6. Dilute the hemolymph with an equal volume of sterilized
water.
7. Streak on NBTA/TSA medium.
8. Isolate bacteria.
9. Select blue colonies (adsorption of dyes) or green colonies.
10. Analyze for the phenotypic characteristics.
11. Use nutrient broth to culture the isolated bacteria at a temperature of 25
C for 48 h.
12. Store at À80
C in nutrient broth containing 15% glycerol
(vol/vol).
13. Maintain until further use (Fig. 1).
2 Morphological Characterization
Identification of the symbiotic bacteria helps to verify that the
isolates belonged to the genera Xenorhabdus and Photorhabdus.
Morphological and biochemical identification of bacterial isolates
are evaluated according to the methods described by Akhurst and
Boemare [201].
2.1 Diagnostic
Characteristics
2.1.1 Characters of the
Genus Xenorhabdus
l
They are Gram-negative bacteria and have asporogenous rods.
l
Cell size is highly inconstant within and between cultures, ranging from 0.3–2.0 μm to 2.0–10 μm and rarely with filaments
15–50 μm in length.
l
Spheroplasts look like in the last third of exponential
development.
l
Birefringent inclusions happen within cells.
l
Motile, peritrichously flagellated.
l
They are facultative organisms and anaerobic and can have respiratory and fermentative metabolism.
l
Negative for oxidase, o-nitrophenyl p-D-galactopyranoside
(ONPG), p-nitrophenyl p-D-galactopyranoside (PNPG),
Voges–Proskauer, arginine dihydrolase, lysine, and ornithine
decarboxylase tests.
l
Most strains do not reduce nitrate or yield indole.
l
Can hydrolyze casein, liquefaction of gelatin occurs. Glycogen
and starch are not hydrolyzed. Positive for DNAase and lipase
(Tweens 40 and 60).
l
Assimilates N-acetylglucosamine, D-fructose, fumarate, glucose,
L-glutamate, D,L-glycerate, glycerol, L-malate, maltose, mannose, L-proline, pyruvate, succinate, and, most strains, L-alanine
in API LRA medium.
116
Laboratory Techniques for Symbiotic Bacteria
water.
7. Streak on NBTA/TSA medium.
8. Isolate bacteria.
9. Select blue colonies (adsorption of dyes) or green colonies.
10. Analyze for the phenotypic characteristics.
11. Use nutrient broth to culture the isolated bacteria at a temperature of 25
C for 48 h.
12. Store at À80
C in nutrient broth containing 15% glycerol
(vol/vol).
13. Maintain until further use (Fig. 1).
2 Morphological Characterization
Identification of the symbiotic bacteria helps to verify that the
isolates belonged to the genera Xenorhabdus and Photorhabdus.
Morphological and biochemical identification of bacterial isolates
are evaluated according to the methods described by Akhurst and
Boemare [201].
2.1 Diagnostic
Characteristics
2.1.1 Characters of the
Genus Xenorhabdus
l
They are Gram-negative bacteria and have asporogenous rods.
l
Cell size is highly inconstant within and between cultures, ranging from 0.3–2.0 μm to 2.0–10 μm and rarely with filaments
15–50 μm in length.
l
Spheroplasts look like in the last third of exponential
development.
l
Birefringent inclusions happen within cells.
l
Motile, peritrichously flagellated.
l
They are facultative organisms and anaerobic and can have respiratory and fermentative metabolism.
l
Negative for oxidase, o-nitrophenyl p-D-galactopyranoside
(ONPG), p-nitrophenyl p-D-galactopyranoside (PNPG),
Voges–Proskauer, arginine dihydrolase, lysine, and ornithine
decarboxylase tests.
l
Most strains do not reduce nitrate or yield indole.
l
Can hydrolyze casein, liquefaction of gelatin occurs. Glycogen
and starch are not hydrolyzed. Positive for DNAase and lipase
(Tweens 40 and 60).
l
Assimilates N-acetylglucosamine, D-fructose, fumarate, glucose,
L-glutamate, D,L-glycerate, glycerol, L-malate, maltose, mannose, L-proline, pyruvate, succinate, and, most strains, L-alanine
in API LRA medium.
116
Laboratory Techniques for Symbiotic Bacteria