We are detecting Indole acetic acid (IAA) producing actinobacteria according to Bano and Musarrat (2003) method.
The summary of the method is:-
Inoculation of the isolates in LB medium (supplemented with 0.5% glucose and 500 μg/mL tryptophan) -----> Incubation at 28 ◦C for 48 h -----> Centrifugation of the cultures at 10000 rpm for 15 min ----> 2 mL of the supernatant were transferred to a fresh tube to which 100 μL of 10 mM ortho-phosphoric acid and 4 mL of the Salkowski reagent (1 mL of 0.5 M ferrous chloride in 50 mL of 35% perchloric acid) were added ------>incubation of the mixture at room temperature for 25 min and the absorbance of pink color development read at 530 nm -----> Calculation of the IAA concentration in cultures.
Is there any method better than this one? or if any modification?
My dear friend, the method you have mentioned is the best way to evaluate the production of auxin, but sometimes with a few changes in the method, a good result can be achieved, for example, changing the ratio of the reagent (Salkowski) to the sample (supernatant) and also adding or not adding ortho-phosphoric acid to the mix.
The incubation is also better in the dark.
Wishing you success with you dear friend
IF you know a bit more about the pathways involved and the gene, you can design PCR primers. It would be much faster method. Or search for similar already designed primers if already someone did this kind of study.
Hi
One milliliter of
supernatant was mixed with 2 ml of
Salkowski reagent (1ml of 0.5M FeCl3 in
50mL of 35% HClO4) and incubated for 1hr.
Development of pink colour indicated the
production of IAA. The quantification of
IAA was read at 530 nm in a UV- Vis
spectrophotometer. A standard curve was
plotted for quantification of IAA solution
and uninoculated medium with a reagent as
a control. The amount of IAA in the culture
was expressed as μg/ml.
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