Isolation of Azospirillum spp. from natural soils by immunomagnetic separation

Abstract An immunomagnetic separation (IMS) technique was developed to isolate the nitrogen-fixing bacterium, Azospirillum , from soil. Samples were reacted with polyclonal antibodies (pAb) raised in rabbits against whole-cell antigens of a mixture of five strains of Azospirillum brasilense . Following reaction with pAb, samples were treated with immunomagnetic beads (IMB: magnetic beads coated with anti-rabbit IgG antibodies), to allow capture of antibody-coated Azospirillum cells. The IMB were trapped by means of a magnet to permit washing for removal of unbound bacteria and were plated onto Congo Red agar (CRA) to allow growth of azospirilla. The method was optimized by incubating and washing samples in a 10 ml glass test-tube instead of a 1 ml plastic Eppendorf tube and by incubating the CRA plates in plastic bags to control the composition of the gas phase and moisture. Azospirillum spp. were isolated from the rhizospheres of wheat plants grown in soil from seven different regions in New South Wales (NSW). The IMS procedure is more efficient than culturing in nitrogen-free semi-solid medium (Nfb) for detecting and counting azospirilla in natural soils.