oj oj oj commiss direct april establish commun method analysi offici control feedingstuff eec commiss european commun regard treati establish european econom commun regard council direct juli introduct commun method sampl analysi offici control feedingstuff articl thereof direct requir offici control feedingstuff carri commun method sampl analysi purpos check complianc requir aris provis laid law regul administr action qualiti composit feedingstuff commiss direct eec june eec novemb establish number commun method analysi view progress made subsequ work set method adopt measur provid direct accord opinion stand committe feedingstuff adopt direct articl member state requir analys offici control feedingstuff level starch crude protein crude protein dissolv pepsin hydrochlor acid free total gossypol pepsin activ carri method annex direct gener provis set part introduct annex commiss direct eec june establish commun method analysi offici control feedingstuff applic method annex direct articl member state requir analys offici control feedingstuff purpos detect identifi antibiot tetracyclin group determin level chloretracyclin oxytetracyclin tetracyclin oleandomycin tylosin virginiamycin feedingstuff carri method annex ii direct gener provis set part introduct annex commiss direct eec june except prepar sampl analysi applic method annex ii direct articl member state juli bring forc law regul administr provis compli direct forthwith inform commiss thereof articl direct address member state brussel april commiss presid mansholt annex determin starch polarimetr method purpos scope method make determin level starch high molecular weight starch degrad product feedingstuff except feedingstuff beet chip beet pulp dri beet top leav potato pulp dehydr yeast product rice inulin chip meal jerusalem artichok greav principl method compris determin sampl treat hot dilut hydrochlor acid clarif filtrat optic rotat solut measur polarimetri sampl extract ethanol acidifi filtrat hydrochlor acid clarifi filter optic rotat measur determin differ measur multipli factor starch content sampl reagent hydrochlor acid hydrochlor acid concentr check titrat sodium hydroxid solut presenc methyl red ethanol ml ml naoh carrez solut dissolv zinc acet zn ch coo glacial acet acid water make ml water carrez solut ii dissolv potassium ferrocyanid fe cn water make ml water ethanol apparatu ml erlenmey flask standard ground glass joint reflux condens polarimet saccharimet procedur prepar sampl crush sampl fine pass mm round mesh siev determin total optic rotat observ weigh crush sampl nearest mg place ml graduat flask add ml hydrochlor acid shake obtain distribut test sampl add ml hydrochlor acid immers flask boil water bath shake vigor steadili minut prevent format agglomer quantiti water water bath suffici bath remain boil point flask introduc flask bath whilst shaken minut remov bath add ml cold water cool immedi add ml carrez solut shake minut add ml carrez solut ii shake minut make volum water homogen filter filtrat perfectli clear rare repeat determin larger quantiti carrez solut ii ml measur optic rotat solut mm tube polarimet saccharimet determin optic rotat substanc solubl ethanol weigh sampl nearest mg place ml graduat flask add ml ethanol observ leav flask stand hour room temperatur time shake vigor occas test sampl mix ethanol make volum ethanol homogen filter pipett ml filtrat sampl ml erlenmey flask add ml hydrochlor acid shake vigor fit reflux condens erlenmey flask immers boil water bath minut remov erlenmey flask bath transfer content ml graduat flask rins cold water cool clarifi carrez solut ii make volum water homogen filter measur optic rotat paragraph calcul result oj observ sampl carbon calcul term calcium carbon destroi treatment ab quantiti dilut sulphur acid determin total optic rotat case product high lactos content powder milk serum skim milk powder proce ad ml ethanol fit reflux condens flask immers water bath minut leav cool continu analysi determin crude protein purpos scope method make determin convention crude protein content feedingstuff basi nitrogen content determin kjeldahl method principl sampl digest wet combust acid solut alkal sodium hydroxid solut ammonia releas remov distil collect measur quantiti sulphur acid excess titrat solut sodium hydroxid reagent potassium sulphat catalyst cupric