Summary of synthetic media resonses (By request --LONG)

Nobody nobody at hgmp.mrc.ac.uk
Thu Mar 21 14:43:52 EST 2002


Many people have requested that I post the responses I received 
regarding growing C.elegans in a bacteria-free culture. The responses 
follow.

Onan



Hi Onan,

Worms can be grown axenically. We do this frequently in our lab
(Vanfleteren Lab). I will attach a protocol of how to make axenic
medium and grow the worms in a sterile way. Worms will grow somewhat
slower and live twice as long (they will be more slender as well). To
increase growth you can also add 20% of (sterile) skimmed milk to the
medium.

I hope this can help you,

Bart

--
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Bart P. Braeckman, Ph.D.
Department of Biology
Ghent University
K.L.Ledeganckstraat 35
B-9000 Ghent
BELGIUM

<http://allserv.rug.ac.be/~jvfleter>http://allserv.rug.ac.be/~jvfleter
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BASAL MEDIUM: 3% (w/v) dry yeast extract (Difco, Oxoid, or any other 
supplier, it does not matter, but it should not contain very high 
amounts of salt), 3% (w/v) soy peptone (this is essential - other 
peptones won't work well); sterol (e.g. cholesterol) is usually 
sufficiently present as an impurity in YE, SP and the supplement (Hb, 
see below). This basal medium is autoclaved at 121 C (=3D15 psi) for 20 
min.

SUPPLEMENT: Make up 50 mg/ml hemoglobin (I currently use bovine Hb, 
Serva N=F8 24510) in 0.1 N KOH and autoclave this stock solution at 121 
C for no more than 10 min. This treatment will partially cleave the 
protein, so it will no longer form a copious precipitate when added 
to the basal medium, but the fragments are large and haem (which is 
the essential nutrient) apparently remains

attached to them somehow - at least this should explain the sustained 
growth stimulatory activity of the preparation.

Add the supplement aseptically to the autoclaved and cooled (still to 
minimize precipitation) basal medium at a final concentration of 0.5 
mg/ml. The generation time of N2 grown in this medium is

about 6 days (at room temperature) and it will sustain continuous 
population growth. However, reproduction is not optimal as compared 
to monoxenic culture (only 20-60 offspring per hermaphrodite compared 
to 280 in the monoxenic regime). Note that the maximum depth of the 
liquid medium should not exceed some 2-3 mm if the cultures are not 
shaken. The depth of

continuously shaken cultures may be increased to over 5 mm. Both the 
supplement and the complete medium can be stored indefinitely at -18 
=B0C or lower and thawed for further use as needed



AXENIZATION

Standard methods of chloroxing should work, but frequently fail in 
practice (bacterial contamination, or contamination with yeasts or 
fungi). I obtained much better results by chloroxing twice (i.e

chlorox for 7 min, centrifuge for 3 min and add sterile S buffer; 
wash two times more, chlorox again, but now for only about 5 min, 
wash three times). A convenient small scale method is as follows.

Put a small drop (20-25 microliter) of chlorox mixture (5.5 ml water, 
4 ml commercial bleach (8 degrees; should contain no additives) and 
0.5 ml 10 N NAOH, freshly mixed) onto the center of a small sterile 
petri dish (e.g. 5 cm in diameter) and add several (5-10) gravid 
adults using a platina needle and mix gently with the needle. They 
should dissolve in about 7- 10 min, releasing the eggs. 
Alternatively, to better control bleaching time gravid females can be 
transferred into 10 =B5l sterile distilled water followed by 10 =B5l of 
double concentrated bleach. After standing no longer than 10 min (the 
shorter the better, but worm tissue should have dissolved, which 
guarantees that microbial life will also be destroyed), 5 ml of 
complete medium are added. The axenic medium will effectively 
neutralize the bleaching mixture and larvae will hatch and develop to 
adulthood and reproduce. They can then be transferred using standard 
aseptic handling (sterile pipettes as for bacterial or cell culture) 
to fresh culture medium as needed. Small screw capped vials are very 
convenient for maintaining stock cultures.

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Tom,

I'm not sure if by "defined" you mean what others have named CbMM, or
"semidefined", i.e., with crude but bacteria-free ingredients.

The end of this message contains my current protocol for raising C. elegans
in liquid medium. If you ever need a literature reference, I can provide it.

