WPC 6Npqè Y9iؑ 9 P[Y* {RU0ۓ|/ xagRst021,l[gLMwMr0P _Ѡ`›2Z`{?tv?Xc4/H~ۊO wP"U_'ks&1yRz:̘}ֹJ-:g62|+ڴ~hkYdGm{$^1}xx@ %]>_nnNkLmkf:qeRz4օn.W`pP*S5BPWVva]oPm &Uy<<:09k,1ԎyI2> 62K'JP[rbX-pĥѳ 0U<UN % 0(w@'4g{<uj:Default Para  Q\  `$Times NewRomanQQ\  `$Times NewRomanQ\  `$Times NewRoman' WSU-ETCFSOP 100-01-01 4Dr. Barry Forsythe II 0 .   3|x(9 Z 6Times New Roman RegularX($ UUUUUUUUUUn$ !    X  XaEXXXWSUECTF/SOP40003601 X Page1of4EffectiveDate:01December1997  Title: D.PULEXANDD.MAGNACULTURE  ________________________________________________________________________ Author(s):  _______________________________  Date:____________ x  4   Dr.BarryL.ForsytheIISectionLeader: _______________________________  Date:____________  <   4   Dr.MichaelJ.Lydy ________________________________________________________________________ l    1.0OBJECTIVE  D   4 ToprovideaconsistentmethodofcultureforDaphniamagnaandDaphniapulex,  Ѐinordertominimizevariationsinsensitivity.̜ 2.0HEALTHANDSAFETY    3.0PERSONNEL/TRAINING/RESPONSIBILITIES  `  4 3.1 PERSONNEL 8  4  3.1.1 Onlythoseindividualstrainedandhavingthedocumentationof h  4   suchtrainingwillbeallowedtoassumetheresponsibilityofthe T ?+ 4 <DL!X? 4 B.4 <DL!XB  ?+ 4 <DL!X?ӜDaphniasp.colonysmaintenance. @ B.4 <DL!XB 4 3.2 TRAINING   4  3.2.1 AllpersonnelmaintainingDaphniasp.culturesshouldbefamiliar   4   withthepreparationofreconstitutedwaterandwiththepreparation 4   ofDaphniafood.  p! М̛ 4  3.2.2 Allpersonnelshouldcompleteatourandaproceduretraining "H #  4   seminarpresentedbythe colonymanager. 4 3.3 RESPONSIBILITY d% #& ?+ 4 <DL!X? 4  3.3.1 Theprimaryresponsibilityofmaintainingthecolonybelongstothe <'$(  4   colonymanager,andthenfallstosomeonenamedbythe 4   manager,whohasbeendeemedadequatelytrained,incaseofthe 4   managersabsence.B.4 <DL!XBӜ -X+/  4.0REQUIREDANDRECOMMENDEDMATERIALS  X М 4 PreparedyeasttroutchowCerophyll(YTC) 4 Preparedalgaesuspensionfood 4 Organismculturevessels 4 Aerationapparatus 4 Asiphontube 4 Alargefirepolisheddisposablepipette 5.0PROCEDURE  L   ?+ 4 <DL!X? > 5.1CULTUREWATERPREPARATION | $    5.1.1 AlthoughDaphniastockculturescanbesuccessfullymaintainedinsometap T  waters,wellwaters,andsurfacewaters,useofsyntheticwaterastheculturemediumisrecommendedbecauseitiseasilypreparedandisofknownquality.Hardreconstitutedwater(hardness:160to180mg/LCaCO3)isrecommendedfor  D.magna,whereasmoderatelyhardreconstitutedwater(hardness:80to90mg/L  CaCO3)isrecommendedforD.pulex.   > B.4 <DL!XB ?+ 4 <DL!X? >̜ 5.2RECONSTITUTEDWATERS \    5.2.1 Deionizedwaterfromanadequateultrafiltrationsystemmaybecombined 4    withreagentgradechemicalsinarecipemanneraccordingtothose   guidelinessetforthbyEPAandASTM.  5.2.2 Commercialmineralwatermaybemixedwithultrafilteredwater(1:4)asa <    sourceofculturewater.  5.2.3 Waterstoredincarboysshouldbecoveredandoutofdirectlight.Shelflife     isapproximately14days.̜ 5.3FEEDING !X"    5.3.1Feed2.0mLofYTCand1.5mLofSelenastrum(3.5x107algaecells/mL) #0!