Objective:toseparatetheBlastomereatdifferentpointsindevelopmentandobservethegrowthoftheresultingembryos.

Procedure:Weextractrfthegametesfromtheseaurchins,fertilizedtheseaurchineggs,andremovedthefertilizationenvelope.Thecellswerethenbeallowedtocleave,atwhichpointtheywerebeintroducedintoahypertonicseawatersolution.Thiscausedthecellstoshrinkandbreakapart.Oncethemajorityofthecellshadseparated,thewaterwasreplacedwithnormalseawater.Theirgrowthwasbecomparedtothatofthecontrolgroupthatwasnotdivided.

1. TheeggswerewashedseveraltimeswithASWbyallowingthemtosettleandpouringoffthewater.Thiswillremovedsomeofthejellycoat.
2. Enougheggstocoverthebottomofa50mlbeakerweretransferedalongwith10mlof10mMp-aminobenzoicacidinASW.
3. 50mlofspermweredilutedin5mlofASW.2dropswereusedtofertilizeeggs.
4. Thepresenceofthefertilizationenvelopewascheckedafter5and10minutesinthecontrolculture.
5. Theeggswerepouredthroughanylonfiltermountedtotheendofa50mlbeaker.Thiswasrepeateduntilmostofthefertilizationenvelopeshavebeenremoved.
6. Thecellswereallowedtosettleforafewminutes.

BlastomereSeparation

1. Thecellswerethenseparatedinto3100mlbeakersfullofASW.Carewastakensoasnottoaddtoomuchoftheseawater-PABAsolution.
2. Wethenwaitedforthecontrolculturetocleaveforthefirsttime.
3. Thenmostofthewaterwaspouredfromthefirstexperimentalculture.
4. Thebeakerwasgentlyfilledwithhypertonicseawater.Theculturewasthencheckedafter5minutestoseewhetherosmoticshrinkagecausedthecellstoseparate.Thesolutionwasreplacedwithadditionalhypertonicseawateriftheyhadnotseparated.
5. Wewaited10minutesafterthecellsshowosmoticshrinkage.
6. Thehypertonicsolutionwaspouredoffandreplaceditwithnormalseawater.Thiswasrepeated.
7. Steps3-6wererepeatedforthesecondexperimentalcultureoncethecontrolgroupcleavedathirdtime.
8. Theembryoswereallowedtodevelopandobservedatdifferentstagesintheirgrowth.

ResultsWhilethecontrolembryosgrewtoformnormalpluteustheexperimentalembryosdidnotcleaveonceseperatedfromeachother.Thecontrolorganismswerestainedwith5C7,EctoV,andIg8tolookattheirformationeventhoughtherewerenoexperimentalclonestocomparetothem.Anotherprocedureinvolvingacalciumpoorseawatersolutioncanbeusedtoseperatetheembryosandmayproducemoredesirableresults.Clickonthumbnailstoseefullsizepictures.Usethebackbuttonofyourbrowsertoreturntothispage

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