Chapter 9
Laboratory Techniques for Symbiotic Bacteria
Abstract
Xenorhabdus and Photorhabdus belonging to the family of Enterobacteriaceae are symbionts thriving in
commensalism with Steinernema and Heterorhabditis, respectively. Though most of the insect symbionts
are endocytobionts, it is hard to cultivate them. Isolation of bacteria from nematodes can be done by release
of bacteria from intestine or whole nematodes and through insect hemolymph (G. mellonella). Identification of the symbiotic bacteria helps to verify that the isolates belonged to the genera Xenorhabdus and
Photorhabdus. Morphological, biochemical and molecular methodologies are assessed for characterizing the
bacterial isolates. Novel bioactive compound of symbiotic bacteria is evaluated by extraction, purification
and structural identification. The efficacy, virulence and pathogenicity of symbiotic bacteria are analyzed
under laboratory conditions.
Keywords Isolation, Xenorhabdus, Photorhabdus, Hemolymph, Antimicrobial, Mosquitocidal,
Larvicidal
1 Isolation of Symbiotic Bacteria
The genera Photorhabdus and Xenorhabdus belong to the family of
Enterobacteriaceae that are generally the intestinal bacterial symbionts thriving in commensalism with entomopathogenic nematodes (EPNs) of the genera Heterorhabditis and Steinernema,
respectively. Nevertheless, though most of the insect symbionts
are endocytobionts, it is hard to cultivate them. Xenorhabdus and
Photorhabdus have the distinctive benefit of cultivability on typical
bacteriological media.
1.1 Direct Method:
Isolation of Bacteria
from Nematodes
1.1.1 Materials Required
l
Chemicals: Use any one sterilization agents for surface sterilization of nematodes or insect cadaver: sodium hypochlorite/merthiolate/thimerosal/hyamine/benzethonium chloride.
l
Media: Yeast salt broth (YS)/Luria–Bertani (LB) broth/nutrient broth (NB), sterile Ringer’s solution, nutrient bromothymol
blue agar (NBTA) media, MacConkey Agar (MAC) media.
l
Organisms: IJ suspension, G. mellonella larvae, S. litura larvae.
l
Glasswares: Microcentrifuge tube, petri dishes, beaker, pipette,
glass tissue homogenizer.
S. Sivaramakrishnan and M. Razia, Entomopathogenic Nematodes and Their Symbiotic Bacteria, Springer Protocols Handbooks,
https://doi.org/10.1007/978-1-0716-1445-7_9,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021
113
Laboratory Techniques for Symbiotic Bacteria
Abstract
Xenorhabdus and Photorhabdus belonging to the family of Enterobacteriaceae are symbionts thriving in
commensalism with Steinernema and Heterorhabditis, respectively. Though most of the insect symbionts
are endocytobionts, it is hard to cultivate them. Isolation of bacteria from nematodes can be done by release
of bacteria from intestine or whole nematodes and through insect hemolymph (G. mellonella). Identification of the symbiotic bacteria helps to verify that the isolates belonged to the genera Xenorhabdus and
Photorhabdus. Morphological, biochemical and molecular methodologies are assessed for characterizing the
bacterial isolates. Novel bioactive compound of symbiotic bacteria is evaluated by extraction, purification
and structural identification. The efficacy, virulence and pathogenicity of symbiotic bacteria are analyzed
under laboratory conditions.
Keywords Isolation, Xenorhabdus, Photorhabdus, Hemolymph, Antimicrobial, Mosquitocidal,
Larvicidal
1 Isolation of Symbiotic Bacteria
The genera Photorhabdus and Xenorhabdus belong to the family of
Enterobacteriaceae that are generally the intestinal bacterial symbionts thriving in commensalism with entomopathogenic nematodes (EPNs) of the genera Heterorhabditis and Steinernema,
respectively. Nevertheless, though most of the insect symbionts
are endocytobionts, it is hard to cultivate them. Xenorhabdus and
Photorhabdus have the distinctive benefit of cultivability on typical
bacteriological media.
1.1 Direct Method:
Isolation of Bacteria
from Nematodes
1.1.1 Materials Required
l
Chemicals: Use any one sterilization agents for surface sterilization of nematodes or insect cadaver: sodium hypochlorite/merthiolate/thimerosal/hyamine/benzethonium chloride.
l
Media: Yeast salt broth (YS)/Luria–Bertani (LB) broth/nutrient broth (NB), sterile Ringer’s solution, nutrient bromothymol
blue agar (NBTA) media, MacConkey Agar (MAC) media.
l
Organisms: IJ suspension, G. mellonella larvae, S. litura larvae.
l
Glasswares: Microcentrifuge tube, petri dishes, beaker, pipette,
glass tissue homogenizer.
S. Sivaramakrishnan and M. Razia, Entomopathogenic Nematodes and Their Symbiotic Bacteria, Springer Protocols Handbooks,
https://doi.org/10.1007/978-1-0716-1445-7_9,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021
113