Serum sensitivity: comparing motility and growth in normal human serum (NHS) versus heat-inactivated serum (HIS). A. Direct killing assay. Six different Borrelia species were subjected to 50% pooled NHS or 50% pooled HIS: B. afzelii strain PKo, B. garinii strain A87S, B. anserina Ni-NL, B. hermsii HS1 and B. miyamotoi (LB-2001 and HT31). Blinded samples were examined by dark-field microscopy and 100 spirochetes per well were scored as either motile or immotile. Loss of motility in the NHS wells compared to HIS is indicative of complement mediated killing and inactivation of spirochetes. The figure depicts the mean, and error bars represent the standard error of the mean of triplicates from one representative experiment. A Kruskal-Wallis test was performed at t = 1 hour and t = 3 hours (p = 0.02 and 0.003, respectively) and for each strain motility between NHS and HIS incubation was compared using a two-tailed Mann–Whitney test (no significant differences). This experiment is representative of three different experiments. B. Growth inhibition assay. A total of 5x106 spirochetes per well of B. miyamotoi LB-2001, B. miyamotoi HT31 or B. anserina Ni-NL were subjected to 50% NHS or HIS in the presence of a pH indicator (phenol red), cultivated at 33°C and absorbance at 562/630 nm was measured daily. A decrease in OD562/630 indicates decreasing pH due to spirochete growth. Error bars represent mean ± standard error of the mean (triplicates). This experiment is representative of two different experiments. The OD562/630 of spirochetes subjected to NHS versus HIS was compared using a repeated measures analysis of variance (ANOVA), and the p-value for interaction is depicted for each strain.