17.3 Cytometry Techniques
17.3.1 Historical
Cytometry include a set of techniques to characterize and to
measure the physical properties of individual cells and cellular
components. The best known of these techniques and the oldest
is the microscope. It appeared in the seventeenth century and
quickly gave birth to an extraordinary period of discoveries in
biology, in particular under the leadership of two Dutch,
Antonie van Leeuwenhoek (1632–1723) and Jan Swammerdam
(1637–1680), and one Italian, Marcello Malpighi (1628–1694).
Progress in the understanding of the fundamental laws of
optics and in the construction of instruments in the fields of
astronomy and navigation then led to a considerable evolution
of microscopes in the eighteenth century. The increasing interest in biology and the discoveries done in the field of medicine
in the nineteenth century have stimulated the need for observation and for the development of more effective instruments.
The microscope allowed Pasteur to undermine the theory of
spontaneous generation and Koch to discover several
pathogens. In the twentieth century, advances in electronics,
informatics, and optics have enabled the design of new
generations of photonic and electronic microscopes with previously unmatched performance. Meanwhile, progress in the
field of chemistry and methods for fluorescence labeling of
cellular constituents have allowed the development of new
applications of cytometry techniques in biology.
The more and more important need to automate the counting
of cells, especially in the medical field for the analysis of blood
cells, has led to the emergence of a new technique based on flow
cytometry analysis of cells in liquid vein: flow cytometry.
Designed by Moldavan in the early twentieth century, it was
mainly developed in the 1970s by researchers at Los Alamos
and Stanford in the USA who linked methods of measuring
individual volume or fluorescence of cells driven by a stream
with methods for electrostatic cell sorting in vital conditions.
This technique helped to make extraordinary discoveries
in the field of biological oceanography by observing phytoplankton cells on the basis of their natural pigments. Thus,
Sallie Chisholm and Robert Olson teams made the discovery
of two new cyanobacteria in the years 1979–1986,
Synechococcus and Prochlorococcus genera, and were discovered by this technique, and their quantification allowed
to reveal the essential role played by these cyanobacteria in
the functioning of the global ocean and CO 2 fixation.
Flow cytometry was quickly used in biological oceanography by specialists of the phytoplankton under the leadership of Daniel Vaulot in Roscoff (Marine Biological Station,
France) who participated in the work carried out in the USA.
Later with the development of fluorescent cellular markers
that could be applied to non-photosyntetic organisms, this
technique has been developed and used in microbial ecology,
and in the 1990s, it has become a routine technique in many
laboratories for the enumeration and characterization of the
physiological state of bacterial cells. All these instrumental
developments combined with advances in parallel in the field
of molecular biology have revolutionized microbiology, in
general, and particularly environmental microbiology.
17.3.2 The Different Cytometry Techniques
This chapter does not aim to describe the different microscopy
techniques since there are books and websites dedicated to these
techniques. Some of them are listed below to which the reader
should refer for further analysis and more technical details on
instruments. Our goal is to provide the reader with a list of
techniques that are available today to present their principles
and main applications in the field of microbial ecology.
17.3.2.1 Microscopy Techniques
Optical Microscopes
This is the oldest technology used by Renaissance scientists,
but they are still present in laboratories. Light microscopes
can be bright-field, dark-field, polarization, fluorescence,
phase contrast, interference, and confocal scanning laser.
Direct-Light Microscopes
Visible light is passed through the specimen and then through
glass lenses. One of the limitations of the bright-field microscopy is insufficient contrast. The lenses refract the light in such
a way that the image of a specimen is magnified as it is
projected into the eye. The observed structures can be naturally colored, but dyes can also be used to stain cells and
increase their contrast so that they can be more easily seen in
the bright-field microscope. Depending on the intensity of the
colored parts, the light will differently be absorbed and these
parts will appear more or less dark. The coloration is due either
to a dye that binds preferentially to a particular molecule or
family of molecules or to a dark precipitate. This precipitate
generally results from the action of an enzyme and occurs at
the place where the protein is located, which allows to observe
the distribution of the enzyme in the biological structure.
In these techniques, the light rays coming from the condenser can penetrate into the lens (bright-field) or indirectly
through a set of mirrors (dark-field). The black-field is used
for the observation of specimen whose structures have significant variations in refractive index and, because of the
lack of contrast, are only slightly or not visible in bright-field
microscopy (bacteria, nucleus, vacuoles, flagellar, skeletons
of diatoms, etc.). The dark-field microscopy is very
suitable for fresh observations, and its resolution is quite
high. It is also an excellent way to observe the motility of
microorganisms, as bundles of flagella are often resolvable
in bright-field or phase contrast microscopes.
