SB015 Lab Manual

Procedures and Observation

1.1.1 Images under the microscope

1. Observe the ‘e’ prepared slide using the 4x objective lens.

2. What do you observe using the 4x objective lens? Draw what you
have observed.
3. Determine the position of ‘e’ (inverted/original position)
(Figure 1.1).

Figure 1.1: Letter ‘e’ to be observed under microscope

1.1.2 The depth of field

The depth of field refers to the thickness of the plane of focus. With a
large depth of field, all of the threads can be in focused at the same
time. With a smaller or narrower depth of field, only one thread or a
part of one thread can be focused, everything else will be out of focus.
In order to view the other threads, you must focus downward to view
the ones underneath and upward view the ones that are above.

Do the following exercises to determine the depth of field of

microscope.
1. Observe the position of the thread on the slide with your naked
eyes. Identify the colour of thread
a) at the top
b) in the middle
c) at the bottom
2. Observe the crossthreads under the microscope using 4x and 10x
objective lens.
3. Determine what happens to the depth of field when the power of
objective lens increases.

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Bottom: Yellow

Middle: Red

Top: Blue

Figure 1.2 :Cross thread prepared slide

1.1.3 The field of view

The simplest method of estimating linear dimension is to compare the

size of the image to the diameter of the field of view. You can make a
rough estimate of the field diameter by focusing on the millimetre
scale of a transparent ruler using the lowest power objective. To
calculate the field of view, use this formula:

Diameter of field of view under low

magnification power
High magnification power
=
Diameter of field of view under high Low magnification power
magnification power

Do the following exercises to determine the diameter of the field of

view for each of the objective lens on your microscope.
1. Place a transparent ruler on the stage.
2. Observe the transparent rulerusing the 4x, 10x and 40x objective
lenses. (Increase the amount of light by adjusting the control knob
to the maximum).
3. What do you observe using the 4x objective lens? Draw what you
have observed.

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Figure 1.3 : Diameter field of view

The diameter of field of view for the 4x objective lens is mm or m.

Determine the diameter of field of view for the 10x and 40x objective
lens in mm or m.
The diameter of field of view for the 10x = ____ mm = ____ m.
The diameter of field of view for the 40x = ____ mm = ____ m.
4. Using 40x objective lens, determine the size of a cell from a piece
of cork tissue with approximately 20 cells in horizontal position
and 10 cells in vertical position.

Exercise 1.2: Magnification

Procedures and Observation

1. Determine the actual magnification of a specimen by using the

formula below.

Magnification power Magnification

Actual magnification = of objective lens x power
a of ocular lens

2. Calculate the actual magnification in Table 1.1.

Table 1.1 Actual magnification of a specimen

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

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Exercise 1.3: Oil Immersion Objective Lens

Apparatus

Compound microscope

Materials

Prepared slide of bacteria

Lens tissue papers
Immersion oil
Methylated spirit (only for specific use)

Procedures and Observation

1. Observe the prepared slide under the microscope.

(Caution: Use immersion oil only for 100x objective lens).
2. Draw your observation.
(Caution: Draw only the bacteria and not artifacts such as
air bubbles, dust, fibre, etc.)
(Refer to the method in Introduction to Microscopy)

Questions

A) For questions 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?

2. Which objective lens should you use when you begin to focus a
specimen?
3. Which objective lens should be in position before you store a
microscope?
4. Which objective lens will deliver the highest amount of light?
5. Which objective lens requires immersion oil to fill up the space
between the specimen and the lens?

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6. Which objective lens will still remain in focus when placed at the
longest working distance from the specimen?
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) 1000 times of its diameter

B) Answer the following questions.

1. Based on laboratory practices, what do you use to clean the

microscope lenses?
2. While observing a moving microorganism under a microscope,
you found that the organism has moved out of the field of view
to the right. In order to keep observing the microorganism, which
direction do you move your slide (right/left)?
3. How do you adjust the slide when the specimen is out of the field
of view to the top?

C) Complete the following sentences.

1. A microscope is called a compound microscope when it consists

of more than one set of …………………………………
2. Condenser and iris diaphragm are useful to
coordinate…………………………………………...

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