Experiment 1
Use of light microscope and stereomicroscope: measuring microscopic
objects
1.1 Introduction
The microscope is a major tool used by biologists, which was invented about 350 years
ago. It is a device that allows observation of minute organisms and various cell types too
small to be seen by the unaided eye. Since an unaided eye cannot detect anything smaller
than 0.1 mm, a light microscope can extend our view a thousand times so that objects as
small as 0.2 micrometer (2 x 10-7 m) can be seen. Whereas an electron microscope can
further extend our viewing capability down to one nanometer (1 x 10-9 m). With the
advance of technology, such as light scattering technique, today we can view the outlines
of protein and nucleic acid molecule, which are in the range of angstrom (1 x 10-10 m).
Good microscopy involves three main factors: resolution, magnification, and contrast.
The resolution, or resolving power of a microscope, which will increase when the
wavelength of the light source is reduced. Different microscopes will come with a range
of magnifications, such as light microscope ranging from 40 to 1000 and the
stereomicroscope from 7 to 30. Staining with suitable stains will increase the contrast of a
specimen to be observed under a microscope.
1.2 Objectives
1. To learn the parts of a light microscope and a stereomicroscope
2. To learn how to use them
3. To learn how to properly prepare slides for microbiological examination
4. To learn how to measure microscopic objects
1.3 Materials required
Light microscope (compound microscope, binocular microscope)
Stereomicroscope (dissecting microscope)
Glass slides and cover slips
Ocular and stage micrometers
Prepared slides
Lens paper
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Resolving power of different microscopy techniques
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1.4 Parts of a light microscope (compound microscope)
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1.5 Parts of a stereomicroscope
Label the following diagram from A to F
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1.6
Electron microscope (EM)
EM was invented about 50 years ago, which makes use of electrons rather than light
as the illumination source. The theoretical resolution of EM would be 100,000 times
greater than that of the light microscope as electrons have a wavelength of 0.005 nm.
Transmission electron microscope (TEM)
Scanning electron microscope (SEM)
Mosquito head 200X
A section of a bacterial cell of Bacillus subtilus,
scale bar 200 nm
Transmission electron micrograph
Scanning electron micrpgraph
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1.7 Handling and use of a microscope
Microscopes are expensive and precision optical instruments with many delicate working
parts. So they must be handled carefully at all times.
When moved from one place to another, the microscope must be in an upright position
with the base supported.
Never touch the lens of the microscope with your hands. For cleaning, use only special
lens paper.
When viewing a prepared slide, always have the cover slip on the upper side.
Never focus downward while looking through the eyepiece, which might break the glass
slide. Rather, look from the side and lower the objective to slightly touch the slide but
without breaking it, then focus upward.
Always locate the object to be studied with the low power objective before using the high
power ones.
Turn on the light source and fix your slide on the stage with the clips
Bring the substage light condenser up to the highest position and close down the iris
diaphragm until the light is diffused evenly across the entire field of vision.
Use the coarse adjustment knob first to bring the object into focus and then use fine
adjustment knob to view more details.
You might need to adjust the iris diaphragm size to give better object resolution and
contrast.
Adjust the angle of the two eyepieces so that only one image can be viewed comfortably.
One of the eyepieces can be adjusted if the focus of your two eyes is not equal.
1.8 Preparation of a Wet-mount Slide
(a) Add a drop of water to a slide.
(b) Place the specimen (letter e) in the water.
(c) Place the edge of a coverslip on the slide so that it touches the edge of the water.
(d) Slowly lower the coverslip to prevent forming and trapping air bubbles.
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1.9 Measuring microscopic objects
1.9.1 Determination of the magnification of microscopic objects
This method does not actually determine the size of the objects but it is convenient. This
is done by multiplying the ocular lens magnification by the objective lens magnification,
for example, if the ocular lens is 10X and objective lens 40X, then total magnification is
10 x 40 = 400X.
Exercise 1.9.1
What are the minimum and maximum magnification readings that the light microscope
and stereomicroscope in the lab can give?
Light microscope:
Minimum: ocular lens (
Maximum: ocular lens (
) x objective lens (
) x objective lens (
)=
)=
Stereomicroscope: ocular lens (
) x objective lens (
Minimum: ocular lens (
) x objective lens (
)=
Maximum: ocular lens (
) x objective lens (
)=
15
)=
1.9.2 The use of a pair of ocular micrometer and stage micrometer
This is the most accurate method of measuring microscopic objects. The ocular
micrometer is a glass disc with lines uniformly etched on it and the stage micrometer is a
glass slide with etched lines 0.01 mm apart.
The ocular micrometer will serve as a ruler against which the specimen under observation
will be measured, after it has been calibrated against a stage micrometer.
How to calibrate:
1. Match the first line of the ocular disc with the first line of the stage micrometer. Call
this line 1
2. Next look for a second line on the ocular micrometer which lines up with another line
on the stage micrometer. Call this line 2.
3. Count the number of spaces on the stage micrometer between line 1 and line 2. Use
this number as the numerator for the equation below. Count the number of spaces on
the ocular micrometer included between lines 1 and 2. Use this number as the
denominator. Multiply the numerator with 0.01 mm since micrometer stage space is
equal to 0.01 mm. To obtain the value of one ocular micrometer space, use the
following equation:
1 ocular micrometer space =
(No. of stage micrometer spaces) x 0.01 mm
No. of ocular spaces
Sample calculation
Line 1: line 0 of the ocular micrometer and line 0 of the stage micrometer
Line2: line 10 of the ocular micrometer and line 7 of the stage micrometer
No. of ocular spaces = 10
No. of stage micrometer spaces = 7
Hence, 1 ocular micrometer space = (7 x 0.01 mm)/10 = 0.007 mm
Exercise 1.9.2
Calibrate for each of the objectives on your microscope and record below:
1. 4X =
2. 10X =
3.
40X =
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Calibrating an Ocular Micrometer.
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1.10 Lab Exercise
1. Complete the following on units of measurement:
Unit
1 centimeter
1 millimeter
Abbreviation
cm
mm
Value
10-2 m
10-6 m
10-9 m
10-10 m
1 nanometer
1 angstrom
2. What effect does closing the diaphram have on the image brightness?
3. Which objective lens on the light microscope in our lab has the (a) shortest working
distance and (b) longest working distance?
1.11 References
Microscopy: www.southwestschools.org/jsfaculty/Microscopes/microscopeparts.html
Vernier scale:
http://biology.clc.uc.edu/fankhauser/Labs/Anatomy_&_Physiology/A&P203/Circulatory_
System/Vernier_scale/Vernier_Scales.htm
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