Forma series 2 water jacket co2 o2 incubator

B. burgdorferi strain B31-A3 [85 (link)] and derivatives were grown in Barbour-Stoenner-Kelly medium (BSKII) with 6% rabbit serum, while B. hermsii strain DAH [86 (link)] and derivatives were grown in BSKII with 12% rabbit serum (S1 Table). All liquid and plated cultures were incubated microaerophilically (5% CO₂ and 3% O₂) in a Forma Series II Water Jacket CO₂/O₂ incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA). For growth experiments in the presence of increasing levels of exogenously added L-arginine and/or ornithine, 1 M solutions of either L-arginine or ornithine were prepared in sterile water, filter sterilized and added to the growth medium at the indicated concentrations. Spirochetes were generally enumerated by dark-field microscopy. Specifically, 10 μL of cultures were placed on a glass slide (VWR, Radnor, PA) and a wet mount using a 22 x 22 mm coverslip was prepared for counting spirochetes. Escherichia coli was used for cloning and plasmid maintenance. Antibiotics were used at the following concentrations: chloramphenicol and gentamicin at 20 μg ml^-1, streptomycin at 50 μg ml^-1, and spectinomycin at 100 μg ml^-1. All chemicals were purchased from Sigma-Aldrich (St Louis, MO) unless stated otherwise.

Immediately after chamber feeding, B. hermsii densities in individual ticks and from the blood reservoir were determined by colony enumeration in solid medium, as previously described^{26 (link)}. Briefly, engorged, infected ticks were surfaced sterilized by washing sequentially in 3% H₂O₂, 70% ethanol, and sterile water. Ticks were then pulverized using a sterile pestle in a 1.5 mL Eppendorf tube with 0.1 mL BSKII, then raised to 1 mL final volume. Aliquots of this suspension were added to plating medium^{26 (link)} that was allowed to solidify at room temperature prior to incubation at 35 °C in 5% CO₂ and 3% O₂ using a Forma Series II Water Jacket CO₂/O₂ incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Plates were incubated for 10–14 days after which colony forming units were counted and total spirochete densities were calculated for each tick and the blood reservoir. Statistical analysis was performed using GraphPad Prism software version 9.1.1 for macOS (GraphPad Software, San Diego, CA, USA).