crystals appear on plates few days after adding ascorbate


I am trying to make 0.1 M vitamin C (ascorbic acid) solution (dissolved in ddH2O) and add 250 uL of it to 5 mL agar for a final concentration of 5 mM vitamin C in my experiment. I dissolve ascorbic acid powder (Sigma-Aldrich, product number A4544) in double distilled water and then filter-sterilize (filter size 0.20 um) it for use in my experiment. The solubility for this product, as mentioned in its Specification Sheet, is 50 mg/mL. I make 17.6 mg/mL (0.1 M), vortex it for a long time, the powder seems to completely dissolve, but when I try to pass it through the filter, it gets stuck. I also tried to sonicate for 15 min, tried dissolving it in a warm environment, nothing worked. I changed almost every single thing: water, syringe, doesn’t work.

However, when I try to use a different filter, 0.45 um size, the 0.1 M vit C solution does pass through. I add it to agar plates, I seed the plate with OP50 the next day, and put worms the following day. Around 4 days after adding vit C, crystal-like substances begin to appear both on the surface of and inside the agar, with them being most concentrated at the agar-OP50 border (where OP50 concentration is also highest). The plates that contain just the water but no vit C does not have that problem. Also, vit C containing plates that do not have worms in them still will form the crystals after ~ 4 days.

The worms do not seem to mind the crystals, they just move normally and munch on the food. But did anyone have the same observation/problem when using vitamin C? Could anyone speculate what these crystals might be and why they are forming? Should it affect the phenotype on vit C? It would seem as if the vitamin C did not dissolve properly; but it is difficult to accept that as I vortex the solution for a long time, and crystals appear a few days after adding vit C, and in a vertical plane…

Thank you for your generous help.


this feels a little bit like groundhog day… you asked about the crystals in this thread;

unless chemistry has undergone a paradigm shift of epic proportions then the answer given then probably pertains in this instance too.

As for the filter sterilisation, something of low molecular weight like ascorbic acid should pass through the filter ok…but not if you use the same syringe filter many times (or are you bulk filtering?).

Regards from the Far Side


Thank you Steve for your response. Actually vit C-added plates that do not have worms on them still have those crystals after a few days. I always use fresh, sterile filter and I have also tried changing the syringe. I filter only 5 mL vit C solution at a time.

I emailed one lab group who also used ascorbate (vit C) in his experiments, albeit a higher final concentration (8 mM, versus my 5 mM). But they don’t seem to have that problem. And that’s why I’m still trying to find a way to get rid of them, if it is at possible…

the vitamin c will oxidise without worms being present on the plate, so they need to be used immediately and not stored. If there are oxalate crystals present then the vit. C content is reduced and the original concentration is no longer valid.