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AMB Express. 2012; 2: 50.
Published online Sep 18, 2012. doi:  10.1186/2191-0855-2-50
PMCID: PMC3488030
Advantages of functional single-cell isolation method over standard agar plate dilution method as a tool for studying denitrifying bacteria in rice paddy soil
Tomoyasu Nishizawa,corresponding author#1 Kanako Tago,#2 Yusuke Uei,1 Satoshi Ishii,1,3 Kazuo Isobe,1 Shigeto Otsuka,1 and Keishi Senoo1
1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
2Environmental Biofunction Division, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki, 305-8604, Japan
3Present address: Division of Environmental Engineering, Hokkaido University, Sapporo, Hokkaido, 060-8628, Japan
corresponding authorCorresponding author.
#Contributed equally.
Tomoyasu Nishizawa: anisitom/at/mail.ecc.u-tokyo.ac.jp; Kanako Tago: tago/at/affrc.go.jp; Yusuke Uei: yuusukeuei/at/yahoo.co.jp; Satoshi Ishii: s.ishii/at/eng.hokudai.ac.jp; Kazuo Isobe: akisobe/at/mail.ecc.u-tokyo.ac.jp; Shigeto Otsuka: almond/at/mail.ecc.u-tokyo.ac.jp; Keishi Senoo: asenoo/at/mail.ecc.u-tokyo.ac.jp
Received July 24, 2012; Accepted July 27, 2012.
Abstract
We recently established a method for isolating functional single cells from environmental samples using a micromanipulator (Functional single-cell (FSC) isolation), and applied it to the study of denitrifying bacteria in rice paddy soil (Ashida et al. 2010. Appl Microbiol Biotechnol 85:1211–1217). To further examine the advantages and possible disadvantages of the FSC method, we isolated denitrifying bacteria from the same rice paddy soil sample using both FSC and standard agar plate dilution (APD) methods and compared in this study. The proportion of denitrifying bacteria in the total isolates was more than 6-fold larger with FSC isolation (57.1%) compared with the APD method (9.2%). Denitrifying bacteria belonging to Alphaproteobacteria and Bacilli were commonly isolated using both methods, whereas those belonging to Betaproteobacteria, which had been found to be active in the denitrification-inductive paddy soil, were isolated only with the FSC method. On the other hand, Actinobacteria were only isolated using the APD method. The mean potential denitrification activity of the FSC isolates was higher than that of the APD isolates. Overall, FSC isolation was confirmed to be an excellent method for studying denitrifying bacteria compared with the standard agar plate dilution method.
Keywords: 16S rRNA gene, Denitrifying bacteria, Functional single-cell isolation, Phylogenetic analysis, Rice paddy soil
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