Previously (WBG 13#2p12), I described our use of MYOB mix for worm plates. Since then, I have heard from a few people that some of their strains arehappier on NGM than on MYOB. The most extreme case I'm aware of involvesdpy-23(e840
), which Jonathan Hodgkin observed as beingcompletely unable to grow on MYOB. Below, I've listed the recipes for NGMand MYOB for comparison. Probably the biggest difference between the twois that NGM plates are pH6, whereas MYOB plates are pH8. Why was I socavalier as to alter the pH by two whole units? Well, none of my stocksseemed to care very much about the difference in pH. Plus, Tris is not aneffective buffer anywhere near pH6. Another big difference is that MYOBhas no added calcium, magnesium or phosphate. Again, most strains don'tseem to care about this. So, addressing the dpy-23 lon-2
strain, what is the problem here?When I tried supplementing MYOB with combinations of calcium, magnesium ormonobasic phosphate, I found that only the addition of phosphate alloweddpy-23 lon-2
animals to survive on MYOB. Calcium and magnesium not onlydidn't help, but they made the E. coli extremely thick and granular(yuck). Part of the ameliorative effect of the added phosphate probablyresulted from the fact that it brought the pH down near 6. However, evenpH 8 phosphate allowed dpy-23 lon-2
worms to survive (although they didnot grow as well as parallel stocks at pH6). Since this is the onlystrain that I know of that completely fails to grow on MYOB, it appearsthat the addition of calcium and magnesium is dispensable (at least forthe huge majority of strains). This is fortunate, because these are thethings that really cause precipitation problems when autoclavingeverything together. Consequently, I would recommend trying out the NGM-Lite recipe listed below. It has most of the advantages of MYOB, but nowin low pH. If you try it, please let me know if your strains like it ornot. firstname.lastname@example.orgNGMMixed with 1L water before autoclaving: 3.0g NaCl, 2.5g Bactopeptone, 5mg Cholesterol, 17.5g Agar.Added from 1M stocks after autoclaving: 1mM CaCl2, 1mM MgSO4, 25mM Potassium phosphate (pH6).MYOBAll components mixed with 1L water and then autoclaved:2.0g NaCl, 0.55g TrisHCl, 0.24g TrisOH, 4.6g Bactotryptone, 8mgCholesterol, 20g Agar.For convenience, make a large quantity of dry mix (without agar) and thenjust weigh out 7.4 g per liter of medium.NGM-LiteAll components mixed with 1L water and then autoclaved:2.0g NaCl, 4.0g Bactotryptone, 3.0 g KH2PO4, 0.5g K2HPO4, 8mg Cholesterol, 20g Agar.These components can also be premixed (- agar) , as for MYOB, and then9.5g weighed out per liter. You will probably see some particles afterautoclaving, but their density is pretty low and they don't interfere withoptics on the dissecting scope.