Worm Breeder's Gazette 3(2): 15

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

A Conveinient Way of Preparing Radioactive Worms

J. Lewis

We have tried a number of different ways of growing radioactive 
worms including forced aeration, rotary shaking and growth on Petri 
plates made with radioactive sulfate.  The method we like best and 
which seems to give a good yield of healthy worms (mutant as well as 
wild-type) is the following:  We grow [35S]-labelled bacteria (0.5 
mCi/100 ml of 0.05 mM sulfate) by rotary shaking, collect the bacteria 
by centrifugation, resuspend them in 15 ml of S medium and divide the 
concentrated bacteria between two large empty Petri plates.  A chunk 
of worm-filled agar is added and the plates taped shut with masking 
tape (no apparent anoxia for up to two weeks-incomplete taping is used 
to prevent desiccation of stock plates at 15 C).  This method is 
convenient for doing a large number of preparations simultaneously at 
controlled temperatures and the worms are in a reasonably small volume 
for harvesting.  To maximize incorporation, it is best to gauge the 
amount of bacteria left by carefully tilting a plate under a 
dissecting microscope (precocious plates are kept at 4 C up to 24 
hours).  To obtain dauer larvae, the plates are harvested two weeks 
after innoculation.  We have a modified method of harvesting worms.  
Flotation on 35% sucrose results in a substantial loss of worms (
especially young larvae) by osmotic shock.  Flotation on Ficoli 400 of 
equivalent density does not work, either for reasons of viscosity or 
lack of osmotic shock to the bacteria.  We collect our worms by 
spinning them at 300 x g for 5 minutes (1000 rmp in a Sorvall SS-34 
head).  The worms are then layered on a 14.8% w/w Ficoll 400 and 
sedimented for 15 minutes at 300 x g.  Bacteria, worm cuticles and 
almost all dead and grotty alive old worms remain on top.  Healthy 
larvae, adults and bunches of eggs are pelleted.  Fungal contamination 
is not separable from worms by this method.  If the worms are allowed 
to starve very much, adults may fail to sediment (not carefully 
Dauer larvae are harvested by initial collection at 300 x g, as 
above, but purified by flotation for 5 minutes on 30% w/v sucrose at 
1000 x g.  The dauer larvae so obtained are essentially free of other 
worm stages and of contamination.  Dauer larvae are useful to us 
because they are a relatively defined juvenile stage and seem to give 
more reproducible electrophoretic patterns of detergent-extractable 
proteins than mixed populations do.  The patterns of detergent-
extractable proteins from dauer larvae and well-fed worms show many 
quantitative and qualitative differences.