Quick isolation and characterization for the confirmation of a novel Bacillus thuringiensis strains from chicken manure samples

Abstract

Bacillus thuringiensis (Berliner) (Bt) based pesticides has become the greatest successes among the microbial pesticides. This research was focused on the isolation and characterization of Bt from chicken manure samples as a comprehensive effort to contribute to the establishment of native Bt strains from various types of habitats in Malaysia. Initially, selection was done mainly based on colony morphology, Gram staining and phase contrast microscopy. Total of 85 colonies were identified and isolated based on colony morphology, in which off-white, matte and creamy appearance showed the characteristics of the genus Bacillus. The number reduced to 35 colonies after Gram staining was done. Subsequently, total of 8 isolates were confirmed to be B. thuringiensis after Coomassie Brilliant Blue staining was carried out. The presence of B. thuringiensis strain was confirmed when a blue cap-like structure and a darkly stained oval structure, insecticidal crystal proteins (ICP) appeared in the sporulation phase. An isolate was chosen out of the 8 isolates in order to generate the growth curve of B. thuringiensis bacteria. The similarity of the aspects above confirms that the selected isolate was a Bt strain. Isolates were further characterized by SDS-PAGE analysis to determine the molecular masses of the Bt proteins. These isolates produced two major ranges of polypeptides, the Cry protein which corresponds to a range of 70-150kDa and the Cyt protein profile which corresponds to the range of 18-40kDa. The protein profile obtained correlates the strains to the typical Bt strain. A total of 6 isolate yielded SDS-PAGE products that demonstrated major protein bands of approximately 135kDa and 140kDa, possibly encoded by the genes cryI and cryIV, respectively.