17.3.5 A Taxonomic Approach at the Cellular
Level
17.3.5.1 Immunofluorescence
The immunoassay techniques are widely used in microbiology as a diagnostic tool. They can be used to search for a
pathogen, its identification, and counting (especially in
controlling the quality of the water). These techniques are
based on the use of specific antibodies to pathogens. There
are monoclonal antibodies (recognizes only one type of
epitope) which are very specific and reproducible and polyclonal antibodies (mixture of antibodies recognizing different epitopes on the same antigen) that are less specific.
Among the various immunoassays, immunofluorescence
consists of chemically modifying an antibody by adding a
fluorochrome without altering the specificity of the antibody
(Fig. 17.18). The targeted microorganisms are then detected
using an instrument that can detect fluorescent cells
(epifluorescence microscopy, flow cytometry, or solidphase cytometry). These techniques are mainly used in the
field of food microbiology, because antibodies are commercially available for most pathogenic microorganisms. For
taxonomic purposes, these probes are increasingly
substituted by DNA probes whose specificity is often better
and also because these probes can be defined for all
microorganisms, without the need for prior cultivation.
17.3.5.2 In Situ Hybridization with Fluorescent
Probes
In situ hybridization with fluorescent probes (“fluorescent in
situ hybridization” FISH) is a cytogenetic approach based on
the principle of complementary between nucleic acid chains
and allows the specific pairing of a labeled oligonucleotide
probe to an RNA or DNA sequence, ribosomes, or
messengers. So it is an approach conceptually close to the
gene amplification by PCR followed by sequencing, with the
advantage of a possible eye control by microscopy; but since
it is a short sequence, the disadvantage is that the resolution
is lower.
This technique was originally developed for the detection
of mutations in eukaryotic genomes after spreading of the
chromosome on a glass slide. Subsequently adapted to the
bacterial cells, the technique allows to observe cells without
culturing.
Bacterial cells are surrounded by a lipid membrane and a
cellular wall consisting of sugar (peptidoglycan) and
proteins that can be made porous to allow the passage of
small oligonucleotide sequences but not the ribosomes,
much bigger, which therefore remain inside cells.
Ribosomes are made of RNA sequences. Their number
varies from 20,000 per active cell in E. coli to 0 in starved
cells. The effectiveness of the FISH technique is
proportional to the number of ribosomes and ultimately to
the physiological state of the cell.
It is also possible to detect specific mRNA by in situ
hybridization (Pernthaler and Amann 2004). In the latter
case, the situation will depend on the level of expression of
the selected gene, considering that some genes do not have
enough expression to be detected by FISH. The probes are
oligonucleotide sequences of about twenty bases. It is necessary that they have a short size to allow their penetration
into the cells and also to have a melting temperature close to
the room temperature to protect the cell structures. The
sequences of the target group are aligned and analyzed, for
example, using the ARB software (Ludwig et al. 2004),
which allows to design an oligonucleotide sequence whose
specificity can be checked in the database. When possible,
mismatches are located at the center to increase the instability of hybrid and thus to reduce false positives. Cells undergo
different treatments before hybridization, starting with
diethylpyrocarbonate to inactivate intracellular RNases and
then with lysozyme (which catabolizes the peptidoglycan)
and proteinase K to permeabilize the cellular wall, then with
paraformaldehyde to preserve the subcellular structures. The
cells are incubated in a hybridization solution containing the
labeled oligonucleotides for a few hours and then washed to
remove probes that are unbound to their specific target. A
second nonspecific dye (i.e., DAPI) is used to stain all the
cells. Finally, the cells are observed under a common fluorescence microscope.
It is possible to use two primers in the same experiment to
determine, for example, the proportion of cells belonging to
two nested taxa, which is the case of cells belonging to the
Enterobacteriaceae and to the genus Erwinia. In this case,
two probes are used with different fluorochromes. It is also
possible to use two primers, one targeting ribosomes and the
other a functional gene in the same cell to link taxonomic
and functional informations. Finally, it is possible to have
two primers targeting two different taxa, such as one for E.
coli and one for Pseudomonas fluorescens when the objective is to quantify their co-occurrence in a complex environment. One of the most limiting problems in the use of FISH
is the electrostatic repulsion due to the negative charge of the
phosphodiester groups of the DNA that make some ribosome
regions inaccessible to the classic FISH method. It is then
possible to use, instead of DNA, APN molecules (or acid
peptido-nucleic) where sugar phosphates are replaced by
neutral links based on polyamide and following the
Watson–Crick matching rules, but which hybridize more
rapidly and more efficiently (Stender et al. 1999). Finally,
it is possible to use molecular beacons which operate on
the basis of FRET (fluorescence resonance energy transfer)
and are already used in quantitative PCR to reduce the
background noise (Lenaerts et al. 2007) due to nonspecific
hybridization.
