EXPERIMENT 1 : BASIC TECHNIQUES IN MICROSCOPY

Objectives
i. To obtain accurate images
ii. To determine the depth of field
iii. To determine the field of view
iv. To calculate the actual magnification
v. To apply the use of oil immersion with high magnification (oil immersion lens)
Exercise 1.1: Images, Depth Of Field and Field Of View Of The Microscope
1.1.1 Images under the microscope
Apparatus
Compound light microscope

Materials
‘e’ prepared slide

Procedures
1. The ‘e’ prepared slide is observed using the 4x objective lens
2. Observation under 4x objective lens is drawn
3. The position of ‘e’ is determined

Observation

‘e’ image

Actual magnification: 40x

Conclusion
The letter ‘e’ image is inverted and bigger
Actual magnification: 40X
 1.1.2 The depth of field

Apparatus
Compound light microscope

Materials
‘Cross thread’ prepared slide [3 colours ; yellow, red and blue]

Procedures
1. The position of the thread on the slide is observed with naked eyes. The colour of
the thread at the top, in the middle and at the bottom is observed.
2. The cross thread is observed under the microscope using 4x, 10x and 40x objective
lens.
3. Depth of the field is determined when the power of objective lens increases.

Observation

As for your reference, the above diagram shows the cross thread slide observed with naked
eyes and using 4x, 10x and 40x objective lens.
Follow the procedures given in the lab manual and state your observation in the table below.

Position of the Naked eyes Identify the colour of thread under the
thread on the microscope using
slide
4x objevctive 10x objective 40x objective
lens lens lens
at the top red red red red

in the middle blue blue blue blue

at the bottom yellow yellow yellow yellow

Conclusion
The higher the magnification, the smaller the depth of field.

1.1.3 The field of view

Apparatus
Compound light microscope

Materials
Transparent ruler (10mm size)

Procedures
1. A transparent ruler is placed on the stage. The ruler scale is placed at the centre of
the field of view.
2. The transparent ruler is observed using 4x, 10x and 40x objective lens. The amount
of light is increased by adjusting the control knob to the maximum.
3. The observation at 4x objective lens is captured.
4. The diameter of field of view at 4x, 10x and 40x is determined.
5. The diameter of field of view is calculated using the formula below:

Diameter of field of view under low magnification power = high magnification power
Diameter of field of view under high magnification power low magnification power
6. The result is recorded.

Observation

(Image of transparent ruler under 4x objective lens)

Actual magnification: 40x

Determine

1. Diameter of field of view for the 4x objective lens is 3mm or 3000um

2. Diameter of field of view for the 10x is 1.2mm or 1200um

3. The diameter of field of view for the 40x is 0.3mm or 300um

Conclusion
The higher the magnification, the smaller the size of the field of view

Exercise 1.2. Magnification

Procedure
1. The actual magnification is determined by using the formula below.

Actual magnification = Magnification power of x Magnification power of

objective lens ocular lens

2. The actual magnification in Table 1.1 is calculated.

Observation

Actual magnification
Magnification power of Magnification power of objective lens
ocular lens
4x 10x 40x 100x
10x
40x 100x 400x 1000x

Conclusion

Using the 10x magnification power of ocular lens, the greater the magnification power of
objective lens will cause the actual magnification to increase.

Exercise 1.3 : Oil Immersion Objective Lens

Apparatus
Compound light microscope

Materials
Immersion oil
Lens
Tissue paper
Prepared slide of bacteria
Methylated spirit

Procedures
1. The prepared slide is observed under the microscope using 100x objective lens
• The slide is moved so that the specimen is placed in the centre and under the
objective lens.
• Scanning objective lens (4x) or low-power objective lens (10x) is placed above the
specimen.
• While looking at the slide from the side, the stage is moved upwards until it stops
completely. Coarse adjustment knob is used to do this.
• The specimen is observed through the ocular lens. The specimen will appear blur
because it is not focused. To focus the specimen, the stage is moved downwards
until the specimen comes into sharp focus and clear. The fine adjustment knob is
used to do this.
• The nosepiece is rotated to bring the high-power objective lens (40x) halfway.
 • While holding the nosepiece in this position, a single small droplet of immersion oil is
applied to the illuminated spot on the slide.
• The nosepiece is rotated again to move the high-power objective lens into position
until a clicking sound is heard. The objective lens is now right above the specimen
and will be immersed in the oil.
• The iris diaphragm is opened up to increase the amount of light.
• While looking at the specimen through the ocular lens, the fine adjustment knob is
used until the specimen comes into sharp focus and become clear.

2. The image appeared is captured.

Observation

E.coli

Actual magnification: 1000x

Conclusion
Oil immersion techniques using higher magnification (100x objective lens), where oils
reduce light refraction. The higher the resolution of the microscope, the higher is the ability
to distinguish details of the object
Questions:
Part A
For question 1 to 7, choose the correct answer from the following list:
A Scanning objective lens (4X)
B Low-power objective lens (10X)
C High-power objective lens (40X)
D Oil immersion objective lens (100x)

1. Which is the shortest objective lens? (A)

2. Which objective lens should you use when you begin to focus a specimen? (A)
3. Which objective lens should be in position before you store a microscope? (A)
4. Which objective lens will deliver the highest amount of light? (D)
5. Which objective lens requires immersion oil to fill up the space between the
specimen and the lens? (D)
6. Which objective lens will remain in focus when placed at the longest working
distance from the specimen? (C)
7. When using an ocular lens with 10X magnification power, which objective lens
should be used to obtain the following actual magnification?
(a) 100 times of its diameter (B)
(b) 1000 times of its diameter (D)

Part B

1. Using 40X objective lens, determine the length and width of a cell from piece of cork
tissue with approximately 20 cells in horizontal position and 10 cells in vertical
position

Figure 1.4 Cork tissue

Therefore, the length = 15.00 um and the width is 30.00 um

 2. Based on laboratory practices, what do you use to clean the microscope lenses?
I use lens cleaning paper to clean the microscope lens.

3. While observing a moving microorganism under a microscope, you found that the
organism has moved out of the field of view to the right.
If the organism has moved out of the field of view to the right, the slide should be
moved to the right in order to keep observing the microorganism.

4. How do you adjust the slide when the specimen is out of the field of view to the top?
I will adjust the slide using the slide adjustment knob to rotate and adjust the slide
to the top.

Part C

1. Complete the following sentences

a. A microscope is called a compound microscope when it consists of more than one

set of lens.
b. Condenser and iris diaphragm are useful to coordinate the amount of light that
enters the specimen.

References:

(2022, January 3). How to use a microscope. https://www.microscopeworld.com/p-3561-

how-do-i-use-a-microscope.aspx

Rachel. (2021, September 15). Handling oil immersion microscope

https://rsscience.com/how-to-use-microscope-immersion-oil-to-get-higher-resolution-

Connor Rid. (July, 21, 2021). Microscope calculation: field of view, depth of field
https://dovermotion.com/applications-capabilities/automated-imaging/microscope-
calculations/