## 4 In Vitro Susceptibility Testing Methods for _B. burgdorferi_ s.l.
### Microdilution in Combination with Colorimetric MIC Determination
has been established that the growth of spirochetes in a microtitre well results in a 10% decrease in absorbance when compared to the initial absorbance values of the microtitre well after 72 h [51; 55; 56; 57; 58]. Recently, a slightly modified assay also proved suitable for testing relapsing fever spirochetes, including _B. hermsii_ and _B. miyamotoi_[48; 49]. However, MKP medium was used instead of BSK medium and there have been reported challenges with variation of initial absorbance values throughout the experiments as well as with detecting the smaller decrease in absorbance compared to the initial absorbance values in some _Borrelia_ strains with low replication rates [48; 49]. As outlined above, this can happen due to the variability of the serum batches used for the medium preparation and/or inconsistencies in the timely preparation and transfer of the medium to the test plates, as well as the lack of a precise pH-adjustment before the experiment. Consequently, in this study, an alternative MIC calculation has been suggested. Here, the colorimetric MIC is calculated by comparing the drop in absorbance (Et\({}_{0}\)-Et\({}_{72}\)) to the drop in absorbance of the positive control (no antibiotics) (E\({}_{\text{POS,0}}\)-E\({}_{\text{POS,0}}\),\({}_{\text{72}}\)). The MIC is set arbitrarily at 25% [48; 49]. Thus, in this study, the colorimetric MIC was defined as the lowest concentration of antibiotics where (E\({}_{\text{t0}}\)-E\({}_{\text{t72}}\)) is 25% of (E\({}_{\text{POS,t0}}\)-E\({}_{\text{POS,t72}}\)). The authors suggest the use of this formula as a more robust alternative when calculating colorimetric MIC values but fail to demonstrate clinically significant differences between the colorimetric MIC values as calculated by the two alternative approaches and by dark-field microscopy [48; 49]. Also, no reason is given for the arbitrarily chosen threshold, and no ATCC reference strains were used in these investigations to control and adjust for possible negative interference of antibiotics, test medium or test methodology. Bearing in mind also the small number of strains and substances tested, significant differences in these two approaches are not apparent and we would strongly suggest the use of the original method using modified BSK medium for experienced laboratories. In summary, colorimetric microdilution testing is well-established as the testing method of choice for borreliae and offers the benefits of reliability, reproducibility, convenience and standardization. Moreover, it can handle large numbers of isolates and antibiotics under standardized conditions. It is important to note, though, that, in addition to a timely and accurate preparation of the BSK medium, antibiotics and inoculation of the plates, the pH of the final medium must be carefully adjusted in order to achieve comparable results. Moreover, for purposes of quality control, reference strains, such as _Staphylococcus aureus_ ATCC 29213, _Escherichia coli_ ATCC 25922, and _Pseudomonas aeruginosa_ ATCC 27853, should be examined with the same assay, in the same medium and under the same test conditions after 24 h of incubation according to NCCLS guidelines [51; 55; 56; 57; 58; 59].