$ per1,000mLofDaphniasp.culture. t$"%   5.3.2Culturesshouldbefedoncedaily. L&#'  5.4CULTURING $(%)    5.4.1 CultureTemperature )'+ М   5.4.1.1 N   Daphniacanbeculturedsuccessfullyoverawiderangeof +|)-     N   temperatures,butshouldbeprotectedfromsuddenchangesin    N   temperature,whichmaycausedeath. -T+/ М   5.4.1.2 N   Theoptimumtemperatureisapproximately20$C. D М  5.4.2 Illumination     5.4.2.1 N   Thevariationsinambientlightintensities(50to100ftc)and      N   prevailingday/nightcyclesinmostlaboratoriesdonotseem    N   toaffectDaphniagrowthandreproduction.  t     5.4.2.2 N   However,aminimumof16hofilluminationshouldbe  L      N   providedeachday  5.4.3 CultureVessels h      5.4.3.1 N   Culturevesselsofclearglassarerecommendedsincethey @      N   alloweasyobservationoftheDaphnia. ,     5.4.3.2 N   Apracticalculturevesselisa1Lbeakerfilledwith      N   reconstitutedwater.   5.4.3.3 N   Initially,allculturevesselsshouldbewashedwell. p    5.4.3.4 N   Aftertheculturehasbeenestablished,cleaneachbeakerwell H     N   withdistilledordeionizedwaterandwipewithaclean    N   spongetoridthevesselofaccumulatedfoodanddead x      N   Daphnia. d     5.4.3.5 N   Oncepermonthwasheachvesselwithdetergentduring <     N   mediumreplacement.Rinsethreetimeswithtapwaterand    N   thenwithculturemediumtoremovealltracesofdetergent.  5.4.4 CultureInitiation     5.4.4.1 N   Fourculturevesselsshouldbeinitiatedeachweekwith  l!     N   Daphnianeonates,obtainedfromweeklyculture !X" 񀀀񀀀maintenance.   5.4.4.2 N   Gentlypipette30uniformsizedDaphnianeonatestoeach t$"%     N   culturevesselcontaining1Lofmedium,2mLofYTC,and    N   1.5mLofalgalsupplement.   5.4.4.3 N   Thetipofthepipetteshouldbekeptunderthesurfaceofthe $(%)     N   liquidwhentheDaphnianeonatesarereleased. )&* М  5.4.5 WeeklyCultureMaintenance *(,    5.4.5.1 N   Culturesshouldbemaintainedforfourweeks,afterwhich ,h*.     N   theymaybediscarded. -T+/    5.4.5.2 N   Gentlypipette10to30adultsofsimilarsizefromtheold X     N   vesselintoacleanvesselfilledwith1,000mLof    N   reconstitutedwater,2mLofYTC,and1.5mLofalgal    N   supplement.   5.4.5.3 N   Cleantheoldculturevessel.    5.4.6 ObtainingNeonatesforaTest t    5.4.6.1 N   GenerallyDaphnia24hoursoldorless(neonatesorfirst  L      N   instars)areusedintesting.   5.4.6.2 N   Removegravidfemalesfromthestockculturesontheday h       N   priortotestinitiationandplacetheminasuitablecontainer    N   with2mLofYTCand1.5mLofalgalsupplementper1,000    N   mLofmedia.   5.4.6.3 N   Daphniashouldbeisolatedattesttemperatureinthedilution      N   watertobeusedforthetest.   5.4.6.4 N   Onthedayoftestinitiation,collecttheneonatesproduced p     N   overnight.   5.4.6.5 N   Toinsurethatneonatesaretherequiredage,thetestmustbe 4     N   initiatedwithin24hoursofisolation. 6.0QUALITYCONTROLCHECKSANDACCEPTANCECRITERIA  P   ThequalityAssuranceUnitwillperiodicallyreviewtheseprocedures. 7.0LITERATURECITED   ̜DaphniamagnaandDaphniapulexculturinginformationisrecordedonFCETLQAform  No.029(AppendixA)and)#)(AppendixB).