Allegra x 14r centrifuge

An MDR S. Heidelberg isolate from the 2011 ground turkey outbreak in the United States (kindly donated by Dr. Irene Hanning, College of Genome Sciences and Technology, University of Tennessee and Dr. Venkitanarayanan, University of Connecticut), was used for the study. Working cultures of S. Heidelberg was prepared from the glycerol stock cultures stored at -80°C. S. Heidelberg was made resistant to 50 μg/ml nalidixic acid sodium salt (NA; CAS. no. 3374-05-8, Alfa Aesar, Haverhill, MA, United States) for selective enumeration. The growth of S. Heidelberg (overnight culture) in tryptic soy broth (TSB; catalog no.C7141, Criterion, Hardy Diagnostics, Santa Maria, CA, United States) was determined on xylose lysine desoxycholate agar plates (XLD; catalog no. C 7322, Criterion, Hardy Diagnostics, Santa Maria, CA, United States) containing, 50 μg/ml NA and incubating at 37°C for 24 h. Then, final inoculum levels of 4 and 7 log₁₀ CFU/ml were prepared from overnight broth culture (∼9 log₁₀ CFU/ml) after centrifugation (3,600 × g, 15 min, 4°C) (Allegra X-14 R centrifuge, Beckman Coulter; 5350 Lakeview Parkway S Drive, Indianapolis, IN, United States) and suspending the pellets in sterile phosphate-buffered saline (PBS; pH 7.2) (Kollanoor-Johny et al., 2012a (link)).

The CRFK-derived EVs were isolated and purified from EMEM exosome-free cell culture media as described previously in [30 (link)]. Previously collected cell-free media were spun down at 1300 revolutions per minute (rpm) for 10 min using an Allegra X-14R Centrifuge (Beckman Coulter, Brea, CA, USA). The supernatant was further spun down at 3900 rpm for 10 min and filtered through a 0.22 µm pore size filter. The filtrate was transferred to an ultracentrifuge tube, and the remaining tube was filled with 6–10 mL of 1X PBS to avoid sample spillage during ultracentrifugation. The mixture was then centrifuged at 10,800 rpm for 45 min at 4 °C in an SW41T1 swinging rotor using a Beckman Coulter Optima L-70K ultracentrifuge. The supernatant was further centrifuged at 32,000 rpm for 70 min at 4 °C in the same L-70K ultracentrifuge. Finally, the supernatant was discarded, and approximately 500 µL of resuspended EV pellets was collected from each tube from below the meniscus of each centrifuge tube. The protease inhibitor (Halt Protease Inhibitor Single Use Cocktail, Thermo Scientific) was added to the collected EVs at the concentration of 10 µL/mL for preventing rapid protein degradation. The isolated EVs were stored at −80 °C for future experimentation.

The ActiveCopper material is washed with isopropanol and pentane and ultrasonically agitated using a Branson Ultra Sonifier model #550 at 70% power for 25 sec each followed by centrifugation using a Beckman Coulter Allegra X-14R Centrifuge at 870 RCF for 6 min. The washed ActiveCopper is added to isopropanol at 1.16% concentration by weight. This solution is again sonicated and then homogenized using Omni International homogenizer model #GLH850 at 10,000 rpm for 5 min resulting in a uniform dispersion that is then used to coat the various types of fabric.