Bacterioplankton – 02 Materials and Methods

Representative Freshwater Bacterioplankton Isolated from Crater Lake, Oregon

Kathleen A. Page, Stephanie A. Connon, and Stephen J. Giovannoni

Materials and Methods

Cultures. Samples of Crater Lake water were obtained aboard the NPS research vessel Neuston moored over the deepest part of the lake. Niskin containers were deployed at 15, 130, 300, and 500 m to obtain bacterial isolation samples and at 300 m to obtain natural water as a base to prepare culture media. Samples for bacterial isolation were kept at 4°C and inoculated into culture medium within 72 h of collection. Acid-washed polycarbonate containers were used for water, live samples, and medium preparation whenever possible. Crater Lake water to be used for medium was filtered through a 0.2-m-pore-size membrane filter and immediately autoclaved. To prepare media, sterile, concentrated stocks of nutrients were added to sterile Crater Lake water. Mixed carbon medium consisted of 0.001% D-glucose, 0.001% D-ribose, 0.001% pyruvate, 0.001% succinate, 0.001% glycerol, 0.001% N-acetylglucosamine, 0.002% ethanol, 1 M NH4Cl, 0.1 M K2HPO4 3H2O, 100 g of inositol/liter, 200 g of thiamine HCl/liter, 0.1 g of cyanocobalamin/liter, 0.1 g of biotin/liter, 0.2 g of folic acid/liter, 1 g of p-aminobenzoic acid/liter, 10 g of niacin/liter, 20 g of calcium pantothenate/liter, and 10 g of pyridoxine/liter. Yeast extract-acetate medium consisted of 0.001% yeast extract, 0.001% sodium acetate, 325 M (NH4)2SO4, 20 M KCl, 10 M K2HPO4, 7 M MgSO4 7H2O, and 0.6 M Ca(NO3)2 4H2O. To determine bacterioplankton densities for inoculation, direct cell counts were done by collecting 5-ml samples of Crater Lake water onto 13-mm-diameter, 0.2-m-pore-size polycarbonate filters. The filters were fixed with 1% formaldehyde and stained for 5 min with 5 g of DAPI (4 ,6 -diamidino-2-phenylindole)/ml. Membrane filters were mounted on oiled slides and viewed by fluorescence microscopy. Forty-eight-well culture plates were inoculated with 25 or 50 cells per well in a 1-ml volume of medium. Plates were incubated in the dark at 4, 10, or 15°C for 3 to 22 weeks. To detect cell growth, 48-well plate cell culture arrays were prepared, DAPI stained, and observed by fluorescence microscopy as previously described (7). Percent culturability was determined by the equation for estimation of culturability, V ln(1 p)/X, as described by Button et al. (6), where V is the estimated culturability, p is the proportion of wells positive for growth (wells positive for growth/total inoculated wells), and X is the initial inoculum of cells added per well.

Wells positive for growth were identified, and cultures were transferred to 10 ml of fresh medium for 3 to 6 weeks of additional incubation. Ten-milliliter cultures were screened with cell array technology, and genomic DNA was isolated from positive cultures demonstrating a single morphological type. For long-term storage, glycerol was added to cultures to a final concentration of 10%, and the cells were frozen at 70°C. A representative from each of the 16 different isolates was recoverable from the frozen state and verified morphologically. To determine culturability by traditional methods, inocula of 20 l of viable culture were applied to spread plates of a 0.1 dilution of R2A (10% R2A) (34).

Imaging. Two hundred microliters of bacterial culture was fixed and stained with 1% formaldehyde and 5 g of DAPI/ml followed by filtration onto 0.2-mpore-size polycarbonate filters as described by Connon and Giovannoni (7). Filters were mounted on oiled microscope slides and viewed at 1,000 magnification with a Leica DMLS epifluorescence microscope. Digital images were acquired with a Spot Jr. camera (Diagnostic Instruments, Inc.). Size measurements were made using Spot imaging software version 3.3.2.