oxid cuo crystal cupric sulphat cuso mercuri mercur oxid hgo granul zinc sulphur acid sulphur acid sulphur acid methyl red indic dissolv mg methyl red ml ethanol solut sodium hydroxid sodium hydroxid solut sodium hydroxid solut satur solut sodium sulphid solut sodium thiosulph na granul pumic stone wash hydrochlor acid ash apparatu apparatu digest combust distil kjeldahl method observ procedur digest weight sampl nearest mg place flask digest apparatu add potassium sulphat quantiti catalyst cupric oxid cupric sulphat drop mercuri mercur oxid ml sulphur acid granul pumic stone homogen heat flask moder shake time time mass carbon foam disappear heat intens liquid boil steadili prevent side overh organ particl stick solut clear colourless light green copper base catalyst continu boil hour leav cool distil carefulli add ml water stir dissolv sulphat complet leav cool add granul zinc place collect flask distil apparatu measur quantiti ml sulphur acid depend presum nitrogen content observ add drop methyl red indic connect flask condens distil apparatu immers end condens liquid contain collect flask depth cm observ slowli pour ml sodium hydroxid solut flask drop funnel mercuri base catalyst add ml sodium sulphid solut ml sodium thiosulph solut heat flask approxim ml liquid distil minut end time check ph result distil litmu paper reaction alkalin continu distil discontinu distil neutral litmu paper distil colour observ shake content collect flask time time liquid turn yellow immedi add measur volum sulphur acid titrat collect flask titrat excess sulphur acid sodium hydroxid solut depend normal sulphur acid colour turn pale yellow verif method establish reagent free nitrogen carri blank test distil titrat omit sampl analyz check accuraci method carri analysi digest distil titrat acetanilid presenc nitrogen free sucros acetanilid consum ml sulphur acid calcul result determin volum sulphur acid consum ml sulphur acid correspond mg nitrogen multipli quantiti nitrogen factor express result percentag sampl repeat differ result parallel determin carri sampl exce absolut crude protein content rel content absolut content observ apparatu requir transfer digest distil apparatu transfer carri loss product low nitrogen content volum sulphur acid collect flask reduc ml made ml water flask distil apparatu fit drop funnel add sodium hydroxid immedi connect flask condens pour liquid slowli side condens mix acid solut determin crude protein dissolv pepsin hydrochlor acid purpos scope method make determin fraction crude protein dissolv pepsin hydrochlor acid defin condit applic feedingstuff principl sampl heat hour solut pepsin hydrochlorid suspens filter nitrogen content filtrat determin method determin crude protein reagent hydrochlor acid hydrochlor acid mg pepsin pepsin activ defin method part annex establish method freshli prepar solut pepsin hydrochlor acid activ anti foam emuls silicon reagent list method determin crude protein apparatu water bath incub set kjeldahl digest distil apparatu procedur prepar solut observ weigh sampl mearest mg place ml graduat flask add ml pepsin hydrochlorid solut previous heat shake prevent format agglomer check ph suspens place flask water bath incub leav hour shake hour hour add ml hydrochlor acid cool make volum water filter digest ml filtrat place flask distil apparatu add reagent digest sentenc method determin crude protein homogen bring boil foam form add drop anti foam emuls continu boil vigor water complet evapor reduc heat carefulli elimin trace water solut clear colourless light green copper base catalyst continu boil hour leav cool distil titrat proce method determin crude protein blank test carri blank test appli procedur omit sampl analyz calcul result subtract volum sulphur acid consum blank test consum test sampl ml sulphur acid correspond mg nitrogen multipli quantiti nitrogen factor express result percentag sampl repeat differ result parallel determin carri sampl exce absolut content relat content absolut content observ valu obtain method direct connect digest vivo product oil fat content exceed defat extract petroleum ether estim pepsin activ purpos scope method make establish activ pepsin determin crude protein dissolv pepsin hydrochlor acid principl haemoglobin treat pepsin hydrochlor acid medium defin condit hydrolyz fraction protein precipit trichloroacet