l
L-Fucose, D,L-lactate, or mannitol not used, protoplasmic inclusions produced below par.
l
Grows well at 37–38
C.
2.2 Culture
Characteristics
2.2.1 NBTA Media
1. Streak each isolate on petri plate containing NBTA medium
and incubate for 4 days.
2. Observe the colonies for absorption of bromothymol blue dye
from the media and formation of typical bluish green colonies.
2.2.2 MacConkey Agar
1. Streak each isolate on petri plate containing MacConkey agar
media and incubate for 5 days.
2. Observe the symbiotic bacterial isolates for absorption of neutral red from MacConkey agar media and formation of characteristic brown or red colonies.
2.2.3 Nutrient Agar
1. Streak each isolate on petri plate containing nutrient agar
medium and incubate for 2 days.
2. Observe the symbiotic bacterial isolates for formation of characteristic buff or cream colored colonies on nutrient agar
media.
2.3 Measurement of
Growth
Cell biomass of symbiotic bacteria—Xenorhabdus and Photorhabdus—is carried out by measuring the absorbance of cultures at
600 nm using a spectrophotometer.
2.3.1 Materials Required
l
Nutrient broth.
l
Sterile petri plates.
l
Micropipettes.
l
Polystyrene cuvette.
l
Conical flask.
l
Sterile tips.
l
Culture—overnight culture of Xenorhbdus and Photorhabdus.
l
Spectrophotometer.
2.3.2 Procedure
1. Inoculate an isolate colony of the symbiotic bacteria into 15 ml
nutrient broth.
2. Keep for overnight and incubate at 37
C in shaking incubator
at 220 rpm.
3. Dispense 1 ml of each overnight culture to a polystyrene
cuvette.
4. Dispense 1 ml of fresh media to a second cuvette (use zero to
the spectrophotometer) as control.
120
Laboratory Techniques for Symbiotic Bacteria
L-Fucose, D,L-lactate, or mannitol not used, protoplasmic inclusions produced below par.
l
Grows well at 37–38
C.
2.2 Culture
Characteristics
2.2.1 NBTA Media
1. Streak each isolate on petri plate containing NBTA medium
and incubate for 4 days.
2. Observe the colonies for absorption of bromothymol blue dye
from the media and formation of typical bluish green colonies.
2.2.2 MacConkey Agar
1. Streak each isolate on petri plate containing MacConkey agar
media and incubate for 5 days.
2. Observe the symbiotic bacterial isolates for absorption of neutral red from MacConkey agar media and formation of characteristic brown or red colonies.
2.2.3 Nutrient Agar
1. Streak each isolate on petri plate containing nutrient agar
medium and incubate for 2 days.
2. Observe the symbiotic bacterial isolates for formation of characteristic buff or cream colored colonies on nutrient agar
media.
2.3 Measurement of
Growth
Cell biomass of symbiotic bacteria—Xenorhabdus and Photorhabdus—is carried out by measuring the absorbance of cultures at
600 nm using a spectrophotometer.
2.3.1 Materials Required
l
Nutrient broth.
l
Sterile petri plates.
l
Micropipettes.
l
Polystyrene cuvette.
l
Conical flask.
l
Sterile tips.
l
Culture—overnight culture of Xenorhbdus and Photorhabdus.
l
Spectrophotometer.
2.3.2 Procedure
1. Inoculate an isolate colony of the symbiotic bacteria into 15 ml
nutrient broth.
2. Keep for overnight and incubate at 37
C in shaking incubator
at 220 rpm.
3. Dispense 1 ml of each overnight culture to a polystyrene
cuvette.
4. Dispense 1 ml of fresh media to a second cuvette (use zero to
the spectrophotometer) as control.
120
Laboratory Techniques for Symbiotic Bacteria