Culturing the Uncultured in the Ocean

Epifluorescence microscopy and direct viable counting methods have shown that only 0.01-0.1% of all the microbial cells from marine environments form colonies on standard agar plates. To culture novel marine microorganisms, high throughput culturing (HTC) techniques were developed to isolate cells in very low nutrient media. This approaches was designed to address microbial metabolic precesses that occur at natural substrate concentrations and cell densities, which are typically about three orders of magnitude less than in common laboratory media. Approximately 5000 cultures of pelagic marine bacteria were examined over the course of 3 years. Up to 14% of cells from coastal seawater were cultured using this method, a number that is 1400 to 140-fold higher than obtained by traditional microbiological culturing techniques. Among the cultured organisms are many unique phylogenetic lineages that have been named as new phyla (7), orders (2, 5, 12), families (3), and genera (1, 4, 6). Over 90% of the cells recovered by this method do not replicate in standard agar plating, the most common method of microbial cell cultivation.