Isolation of functional single cells from environments using a micromanipulator: application to study denitrifying bacteria.
We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.
Ashida
Naoaki
N
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Ishii
Satoshi
S
Hayano
Sadakazu
S
Tago
Kanako
K
Tsuji
Takashi
T
Yoshimura
Yoshitaka
Y
Otsuka
Shigeto
S
Senoo
Keishi
K
D016949:Azospirillum / Q000145:classification / Q000302:isolation & purification; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005452:Fluoresceins; D009566:Nitrates / Q000378:metabolism; D042041:Ochrobactrum / Q000145:classification / Q000302:isolation & purification; D012275:Oryza; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012988:Soil Microbiology
C505935:5-carboxyfluorescein diacetate acetoxymethyl ester; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D005452:Fluoresceins; D009566:Nitrates; D012336:RNA, Ribosomal, 16S