Quantification of ammonia oxidizing bacteria in soil using microcolony technique combined with fluorescence in situ hybridization (MCFU–FISH)

Abstract

A new technique was developed for quantification of viable ammonia oxidizing bacteria (AOB) extracted from soil. Extracted bacteria were grown to microcolony-forming units (MCFU) on membrane filters and selectively stained with 16S rDNA probes using fluorescence in situ hybridization (FISH). The MCFU–FISH technique was used to enumerate a Nitrosomonas europaea population after inoculation to soil and the indigenous AOB populations in native and N-enriched soils. A low extraction efficiency (0.3–3%) of AOB in the native soil indicated predominance of a sessile AOB cell type. In contrast, N enrichments increased extractability of AOB from soil particles. Comparing all soils, a close correlation ( R 2=0.98) existed between extractable AOB numbers (MCFU–FISH counts) and potential ammonia oxidation activities covering more than three orders of magnitude, which indicated that MCFU–FISH counts reflected the active AOB extracted from the soil. By comparison, the correlation between AOB number estimated by the most-probable number (MPN) method and potential ammonia oxidation was considerably lower ( R 2=0.56), suggesting that MCFU–FISH counts better reflected the number of active AOB extracted from soil. Hence, the MCFU–FISH technique seems suitable for enumeration of viable and active AOB populations in soils, as long as the extractable population of microcolony-forming AOB is above a current minimum threshold for detection of 10 3–10 4 MCFU g −1 dry wt. soil.