I am usually not working with shaken culture, but with 20, 40 or 100
large petri
dishes (10 cm diameter). These are inoculated with about 1 sqcm agar blocks =
of
overgrown plates of worms. After about 3 or 4 days you have the
plates full with
gravid adults, that I wash away with about 4 ml of cold M9. Then I wash the
worms 6 times to get rid of the bacteria (2x 1200 rpm, 2x 1000 rpm,
2x 800 rpm).
By bleaching it became good to degas the bleach solution for about 15 min ( =
12
ml NaOCl, 2,5 ml 5 M KOH ad 50 ml).
By bleaching I use not more than 1 min with 8-9 ml of bleach sol. by vigorou=
sly
shaking at RTin a 15 ml Falcon tube, I spin down the now dead worms 1min 150=
0
rpm RT. I wash the pellet once with RT M9, bleach again for ~1 min to crack =
the
worms. Now I spin down the eggs, and wash the egg pellet 5 times with ice co=
ld
M9. The eggs are then incubated 24h by shaking in 10-20 ml S-Basal w/o food.
This is my starting point for doing synchronous culture of normally producin=
g
dauers.
Exakt times for bleaching depend on the centrifuge, for wt I use first 40 se=
c.
then 20 sec.
By producing dauers with daf-1 (DR40) or daf-2 (DR1564) I use 2 times
1 min. THe
adults were preincubated 12 h at 25=B0C before bleaching. Eggs and starved L=
1's
are incubated for dauer development completely at 25=B0C. Only spinning down=
L1's
is done at 4=B0C. OP50 plates are prewarmed to 25=B0C.
Nobody wrote:
> I am trying to grow large numbers of gravid adults in liquid culture, to
> isolate eggs. Apart from growing a synchronized culture (starting with
> eggs/L1) does anyone know of a trick to increase the proportion of gravid
> adults in liquid cultures?
>> Is it possible, for example, to start cultures with less food (I usually =
use
> 500 mL worth of NA22 per 500 mL of S-basal), so that they starve & form
> dauer, and then to start them up again, so they all hit adulthood but the
> same time?
>> Any help would be greatly appreciated
>> Dan Jeffares
>> Dan Jeffares
>> Department of Evolutionary Biology
> Zoological Institute
> University of Copenhagen
> Universitetsparken 15
> DK-2100 Copenhagen =3DD8
> Denmark
> Phone +45 35321278 (W)/38193924 (H)
> =3D46ax +45 35321300
> Email: dcjeffares at zi.ku.dk>> ---
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