acid sodium hydroxid folin ciocalteu reagent ad filtrat optic densiti solut measur nm quantiti tyrosin read calibr curv definit unit pepsin defin quantiti enzym condit method liber minut quantiti hydroxyaryl group stain folin ciocalteu reagent optic densiti umol tyrosin stain manner reagent hydrochlor acid hydrochlor acid hydrochlor acid solut trichloroacet acid sodium hydroxid solut folin ciocalteu reagent place sodium tungstat na wo sodium molybd na moo ml water litr round bottom flask fit standard ground glass joint add ml phosphor acid ml concentr hydrochlor acid connect reflux condens flask bring boil solut gentli boil hour leav cool detach reflux condens add lithium sulphat li ml water ml bromin boil minut elimin excess bromin leav cool transfer solut litr graduat flask make volum water homogen filter greenish color remain dilut volum reagent volum water haemoglobin solut weigh quantiti haemoglobin approx protein substratum determin anson mg nitrogen place ml flask fit standard ground glass joint add ml hydrochlor acid connect flask vacuum pump shake haemoglobin complet dissolv releas vacuum shake add hydrochlor acid make ml prepar immedi standard tyrosin solut dissolv mg tyrosin hydrochlor acid make litr acid stick solut ml dilut ml hydrochlor acid ml solut umol tyrosin apparatu water bath set ultrathermostat spectrophotomet chronomet accuraci ph meter procedur prepar solut observ dissolv mg pepsin ml hydrochlor acid pipett ml solut ml graduat flask make volum hydrochlor acid ph check ph meter immers flask water bath hydrolysi pipett ml haemoglobin solut test tube heat water bath add ml pepsin solut obtain mix glass rod thicken end back movement leav test tube water bath minut time addit pepsin solut durat temperatur strictli observ add ml trichloroacet acid solut previous heat homogen filter dry filter develop color measur optic densiti pipett ml filtrat ml erlenmey flask add ml sodium hydroxid solut shake constantli ml dilut folin ciocalteu reagent minut determin optic densiti solut spectrophotomet nm cell cm thick water blank test determin carri blank test pipett ml haemoglobin solut test tube heat water bath add ml trichloroacet acid solut previous heat homogen add ml pepsin solut obtain mix glass rod leav test tube water bath minut homogen filter dry filter follow procedur calibr curv place ml aliquot standard tyrosin solut umol tyrosin ml erlenmey flask complet seri refer solut free tyrosin make volum ml hydrochlor acid add ml sodium hydroxid solut shake constantli ml dilut folin ciocalteu reagent measur optic densiti sentenc trace calibr curv plot optic densiti quantiti tyrosin calcul result oj observ quantiti pepsin dissolv final photometr measur optic densiti obtain unit mg obtain method correspond anson milliunit mg umol tyrosin mg min commerci unit umol tyrosin min determin free gossypol purpos scope method make determin level free gossypol total gossypol chemic relat substanc cottonse cottonse meal cottonse cake compound feedingstuff substanc ppm present principl gossypol extract presenc aminopropan ol mixtur propan ol hexan determin free gossypol dimethylformamid determin total gossypol gossypol convert anilin gossypol dianilin optic densiti measur nm reagent propan ol hexan mixtur mix part volum propan ol part volum hexan solvent place litr graduat flask approxim ml propan ol hexan mixtur ml aminopropan ol ml glacial acet acid ml water make volum propan ol hexan mixtur reagent stabl week solvent pipett ml aminopropan ol ml glacial acet acid ml graduat flask cool room temperatur make volum dimethylformamid reagent stabl week anilin optic densiti blank test exce distil anilin zinc dust discard fraction distil refriger store brown stopper glass flask reagent month standard gossypol solut place mg gossypol acet ml graduat flask dissolv make volum solvent pipett ml solut ml graduat flask make volum solvent gossypol concentr solut mg ml leav stand hour room temperatur standard gossypol solut place mg gossypol acet ml graduat flask dissolv make volum solvent gossypol concentr solut mg ml standard gossypol solut remain stabl hour protect light apparatu mixer tumbler approxim rpm spectrophotomet procedur test sampl amount test sampl depend presum gossypol content sampl prefer work small test sampl larg aliquot part filtrat obtain suffici gossypol precis photometr