The screws were added to test tubes containing the bacterial suspension with and without 0.8 μg/ml FOS—the MIC for the strain—and incubated at 35°C. At 4 and 24 h of incubation the screws were washed with PBS, fixed with 2.5% glutaraldehyde for 24 h and rinsed in Sorensen’s phosphate

buffer for 15 min three times. This was followed by post-fixation in 1% osmium tetraoxide for 30 min at room temperature, washing in Sorensen’s phosphate buffer for 15 min two times, dehydration through an ethanol gradient (50-100%), critical-point drying, and finally sputter coating with gold. Samples treated with and without 0.8 μg/ml FOS were imaged at Roxadustat price 4 levels (3, 10, 30, and 100 μm) at two locations —along the head and between the threads of the orthopaedic screws—using a Hitachi S-570 scanning electron microscope. Image acquisition location was standardized across all replicates in relation to the detector beam, with images taken in the top-right quadrant of the screw head, and second screw thread

along the minor diameter. Percent particulate coverage of the surface of titanium orthopaedic screws was determined AZD6244 price from multiple SEM images of the same region of interest using ImageJ image analysis program (National Institute of Health, Bethesda, USA). The gray-scale SEM images were converted to binary format and the percent white-to-black pixels were calculated for each of the images. The SEM images were also visually ranked for microbial biofilm morphology. Enumeration of biofilm on screws Enumeration of adherent biofilm grown on titanium screws was completed after removal by sonication. The same high biofilm-forming strain from the population was grown over night before inoculation at a 0.5 McFarland standard suspension in 5 ml of TSB-G + 25 μg/ml G6P. Titanium screws were added to the inoculated media with and without 0.8 μg/ml of Ergoloid FOS and incubated for 24 h. Following incubation,

the screws were removed from the inoculum, washed to remove non-adherent bacteria and then transferred to tubes containing fresh TSB-G. Samples were then sonicated for 2 min (Branson Ultrasonic Cleaner Model 2510, Emerson Industrial Automation, Danbury, USA) and vortexed for 15 s to disperse previously adhered biofilm amongst the media. Serial dilutions of 10-1 through 10-5 for each screw were plated and colony forming units (CFU) counted (n = 3) after overnight growth. Atomic Force Microscopy (AFM) For morphological studies, one strong biofilm producing isolate as determined from the MPA study was chosen from the population and inoculated at a 0.5 McFarland standard suspension in 10 ml of TSB-G + 25 μg/ml G6P and grown to late mid-log phase. The cells in a 1 ml sub-sample were centrifuged in a Scilogex Model D3024 microfuge at 5000 g for 3 min at room temperature, and washed 3 times with sterile analytical-grade water. The pellet was again suspended in deionized distilled water and the concentration of the bacteria was measured by a spectrophotometer at 540 nm.