*4.3. Antimicrobial Bioassays*

Antimicrobial activity of the crude sponge extracts was determined by micro broth dilution assays in 384 well microtiter plates (Greiner, Kremsmünster, Austria). A Cybi Liquid handling system (Analytic Jena, Jena, Germany) was used to distribute 0.5, 0.25, and 0.125 μL (in duplicate, corresponding to 10.0, 5.0, and 2.5 μg/mL extract concentration) of each extract to the assay plates. A dilution series of gentamycin (64–0.002 μg/mL, Sigma Aldrich, St. Louis, MS, USA) was added to the antibacterial assays as positive control, while wells containing only medium or only bacterial suspension were used as sterile and growth control respectively. Pre-cultures of *E. coli* ATCC35218*, S. aureus* ATCC33592, and *P. aeruginosa* ATCC27853 were incubated (overnight, 37 ◦C, 180 rpm) in cation adjusted Mueller Hinton II medium (Becton Dickinson, Sparks, NV, USA) before the cell density was adjusted to <sup>2</sup> <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL and 50 <sup>μ</sup>L bacterial suspension was added to each well (except the sterile control) using a multi-well dispenser (Multidrop; Thermo Labsystems, Waltham, MA, USA). After incubation (18 h, 37 ◦C, 180 rpm, 80% rH), cell growth was assessed by turbidity measurement with a microplate spectrophotometer at 600 nm (LUMIstar® Omega BMG Labtech, Ortenberg, Germany).

The pre culture of *C. albicans* FH2173 was incubated for two days at 27 ◦C. Cell density was diluted to 1 <sup>×</sup> 105 cells/mL in Mueller Hinton II medium before the assay plates were incubated for 48 h at 37 ◦C, 180 rpm, and 80% rH. For *S. tritici* MUCL45407, a previously prepared spore solution was used to adjust the assay inoculum to 1 <sup>×</sup> 10<sup>5</sup> spores/mL in YM medium (yeast extract 4 g <sup>×</sup> L−1, malt extract 4 g <sup>×</sup> L−1, sucrose 4 g <sup>×</sup> L−1). *Septoria* assay plates were incubated for 72 h at 24 ◦C, 180 rpm, and 80% rH. Nystatin (Sigma Aldrich) was used as positive control for both, yeast and mold assays. Cell viability was evaluated via ATP quantification (BacTiter-Glo™, Promega, Madison, WI, USA) according to the manufacturer's instructions.
