ninth commiss direct juli establish commun method analysi offici control feedingstuff eec commiss european commun regard treati establish european econom commun regard council direct eec juli introduct commun method sampl analysi offici control feedingstuff amend act access greec articl thereof direct requir offici control feedingstuff purpos check complianc requir provis laid law regul administr action qualiti composit feedingstuff carri commun method sampl analysi commiss direct eec eec eec eec eec eec eec eec amend direct juli establish number commun method analysi progress work make advis adopt ninth set method measur provid direct accord opinion stand committe feedingstuff adopt direct articl member state requir analys offici control feedingstuff content avoparcin monensin sodium carri accord method annex articl member state bring forc law regul administr provis compli direct decemb forthwith inform commun thereof articl direct address member state brussel juli commiss presid gaston thorn oj oj oj oj oj oj oj oj oj annex determin avoparcin diffus agar medium purpos scope method determin avoparcin feedingstuff premix lower limit determin mg kg ppm presenc polyeth antibiot interfer determin principl sampl extract mixtur aceton water hydrochlor acid antibiot activ extract determin measur diffus avoparcin agar medium inocul bacillu subtili diffus shown format zone inhibit micro organ diamet zone direct proport logarithm antibiot concentr rang antibiot concentr emploi micro organ bacillu subtili atcc ncib mainten stock cultur inocul tube slope cultur medium bacillu subtili incub overnight ºc store cultur refriger ºc reinocul month prepar spore suspens harvest growth recent prepar agar slope mean ml steril water suspens inocul ml cultur medium contain roux flask incub dai ºc harvest growth ml ethanol check sporul microscop mix suspens month ºc cultur media reagent cultur medium gt pic file quot quot gt ethanol dilut ml ethanol ml water hydrochlor acid method provid establish give similar spore suspens commerci cultur medium similar composit give result sodium hydroxid solut phosphat buffer potassium dihydrogen phosphat kh po water ml adjust ph mixtur aceton water hydrochlor acid standard substanc avoparcin sulphat activ standard solut dissolv accur weigh quantiti approxim mg standard substanc phosphat buffer dilut buffer give stock solut avoparcin millilitr store stopper flask ºc solut stabl dai premix stock solut prepar success dilut buffer solut gt pic file quot quot gt feedingstuff stock solut prepar success dilut buffer solut gt pic file quot quot gt prepar extract assai solut premix weigh nearest mg suffici sampl mg avoparcin transfer ml graduat flask ml mixtur shake minut mechan shaker check ph adjust ph hydrochlor acid make volum mixtur mix filter portion suitabl filter paper whatman discard ml filtrat aliquot adjust ph sodium hydroxid solut dilut solut buffer obtain expect avoparcin concentr ml solut prepar solut expect content ml expect content ml expect content ml mean success dilut buffer feedingstuff weigh quantiti sampl shake ml mixtur minut mechan shaker clarifi extract centrifug stopper centrifug tube aliquot clarifi extract tabl adjust ph sodium hydroxid solut dilut aliquot buffer provid tabl solut prepar solut expect content ml expect content ml expect content ml mean success dilut buffer gt pic file quot quot gt assai procedur inocul assai medium inocul assai medium spore suspens ºc preliminari trial plate assai medium determin quantiti spore suspens requir give largest clearest zone inhibit concentr avoparcin prepar plate diffus agar carri plate concentr standard solut concentr assai solut concentr extract standard necessarili plate effect select plate big hole diamet mm mm centr made agar medium test carri plate consist sheet glass face aluminium plastic ring top mm diamet mm high pour plate quantiti medium inocul give layer mm thick ml plate mm diamet set level posit bore hole place measur volum assai standard solut ml hole diamet appli concentr time determin subject evalu zone inhibit incub incub plate hour ºc evalu measur diamet zone inhibit nearest mm record measur concentr semi logarithm graph paper show logarithm concentr relat diamet zone inhibit plot quot fit quot line standard solut extract determin quot fit quot point standard highest level sl formula gt pic file quot quot gt determin quot fit quot point standard highest level sh formula gt pic file quot quot gt similarli calcul quot fit quot point extract lowest level ul extract highest level uh substitut formula record calcul sl sh valu graph paper join give quot fit quot line standard solut similarli record ul uh join give quot fit quot line extract absenc interfer line parallel practic purpos line consid parallel valu sh sl uh ul differ line found parallel discard sl sh ul uh calcul altern formula give altern quot fit quot line gt pic file quot quot gt similarli ul uh altern quot fit quot line check parallel fact result calcul level note final report line consid parallel calcul logarithm rel activ log mean formula gt pic file quot quot gt real activ suppos activ rel activ rel activ found rang repeat assai make adjust extract concentr standard solut rel activ brought requir rang result obtain consid approxim note final report line consid parallel repeat determin parallel achiev determin consid unsatisfactori repeat differ result determin carri sampl