780
F. Joux et al.
Level
17.3.5.1 Immunofluorescence
The immunoassay techniques are widely used in microbiology as a diagnostic tool. They can be used to search for a
pathogen, its identification, and counting (especially in
controlling the quality of the water). These techniques are
based on the use of specific antibodies to pathogens. There
are monoclonal antibodies (recognizes only one type of
epitope) which are very specific and reproducible and polyclonal antibodies (mixture of antibodies recognizing different epitopes on the same antigen) that are less specific.
Among the various immunoassays, immunofluorescence
consists of chemically modifying an antibody by adding a
fluorochrome without altering the specificity of the antibody
(Fig. 17.18). The targeted microorganisms are then detected
using an instrument that can detect fluorescent cells
(epifluorescence microscopy, flow cytometry, or solidphase cytometry). These techniques are mainly used in the
field of food microbiology, because antibodies are commercially available for most pathogenic microorganisms. For
taxonomic purposes, these probes are increasingly
substituted by DNA probes whose specificity is often better
and also because these probes can be defined for all
microorganisms, without the need for prior cultivation.
17.3.5.2 In Situ Hybridization with Fluorescent
Probes
In situ hybridization with fluorescent probes (“fluorescent in
situ hybridization” FISH) is a cytogenetic approach based on
the principle of complementary between nucleic acid chains
and allows the specific pairing of a labeled oligonucleotide
probe to an RNA or DNA sequence, ribosomes, or
messengers. So it is an approach conceptually close to the
gene amplification by PCR followed by sequencing, with the
advantage of a possible eye control by microscopy; but since
it is a short sequence, the disadvantage is that the resolution
is lower.
This technique was originally developed for the detection
of mutations in eukaryotic genomes after spreading of the
chromosome on a glass slide. Subsequently adapted to the
bacterial cells, the technique allows to observe cells without
culturing.
Bacterial cells are surrounded by a lipid membrane and a
cellular wall consisting of sugar (peptidoglycan) and
proteins that can be made porous to allow the passage of
small oligonucleotide sequences but not the ribosomes,
much bigger, which therefore remain inside cells.
Ribosomes are made of RNA sequences. Their number
varies from 20,000 per active cell in E. coli to 0 in starved
cells. The effectiveness of the FISH technique is
proportional to the number of ribosomes and ultimately to
the physiological state of the cell.
It is also possible to detect specific mRNA by in situ
hybridization (Pernthaler and Amann 2004). In the latter
case, the situation will depend on the level of expression of
the selected gene, considering that some genes do not have
enough expression to be detected by FISH. The probes are
oligonucleotide sequences of about twenty bases. It is necessary that they have a short size to allow their penetration
into the cells and also to have a melting temperature close to
the room temperature to protect the cell structures. The
sequences of the target group are aligned and analyzed, for
example, using the ARB software (Ludwig et al. 2004),
which allows to design an oligonucleotide sequence whose
specificity can be checked in the database. When possible,
mismatches are located at the center to increase the instability of hybrid and thus to reduce false positives. Cells undergo
different treatments before hybridization, starting with
diethylpyrocarbonate to inactivate intracellular RNases and
then with lysozyme (which catabolizes the peptidoglycan)
and proteinase K to permeabilize the cellular wall, then with
paraformaldehyde to preserve the subcellular structures. The
cells are incubated in a hybridization solution containing the
labeled oligonucleotides for a few hours and then washed to
remove probes that are unbound to their specific target. A
second nonspecific dye (i.e., DAPI) is used to stain all the
cells. Finally, the cells are observed under a common fluorescence microscope.
It is possible to use two primers in the same experiment to
determine, for example, the proportion of cells belonging to
two nested taxa, which is the case of cells belonging to the
Enterobacteriaceae and to the genus Erwinia. In this case,
two probes are used with different fluorochromes. It is also
possible to use two primers, one targeting ribosomes and the
other a functional gene in the same cell to link taxonomic
and functional informations. Finally, it is possible to have
two primers targeting two different taxa, such as one for E.
coli and one for Pseudomonas fluorescens when the objective is to quantify their co-occurrence in a complex environment. One of the most limiting problems in the use of FISH
is the electrostatic repulsion due to the negative charge of the
phosphodiester groups of the DNA that make some ribosome
regions inaccessible to the classic FISH method. It is then
possible to use, instead of DNA, APN molecules (or acid
peptido-nucleic) where sugar phosphates are replaced by
neutral links based on polyamide and following the
Watson–Crick matching rules, but which hybridize more
rapidly and more efficiently (Stender et al. 1999). Finally,
it is possible to use molecular beacons which operate on
the basis of FRET (fluorescence resonance energy transfer)
and are already used in quantitative PCR to reduce the
background noise (Lenaerts et al. 2007) due to nonspecific
hybridization.
780
F. Joux et al.