measur determin free gossypol cottonse cottonse meal cottonse cake test sampl exce compound feedingstuff ml aliquot part filtrat suitabl case ug gossypol determin total gossypol test sampl ml aliquot part filtrat ug gossypol analysi carri room temperatur determin free gossypol place test sampl ground neck ml flask bottom flask cover crush glass pipett add ml solvent stopper flask mix hour mixer filter dry filter collect filtrat small ground neck flask filtrat cover funnel watch glass pipett ident aliquot part filtrat ug gossypol ml graduat flask necessati make volum ml solvent make content flask volum propan ol hexan mixtur solut refer solut measur sampl solut pipett ml solvent eqch ml graduat flask make content flask volum propan ol hexan mixtur solut refer solut measur blank test solut add ml anilin flask heat minut boil water bath develop colour cool room temperatur make volum propan ol hexan mixtur homogen leav stand hour determin optic densiti blank test solut comparison refer solut optic densiti sampl solut comparison refer solut spectrophotomet nm cm glass cell substract optic densiti blank test solut sampl solut correct optic densiti calcul free gossypol content determin total gossypol place test sampl mg gossypol ml graduat flask add ml solvent time prepar blank test place ml solvent ml graduat flask heat flask minut boil water bath cool room temperatur make content flask volum propan ol hexan mixtur homogen leav settl minut filter collect filtrat ground neck flask pipett ml sampl filtrat ml graduat flask ml blank test filtrat ml flask make content flask seri ml propan ol hexan mixtur solut refer solut add ml anilin flask heat minut boil water bath develop colour cool room temperatur make ml propan ol hexan mixtur homogen leav stand hour determin optic densiti free gossypol calcul total gossypol content calcul result oj determin nitrogen content semi micro kjeldahl method theoret content nitrogen annex ii detect identif antibiot tetracyclin group purpos scope method make detect identifi antibiot tetracyclin group feedingstuff ppm antibiot concentr premix principl sampl extract mixtur methanol hydrochlor acid extract refer solut comparison subject ascend paper chromatographi antibiot detect identifi compar rf valu standard substanc fluoresc uv light high antibiot content bioautographi agar medium inocul cereu reagent cultur medium buffer solut ph citric acid monohydr disodium hydrogen phosphat na hpo aceton ml distil water ml phosphat buffer solut ph potassium dihydrogen phosphat kh po disodium hydrogen phosphat na hpo distil water ml eluent mixtur pure nitromethan pure chloroform dichloropropan ol volum prepar immedi eluent ii mixtur pure nitromethan pure chloroform picolin volum prepar immedi mixtur pure methanol hydrochlor acid volum hydrochlor acid ammonia standard substanc chlortetracyclin oxytetracyclin tetracyclin activ express term hydrochlorid micro organ cereu atcc mainten parent strain prepar spore suspens inocul cultur medium follow direct method determin chlortetracyclin oxytetracyclin tetracyclin content diffus agar part annex cultur medium glucos tryptic pepton meat extract yeast extract agar distil water ml adjust ph immedi triphenyltetrazolium chlorid solut glucos solut apparatu apparatu ascend paper chromatographi height paper cm schleicher schuell paper equival centrifug incub set lamp detect fluoresc glass plate approxim cm bioautographi standard solut stock solut hydrochlor acid prepar standard substanc solut concentr ug ml chlortetracyclin hcl oxytetracyclin hcl tetracyclin hcl refer solut detect uv light dilut solut phosphat buffer solut obtain solut concentr ug ml chlortetracyclin hcl oxytetracyclin hcl tetracyclin hcl refer solut detect bioautographi dilut solut phosphat buffer solut obtain solut concentr ug ml chlortetracyclin hcl oxytetracyclin hcl tetracyclin hcl extract presum antibiot content ppm homogen sampl finest fraction separ siev antibiot found fraction suspend sampl mixtur centrifug collect supernat liquid directli dilut mixtur obtain antibiot concentr approxim ug ml ug ml detect identif chromatographi immers paper buffer solut ph remov excess liquid press paper sheet dry filter paper place volum ml refer solut extract paper give good separ paper correct moistur content leav dry develop ascend chromatographi eluent detect bioautographi