analyst exce mg kg absolut content avoparcin mg kg relat highest content mg kg mg kg absolut content mg kg relat highest content mg kg determin monensin sodium diffus agar medium purpos scope method determin monensin sodium feedingstuff premix lower limit determin mg kg ppm principl sampl extract methanol extract submit procedur monensin sodium content sampl antibiot activ determin measur diffus monensin sodium agar medium inocul bacillu subtili diffus shown format zone inhibit micro organ diamet zone direct proport logarithm antibiot concentr rang antibiot concentr emploi sensit assai system reduc presenc sodium ion micro organ bacillu subtili atcc ncib mainten stock cultur inocul tube slope cultur medium bacillu subtili incub overnight ºc store cultur refriger ºc reinocul month prepar spore suspens harvest growth recent prepar agar slope mean ml steril water suspens inocul ml cultur medium contain roux flask incub dai ºc harvest growth ml ethanol check sporul microscop mix suspens month ºc cultur media reagent cultur medium gt pic file quot quot gt assai medium gt pic file quot quot gt mg monensin sodium equival uk unit method provid establish give similar spore suspens commerci cultur medium similar composit give result ethanol dilut ml ethanol ml water methanol anhydr methanol dilut ml methanol ml water methanol dilut ml methanol ml water aluminium oxid granul alcoa mesh activ alumina ug lancast equival standard substanc monensin sodium activ intern laboratori biolog standard central veterinari laboratori weybridg uk surrei kt nb apparatu rotari vacuum evapor ml round bottom flask glass tube chromatographi intern diamet mm length mm open end mm diamet glass tube chromatographi intern diamet mm length approxim mm open end mm diamet standard solut dissolv accur weigh quantiti standard substanc methanol dilut give stock solut monensin sodium ml store stopper flask ºc solut stabl week stock solut prepar success dilut methanol solut gt pic file quot quot gt prepar extract extract premix weigh quantiti sampl add ml methanol homogen centrifug minut dilut supernat solut methanol obtain expect monensin sodium content ml feedingstuff level monensin sodium lower ppm weigh quantiti sampl add ml methanol homogen minut leav settl insert cotton wool plug narrow end glass tube add aluminium oxid gentl tap column reach mm high decant extract aluminium oxid column collect filtrat dilut ml filtrat ml water make subsequ dilut methanol obtain expect monensin sodium content ml feedingstuff level monensin sodium lower ppm limit ppm weigh quantiti sampl add ml methanol homogen minut centrifug till clear sampl ppm monensin sodium ml supernat liquid sampl ppm ml evapor dryness vacuum rotari evapor ºc dissolv residu ml methanol insert cotton wool plug narrow end glass tube add aluminium oxid gentl tap column reach mm high decant methanol solut residu aluminium oxid column collect filtrat wash column ml methanol combin wash filtrat evapor solut dryness vacuum rotari evapor ºc dissolv residu ml anhydr methanol make ml water centrifug solut min minut make subsequ dilut methanol obtain expect monensin sodium content ml assai solut solut prepar solut expect content ml expect content ml expect content ml mean success dilut methanol assai procedur inocul assai medium inocul assai medium spore suspens ºc preliminari trial plate assai medium determin quantiti spore suspens requir give largest clearest zone inhibit concentr monensin sodium prepar plate diffus agar carri plate concentr standard solut concentr assai solut concentr extract standard necessarili plate effect select plate big hole diamet mm mm centr made agar medium test carri plate consist sheet glass face aluminium plastic ring top mm diamet mm high pour plate quantiti medium inocul give layer mm thick ml plate mm diamet set level posit bore hole place measur volum assai standard solut ml hole diamet appli concentr time determin subject evalu zone inhibit incub incub plate approxim hour ºc evalu measur diamet zone inhibit nearest mm record measur concentr semi logarithm graph paper show logarithm concentr relat diamet zone inhibit plot quot fit quot line standard solut extract determin quot fit quot point standard lowest level sl formula gt pic file quot quot gt determin quot fit quot point standard highest level sh formula gt pic file quot quot gt similarli calcul quot fit quot point extract lowest level ul extract highest level uh substitut formula record calcul sl sh valu graph paper join give quot fit quot line standard solut similarli record ul uh join give quot fit quot line extract absenc interfer line parallel practic purpos line consid parallel valu sh sl uh ul differ line found parallel discard sl sh ul uh calcul altern formula give altern quot fit quot line gt pic file quot quot gt similarli ul uh altern quot fit quot line check parallel fact result calcul level note final report line consid parallel calcul logarithm rel activ log mean formula gt pic file quot quot gt real activ suppos activ rel activ rel activ found rang repeat assai make adjust extract concentr standard solut rel activ brought requir rang result obtain consid approxim note final report line consid parallel repeat determin parallel achiev determin consid unsatisfactori repeat differ result determin carri sampl analyst exce relat highest content monensin sodium mg kg mg kg absolut content mg kg relat highest content mg kg