6.1 Preparation of
Whole Cell Suspension
and Cell-Free
Supernatant
6.1.1 Materials Required
l
Symbiotic bacterial stock (Xenorhabdus/Photorhabdus).
l
NBTA medium (nutrient bromothymol blue agar).
l
LB (Luria–Bertani) broth.
l
Phosphate-buffered saline solution.
l
Paper disc.
l
Antibiotic disc.
l
MHA (Mueller–Hinton agar) plate.
l
Ethyl acetate.
l
DMSO (dimethyl sulfoxide).
l
MH (Mueller–Hinton) broth.
l
Test insect larvae.
l
Ruler.
l
Toothpick.
6.1.2 Procedure
1. Streak a symbiotic bacterial stock (Xenorhabdus/Photorhabdus)
on NBTA medium.
2. Incubate at 28
C without light for 4 days.
3. Transfer individual colony and grow in 25 ml LB broth.
4. Incubate in shaker at 180 rpm for 24 h.
5. Adjust the concentration of entire cell suspension to 10
7 –
10
8 CFU/ml using phosphate buffer saline solution.
6. Take 1 ml of whole cell suspension.
7. Centrifuge at 12,000 rpm for 5 min.
8. Collect the supernatant.
9. Filter the supernatant using a 0.22 μm filter. The flow-through
is used as the cell-free supernatant.
6.2 Antimicrobial
Activity
To monitor Xenorhabdus and Photorhabdus against drug-resistant
bacteria:
1. Place 20 μl of whole-cell suspension and of cell-free supernatant onto a sterile 6 mm paper disc.
2. Allow the disc to dry for 3–5 min.
3. Relocate the dried disc onto the surface of MHA plates with
drug-resistant bacteria. Use commercial antibiotic disc as positive control.
4. Incubate plates at 37
C for 24 h.
5. Measure the zone of inhibition.
6. A clear zone from the edge of growth colony of isolates indicate
as positive.
140
Laboratory Techniques for Symbiotic Bacteria