11/24/22

Dear Margarida,

You have an interesting problem. The observation that you get the normal (perfect) response with other bacterial isolates makes it challenging.

There are a couple of possibilities. You could try pre-heating the culture supernatant (e.g., 100C for ~10-15 min). The supernatant, even though clear, probably contains soluble protein which may interfere (?). The opacity and precipitate (ppt) you see could be protein ppt'd by HClO4. Heating the supernatant in advance might denature and precipitate the protein and allow you to remove it by centrifugation prior to addition of the Salkowski reagent. The other culture supernatants you have tested probably also had soluble protein, but you got normal results, so a heating step may or may not solve your problem; however, it is easy to try and won't take long.

You mention that you observed a yellowish coloration with your problem sample, though the normal color observed w/ IAA + Salkowski reagent is pink. The absence of a pink color could be due to 2 things: 1) The supernatant contains no IAA; 2) IAA is present, but something interferes w/ the detection. One way to determine this is to "spike" the sample with a known amount of authentic IAA, then run the test. If you don't see the normal pink color, it indicates that something interferes w/ the reaction. If you get the normal pink color, that indicates that the reagent does work and detects IAA in spite of the opacity and ppt'n problem you observed, and suggests that the supernatant is negative for IAA.

Another option is to try Kovac's reagent. This uses p-dimethyl-amino-benzaldehyde (PDMA). It reacts w/ indole and indole derivatives, e.g., indole-pyruvic acid, so it should react w/ IAA. Below is a reference that you can check:

https://microbiologyinfo.com/indole-test-principle-reagents-procedure-result-interpretation-and-limitations/

I hope this information helps you.

Bill Colonna, Iowa State University, Ames, Iowa, USA [email protected]

Thank you so much. I will try it!