hold sides in place in the stage.
13. Mechanical stage: A moveable stage used in accurate positioning of the slide.
14. Mechanical stage control knob: Two knobs that are usually located below the stage. One
knob controls forward/reverse movement, and the other controls right/left movement.

Focusing the Microscope – Lowest Power


1. Turn the nosepiece so that the lowest power objective in straight alignment over the stage.
2. Always begin focusing with the lowest power object (4x or 10x).
3. With the coarse –adjustment knob, lower the stage until it stops.
4. Place the letter ‘e’ on the stage and stabilize it with the clips (if the microscope has a
mechanical stage, pinch the spring of the slide arms on the stage and insert the slide).
Centralise the ‘e’ as best as it can on the stage.
5. Again, be sure that the lowest power objective is in place. Then as you look from the side,
decrease the distance between the stage and the tip of the objective lens until the lens
comes to an automatic stop or is no closer than 3mm above the slide.
6. While looking into the eyepiece, rotate the diaphragm (or diaphragm lever) to give the
maximum amount of light.
7. Slowly increase the distance between the stage and the objective lens, using the coarse
adjustment knob until the object comes into view or focus.
8. Once the object is seen, some adjustment of light may be needed. To increase or decrease
the contrast, rotate the diaphragm slightly.
9. Use the fine-adjustment knob to sharpen focus if necessary.
10. Practice having both eyes open when looking through the eyepiece, as it greatly reduces
eyestrain.
Focusing the Microscope – Higher Power
Compound light microscope are parfocal; that is, once the object is in focus with lowest
power, it should also be almost in focus with the higher power.

1. Bring the object into focus under the lowest power by following the instructions in the
previous section.
2. Make sure that the letter ‘e’ is centred in the field of the objective.
3. Move to the next higher objective (low power [10x] or high power [40x]) by turning the
nosepiece until you hear it click into place. Do not change the focus; parfocal
microscope objective will not hit normal slides when changing the focus if the lowest
objective is initially in focus. If you are on low power [10x], proceed ti high power [40x]
before going on to step 4.
4. If any adjustment is needed, use only the fine-adjustment knob. Always use only the
fine-adjustment knob with high power. When you have finished your observations of
this slide (or any slide), rotate the nosepiece until the lowest power objective click into
place, and then remove the slide.

STEREOMICROSCOPE/DISSECTING MICROSCOPE
MATERIALS
 Stereomicroscope/dissecting microscope.
 Letter ‘e’ from newspaper.

PROCEDURE
Identification of parts
1. A stereomicroscope is obtained and various part mentioned below are identified.
2. Binocular head: holds two eyepiece lenses that move to accommodate the various distances
between different individuals’ eyes.
3. Eyepiece lenses: the two lenses located on the binocular head.
4. Focusing knob: A large black or gray knob located on the arm used for changing the focus of
both eyepieces together.
5. Magnification changing knob: A knob often built into the binocular head that is used to
change magnification in both eyepieces simultaneously.
6. Illuminator: used to illuminate object from above.

Focusing the Stereomicroscope Dissecting Microscope


1. Place a letter ‘e’ as an object on the centre of the stage.
2. Adjust the distance between the eyepieces on the binocular head so that they comfortably
fit the distance between your eyes.
3. Use the focusing knob to bring the object into focus.
4. Turn the magnification changing knob to the lowest magnification and draw the object.
5. Experiment with various magnifications until you are comfortable with the use of the
stereomicroscope.
Total Magnification
Total magnification is calculated by multiplying of the ocular lens (eyepiece) by the
magnification of the objective lens.

Diameter of field
The diameter of the field (the circle visible through the lens) is the length of the field. This
can be measured by placing a transparent ruler on the stage while viewing through the eyepieces
with different objective lenses.

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