Course: MCB 103 Lab
Section: 1
Name: Noor-E-Khadiza Shama
ID: 1921168
Date: 28/01/20
Experiment 2
MICROSCOPY
PURPOSE
1. To learn how to use a compound light microscope and practice focusing the specimen using four
objective lenses at magnification 4X, 10X, 40X and 100X.
2. To learn how to use oil immersion when magnified under x100 power objective lens
PRINCIPLE
A compound light microscope magnifies a specimen using two lenses: objective lens and ocular lens.
Objective lenses located on the rotary nosepiece, achieve 4 degrees of magnification:
Characteristic Magnification
Red stripe 4X
Yellow stripe 10 X
Blue stripe 40 X
Black stripe 100 X
Ocular lens located at the end of the body tube has a magnification power of 10 X. So total magnification
is determined by multiplying the power of objective by the power of ocular. For example, 4X times 10X =
40X TM. The light source used is visible light that is why it is referred as “Light Microscope”. An iris
diaphragm is located under the stage to control the amount of light entering the condenser. The
condenser, located just above the diaphragm, refract the light rays to condense it on the specimen. As
the light rays passes through the slide, it refracts and enters the objective lens. From there, the rays
travel through a prism and enters the ocular lens. In order to focus the image, the coarse adjustment
knob and fine adjustment knob is used.
At 100 X power objective lens, refraction occurs as light passes from glass slide to air. Due to refraction,
refracted light is lost and do not enter the objective lens, so the specimen is not seen. To prevent
refraction, immersion oil is placed between the lens and the slide. As the refractive index of glass and
immersion oil is same (1.5) ,no refraction occurs and a clear image is seen.
MATERIALS & EQUIPMENT
1. A Light Microscope
2. Permanent slides (it already has a cover slip fixed with it)
3. Immersion Oil
PROCEDURE
1. At first, a permanent slide is placed on the stage and secured using the stage clip.
2. Then the objective lens of power 4X is set into the nosepiece
3. Stage control is used to position the specimen over the light source
4. The coarse adjustment knob is used to raise the stage to its highest position and the specimen is seen
through the ocular lens
5. The coarse adjustment knob is slowly rotated, lowering the stage until the specimen comes into focus.
6. Then the fine adjustment knob is rotated for further focusing
7. At last the clip of the diaphragm is turned to control brightness.
8. The above procedure is repeated for 10X and 40X power objective lens
9. When a permanent slide is observed under 100X power objective lens, no cover slip is used because
permanent slides already has some immersion oil and a cover slip fixed to it.
10. So at first, a drop of immersion oil is placed over the permanent slide.
11. Then the stage is slowly moved upwards using the coarse adjustment knob until the objective lens
touches the oil.
12. Then the fine adjustment knob is used to focus the specimen.
13. After using immersion oil, the lens must be cleaned using tissue paper.
RESULT & OBSERVATION
Some permanent slides of algae ,fungi , protozoa and bacteria were observed under 10X and 40X power objective
lenses and the results are shown below.
DISCUSSION
Out of all the specimen, bacteria were the smallest in size. Algae looked like filaments. The specimens were more
clearly seen when observed under 40 X power objective lens and 100X power oil immersion.
LIMITATIONS
1. It can magnify up to 1000 X only, so structures like flagella cannot be seen
2. 3D image of a specimen cannot be seen
3. No internal structures like nucleus can be seen
4. Usually live samples cannot be seen
PRECAUTION
1. Glass slide must be handled carefully
2. The 100 X power objective lens must be cleaned with a tissue paper to remove oil and prevent
contamination.
