Enumerating Borrelia hermsii in Ticks

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).