Determinants and Fate C. elegans embryos

Materials

• CaenorhaBDitiselegansculturesonagarplates
• E.colistrainOP50
• NGagarplates
• C.elegansRinger"ssolution
• Microscope(preferrablyNomarskiinterferenceorphase)
• Slidesandcoverslips

Procedure

1. PlaceacultureofC.elegansonthestageofadissectingmicroscopeandfocusonthesurfaceoftheagarplate.Theculturescontainbothsmallmalesandthelargerhermaphrodites.Locateaspecimenofeach.

2. Forembryologicalstudies,selectalargehermaphroditeandidentifythevariousorganswithintheworm.Withawoodenapplicator,selectahermaphroditeandtransferittoaslidecontainingadropofRinger"ssolution.Makeawetmount.
3. Thecoverslipshouldholdthewormdown,butwillallowittocontinuemoving.Itwillnormallybeveryactiveatfirst,butthenquietdown.Sincethewormcanonlyflexitsbodyinadorsal/ventralplane,itwillalsonormallybeonitsside.

4. Placetheslideonamicroscopelocatetheworminthefieldofview.Identifytheeggsanddevelopingembryoswithintheovaryoftheworm.Carefuladjustmentofthelightwillbenecessary.Astheslidedriesout,itwillbenecessarytoaddadropofRinger"stotheedgeofthecoverslip.

5. Drawthevariousstagesofdevelopmentandlabeleachcellaccordingthetheschemein.

Optional

Selectseveralgravidworms(hermaphrodites)andtransferthemtoafreshplateofNGagarpre-seededwithalawnofE.coli.Allowthewormstolayeggsontheagar.Aftertheeggsaresown,useRinger"ssolutiontorinsetheadultwormsfromtheagarplate.Anyremainingadultsshouldbepickedofftheagar,buttheeggswillremainstuckintheagar.Thisprocesswillnearlysynchronizetheeggsandtheirdevelopmentcanthenbeobservedovertime.L1larvacanbewashedfromtheplatesinabouttwohoursandthesubsequentdevelopmentoftheselarvamonitoredasasynchronouspopulation.