Isolation of Typical Marine Bacteria by Dilution Culture: Growth, Maintenance, and Characteristics of Isolates under Laboratory Conditions

Abstract

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 x 10 to 1.07 x 10 cells per liter. The mean cell volume varied between 0.042 and 0.074 mum, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions 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 colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 x 10 times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 mum and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity 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 of carbon and energy (a degrees (A), +/- 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.