The M9 issue

I’ve found several recipe M9 out there, which is unfortunate. The recipe we use seems to work for everything, including freezing; I’d assume that freezing would be the most demanding. But it our M9 recipe variably results in precipitation after autoclaving, even when I do just 30 minutes and remove it promptly. The precipitate goes back into solution over the next few days. This phenomenon makes me twitchy.

The recipe we are using (for 1 L):
KH2PO4 3g
Na2HPO4 6g
NaCl 5g
MgSO4 1M 1mL

I know there are other M9 recipes out there, so it might be worth a discussion of their relative merits by people who know better than I.

The recipe you are using is the recipe in the back of Worm I, and it’s the recipe in Brenner’s 1973 Genetics paper (except for the trivial difference that Brenner’s paper uses 0.001 mol of solid MgSO4•7H2O per liter instead of 1 ml of a 1M MgSO4 solution per liter). I suppose there might be a buffer recipe out there that works better in one way or another, but this is the official recipe for M9.


I use the same recipe and I have the same problem after autoclave: the buffer précipitates. Because of that, I sterilise it by filtration instead of autoclave.

Thanks for the replies. I’m tempted to play the “what was good enough for Brenner is good enough for me” card, but I don’t want to be hidebound if there’s a better solution (pun intended). Anybody familiar with the merits of other M9 formulations?

if you add the MgSO4 after autoclaving there should be no precipitate.

I guess the point here is more along the lines of …'if the recipe in the Wormbook works then why look for alternatives?`That’s the great advantage of having something like the Wormbook where protocols, recipes data etc are made public and open to scrutiny.

The M9 recipe works…if you add MgSO4 when the phosphate/chloride solution is too hot or autoclave everything together you get a precipitate of magnesium phosphate. This only happens when the solution is hot enough because at lower temperatures the enthalpy equations are stacked against such a reaction taking place.

So, if we take it that the Wormbook M9 works…and the reason for adding the MgSO4 afterwards is because of the basic chemical rules involved then I guess we don’t need to find a better looking wheel?


I agree with Steve and Marlon. The MgSO4 causes the preciptate to form. Just adding it from an autoclaved stock solution after autoclaving the M9 works well. I’d guess thats why you add 1mL of the MgSO4 instead of in crystal form is it needs to be added as a liquid to a liquid to avoid this.

That is what we do in our lab as well. And M9 just works fine all the time :slight_smile: