An evaluated improvement of the extinction dilution method for isolation of ammonia-oxidizing bacteria

An improved method for isolation of ammonia-oxidizing bacteria (AOB) by the extinction dilution technique is described. It is important to prevent the growth of heterotrophic organisms, which may easily outnumber the AOB in m ixed cultures. This was achieved by careful elimination of C sources in the medium and by sealing the cultures from contact with the atmosphere, thus excluding air-bome, volatile compounds which support growth of heterotrophs . The sealing of the cultures reduced the number of heterotrophs by a facto r of 10, thus grossly increasing the chances of obtaining pure AOB cultures . Another important factor is to use actively growing 'late log' cultures d uring the final isolation step. This was achieved by adjusting the buffer c apacity to ensure a clearly visible pH indicator shift at a stage when one- third to one-half of the ammonia had been oxidized. By this improved isolat ion procedure, AOB were isolated from three different locations: an arable soil, a lead-contaminated soil and an animal house. For an unknown reason, several attempts to isolate pure cultures from a forest soil were unsuccess ful, despite the presence of AOB in the primary extinction dilution culture s. The isolates from soils were all Nitrosospira spp. For isolation of AOB from the animal house, two growth media were used, one containing ammonium sulfate, and one containing urea. From the cultures with ammonium sulfate, Nitrosomonas spp. were isolated, whereas Nitrosospira spp. were isolated fr om the cultures with urea as the main ammonia source. The identifications o f all isolates are based on morphology and 16S rDNA sequences. (C) 1999 Els evier Science B.V. All rights reserved.