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Methane in the atmosphere is an important greenhouse gas, and the global increase in methane emissions from wetland rice fields has been thought to be a major cause of the increase in atmospheric methane1,13). When rice fields are flooded, anoxic and reduced conditions develop in the soil and methane is actively produced by methanogens10). It has been shown that application of rice straw to rice fields causes an increase in methane emission from the paddies3,7,8,11,12,14), and rice roots provide various substrates that enhance microbial activities in the soil6). Investigations on microbes in paddy soil are important to understand methane emission from rice fields. Bulk soil samples collected from paddies are heterogeneous and usually contain various materials such as rice roots, plant residue and small pebbles. Furthermore, two distinct layers, an oxidized surface layer and a reduced layer, develop in the plow layer of flooded paddy soil10). The heterogeneity of paddy soil makes it very difficult to ensure reproducibility and reliability in microbial investigations of the soil. In view of the heterogeneity, in our previous studies on the population densities of microbes in paddy soil4,5), we fractionated the bulk soil sample into three parts; soil fractions, living rice roots and plant residue. The heterogeneity of the soil should also be taken into consideration in sample preparations for the determination of microbial activities of flooded rice soil. Thus, in this study, we examined methods of pre-

Sample Preparation for Methanogenic Activities of Heterogeneous Flooded Rice Soils.