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growing large numbers of worms

Clegg, Eric D Dr USACEHR Eric.Clegg at DET.AMEDD.ARMY.MIL
Mon Dec 2 10:31:48 EST 2002

Previously existing axenic liquid culture media for worms were not 
particularly satisfactory in our hands either. However, the CeHR 
medium works well. It is a hassle assembling the ingredients 
initially, but assembly of the medium from the stock solutions is 
easy. We are exploring now the possibility of a commercial source of 
the medium. (Note: the glycogen is not necessary)

In my lab, nearly all of our work is done with axenic worms; 10-20 
million per week. Time to develop from release of L1s from M9 to 
early gravid adults is 3 - 3.5 days under the appropriate culture 
conditions. Our routine is to bleach an axenic culture on Monday or 
Thursday and place the embryos into M9 overnight. The next day, the 
worms are placed in CeHR (8,000 per ml) and incubated with shaking at 
22C. The worms reach early gravid adult stage on the third day after 
release. Obviously, good sterile technique is essential. Aeration 
conditions are important. Some labs may have trouble obtaining the 
refrigerated ultrapasteurized skim milk that is essential. 
Information is available at the Clegg lab web site and in our 2002 
East Coast Worm Meeting abstract (C. elegans WWW Server).

Eric Clegg

Eric D. Clegg, Ph.D.
Reproductive Hazards Laboratory
U.S. Army Center for Environmental Health Research
568 Doughten Dr.
Fort Detrick, MD 21702-5010
301-619-7237 ph
301-619-7606 fax
eric.clegg at amedd.army.mil

-----Original Message-----
From: leon at eatworms.swmed.edu 
[<mailto:leon at eatworms.swmed.edu>mailto:leon at eatworms.swmed.edu]
Sent: Sunday, December 01, 2002 6:11 PM
To: celegans at net.bio.net
Subject: Re: growing large numbers of worms

100,000 is not a large number of worms.  There are about that many
worms on a single starved 6 cm plate (but almost all are L1s).  If
you need adults, you could easily get that many from 20 10 cm
enriched plates.  By growing them in liquid (which is a bit more of a
chore, but still routine) you can get millions.

"Totally free" of bacteria is pretty demanding -- what are you
planning that would be screwed up by 1 bacterium mixed in with
100,000 worms?  You can get eggs free of bacteria by treatment with
basic hypochlorite.  You can get newly hatched L1s free of bacteria
by letting the eggs hatch in sterile buffer.  In principle you could
grow those worms in axenic medium -- such media exist, but they are
really crummy.  You can also grow up a lot of worms in bacteria, then
wash them in sterile buffer as many times as you like, to approximate
a population free of bugs.  You could use bacteria that were
sensitive to streptomycin and kanamycin, wash away almost all the
bugs, then do whatever experiment you have in mind in buffers
containing strep and kan, to kill the few remaining ones.

Leon Avery                                        (214) 648-4931 (voice)
Department of Molecular Biology                            -1488 (fax)
University of Texas Southwestern Medical Center
6000 Harry Hines Blvd                            leon at eatworms.swmed.edu
Dallas, TX  75390-9148                  

>I've scanned previous posts to this group and lots of websites pretty
>thoroughly but can't find answers to a couple of questions that I
>have, so I hope someone can either help me or point me to a suitable
>site which has the info I need.
>1. How easy is it to grow up large numbers of worms - say in the
>100,000 or more range?
>2. Ia it possible to get this number of worms totally free from
>contaminating bacteria (for the experiment I have in mind they would
>need to have been feeding on bacteria for growth, which I assume is
>essential anyway) or does one have to go through a cycle of egg
>Thanks for any help - I'm not a C. elegans researcher (yet!) hence the
>possible naivety of these questions.
>Pete Lund


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