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. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.