The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of β-galactosidase activity constitutively. Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of β-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to β-galactosidase activity. The method was found to be sensitive enough to determine 1.7 × 10(6) CFU mL(-1) in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.