Dave


David J. Chitwood
Research Leader
Nematology Laboratory
Bldg. 011A, room 165B, BARC-West
Beltsville, MD 20705-2350 USA
Telephone 301-504-8634
=46ax: 301-504-5589
Internet: <mailto:chitwood at ba.ars.usda.gov>chitwood at ba.ars.usda.gov

Culture of Caenorhabditis elegans in sterile medium

Autoclaved basal medium

90 ml distilled water
3.0 g yeast extract (Difco cat. no. 0127-01)
3.0 g soy peptone (Sigma cat. no. P-0521)
1.0 g dextrose
200 ul of a cholesterol stock solution (stock solution is 5.0 mg/ml
in ethanol)

Autoclave 15 minutes at 121 C at 15 psig. Also autoclave a small
polypropylene bottle
or flask to receive the sterile hemoglobin stock solution (unless a filter
unit with a
detachable reservoir is used). Culture vials, tubes and flasks should also
be oven
sterilized; alternatively, sterile disposable culture vessels can be
used. (I presently use
20-ml glass scintillation vials from Wheaton, to which I add 1 ml of final
medium.)


Hemoglobin stock solution

Dissolve 0.5 g hemoglobin (Sigma cat. no. H-2500) by weighing 0.5 g and
immediately transferring it to 100 ml distilled water vigorously stirred
with a magnetic
stirrer. (At one time, this source of hemoglobin contained sufficient
sterol to satisfy the
sterol requirement in C. elegans, but more recent lots have not). Note
that improper
storage of hemoglobin or exposure of cold hemoglobin to air will result in
a poorly
soluble material that will clog the filter unit when filter sterilization
is begun. In some
cases, use of weak (0.001 M) KOH may facilitate solution of partially
denatured
hemoglobin.) After the hemoglobin has dissolved, sterilize it by
filtration through a
0.45- m-pore bacteriological filter unit (e.g., Nalge cat. no. 245-0045).

When basal medium has cooled, add 10 ml of hemoglobin stock and dispense 1-m=
l
portions into culture vials or tubes. The remaining hemoglobin stock may
be frozen for
future use. Unused final medium may also be frozen.

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Hi there,
you may want to check out the homepage from the Clegg Lab. You find the URL
on the Wormserver under the Lab-Listings.

Good luck,
Martin Victor



************************************
Martin Victor, Ph.D.
Project Leader Cancer

EleGene AG
Lochhamer Schlag 17
D-82166 Gr=E4felfing
GERMANY

Phone: ++49-(0)89-700 765-23
=46ax: ++49-(0)89-700 765-11
E-mail: <mailto:m.victor at elegene.com>m.victor at elegene.com

<http://www.elegene.com/>http://www.elegene.com
************************************

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=46rom the C. elegans WWW server, go to The Clegg Lab. The home page to 
which that leads describes a recently developed axenic medium for 
liquid culture that uses ultra-pasteurized skim milk added to a base 
medium. That medium supports worm development and reproduction well 
at quite high worm concentrations - 10,000 worms/ml. Obviously, with 
milk present, the medium cannot be called "defined". This CeHR medium 
was introduced in the last issue of the WBG.

Eric Clegg

Eric D. Clegg, Ph.D.
Reproductive Hazards Laboratory
U.S. Army Center for Environmental Health Research
568 Doughten Dr.
=46ort Detrick, MD 21702-5010
301-619-7237 ph
301-619-7606 fax
eric.clegg at amedd.army.mil



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Absolutely -- it reproduces more slowly, but lives longer! Such "axenic"
media are favored by some: see refs by J. Vanfleteren and others.

Robert J. Shmookler Reis
Professor, Depts. of Geriatrics, Medicine, and Biochemistry/Molecular
Biology
University of Arkansas for Medical Sciences and
Central Arkansas Veterans Health Care System
4300 West 7th Street
Little Rock, AR 72205 (USA)
Phone: 501-257-5560 Fax: 501-257-5578
Email: <mailto:ReisRobertJS at uams.edu>ReisRobertJS at uams.edu

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Hi Onan,

Depends on what you mean by "defined, synthetic media, i.e. no bacteria"
-- if you don't want to use bacteria per se, you can grow worms on egg
plates (see worm Methods book for details)-- the yield from this is
amazing. If the idea is to grow them axenically, there are protocols out
there for this (and a recent WBG article about it too, if I remember
right).

Hope this helps,

Peter




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