ISOLATION OF TYPICAL MARINE-BACTERIA BY DILUTION CULTURE - GROWTH, MAINTENANCE, AND CHARACTERISTICS OF ISOLATES UNDER LABORATORY CONDITIONS

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the ce ntral North Sea off the Dutch coast were cultured in filtered autoclav ed seawater following dilution to extinction. The populations present before dilution varied from 0.11 x 10(9) to 1.07 x 10(9) cells per lit er. The mean cell volume varied between 0.042 and 0.074 mum3, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DN A per cell. All three parameters were determined by high-resolution fl ow cytometry. All 37 strains that were obtained from very high dilutio ns of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible c olonies on any of the agar media tested, indicating that these culture s contained obligately oligotrophic bacteria. It was concluded that th e cells in these 15 dilution cultures had adapted to growth under labo ratory conditions after several months of nutrient deprivation prior t o isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total popu lation was less than 1% and that while more than half of the populatio n behaved as obligately oligotrophic bacteria upon first cultivation i n the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution c ultures obtained from samples diluted more than 2.5 x 10(5) times cons isted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 mum3 and an apparent DNA content of 1 to 1.5 fg per cell. This cell ty pe had the dimensions of ultramicrobacteria. Isolates of these ultrami crobacterial cultures that were eventually obtained on relatively high -nutrient agar plates were, with respect to cell volume and apparent D NA content, identical to the cells in the initially obligately oligotr ophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific af finity for the uptake of mixed amino acids (a-degrees(A) 1,860 liters/ g of cells per h), but not for glucose or alanine as the sole source o f carbon and energy (a-degrees(A) +/- 200 liters/g of cells per h). Th e ultramicrobial strains obtained are potentially a very important par t of picoplankton biomass in the areas investigated.