l
Symbiotic bacteria.
l
Bacterial suspension.
l
Distilled water.
l
24-Well plate.
l
Fine sterile toothpick.
6.5.2 Procedure
1. First restore the larvae in dechlorinated water for 1 day previous to the test.
2. Use symbiotic bacteria isolate (Xenorhabdus and Photorhabdus)
and distilled water as negative controls.
3. Take approximately 30 larvae (10 larvae/well) and transfer to
three wells of a 24-well microtiter plate.
4. Add 2 ml bacterial suspension of each isolate containing
10
8 CFU/ml into each well.
5. Keep the plates at room temperature.
6. Monitor the mortality of the mosquito larvae daily for 4 days at
24, 48, 72, and 96 h exposure to the bacterial suspensions.
7. Consider the larvae dead if larvae show no movement response
after teasing with a fine sterile toothpick.
8. Analyze the mortality rate (%) of the larvae in the control
groups and in the treatment group (each bacterial isolate)
using SPSS version 17 (Fisher’s exact test; P < 0.05).
9. Perform Fisher’s exact test to determine the difference in mortality rates between the fed and unfed groups.
6.6 Insecticidal
Toxin Activity:
Larvicidal Activity
6.6.1 Materials Required
l
Larva (insect).
l
Symbiotic bacteria.
l
Phosphate buffer.
l
NBTA agar plate.
6.6.2 Procedure
1. Larvicidal activity test is carried out to identify the insecticidal
value of the bacterial species [205].
2. Take two different groups, each containing five larvae to identify the insecticidal value by injecting the X. poinarii bacterial
cells and the cell supernatant.
3. Centrifuge the culture to separate the cell supernatant from
bacterial cells.
4. Suspend the separated cells in 1Â phosphate-buffered saline of
pH 7.4 for further injection into larva.
5. Inject the third instar larva and keep on ice for 10–20 s to
anesthetize the larva for injection.
142
Laboratory Techniques for Symbiotic Bacteria
Symbiotic bacteria.
l
Bacterial suspension.
l
Distilled water.
l
24-Well plate.
l
Fine sterile toothpick.
6.5.2 Procedure
1. First restore the larvae in dechlorinated water for 1 day previous to the test.
2. Use symbiotic bacteria isolate (Xenorhabdus and Photorhabdus)
and distilled water as negative controls.
3. Take approximately 30 larvae (10 larvae/well) and transfer to
three wells of a 24-well microtiter plate.
4. Add 2 ml bacterial suspension of each isolate containing
10
8 CFU/ml into each well.
5. Keep the plates at room temperature.
6. Monitor the mortality of the mosquito larvae daily for 4 days at
24, 48, 72, and 96 h exposure to the bacterial suspensions.
7. Consider the larvae dead if larvae show no movement response
after teasing with a fine sterile toothpick.
8. Analyze the mortality rate (%) of the larvae in the control
groups and in the treatment group (each bacterial isolate)
using SPSS version 17 (Fisher’s exact test; P < 0.05).
9. Perform Fisher’s exact test to determine the difference in mortality rates between the fed and unfed groups.
6.6 Insecticidal
Toxin Activity:
Larvicidal Activity
6.6.1 Materials Required
l
Larva (insect).
l
Symbiotic bacteria.
l
Phosphate buffer.
l
NBTA agar plate.
6.6.2 Procedure
1. Larvicidal activity test is carried out to identify the insecticidal
value of the bacterial species [205].
2. Take two different groups, each containing five larvae to identify the insecticidal value by injecting the X. poinarii bacterial
cells and the cell supernatant.
3. Centrifuge the culture to separate the cell supernatant from
bacterial cells.
4. Suspend the separated cells in 1Â phosphate-buffered saline of
pH 7.4 for further injection into larva.
5. Inject the third instar larva and keep on ice for 10–20 s to
anesthetize the larva for injection.
142
Laboratory Techniques for Symbiotic Bacteria