C76 water bath

Two to five milliliter of fresh infected blood or 10 ml of human blood spiked with bacteria at defined concentrations was incubated in BACTEC Plus Aerobic/F Culture vial (BD, Sparks, MD, USA, #456005). The blood cultures were shaken at 150 rpm at 37°C in a New Brunswick Scientific C76 water bath for the indicated time periods.

Ten milliliters of human blood obtained from the National Blood Services, MDA, Israel, under MDA research permit 08-0122 was inoculated with different concentrations of B. anthracis, Y. pestis, and F. tularensis. The inoculated blood was transferred to BACTEC^TM Plus Aerobic/F Culture vials (BD, Cat# 442192). The blood cultures (10 ml blood) or whole blood (1 ml) inoculated with bacteria were shaken at 150 rpm at 37°C in a New Brunswick Scientific C76 water bath for various time periods.

Yersinia strains were grown on BHIA (Brain heart infusion agar, BD Difco, Sparks, MD, USA, #241830) plates at 28 °C for 48 h. E. coli, P. auraginosa and S. typhimurium were grown at 37 °C for 24 h. Colonies were suspended in sterile phosphate buffered saline (PBS, Biological industries, Beth haemek, Israel, #02-023-1A) and added at a defined concentration into BACTEC plus aerobic/F culture vials (BD, MD, USA, #442192) supplemented with 10 mL of naïve human blood containing Citrate-phosphate-dextrose solution with adenine (CPDA) as an anticoagulant. The inoculated blood culture vials were then shaken at 150 rpm at 37 °C in a New Brunswick Scientific C76 water bath for different durations. Final colony forming units (CFU) counts for each vial were determined by plating 0.1 mL of serial 10-fold dilutions on BHIA plates and incubating for 48 h at 28 °C. F. tularensis subsp. holarctica vaccine strain LVS and B. anthracis Vollum strain were grown, as described in Reference [62 (link)]. The resulting blood cultures were processed, as described in the next section.