17 Methods for Studying Microorganisms in the Environment
773
17.3.1 Historical
Cytometry include a set of techniques to characterize and to
measure the physical properties of individual cells and cellular
components. The best known of these techniques and the oldest
is the microscope. It appeared in the seventeenth century and
quickly gave birth to an extraordinary period of discoveries in
biology, in particular under the leadership of two Dutch,
Antonie van Leeuwenhoek (1632–1723) and Jan Swammerdam
(1637–1680), and one Italian, Marcello Malpighi (1628–1694).
Progress in the understanding of the fundamental laws of
optics and in the construction of instruments in the fields of
astronomy and navigation then led to a considerable evolution
of microscopes in the eighteenth century. The increasing interest in biology and the discoveries done in the field of medicine
in the nineteenth century have stimulated the need for observation and for the development of more effective instruments.
The microscope allowed Pasteur to undermine the theory of
spontaneous generation and Koch to discover several
pathogens. In the twentieth century, advances in electronics,
informatics, and optics have enabled the design of new
generations of photonic and electronic microscopes with previously unmatched performance. Meanwhile, progress in the
field of chemistry and methods for fluorescence labeling of
cellular constituents have allowed the development of new
applications of cytometry techniques in biology.
The more and more important need to automate the counting
of cells, especially in the medical field for the analysis of blood
cells, has led to the emergence of a new technique based on flow
cytometry analysis of cells in liquid vein: flow cytometry.
Designed by Moldavan in the early twentieth century, it was
mainly developed in the 1970s by researchers at Los Alamos
and Stanford in the USA who linked methods of measuring
individual volume or fluorescence of cells driven by a stream
with methods for electrostatic cell sorting in vital conditions.
This technique helped to make extraordinary discoveries
in the field of biological oceanography by observing phytoplankton cells on the basis of their natural pigments. Thus,
Sallie Chisholm and Robert Olson teams made the discovery
of two new cyanobacteria in the years 1979–1986,
Synechococcus and Prochlorococcus genera, and were discovered by this technique, and their quantification allowed
to reveal the essential role played by these cyanobacteria in
the functioning of the global ocean and CO 2 fixation.
Flow cytometry was quickly used in biological oceanography by specialists of the phytoplankton under the leadership of Daniel Vaulot in Roscoff (Marine Biological Station,
France) who participated in the work carried out in the USA.
Later with the development of fluorescent cellular markers
that could be applied to non-photosyntetic organisms, this
technique has been developed and used in microbial ecology,
and in the 1990s, it has become a routine technique in many
laboratories for the enumeration and characterization of the
physiological state of bacterial cells. All these instrumental
developments combined with advances in parallel in the field
of molecular biology have revolutionized microbiology, in
general, and particularly environmental microbiology.
17.3.2 The Different Cytometry Techniques
This chapter does not aim to describe the different microscopy
techniques since there are books and websites dedicated to these
techniques. Some of them are listed below to which the reader
should refer for further analysis and more technical details on
instruments. Our goal is to provide the reader with a list of
techniques that are available today to present their principles
and main applications in the field of microbial ecology.
17.3.2.1 Microscopy Techniques
Optical Microscopes
This is the oldest technology used by Renaissance scientists,
but they are still present in laboratories. Light microscopes
can be bright-field, dark-field, polarization, fluorescence,
phase contrast, interference, and confocal scanning laser.
Direct-Light Microscopes
Visible light is passed through the specimen and then through
glass lenses. One of the limitations of the bright-field microscopy is insufficient contrast. The lenses refract the light in such
a way that the image of a specimen is magnified as it is
projected into the eye. The observed structures can be naturally colored, but dyes can also be used to stain cells and
increase their contrast so that they can be more easily seen in
the bright-field microscope. Depending on the intensity of the
colored parts, the light will differently be absorbed and these
parts will appear more or less dark. The coloration is due either
to a dye that binds preferentially to a particular molecule or
family of molecules or to a dark precipitate. This precipitate
generally results from the action of an enzyme and occurs at
the place where the protein is located, which allows to observe
the distribution of the enzyme in the biological structure.
In these techniques, the light rays coming from the condenser can penetrate into the lens (bright-field) or indirectly
through a set of mirrors (dark-field). The black-field is used
for the observation of specimen whose structures have significant variations in refractive index and, because of the
lack of contrast, are only slightly or not visible in bright-field
microscopy (bacteria, nucleus, vacuoles, flagellar, skeletons
of diatoms, etc.). The dark-field microscopy is very
suitable for fresh observations, and its resolution is quite
high. It is also an excellent way to observe the motility of
microorganisms, as bundles of flagella are often resolvable
in bright-field or phase contrast microscopes.
17 Methods for Studying Microorganisms in the Environment
773