Extraction efficiency, size distribution, colony formation and [3H]-thymidine incorporation of bacteria directly extracted from soil

Abstract

The direct extraction method for bacteria in soil by the application of the dispersion-centrifugation technique was evaluated based on the extraction efficiency, cell size distribution, colony formation and rate of [3H]-thymidine incorporation. The extraction efficiency increased by sonication (20 W, 3 min) and by the chelating agents used as extractants: sodium oxalate, sodium citrate, Tris-HCl/EDTA-disodium salts and an Amberlite® IRC-718 resin. Detergents, sodium cholate, tween 80 and triton X-100, also increased the efficiency. Four consecutive extractions using the above-listed four chelating agents with sodium cholate led to the extraction efficiency of 24% to 37% in the Anjo, Nagano, and Ibaraki soils. In the purification procedure of extracted cells by a density-gradient centrifugation using NycodenzlRl, the recovery was 19%. The size distribution of the purified cells was almost identical with that of the total bacterial cells in soil, reflecting the fact that the purified cells were representative of the soil bacteria. The extraction and purification procedures decreased the rates of colony formation and [3H] -thymidine incorporation by bacterial cells and the addition of detergents to extractants enhanced the decrease. Without detergents, the decrease in the rate of [3H]-thymidine incorporation was 52%. The decrease was almost similar between the total extracted cells and a subfraction of cells with smaller sizes. It was suggested that representative bacterial cells would be obtained satisfactorily by consecutive extraction with chelating agents although the bacterial physiology was adversely affected.