eluent ii detect uv light solvent front climb cm approx hour minut stop chromatographi dry paper detect uv light antibiot level greater ug cm chromatogram treat ammonia vapour golden yellow fluoresc spot irradi uv lamp detect bioautographi pour cultur medium previous inocul cereu glass plate place paper cultur medium minut contact detach paper place spot cultur medium remain incub period incub overnight antibiot tetracyclin group present light inhibit zone cloudi cultur medium fix chromatogram solut vapor paper incub identif rel rf valu antibiot tetracyclin group valu vari slightli qualiti paper moistur content chlortetracyclin ctc tetracyclin tc oxytetracyclin otc epi ctc epi tc epi otc antibiot activ quot epi quot compound normal compound determin chlortetracyclin oxytetracyclin tetracyclin diffus agar purpos scope method make determin level chlortetracyclin ctc oxytetracyclin otc tetracyclin tc feedingstuff concentr premix ppm present content ppm estim graphic interpol principl content ppm sampl extract dilut formamid content greater ppm extract mixtur aceton water hydrochlor acid determin ctc mixtur methanol hydrochlor acid determin otc tc extract dilut antibiot activ determin measur diffus ctc otc tc agar medium seed cereu diffus made evid format inhibit zone presenc micro organ diamet zone directli proport logarithm antibiot concentr micro organ cereu atcc mainten parent strain inocul cereu tube slope agar cultur medium free methylen blue boric acid incub overnight approxim cultur refriger inocul slope agar dai prepar spore suspens collect bacteria tube slope agar ml physiolog salin suspens seed roux flask ml cultur medium free methylen blue boric acid agar concentr incub dai collect spore ml ethanol check sporul microscop homogen suspens refriger month preliminari test plate basic medium determin establish quantiti inoculum concentr antibiot give largest inhibit zone clear quantiti ml ml cultur medium inocul cultur media reagent basic medium determin glucos tryptic pepton meat extract yeast extract agar qualiti quot tween quot ml phosphat buffer solut ph ml boric acid solut ml solut methylen blue ethanol ml distil water ml adjust ph phosphat buffer solut ph potassium dihydrogen phosphat kh po disodium hydrogen phosphat na hpo distil water ml phosphat buffer solut ph dilut phosphat buffer solut ph potassium dihydrogen phosphat kh po disodium hydrogen phosphat na hpo distil water ml steril physiolog salin ethanol hydrochlor acid formamid prepar fresh adjust ph sulphur acid approxim mixtur pure aceton water hydrochlor acid volum mixtur pure methanol hydrochlor acid volum standard substanc ctc otc tc activ express term hydrochlorid standard solut chloretracyclin hydrochlor acid prepar standard solut stock solut concentr ug ml chlortetracyclin hcl solut week refriger stock solut prepar standard work solut concentr ug ml chlortetracyclin hcl dilut carri phosphat buffer solut ph dilut amido black ad prepar success dilut buffer solut concentr ug ml ug ml ug ml oxytetracyclin proceed prepar stock solut concentr ug ml oxytetracyclin hcl standard work solut ug ml oxytetracyclin hcl concentr ug ml ug ml ug ml tetracyclin proceed prepar stock solut concentr ug ml tetracyclin hcl standard work solut ug ml tetracyclin hcl concentr ug ml ug ml ug ml extract content ppm test sampl add formamid quantiti tabl shake minut shake platform dilut immedi phosphat buffer solut indic tabl obtain concentr formamid concentr solut exce centrifug decant obtain clear solut prepar concentr success dilut phosphat buffer solut content greater ppm chlortetracyclin test sampl depend presum antibiot content sampl manufactur guarante add time volum mixtur shake minut shake platform ph remain extract readjust ph acet acid miner compound aliquot part extract adjust ph phosphat buffer solut ph presenc bromocresol green turn yellow blue dilut phosphat buffer solut ph dilut obtain concentr prepar concentr success dilut phosphat buffer solut oxytetracyclin tetracyclin proce mixtur mixtur determin method inocul cultur medium inocul basic medium determin spore suspens prepar trai diffus agar carri trai concentr standard solut concentr extract concentr extract standard solut trai choos trai larg hole mm diamet made agar medium calcul quantiti inocul cultur medium need provid uniform cover approxim mm thick test prefer carri