3.1. Selection of the analysis time for important bioautographicsteps3.1.1. The cultivation timeThe LB and HB broths, which were already used for B. s. culti-vation, were compared to a further self-designed broth (MLB). Thelatter consisted of peptone from meat and yeast, similar to the LBbroth. PBS were added to stabilize pH influences, glucose for growthand glycerol for viscosity adjustment.The routinely used pre-cultivation of B. s. was omitted in thisstudy. This was time-saving and led to a fast HPTLC-B. s. protocol,if compared to literature [21,24]. The B. s. spore suspension wasdirectly used to inoculate the broth. For monitoring of the bacterialgrowth, the measured value of OD600 nmwas plotted versus time (t)every 30 min over 15 h for three growing broths, i.e. HB, MLB andLB (Fig. S-1). By doing so, the exponential phase of the bacteria wasdetermined. HPTLC plates silica gel 60 were immersed in the bacte-ria broth at different OD600 nm≥ 0.4. Our observation showed thatdipping in B. s. broth with ODs calculated in the range of 0.7–0.9yielded homogeneous and intensive backgrounds. Hence, brothcultivation times of 6–8 h were recommended for our workflow.