Seperating Dauers from Dead Worms

WBLanier wblanier at aol.com
Sat Dec 9 20:04:03 EST 1995


Ron,

Take advantage of the fact that dauers are alive and wiggling.  They will,
for example, wiggle through sieves that dead 'uns can pass through and
they will wiggle over barriers.

METHOD 1.  Large quantities of dauers can be harvested by means of ATM
Test Sieves (Thomas Catalog, pages 1407 - 1414).  These sieves come in
diameters from 5-in to 12-in.  I think they are normally used in soil
samples.  In any case, we used these sieves to separate the infective
dauers of Heterorhabditis heliothidis and Neoaplectana carpocapsae from
the adult stages and other larval stages (as well as culture junk).  Since
Caenorhabditis elegans dauers are slightly smaller, the mesh count would
be different, but can be derived experimentally.  Usually we used two or
more separations, the first with a relatively open mesh to get rid of
large adults, culture garbage, etc., and the second to obtain a suspension
of essentialy pure dauers.

We made a distilled-water suspension of the culture.  This killed adults
and many non-dauer larvae (which lack protective cuticle).  We poured the
suspension through the sieve, discarding the liquid and keeping the worms.
 We then placed the sieve over a tray of water (about 5-mm or less from
the bottom of the sieve to the water).  Within about a few hours all of
the dauers will have wiggled through the sieve mesh and dropped down into
the water.  At one time, we made a 48-in X 48-in sieve built over a giant
bubbled tray of water for kilogram-amount separations.

METHOD 2.  Small quantities of dauers can be harvested by collecting the
culture on a coarse filter paper disk.  Place the filter paper over a damp
filter pad in a Petri plate.  We used 60-mm plastic Petri plate bottoms
glued into 100-mm Petri plates.  We then filled the "moat" formed between
the small and large Petri plate bottoms with distilled water.  The dauers
would crawl off the filter paper, over the edge of the small Petri plate,
and into the moat.  Quantitatively, we found that about 80% of 1-million
H. heliothidis infective dauers collected on a Millipore filter (you can
use fiber filters, paper filters, whatever works) would migrate into the
moat within 2-hr to 3-hr.  You may find that C. elegans dauers are more or
less active, so be prepared to run the first migrations for several hours.

Send me an e-mail if you have any questions or want more details:  I am at
wblanier at aol.com during the evenings and at lanier at resbiom.com during the
weekdays.

Wayne
______________
Wayne Lanier, PhD



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