trai consist flat glass plate fit perfectli level aluminium plastic ring mm diamet mm high pipett hole accur measur quantiti ml antibiot solut depend diamet hole sampl repeat diffus time concentr determin compris evalu inhibit zone incub incub trai approxim hour evalu measur diamet inhibit zone prefer project record measur semi logarithm paper plot logarithm concentr diamet inhibit zone trace line standard solut extract provid interfer line parallel logarithm rel activ calcul formula oj repeat differ result parallel determin carri sampl exce rel turbidimetri purpos scope method make determin level chlortetracyclin ctc oxytetracyclin otc tetracyclin tc concentr greater kg provid interfer substanc cloud extract method quicker diffus agar principl determin ctc sampl extract mixtur aceton water hydrochlor acid mixtu methanol hydrochlor acid determin otc tc extract dilut antibiot effect determin measur light transmiss cultur medium seed staphylococcu aureu antibiot ad light transmiss depend antibiot concentr micro organ staphylococcu aureu mainten parent strain inocul aureu tube slope agar cultur medium agar ad depend qualiti incub overnight cultur refriger inocul slope agar week time prepar cultur laboratori prepar inoculum hour inocul slope agar cultur incub overnight suspend cultur contain tube agar approxim ml basic medium transfer suspens steril condit approxim ml basic medium incub water bath growth strain enter logarithm phase hour minut hour cultur media reagent basic medium determin pepton yeast extract meat extract sodium chlorid glucos potassium dihydrogen phosphat kh po di potassium hydrogen phosphat hpo distil water ml ph steril phosphat buffer solut ph potassium dihydrogen phosphat kh po distil water ml hydrochlor acid mixtur pure aceton water hydrochlor acid volum mixtur pure methanol hydrochlor acid volum approxim formaldehyd solut standard substanc ctc otc tc activ express term hydrochlorid standard solut hydrochlor acid prepar standard substanc stock solut concentr ug ml ctc hcl otc hcl tc hcl solut week refriger extract chlortetracyclin place test sampl ml graduat flask add approxim ml mixtur shake minut shake platform make volum phosphat buffer solut ph homogen leav settl oxytetracyclin tetracyclin place test sampl ml graduat flask add approxim ml mixtur shake minut shake platform make volum phosphat buffer solut ph homogen leav settl determin method prepar standard seri extract dilut standard solut extract phosphat buffer solut ph obtain seri concentr determin calibr curv drawn respect concentr permit interpol valu relat extract dilut chosen condit strain grown vari laboratori procedur gener chlortetracyclin dilut standard solut phosphat buffer solut obtain standard work solut concentr ug ml ctc hcl phosphat buffer solut prepar test tube dilut dilut duplic dilut extract phosphat buffer solut obtain presum ctc hcl concentr ug ml place ml solut tube ml ug tube make volum tube ml phosphat buffer solut oxytetracyclin tetracyclin dilut standard solut phosphat buffer solut obtain standard work solut concentr ug ml otc hcl tc hcl phosphat buffer solut prepar test tube dilut dilut duplic dilut extract phosphat buffer solut obtain presum otc hcl tc hcl concentr ug ml place ml solut tube ml ug tube make volum tube ml phosphat buffer solut inocul cultur medium inocul basic medium determin inoculum obtain photomet nm light transmiss cm cell transmiss cm cell apparatu set transmiss inocul basic medium seed place ml inocul cultur medium tube tube fill clean necessarili steril condit incub incub carri water bath temperatur uniform stir incub period chosen gener hour minut hour trace transmiss curv gradient suitabl accur measur block growth rapidli inject ml formaldehyd solut tube measur growth measur transmiss photomet nm set apparatu transmiss clearest standard solut highest antibiot content tube show slight differ turbid cm prefer cm cell calcul result trace calibr curv millimetr graph paper plot photometr transmiss antibiot concentr interpol curv transmiss valu extract calcul antibiot content sampl repeat differ result parallel determin carri sampl exce rel determin oleandomycin diffus agar purpos scope method make presenc tetracyclin determin oleandomycin content feedingstuff concentr premix ppm present principl sampl extract dilut methanol solut tri hydroxymethylamino methan