Bacterial cultures growing in TY medium for 48 h to an OD600 of 0.6 were diluted 1000-fold in M1 minimal medium supplemented with Dilworth’s vitamins, and 100 μl of Selleckchem Tipifarnib diluted cultures were added to each well and grown under static conditions at 28°C for up to 4 days. After 2 and 4 days, the contents of the wells were removed and each well was washed two times with 150 μl of sterile physiological saline solution, and then stained for 30 min with 100 μl of 25 μg/ml Calcofluor or 100 μl of 0.85% NaCl containing 5 μM Syto-9 and 30 μM propidium iodide. Next, dye solutions were removed and the wells were washed three times

with 150 μl of 0.85% NaCl, covered by 30 μl of fresh portion of physiological saline solution, and observed in a microscope. This experiment was repeated two times. To analyze different parameters of biofilm, 36 images from 3 wells of individual strain were collected. The ratio of live to dead cells was calculated using the ImageJ 1.43e software

(Wayne Rasband, NIH, USA). Images of biofilms stained with Syto-9 were analyzed to calculate several morphological parameters. The percentage of area covered by biofilm, a fractal dimension, and the length 17-AAG of coastline were calculated using ImageJ 1.43e software according to [76, 77]. Three-dimensional images were reconstructed using the Laser Scanning Confocal Microscope LSC 5 PASCAL (Carl Zeiss, Germany) with 200x magnification. Plant tests Red clover (Trifolium pratense cv. Diana) seeds were surface sterilized, germinated and grown on Fåhraeus medium [66] slants. 5-day-old seedlings were inoculated with bacterial suspensions at an OD600 of 0.2 (200 μl/plant), and grown under natural light supplemented with artificial light (14 h day at 24°C and 10 h night at 18°C) in a greenhouse. The clover plants were inspected for root nodule formation and harvested after 4 weeks. Wet and dry masses of clover shoots were estimated. Plant competition assay For the competition assay, the Rt2472 and Rt2441 mutants, and the Megestrol Acetate wild type Rt24.2 were collected from TY

agar medium into sterile water to an OD600 of 0.1. The mutants and wild type suspensions were mixed in 1:1, 10:1, 100:1, and 1000:1 ratios, and 200 μl of each mixture were added per plant. Twenty seedlings were used for each treatment. 28 days after infection, nodules were surface sterilized, crushed in 20 μl of saline solution, and 10 μl selleck chemicals portions were plated on 79CA agar plates supplemented with nalidixic acid or kanamycin, and incubated at 28°C for 3 days. Ninety nodules per each mixture were examined. Bacteria growing exclusively on the medium supplemented with nalidixic acid corresponded to the wild type strain, and those growing on the medium supplemented with kanamycin corresponded to the rosR mutants. The competitive ability of rhizobia was expressed as the percentage of the particular strain in the analyzed nodules. Assays for root attachment and growth on the root surface Root attachment of the Rt2472 and the Rt24.