Quick isolation and characterization of novel Bacillus<br>thuringiensis strains from mosquito breeding sites in Malaysia

Abstract

Bacillus thuringiensis (Bt.) is an insecticidal, gram-positive and spore forming bacterium which has capabilities infecting the insect vectors belonging to the order Diptera (mosquitoes) especially dengue causing Aedes species. Bacterial strains with combination of Cry and Cyt proteins are useful in controlling and management of resistance in mosquito population. The objective of this research is to isolate and characterize Bacillus thuringiensis from mosquito breeding sites and also study expression of Crystal (Cry) and Cytolytic (Cyt) proteins of these isolates by SDS-PAGE analysis. In this study, 50 samples were collected from mosquito breeding sites which include soil, larvae and water. Samples were processed by initially giving heat shock treatment, serial dilution and followed by plating onto culture medium. Initial selection was based on colony morphology, gram staining, phase contrast microscopy and coomassie blue staining. Total of 144 isolates was identified based on colony morphology and this number was reduced to 69 after gram staining. Total of 15 isolates were confirmed to be Bacillus after phase contrast microscopy. Through coomassie blue staining, a blue cap-like appearance in the sporulation phase and darkly stained oval shaped candidate in autolysis phase can be observed. Growth curve analysis was done with one chosen isolate to determine the generation time. Further protein level characterization based on the SDS-PAGE gel showed the banding patterns range from 65-274 kDa (Cry proteins) and 21-25 kDa (Cyt proteins).