A selective and diagnostic medium for ammonia oxidising bacteria

We present a method for extraction of active methane (CH,)-oxidizing bacteria from soil samples. The method is based on physical dispersion of bacteria from the soil particles followed by separation of bacteria and soil particles by floatation in the density media Nycodenz or Percoll. Separation on Nycodenz produced very pure bacterial suspensions while separation on Percoll produced rather impure suspensions. However, more than 60% of the methane-oxidizing activity was irreversibly inhibited in the procedure using Nycodenz compared to less than 10% irreversible inhibition when Percoll was employed.The bacterial suspensions extracted from soil can be used to study the physiology and ecology of soil bacteria that oxidize methane at atmospheric concentrations. Our data indicated that these bacteria are extremely difficult to dislodge from particles compared to the majority of bacteria in soil. Tentatively, we interpret the strong attachment to long residence time (i.e. slow turnover) of the methane-oxidizing bacteria. A slow turnover/growth rate would explain why soil disturbances, like cultivation, have a long lasting effect on the oxidation of atmospheric methane in soil.

“…were sterilized by autoclaving. The buffer mentioned later in the text is a medium normally used for ammonium-oxidizing bacteria [23] but in this case without ammonium.…”

Section: Methodsmentioning

confidence: 99%

Extraction of methane-oxidizing bacteria from soil particles

Priemé

1996

“…The buffer mentioned later in the text is a medium normally used for ammonium-oxidizing bacteria [23] but in this case without ammonium. were sterilized by autoclaving.…”

Section: Methodsmentioning

confidence: 99%

Extraction of methane-oxidizing bacteria from soil particles

Priemé

Sitaula

Klemedtsson

et al. 1996

We present a method for extraction of active methane (CH4)‐oxidizing bacteria from soil samples. The method is based on physical dispersion of bacteria from the soil particles followed by separation of bacteria and soil particles by floatation in the density media Nycodenz or Percoll. Separation on Nycodenz produced very pure bacterial suspensions while separation on Percoll produced rather impure suspensions. However, more than 60% of the methane‐oxidizing activity was irreversibly inhibited in the procedure using Nycodenz compared to less than 10% irreversible inhibition when Percoll was employed. The bacterial suspensions extracted from soil can be used to study the physiology and ecology of soil bacteria that oxidize methane at atmospheric concentrations. Our data indicated that these bacteria are extremely difficult to dislodge from particles compared to the majority of bacteria in soil. Tentatively, we interpret the strong attachment to long residence time (i.e. slow turnover) of the methane‐oxidizing bacteria. A slow turnover/growth rate would explain why soil disturbances, like cultivation, have a long lasting effect on the oxidation of atmospheric methane in soil.

“…Inoculum of N. europaea NCIMB 11850 was grown in autotrophic liquid medium (LM medium) modi¢ed from MacDonald and Spokes [27] [28], 1 ml. The medium was adjusted to pH 7.5 with 10 M NaOH and autoclaved.…”

Section: Preparation Of Aob Culture For Inoculation In Soilmentioning

confidence: 99%

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

Hesselsøe¹

2001

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 3^1 0 4 MCFU g 31 dry wt. soil. ß (M. HesselsÖe).Abbreviations : MCFU, microcolony-forming unit; FISH, £uorescence in situ hybridization; AOB, ammonia oxidizing bacteria; MPN, mostprobable number FEMS Microbiology Ecology 38 (2001) 87^95 www.fems-microbiology.org