Comparison of different strategies for isolation and preliminary identification of Azotobacter from soil samples

Due to the role of azotobacteria in nitrogen fixation and to their potential biotechnological applications, there is some challenge in developing an effective strategy for the selective isolation of these micro-organisms from soil. One hundred ninety-six Gram-negative strains were isolated from 35 soils sampled in central Italy, by using and comparing three different methods. The screening of soil samples by means of soil paste–plate method combined with isolation on mannitol-agar proved to be the best strategy in terms of reliability and selectivity. Moreover, preliminary recognition of free-living nitrogen-fixing isolates on differential LG medium revealed to be extremely accurate, since the majority of the isolates with Azotobacter-like morphology on such a medium were presumptively identified as members of the family Azotobacteraceae, by means of amplified ribosomal DNA restriction analysis.