I am currently isolating diazotrophic endophytes from rice cultivars in North East India using nitrogen- free semi- solid media with bromothymol blue as a pH indicator in glass vials. Despite slight turbidity after inoculation, there has been no pellicle formation or colour change even after four days. The bacterial growth seems to be concentrated below the surface of the medium.

Given these observations, what is the recommended method for subculturing these diazotrophic endophytes? Should the subculturing be done on solid or semi solid media, and what protocols should be followed to preserve the anaerobic conditions during transfer? Additionally, are there any other techniques or modifications you would recommend that could enhance the success of isolating and characterizing these bacteria in my future research?

Any guidance on optimizing these procedures would be greatly appreciated .