We are recently experiencing problems with the preparation of NGM plates because many precipitates appear over the solidified medium (like the ones in the photos).

We have never had this problem before (we work with C. elegans and NGM plates since 2015), but since it started few months ago it is increasing in its frequency and when we are using small plates (like the ones for the brood size, 35mm), it is impossible to use the plates for experiments because the precipitates are too many.

We realized that precipitates appear only after OP50 bacteria were seeded on the plates and these were let overnight at 37°C to let bacteria grow. We also noticed that when the K2HPO4 buffer was freshly prepared using a new powder the precipitates were absent (at least for a certain amount of time), but then they started to appear again. Apparently filtering the KPO4 buffer before reusing it does not solve the problem.

All other buffers that we use (CaCl2, Mg2SO4) and cholesterol are clear, without visible precipitates or crystals.

Considering the powders, NaCl and peptone are in use from at least 3 years, agar was recently changed.

We are now using milliQ water to prepare the NGM medium, but we used as well deionized one for many years without having problems. Media are let in a water bath set to 55°C for at least 30 minutes before pouring plates (so the medium is not really hot).

Bacteria are frequently streaked from frozen aliquots at -80°C, liquid cultures are always freshly prepared and we also measure the OP50 OD before using them (we noticed that sometimes they can overgrow). The LB for bacteria is prepared also with common stocks of powders that are in use since a long time.

Has anyone experienced this problem before? If so, could you give us some suggestions or tips to solve it?

Thank you