Siderophore Production Assessment in Bacterial Cultures

Analysis of siderophore production was performed following the protocols of Pérez-Miranda et al. (2007 (link)) and Lynne et al. (2011 (link)), with modifications as described in Kuhl et al. (2021 (link)). Briefly, 25 μL of an SCA7 overnight-grown culture was pipetted on NB agar plates and incubated for 48 h. Dye solutions [chrome azurol blue S (Sigma), FeCl3 (Fluka Biochemika) and HDTMA (Hexadecyltrimethylammonium bromide, Sigma)] were prepared and mixed according to Lynne et al. (2011 (link)). Piperazin-N,N'-bis-(2-ethanesulfonic acid) (Pipes, Roth) solution was prepared with 0.9% agar and pH 6.8. The dye solutions were autoclaved separately and then slowly mixed with the Pipes-Agar mix. Cooled but still liquid overlay agar (10 ml) was poured on plates with bacteria. Color change from blue to orange after 2 h incubation indicated siderophore production. Siderophore-producing R. qingshengii RL1 was used as positive control. The experiment was performed three times.