6. Then prepare bacterial cells and the cell supernatants and inject
separately into the larva via the left proleg through the skin
without affecting the integrity of the larva. Larva injected with
phosphate buffer is considered as negative control.
7. After injection observe the larvae for morphological changes
for 48 h.
8. At the end of 48 h, count the dead larvae and calculate the
mortality rate. If the larvae turn to black color, it denotes
infection and their immobility denotes their death.
9. Confirm larvae death by the bacteria on NBTA agar
plate [206].
(a) Plate the hemocoel collected from the dead larvae on NBTA
agar plates (nutrient agar containing bromophenol blue
25 mg/L, triphenyl tetrazolium chloride 4 mg/L).
(b) Xenorhabdus bacterial cells produce blue color colonies (catalase test—negative).
(c) Photorhabdus bacterial cells produce green color colonies (catalase test—positive).
Biological Assay
143