culture media

Leon Avery leon at EATWORMS.SWMED.EDU
Sat Nov 11 12:28:01 EST 1995


In article <482hqo$nof at elna.ethz.ch>, you write:
|> 
|> Leon,
|> I am also a newcomer to the field, and the protocols from the Cold
|> Spring Harbor books look somethat peculiar to me, where it concerns
|> the media. I have quite some experience with many E.coli strains,
|> and we usually grow it on 2XTY with supplements or on NZY. While
|> OP50 is just another stain, somethat vigorous compared to many
|> newer ones, I would like to know, is it intended to feed a slow
|> growing E.coli or a fast growing one to worms? It is no problem to
|> follow to CSH recipe, but in many other areas, where I have a
|> longer experience, like in cloning, blotting or sequencing, I have
|> been able to bring many protocols to a common denominator. After
|> all, there is quite some logic in all these procedures not just
|> cookbook recipes... A comprehensive FAQ on C.elegans is very good
|> idea.
|> Cheers,

Well, the chance of me writing a *comprehensive* FAQ is, frankly,
zero.  It's a big job, and there are lots of protocols out there.
They may not be all that you would desire, but I have no reason to
think I could do better, and I certainly don't have the time.  I just
meant that it would be nice to add a new question or two to the FAQ
telling people how to get hold of worms, and where to find protcols.

Your question about how to grow the E coli: it doesn't really matter
very much how you grow your bug juice.  ("Bug juice" is what many worm
people call the E coli culture that you spread on the plates.)  That's
just an inocolum.  You only put a few E coli on the NGM plate--they
then grow on the small amount of nutrient that NGM contains.  So what
the worms end up eating is pretty much independent of how you grow the
bug juice.  The only exception is this: if you grow your bug juice in
a very rich medium such as 2xYT, and if you spread a large volume
(say, 0.3 ml) on each 12 ml plate, that will significantly add to the
total nutrient concentration and you'll get heavier growth.  I grow my
bug juice in 10X diluted LB to avoid this possible source of
variation.

People use lots of different E coli strains.  OP50 was chosen
initially because it's a leaky uracil auxotroph--the idea was that
this would cause slow growth, so the bugs would not overwhelm the
worms.  In fact, OP50 grows rather better than some prototrophic
strains, and some quick and dirty experiments I've done suggest that
it isn't actually uracil starvation that limits OP50 growth on NGM
plates.  You can use just about anything, really.  The only important
variables I've identified are:

(1) HB101 vs all other E coli (and some Salmonella) strains I've
    tried.  HB101 makes a liquid lawn that is easier for worms to
    eat--all worm strains, but especially those with feeding defects,
    will grow better on HB101.

(2) If you're growing up worms to prepare DNA, you don't want to grow
    them on an E coli strain that has lots of plasmids.  Some of the E
    coli strains that used to be popular, e.g. NA22 (a prototrophic E
    coli that grows better than OP50 on NGM, which was therefore used
    for growing up large numbers for DNA preps) have this problem.

-- 
Leon Avery					   (214) 648-2420 (office)
Department of Biochemistry			            -2768 (lab)
University of Texas Southwestern Medical Center             -8856 (fax)
5323 Harry Hines Blvd				   leon at eatworms.swmed.edu
Dallas, TX  75235-9038			  http://eatworms.swmed.edu/~leon/



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