K2 edta minicollect blood collection system tubes

On day 6 post-inoculation approximately 50 μl of blood was collected from the submandibular vein of each mouse into K2 EDTA MiniCollect blood collection system tubes (Greiner). For each blood sample, two volumes of blood, approximately 38 μl and 4.8 μl, were each added to 40 ml of liquid BSKII containing RPA cocktail (60 μM rifampicin, 110 μM phosphomycin, and 2.7 μM amphotericin B) and plated for colony forming units (CFU) across two 96-well plates. Plates were incubated at 35°C with 2.5% CO₂ for 14 days and scored for the presence or absence of spirochete growth. Each positive well was considered a single CFU. B. burgdorferi/ ml of blood was calculated as the number of CFUs/ volume of blood analyzed multiplied by 100 for each sample and the average of the two measurements for each sample was determined.

On day 6 post-inoculation approximately 50 μl of blood was collected from each mouse into K2 EDTA MiniCollect blood collection system tubes (Greiner). Each 50 μl whole blood sample was added to 1 ml BSKII then plated in solid BSK-agarose medium plus RPA cocktail (60 μM rifampicin, 110 μM phosphomycin, and 2.7 μM amphotericin B) and plates incubated at 35°C under 2.5% CO₂ for 7–14 days. Colony-forming units (CFU) were enumerated and B. burgdorferi/ml of blood was calculated as the CFU/volume of blood analyzed multiplied by 100 for each sample (Aranjuez et al., 2019 ).