## 4 In Vitro Susceptibility Testing Methods for _B. burgdorferi_ s.l.
### Microdilution in Combination with Colorimetric MIC Determination  
As outlined above, early antimicrobial susceptibility testing of _B. burgdorferi_ s.l. resulted in variable MIC values due to the lack of a standardized method and issues with endpoint determination. As a result, a colorimetric microdilution assay using modified BSK was developed as an alternative to microscopic MIC determination. Here the general principles and practices of conventional antimicrobial susceptibility testing that are established for other microorganisms are used to analyze the activity of antimicrobial agents against _B. burgdorferi_ s.l. isolates [51; 55]. The method has also been used to investigate in vitro antimicrobial resistance patterns of _B. spielmanii_ and _B. valaisiana_ strains as well as a _B. bissetitiae_ tick isolate [55; 56]. The test system is based on color changes that occur in BSK medium when phenol red as a pH indicator is added. The color change indicates an accumulation of non-volatile acids produced by actively metabolizing spirochetes. Final concentrations of the lyophilized antibiotics of the preloaded microtiter plates are reconstituted by adding 200 \(\upmu\)L of the final inoculum suspension (5 \(\times\) 10\({}^{6}\) cells) in BSK medium containing phenol red (25 mg/mL) as the growth indicator. The same batch of BSK medium derived from commercially available or self-composed components should be used throughout the experiments [51; 55]. It is very important that the pH be adjusted to \(\sim\)7.6 before testing. Microtitre plates containing spirochetes and growth controls must then be immediately sealed with sterile adhesive plastic and cultured at 33 \({}^{\circ}\)C with 5% CO\({}_{2}\). The presence or absence of growth is then examined after 0, 24, 48, and 72 h by kinetic measurement of the indicator color shift at 562/630 nm using a commercially available reader (e.g. PowerWave 200; Bio-Tec Instruments, Winooski, VT, U.S.A.) in combination with a software-assisted calculation program (e.g. Microwin 3.0; Microtek, Germany; Figure 2a,b) [51; 55]. Finally, growth in the samples and controls is determined for each well based on a decrease in absorbance after 72 h (E\({}_{\text{t72}}\)) over the initial absorbance values (E\({}_{\text{t0}}\)) by performing a software-assisted calculation of the growth curves (Figure 2). A determination is made mathematically as follows: If, at 72 h, the absorbance values have decreased by 10% or more over the initial absorbance values, the well is considered positive for growth of borreliae (E\({}_{\text{t72}}\)\(\sim\) E\({}_{\text{t0}}\) minus 10%). In order to determine the endpoints for each isolate tested, the growth characteristics of the test and control wells are compared. The lowest concentration of antibiotic for which no significant color shift can be detected is considered the MIC. It  
Figure 2: (**a**). Sensitivity of the MIC method as determined for _B. burgdorferi_ is.s. reference strain B31 by investigation of growth using conventional cell counts and colorimetric examinations. Experiments were performed in duplicate on different days (modified from [51]). (**b**). Kinetic measurement of MIC as performed by software-assisted colorimetric microdilution method for reference strain B31 (modified from [51]). Rows: A, tetracycline (0.015–32 mg/L); B, amikacin (0.06–128 mg/L); C, piperacillin (0.06-128 m/L); D, growth control. Growth of samples and controls was determined for each well based on the decrease of absorbance (A562:630) after 72 h (E\({}_{\text{t72}}\)) in comparison to the initial absorbance values (E\({}_{\text{t0}}\)). The lowest concentration at which no or a minor color shift (decline of the curve) could be detected in comparison to the growth control was interpreted as the MIC: Tetracycline: row A, well no. 5 (MIC: 0.25 mg/L); amikacin: row B, well no. 11 (MIC: 64 mg/L); piperacillin: row C, well no. 1 (MIC: 0.06 mg/L).