centrifug extract dilut antibiot activ determin measur diffus oleandomycin agar medium seed cereu diffus made evid format inhibit zone presenc micro organ diamet zone directli proport logarithm antibiot concentr micro organ cereu tr resist tetracyclin mainten parent strain inocul cereu tube slope agar cultur medium ug ml oxytetracyclin ad incub overnight approxim cultur refriger inocul slope agar week prepar spore suspens collect bacteria tube slope agar approxim ml physiolog salin suspens seed roux flask ml cultur medium agar concentr incub dai collect spore ml ethanol check sporul microscop homogen suspens refriger month preliminari test plate basic medium determin establish quantiti inoculum concentr oleandomycin give largest inhibit zone clear quantiti ml ml cultur medium inocul cultur media reagent medium mainten parent strain glucos tryptic pepton meat extract yeast extract agar qualiti distil water ml adjust ph immedi basic medium determin medium adjust ph steril physiolog salin ethanol pure methanol solut tri hydroxymethylamino methan extract solut pure methanol ml distil water ml tri hydroxymethylamino methan standard substanc oleandomycin activ standard solut dissolv standard substanc ml methanol dilut solut obtain oleandomycin concentr ug ml stock solut prepar standard work solut ug ml oleandomycin dilut solut prepar success dilut solut concentr ug ml ug ml ug ml extract test sampl depend presum oleandomycin content sampl add ml solut shake minut shake platform centrifug aliquot part extract dilut solut obtain presum oleandomycin concentr ug ml prepar concentr success dilut solut determin method inocul cultur medium inocul basic medium determin spore suspens prepar trai diffus agar carri trai concentr standard solut concentr extract concentr standard solut extract trai choos trai larg hole mm diamet made agar medium calcul quantiti inocul cultur medium need provid uniform cover approxim mm thick test prefer carri trai consist flat glass plate fit perfectli level aluminium plastic ring mm diamet mm high pipett hole accur measur quantiti ml antibiot solut depend diamet hole sampl repeat diffus time concentr determin compris evalu inhibit zone incub incub trai approxim hour evalu measur diamet inhibit zone prefer project record measur semi logarithm paper plot logarithm concentr diamet inhibit zone trce line standard solut extract provid interfer line parallel logarithm rel activ calcul formula oj repeat differ result parallel determin carri sampl exce rel determin tylosin diffus agar purpos scope method make determin tylosin content feedingstuff concentr premix ppm present principl sampl treat ph phosphat buffer solut previous heat extract methanol centrifug extract dilut antibiot activ determin measur diffus tylosin agar medium seed sarcina lutea diffus made evid format inhibit zone presenc micro organ diamet zone directli proport logarithm antibiot concentr micro organ sarcina lutea atcc mainten parent strain inocul sarcina lutea tube slope agar cultur medium adjust ph incub overnight approxim cultur refriger inocul slope agar month prepar bacteria suspens collect bacteria recent prepar tube slope agar ml physiolog salin suspens seed roux flask ml cultur medium adjust ph incub hour collect bacteria ml physiolog salin homogen dilut suspens obtain approxim light transmiss nm refriger suspens week preliminari test plate basic medium determin establish quantiti inoculum concentr tylosin give largest inhibit zone clear cultur medium inocul cultur media reagent basic medium determin glucos tryptic pepton meat extract yeast extract agar qualiti distil water ml adjust immedi ph mainten parent strain prepar bacteria suspens ph determin phosphat buffer solut ph potassium dihydrogen phosphat kh po dipotassium hydrogen phosphat hpo distil water ml phosphat buffer solut ph potassium dihydrogen phosphat kh po di potassium hydrogen phosphat hpo distil water ml steril physiolog salin pure methanol methanol mixtur phosphat buffer solut pure methanol volum standard substanc tylosin activ standard solut dry standard substanc hour vacuum oven mm mercuri weigh mg graduat flask dissolv ml methanol dilut solut phosphat buffer solut ph obtain tylosin base concentr ug ml prepar standard work solut ug ml tylosin base stock solut dilut mixtur prepar success dilut mixtur concentr ug ml ug ml ug ml extract