3.16.1 Materials
Required
l
Reagent: API 50 CHL medium.
l
Others: pH indicator.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.16.2 Procedure
1. Inoculate the fermentation tests with API 50 CHL medium
which rehydrates the substrates.
2. Use sterile water to provide moisture.
3. Incubate at 28
C for 24 h.
4. Fermentation reaction is exposed by a color change in the tube,
triggered by the anaerobic production of acid.
5. Read the color changes using the pH indicator existing in the
chosen medium.
6. Use the first tube which blank without any active ingredient as
a negative control (Fig. 2).
4 Molecular Characterization
The biochemical and molecular methodologies are very beneficial
for segregating the species, subspecies, or strains, providing insights
of their phylogenies and evolution, identification of species and
strains. Numerous DNA-based molecular approaches have been
used to classify, diagnose the species, and evaluate the phylogenetic
relationships of EPN associated symbiotic bacteria. The most comprehensively applied method is sequencing of specific amplified
fragment of ribosomal DNA. Currently, DNA sequence analysis is
being functional widely in bacterial systematics, as it produces more
evidence about variations at intra- as well as inter-specific level, and
for evaluating their phylogenetic relationships. Molecular methodologies have transfigured the EPN systematic. The taxonomic
level at which diverse degrees of sequence divergence function
necessitates an in-depth understanding of inter- and intra-specific
variations in DNA sequencing, which is still a major constraint in
sole dependence on this approach in taxonomy.
4.1 Isolation
of Genomic DNA [203]
The isolation of DNA from bacteria is a comparatively trouble-free
process. The organism to be utilized should be grown in a
promising medium at an ideal temperature and should be harvested
in late log to early stationary phase for increased yield. The genomic
DNA isolation needs to separate total DNA from RNA, protein,
lipid, etc. Primarily, the cell membranes must be disrupted in order
to release the DNA in the extraction buffer. SDS (sodium dodecyl
sulfate) is used to damage the cell membrane. Once cell is disrupted, the endogenous nucleases have a tendency to cause extensive hydrolysis. DNA can be safeguarded from endogenous
nucleases by chelating Mg
2++ ions using EDTA. Nucleoprotein
Molecular Characterization
131
Required
l
Reagent: API 50 CHL medium.
l
Others: pH indicator.
l
Organisms: Bacteria Xenorhabdus and Photorhabdus.
3.16.2 Procedure
1. Inoculate the fermentation tests with API 50 CHL medium
which rehydrates the substrates.
2. Use sterile water to provide moisture.
3. Incubate at 28
C for 24 h.
4. Fermentation reaction is exposed by a color change in the tube,
triggered by the anaerobic production of acid.
5. Read the color changes using the pH indicator existing in the
chosen medium.
6. Use the first tube which blank without any active ingredient as
a negative control (Fig. 2).
4 Molecular Characterization
The biochemical and molecular methodologies are very beneficial
for segregating the species, subspecies, or strains, providing insights
of their phylogenies and evolution, identification of species and
strains. Numerous DNA-based molecular approaches have been
used to classify, diagnose the species, and evaluate the phylogenetic
relationships of EPN associated symbiotic bacteria. The most comprehensively applied method is sequencing of specific amplified
fragment of ribosomal DNA. Currently, DNA sequence analysis is
being functional widely in bacterial systematics, as it produces more
evidence about variations at intra- as well as inter-specific level, and
for evaluating their phylogenetic relationships. Molecular methodologies have transfigured the EPN systematic. The taxonomic
level at which diverse degrees of sequence divergence function
necessitates an in-depth understanding of inter- and intra-specific
variations in DNA sequencing, which is still a major constraint in
sole dependence on this approach in taxonomy.
4.1 Isolation
of Genomic DNA [203]
The isolation of DNA from bacteria is a comparatively trouble-free
process. The organism to be utilized should be grown in a
promising medium at an ideal temperature and should be harvested
in late log to early stationary phase for increased yield. The genomic
DNA isolation needs to separate total DNA from RNA, protein,
lipid, etc. Primarily, the cell membranes must be disrupted in order
to release the DNA in the extraction buffer. SDS (sodium dodecyl
sulfate) is used to damage the cell membrane. Once cell is disrupted, the endogenous nucleases have a tendency to cause extensive hydrolysis. DNA can be safeguarded from endogenous
nucleases by chelating Mg
2++ ions using EDTA. Nucleoprotein
Molecular Characterization
131