MIB151S: Microscopy and Staining (Microbiology 1)
Cape Peninsula University of Technology
Lecture 2 Notes
Microbiology 1
2.1 Lenses and bending light
2.2 The light microscope
2.3 Microscope resolution
2.4 Preparation and staining of specimens
2.5 Fixation
2.6 Dyes and simple staining
2.7 Differential staining and staining specific structures
_______________________________________________________
Microscopy and Staining
The microscope is an indispensable tool in microbiology that enables us to see micro-
organisms that are normally invisible with the naked eye.
The first compound microscope was made by Jansen (1590) and was able to magnify
objects 50X.
In 1674, Antony Van Leeuwenhoek
constructed a simple microscope
with a single lens, which was able to
magnify an object 300X.
Remember!!!! Van Leeuwenhoek
was the first to observe and identify
microorganisms!
2.1 Lenses and the bending of light
Magnification of an object is possible because lenses can bend light;
The different types of microscopy, depends on the type of condenser fitted and the light
source;
To understand how a light microscope works, know something about the way in which
lenses bend and focus light to form images;
When a ray of light passes from one medium to another, e.g. from air to glass, refraction
occurs, i.e. the ray is bent at the interface;
1
, MIB151S: Microscopy and Staining (Microbiology 1)
Cape Peninsula University of Technology
Lecture 2 Notes
The refractive index is a measure of how greatly a substance slows the velocity of light –
the direction and magnitude of bending is determined by the refractive indices of the two
media forming the interface.
2.2 The light microscope
There are two types of microscopy:
a) Light Microscopy (optical) and
b) Electron microscopy (EM).
There are two types of light microscopes – Simple and Compound Microscopes.
1) The simple microscope – made use of only one lens, as used by Van Leeuwenhoek
2) The compound microscope – makes use of a double lens system – maximum 1000X
magnification.
There are different types of compound microscopes:
Bright Field
Dark Field
Phase Contrast
Fluorescence
Bright Field Microscopy is the most commonly used form of microscopy - Light passes
through the specimen.
A special condenser is used for dark field microscopy. Light reflects off the specimen,
instead of passing through it.
Phase-contrast microscopy – It is used to observe living organisms. It picks up small
differences in the refractive indexes of structures and cells, so no staining is necessary. A
special condenser is fitted.
Fluorescence Microscopy – Uses UV, instead of white light, which causes dyes in the
organisms to fluoresce.
Electron Microscopy was invented in 1932. It uses an electron beam instead of a light
beam and electron magnets instead of glass lenses to focus on small objects.
Transmission electron microscope – shows the internal structure of micro-organisms
and can magnify up to 500 000X (viruses, protein, DNA).
2
Cape Peninsula University of Technology
Lecture 2 Notes
Microbiology 1
2.1 Lenses and bending light
2.2 The light microscope
2.3 Microscope resolution
2.4 Preparation and staining of specimens
2.5 Fixation
2.6 Dyes and simple staining
2.7 Differential staining and staining specific structures
_______________________________________________________
Microscopy and Staining
The microscope is an indispensable tool in microbiology that enables us to see micro-
organisms that are normally invisible with the naked eye.
The first compound microscope was made by Jansen (1590) and was able to magnify
objects 50X.
In 1674, Antony Van Leeuwenhoek
constructed a simple microscope
with a single lens, which was able to
magnify an object 300X.
Remember!!!! Van Leeuwenhoek
was the first to observe and identify
microorganisms!
2.1 Lenses and the bending of light
Magnification of an object is possible because lenses can bend light;
The different types of microscopy, depends on the type of condenser fitted and the light
source;
To understand how a light microscope works, know something about the way in which
lenses bend and focus light to form images;
When a ray of light passes from one medium to another, e.g. from air to glass, refraction
occurs, i.e. the ray is bent at the interface;
1
, MIB151S: Microscopy and Staining (Microbiology 1)
Cape Peninsula University of Technology
Lecture 2 Notes
The refractive index is a measure of how greatly a substance slows the velocity of light –
the direction and magnitude of bending is determined by the refractive indices of the two
media forming the interface.
2.2 The light microscope
There are two types of microscopy:
a) Light Microscopy (optical) and
b) Electron microscopy (EM).
There are two types of light microscopes – Simple and Compound Microscopes.
1) The simple microscope – made use of only one lens, as used by Van Leeuwenhoek
2) The compound microscope – makes use of a double lens system – maximum 1000X
magnification.
There are different types of compound microscopes:
Bright Field
Dark Field
Phase Contrast
Fluorescence
Bright Field Microscopy is the most commonly used form of microscopy - Light passes
through the specimen.
A special condenser is used for dark field microscopy. Light reflects off the specimen,
instead of passing through it.
Phase-contrast microscopy – It is used to observe living organisms. It picks up small
differences in the refractive indexes of structures and cells, so no staining is necessary. A
special condenser is fitted.
Fluorescence Microscopy – Uses UV, instead of white light, which causes dyes in the
organisms to fluoresce.
Electron Microscopy was invented in 1932. It uses an electron beam instead of a light
beam and electron magnets instead of glass lenses to focus on small objects.
Transmission electron microscope – shows the internal structure of micro-organisms
and can magnify up to 500 000X (viruses, protein, DNA).
2