concentr test sampl premix feedingstuff test sampl add ml phosphat buffer solut ph previous heat homogen minut domest mixer ultra turrax leav stand minut add ml methanol homogen minut centrifug extract dilut aliquot part mixtur obtain presum tylosin concentr ug ml prepar concentr success dilut mixtur content ppm evapor extract dry rotari evapor dissolv residu methanol determin method inocul cultur medium inocul basic medium determin adjust ph bacteria suspens prepar trai diffus agar carri trai concentr standard solut concentr extract concentr standard solut extract trai choos trai larg hole mm diamet made agar medium calcul quantiti inocul cultur medium need provid uniform cover approxim mm thick test prefer carri flat trai consist glass plate fit perfectli level aluminium plastic ring mm diamet mm high pipett hole accur measur quantiti ml antibiot solut depend diamet hole sampl repeat diffus time concentr determin compris evalu inhibit zone incub incub trai overnight evalu measur diamet inhibit zone prefer project record measur semi logarithm paper plot logarithm concentr diamet inhibit zone trace line standard solut extract provid interfer line parallel logarithm rel activ calcul formula oj repeat differ result parallel determin carri sampl exce rel determin virginiamycin diffus agar purpos scope method make determin virginiamycin content feedingstuff concentr premix ppm present principl sampl extract quot tween quot methanol solut centrifug filter extract dilut antibiot activ determin measur diffus virginiamycin agar medium seed sarcina lutea diffus made evid format inhibit zone presenc micro organ diamet zone directli proport logarithm antibiot concentr micro organ sarcina lutea atcc mainten parent strain inocul lutea tube slope agar cultur medium incub overnight approxim cultur refriger inocul slope agar dai prepar bacteria suspens collect bacteria recent prepar tube slope agar ml physiolog salin suspens seed roux flask ml cultur medium incub hour collect bacteria ml physiolog salin homogen dilut suspens obtain approxim light transmiss nm refriger suspens week preliminari test plate basic medium determin establish quantiti inoculum concentr virginiamycin give largest inhibit zone clear cultur medium inocul cultur media reagent basic medium determin glucos tryptic pepton meat extract yeast extract agar qualiti distil water ml adjust ph phosphat buffer solut ph potassium dihydrogen phosphat kh po dipotassium hydrogen phosphat hpo distil water ml steril physiolog salin pure methanol mixtur phosphat buffer solut pure methanol volum quot tween quot methanol solut standard substanc virginiamycin activ standard solut prepar methanol solut standard substanc ug ml virginiamycin stock solut prepar standard work solut ug ml virginiamycin dilut mixtur prepar success dilut mixtur concentr ug ml ug ml ug ml extract product virginiamycin content ppm test sampl add ml solut shake minut shake platform centrifug filter ml clear solut evapor dry rotari evapor dissolv residu ml mixtur obtain presum virginiamycin concentr ug ml prepar concentr success dilut mixtur product virginiamycin content greater ppm test sampl add ml solut shake minut shake platform centrifug filter dilut mixtur obtain presum virginiamycin concentr ug ml prepar concentr determin method inocul cultur medium inocul basic medium determin suspens bacteria prepar trai diffus agar carri trai concentr standard solut concentr extract concentr standard solut extract trai choos trai larg hole mm diamet made agar medium calcul quantiti inocul cultur medium need provid uniform cover approxim mm thick test prefer carri flat trai consist glass plate fit perfectli level aluminium plastic ring mm diamet mm high pipett hole accur measur quantiti ml antibiot solut depend diamet hole sampl repeat diffus time concentr determin compris evalu inhibit zone incub incub trai approxim hour evalu measur diamet inhibit zone prefer project record measur semi logarithm paper plot logarithm concentr diamet inhibit zone trace line standard solut extract provid interfer line parallel logarithm rel activ calcul formula oj repeat differ result parallel determin carri sampl exce rel commerci cultur medium similar composit give result amido black make evid inhibit zone standard solut blue ring strain isol lufa kiel grow rapidli aureu atcc strain isol lufa kiel