5. Dry them with liquid CO 2 in a critical point mode, mount on
SEM stubs, coated with gold.
6. Scan at 15 kV accelerating voltage.
Note: Efficacious preparation of nematodes for SEM involves of
several steps. Killing, fixing, and drying are possibly the most
important. Extensively used killing and fixation techniques follow
two generalized patterns. Rapid fixation highlights the necessity to
hastily arrest cellular activity and thereby avoid the degradation of
tissues by autolytic activities. Sequential fixation highlights the
effect of the concentration of the fixative on tonicity of the buffer.
Differences in tonicity between the buffer and tissue can cause
inflammation or shrinkage.
OR
4.2.3 Materials Required
l
TAF.
l
Glycerol.
l
Nail varnish.
4.2.4 Procedure
1. Fix the nematode (IJs, male and female) in TAF and saturate
with glycerol.
2. Drain off excess glycerol by warming faintly and lifting the
nematode from the warm liquid.
3. Place the nematode on a tiny spot of fresh nail varnish, durofix,
or a comparable quick drying adhesive on cover slip.
4. Scrutinize the specimen before the adhesive hardens, under the
highest power dry objective of an optical microscope, for possible distortion and check that the alignment is correct.
5. Stick the cover slip with the specimen on to one of the mounting stubs provided for scanning microscope.
4.2.5
Cross-Breeding Test
To substantiate the identification of Steinernema or Heterorhabditis
isolates, cross-breeding tests are conducted between the isolate
under analysis, and the species are recognized with the help of
hemolymph hanging drop technique [168].
Cross-hybridization assays have been used as an additional tool
for diagnosis of EPNs. The technique provides evidence to support
the “biological species concept,” which describes species in terms of
interbreeding Mayr [169]. According to this concept, “species are
groups of inter-reproducing natural populations that are isolated
reproductively from other similar groups.”
If hybridization assays show there is no offspring as a result of
the crosses between two isolates, then the isolate which species was
identified is being verified is established as a new species. It is
recommended to consider two to four other species [usually
Morphological Characterization
83
SEM stubs, coated with gold.
6. Scan at 15 kV accelerating voltage.
Note: Efficacious preparation of nematodes for SEM involves of
several steps. Killing, fixing, and drying are possibly the most
important. Extensively used killing and fixation techniques follow
two generalized patterns. Rapid fixation highlights the necessity to
hastily arrest cellular activity and thereby avoid the degradation of
tissues by autolytic activities. Sequential fixation highlights the
effect of the concentration of the fixative on tonicity of the buffer.
Differences in tonicity between the buffer and tissue can cause
inflammation or shrinkage.
OR
4.2.3 Materials Required
l
TAF.
l
Glycerol.
l
Nail varnish.
4.2.4 Procedure
1. Fix the nematode (IJs, male and female) in TAF and saturate
with glycerol.
2. Drain off excess glycerol by warming faintly and lifting the
nematode from the warm liquid.
3. Place the nematode on a tiny spot of fresh nail varnish, durofix,
or a comparable quick drying adhesive on cover slip.
4. Scrutinize the specimen before the adhesive hardens, under the
highest power dry objective of an optical microscope, for possible distortion and check that the alignment is correct.
5. Stick the cover slip with the specimen on to one of the mounting stubs provided for scanning microscope.
4.2.5
Cross-Breeding Test
To substantiate the identification of Steinernema or Heterorhabditis
isolates, cross-breeding tests are conducted between the isolate
under analysis, and the species are recognized with the help of
hemolymph hanging drop technique [168].
Cross-hybridization assays have been used as an additional tool
for diagnosis of EPNs. The technique provides evidence to support
the “biological species concept,” which describes species in terms of
interbreeding Mayr [169]. According to this concept, “species are
groups of inter-reproducing natural populations that are isolated
reproductively from other similar groups.”
If hybridization assays show there is no offspring as a result of
the crosses between two isolates, then the isolate which species was
identified is being verified is established as a new species. It is
recommended to consider two to four other species [usually
Morphological Characterization
83