. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. • I * • «,«'• t I . • • • • «« H •*i> • »>•'*>»* • • » > •«:• •3&S8tlS&S&&S^ :» *"******* ' '*' ^iJiWW^wUr^??^. Hybridization -Detection of captured rRNA -Identification of (active) ribotypes Figure 1. Outline of the experimental approach used for the functional diversity array. that the approach is sensitive enough to detect populations at naturally occurring levels. The use of phosphor-imaging screens, to which the entire array is exposed, allows 14C hound to each spot to be quantified. We
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. • I * • «,«'• t I . • • • • «« H •*i> • »>•'*>»* • • » > •«:• •3&S8tlS&S&&S^ :» *"******* ' '*' ^iJiWW^wUr^??^. Hybridization -Detection of captured rRNA -Identification of (active) ribotypes Figure 1. Outline of the experimental approach used for the functional diversity array. that the approach is sensitive enough to detect populations at naturally occurring levels. The use of phosphor-imaging screens, to which the entire array is exposed, allows 14C hound to each spot to be quantified. We have experimentally determined the detection limit for 14C on these screens to be DPM for a spot 150 jam in diameter, which consists of about 108 oligonucleotide probes. Assuming that the rRNA molecules fragment to about 300 bp and that only 1% of the oligonucleotide probes will be bound to templates after hybridization, the detection limit is between 102 and 103 cells. This estimate is based on a cellular rRNA content between 1,000 and 10,000 molecules, which is in the range of slow- and fast-growing cells, respectively. Using this procedure, we have been able to specifically detect and differentiate rRNA from sulfate-reducing strains grown on l4C-labeled lactate (Klepac and Polz, unpubl. data). For the array to represent the actual microbial populations responsible for metabolism of a specific compound, realistic substrate concentrations must be used in the incubations to avoid introducing a major bias in community structure due to selective growth of specific populations. In coastal wa- ters, which we are using as a model ecosystem, we found that even low additions of 14C-labeled organic substrates (representing <3% of the total organic carbon) produced highly radiolabeled rRNA after 7 h incubation at in situ conditions. This incubation time is similar to the average generation time for the entire bacterial community (9 h), and b
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
