l
Apparatus: Stereo zoom microscope or phase contrast microscope, incubator.
1.1.2 Procedure
Sterilization of Nematode
Surface
See Table 1.
1.1.3 Procedure
1. Use fresh infective juveniles (IJs) (20 IJs/0.5 ml) for isolation
purpose.
2. Take 100 IJs/ml in a beaker.
3. Allow it for surface clean in 1% sodium hypochlorite for
10 min.
4. Use sterile distilled water to rinse IJs thrice in order to remove
traces of sodium hypochlorite.
5. Visualize under a microscope to confirm that the cuticle surface
is free of bacteria.
6. Later, rinse nematodes twice with sterile Ringer’s solution.
Extraction of Symbiotic
Bacteria
7. Suspend the surface-sterilized IJs in yeast salt broth (YS) or
Luria–Bertani (LB) broth or nutrient broth (NB) in a 1.5-ml
microcentrifuge tube.
8. Mash the nematodes with a sterile tissue homongenizer.
9. Examine under stereo zoom microscope.
To Observe Release
of Bacteria from Intestine
or Whole Nematodes
10. Streak one loopful of the crushed suspension on the indicator
NBTA media plates.
11. Incubate the plates for 24—48 h at 28
C in the dark
condition.
12. Purify individual colonies by re-streaking them onto fresh
solid media.
Identification of Phase
Variants
13. Use NBTA and MAC agar media to ratify phase variation.
14. Phase I cells may seem as brick/dark red colonies on NBTA,
however as red colonies on MAC agar. Phase II cells may seem
yellow on NBTA and transparent on MAC agar.
1.2 Indirect Method
1.2.1 Materials Required
l
Chemicals: Surface sterilization agents (sodium hypochlorite),
glycerol, sterile distilled water.
l
Media: NBTA media/TSA (tryptic soy agar)/MacConkey/NA
plates, nutrient broth (NB), 24 h nematode-infected larvae
(Galleria).
l
Glass wares: Petri dishes, Whatman No.1 filter paper.
114
Laboratory Techniques for Symbiotic Bacteria
Apparatus: Stereo zoom microscope or phase contrast microscope, incubator.
1.1.2 Procedure
Sterilization of Nematode
Surface
See Table 1.
1.1.3 Procedure
1. Use fresh infective juveniles (IJs) (20 IJs/0.5 ml) for isolation
purpose.
2. Take 100 IJs/ml in a beaker.
3. Allow it for surface clean in 1% sodium hypochlorite for
10 min.
4. Use sterile distilled water to rinse IJs thrice in order to remove
traces of sodium hypochlorite.
5. Visualize under a microscope to confirm that the cuticle surface
is free of bacteria.
6. Later, rinse nematodes twice with sterile Ringer’s solution.
Extraction of Symbiotic
Bacteria
7. Suspend the surface-sterilized IJs in yeast salt broth (YS) or
Luria–Bertani (LB) broth or nutrient broth (NB) in a 1.5-ml
microcentrifuge tube.
8. Mash the nematodes with a sterile tissue homongenizer.
9. Examine under stereo zoom microscope.
To Observe Release
of Bacteria from Intestine
or Whole Nematodes
10. Streak one loopful of the crushed suspension on the indicator
NBTA media plates.
11. Incubate the plates for 24—48 h at 28
C in the dark
condition.
12. Purify individual colonies by re-streaking them onto fresh
solid media.
Identification of Phase
Variants
13. Use NBTA and MAC agar media to ratify phase variation.
14. Phase I cells may seem as brick/dark red colonies on NBTA,
however as red colonies on MAC agar. Phase II cells may seem
yellow on NBTA and transparent on MAC agar.
1.2 Indirect Method
1.2.1 Materials Required
l
Chemicals: Surface sterilization agents (sodium hypochlorite),
glycerol, sterile distilled water.
l
Media: NBTA media/TSA (tryptic soy agar)/MacConkey/NA
plates, nutrient broth (NB), 24 h nematode-infected larvae
(Galleria).
l
Glass wares: Petri dishes, Whatman No.1 filter paper.
114
Laboratory Techniques for Symbiotic Bacteria