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Abstract
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 mixed 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-borne, volatile compounds which support
growth of heterotrophs. The sealing of the cultures reduced the number of heterotrophs
by a factor of 10, thus grossly increasing the chances of obtaining pure AOB cultures.
Another important factor is to use actively growing 'late log' cultures during the
final isolation step. This was achieved by adjusting the buffer capacity 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 isolation 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 unsuccessful, despite the presence of AOB in the primary extinction dilution
cultures. 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 from the cultures with urea
as the main ammonia source. The identifications of all isolates are based on morphology
and 16S rDNA sequences.