Dear Maissa,

You need to first set blank with unionculated media (without adding CAS dye). Then take the reading of reference (uninoculated media+CAS dye). I guess you have set blank with the reference that is why you are getting reference OD 00.

Hope this solves your problem.

MEB medium was prepared and used for siderophore production. 24 hr old culture of microorganisms were used to inoculate for 24 hr at 30o C with constant shaking at 120 1624 S. K. Ghosh et al.: The Qualitative and Quantitative Assay of….r.p.m. Following the inoculation, fermented broth was centrifuged (10,000 r.p.m. for 15 min)and cell free supernatant was subjected to detection and estimation of siderophore. Quantitative estimation was done by CAS – Shuttle assays31,32. In which 0.5 mL of culture supernatant was mixed with 0.5 mL of CAS reagent and absorbance was measured at 630 nm against a reference consisting of 0.5 mL of uninoculated broth and 0.5 mL of CAS reagent. Siderophore content in the liquor were calculated by using following formula :

% Siderophore units = Ar

Ar − As × 100 …(1)

Where Ar = Absorbance of reference at 630 nm (CAS reagent)

As = Absorbance of sample at 630 nm.

Dear Utkarsh singh Rathore, thank you for your reply I used the king B medium to detect the presence of the siderophore, I took 0.5 ml of a supernatant from a 24H culture with 0.5 ml of CAS reagent, the color of the control was blue and that of the essay was yellow green but I found impurities in the tubes (precipitates) and with spectrophotometry it was difficult to read the OD THANKS, excuse me for the late response Best regards

Hello.

Did you try to filter the mixture before reading OD?

Now I got a good result, yes I filtered the supernatant , the problem was the OD of Ar solution, i mean CAS solution which must be higher than that of the tests.