Hyphal Growth Assay of Streptomyces Compounds

RPMI-1640 medium with L-glutamine without sodium bicarbonate (Sigma) was prepared in DI water (15–20 ℃) and 34.53 gr of MOPS (Sigma) was added. The final pH was adjusted with NaOH to a pH of 7 and added additional water to 1 l of the final volume solution. A stock solution of extracted SLs produced by S. riodocensis in dimethyl sulfoxide (DMSO, Sigma) was prepared and kept at −20 °C until use. Hyphal growth assay was formed in 5 ml of RPMI-1640 medium and RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) [18 (link)]. Cell suspension was diluted at 1 × 10⁶ cells ml⁻¹ in medium and incubated with different concentrations of purified SLs (0 μg ml⁻¹, 8 μg ml⁻¹, 16 μg ml⁻¹, 32 μg ml⁻¹, 62.5 μg ml⁻¹, 125 μg ml⁻¹, 250 μg ml⁻¹, 500 μg ml⁻¹, and 1000 μg ml⁻¹) at 37 °C with agitation at 200 rpm for 5 h. Aliquots of samples were visualized under a bright-field using a 40X objective lens by Huma Scope Premium microscope and photographed.