We determined the number of total oligotrophs (obligate and facultative) in the northern basin of Lake Biwa (mesotrophic lake) on May 9th and June 7th, 1995, using the MPN (most probable number) method with two nutrient-poor media; a filter-sterilized lake water medium (LW-medium; ca. 1.7mg C/l) and a diluted peptone medium (LT10^-3; ca. 2mg C/l). The number of oligotrophs was generally 300-fold higher than the number of colony forming units on LT10^-1 agar plates containing 0.5g of peptone per liter (ca. 200mg C/l), and corresponded to more than 25% of the total bacterial count determined by a direct counting method with DAPI staining. Most oligotrophs (97.5%; average) were obligate oligotrophs (OO), growing only at low nutrient concentrations with no growth on LT10^-1 solid or liquid media. Five OO strains were isolated from LT10^-3 and LW-medium in May, and approximately sequences of a region of 500 nucleotides of their 16S rRNA gene (16S rDNA) were determined. Phylogenetic analysis of 16S rDNA showed that the OO isolates were clearly distinguishable from the six strains of platable bacteria, which were isolated from the LT10^-1 agar plate medium at the same time. Moreover, all platable bacteria were closely related to Comamonas testosteroni (β-proteobacteria subgroup), while all OO isolates with the exception of LO-52 were phylogenetically distant from every well-known bacteria in the database and were widely distributed into proteobacterial subgroups. These results strongly suggest that there are many unknown OO living in freshwater environments, and that OO isolates will provide important information regarding microbial ecology.

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There has been a great progress in the methods to analyze microbes, and revealed a more detailed picture of the naturally ocurring microbes. In this review, we describe new methods to detect fluorescent signals, practical application of molecular biological methods, such as PCR, basic principles of these methods, and their possibilities and limitations.

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