Worm Breeder's Gazette 13(5): 11 (February 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Biological Sciences, Dartmouth College, Hanover NH 03755.
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 are
happier on NGM than on MYOB. The most extreme case I'm aware of involves
dpy-23(e840) lon-2(e678), which Jonathan Hodgkin observed as being
completely unable to grow on MYOB. Below, I've listed the recipes for NGM
and MYOB for comparison. Probably the biggest difference between the two
is that NGM plates are pH6, whereas MYOB plates are pH8. Why was I so
cavalier as to alter the pH by two whole units? Well, none of my stocks
seemed to care very much about the difference in pH. Plus, Tris is not an
effective buffer anywhere near pH6. Another big difference is that MYOB
has no added calcium, magnesium or phosphate. Again, most strains don't
seem 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 or
monobasic phosphate, I found that only the addition of phosphate allowed
dpy-23 lon-2 animals to survive on MYOB. Calcium and magnesium not only
didn't help, but they made the E. coli extremely thick and granular
(yuck). Part of the ameliorative effect of the added phosphate probably
resulted from the fact that it brought the pH down near 6. However, even
pH 8 phosphate allowed dpy-23 lon-2 worms to survive (although they did
not grow as well as parallel stocks at pH6). Since this is the only
strain that I know of that completely fails to grow on MYOB, it appears
that the addition of calcium and magnesium is dispensable (at least for
the huge majority of strains). This is fortunate, because these are the
things that really cause precipitation problems when autoclaving
everything together. Consequently, I would recommend trying out the NGM-
Lite recipe listed below. It has most of the advantages of MYOB, but now
in low pH. If you try it, please let me know if your strains like it or
not. eric.j.lambie@dartmouth.edu
NGM
Mixed 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).
MYOB
All components mixed with 1L water and then autoclaved:
2.0g NaCl, 0.55g TrisHCl, 0.24g TrisOH, 4.6g Bactotryptone, 8mg
Cholesterol, 20g Agar.
For convenience, make a large quantity of dry mix (without agar) and then
just weigh out 7.4 g per liter of medium.
NGM-Lite
All 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 then
9.5g weighed out per liter. You will probably see some particles after
autoclaving, but their density is pretty low and they don't interfere with
optics on the dissecting scope.