Why does my CAA Media precipitate when I add Iron Chloride solution to it?

[Jake Daniels]

I'm currently doing an undergrad project where we're trying to create 3 CAA (casaminoacid) media's (one iron limited, one plain CAA, and one iron supplemented). We created an iron supplemented media by adding 10ml of 2.6g/10ml of FeCl₃ (dissolved in ddH₂O) to a bottle of 200ml of CAA media (made up of 4g of CAA, 0.944g of K₂HPO₄•H₂O, and 0.2g of MgSO₄). This worked and the media appeared clear. 

Every subsequent time we've attempted to make this, as soon as the FeCl₃ stock solution is added a precipitate forms and we can't figure out why, we've tried adjusting for a more acidic pH but it still happens and it also occurs in other medias like TSB. This also occurs before the K₂HPO₄•H₂O and the MgSO₄ has been added in.

Any help would be appreciated! Thanks! 

[CharonY]

Ah the pain of controlling iron in media. So as you probably noticed the moment the medium turns neutral, insoluble ferrihydrite will form. So only using only dibasic potassium phosphate won't work (not sure how you acidified it, but you need to titrate with monobasic to ensure that it buffers, for example). However, you mentioned that it also precipates in presence of CAA alone? I would check the pH of the CAA (without buffer) first. In my memory, there wasn't any particularly strong interactions with amino acid mixes, or digested proteins, so I would focus on ensuring that the buffer system maintains a stable pH <7 first.

A second thing I am wondering about the concentration of the FeCl3 solution. It is awfully high isn't it? Are you sure that you really have 2.6g/10 mL (i.e. 260 g/L)? It is possible that that concentrations other reactions might occur (though one could test by drying the sample and run powder x-ray diffraction to check whether it is ferrihydrite, for example.

I will also add that anything with complex additives (CAA, tryptone, peptone, etc.) can have somewhat low iron contents, but they might be less limiting than one might think. Typically one needs to purify it further to ensure reproducible amount of iron in the medium (that is why many standard medium do not add iron, despite the fact that almost all bacteria need it in significant amounts).

[Jake Daniels]

Sorry forgot to add some info! 

We've checked the pH of the media throughout the adding of the Iron stock solution and the pH stays <7. The buffer isn't in the CAA medium already but is what the FeCl₃ is currently dissolved in instead of H₂O. 

2.6g/10ml was what we used to make a concentrated Iron Supplemented CAA medium before and have since tried lower concentrations, currently on 1g/L however no change in concentration seems to change anything. Precipitate seems to form at higher and lower concentrations of FeCl₃ so some reason.

One of my project supervisors says it's common for iron supplementation in CAA media to add FeCl₃ and we are going off of a specific paper, although I am unsure which one. Just a bit strange that it's worked before in exactly the same conditions but we can't figure out why it's happening now or what might have changed.

Thanks for your response!

[CharonY]

1 hour ago, Jake Daniels said:

The buffer isn't in the CAA medium already but is what the FeCl₃ is currently dissolved in instead of H₂O. 

Long shot, but what buffer are you using for the FeCl3?

[Jake Daniels]

The buffer we are using is HEPES buffer, the precipitate forms with it and without it