At the moment I am working on a project and with my group we test whether the bacterial strains obtained are beneficial to plants. In order to characterize the bacterial strains, we want to test whether the bacteria produce siderophores.

I prepared CAS reagent by adding (0.06 g CAS in 50 ml ddH20) to 9 mL of (0.0027 g FeCl3.6H20 in 10 mL 10 mM HCl) and then adding (0.073 g HDTMA in 40 mL ddH 2 O). I inoculated each strain in 10 mL of LB medium and left it overnight. The next day I made a fresh culture of each culture (also in LB). From both the fresh and old cultures ( we did this to see if there is any difference in siderophore produce if OD600 divers) we pipetted 1 ml for each strain in a separate tube and centrifuged at 10000 rpm for 10 minutes. I took 100 µl from each tube and pipetted each strain into a separate well. Above that I pipetted 100 µl cas reagent in each well. However, we didn't see a color change from blue to orange/yellow. Also my positive controls (p.aeruginosa and E.coli) do not show a color change. We incubated the 96-well plate the longest for 10 minutes. Is there something we can change or ad to the experiment to make at least de positive controles show there siderophore production? I would like to hear your comments or help on this question.