Uvavanyo lwegwele ngemethylene blue: ukubala iiseli eziphilayo xa kuthelekiswa nokulinganisa ixesha lokuphefumla

Kukho iityhubhu ezimbini phezu kwetafile yaselabhoratri. Zombini ziluhlaza okwesibhakabhaka. Kwenye, iiseli eziluhlaza okwesibhakabhaka ezibonwa phantsi kwemikroskopu zithetha igwele elifileyo. Kwenye, ityhubhu etshintsha isuke kumbala oluhlaza okwesibhakabhaka iye ekungabinambala ithetha ukuba igwele liyaphila kwaye liyaphefumla. Yidayi enye, kodwa iziphumo zithetha izinto ezichaseneyo. Xa ubhidanisa ezi mvavanyo zimbini, zonke izigqibo ziyajika.

Lo mahluko ngowona mxholo weli nqaku. Imethylene blue yidayi ye-redox: xa ikwimo eneoksideshini iluhlaza okwesibhakabhaka, xa incitshisiwe iba yileucomethylene blue engenambala. Ukubala iiseli eziphilayo kusebenzisa le khemistri ekuzijongeni okwexeshana phantsi kwemikroskopu. Uvavanyo lokuphefumla kweeseli luyisebenzisa ekulinganiseni ixesha lokusabela kwesampulu yonke. Nganye kwezi mvavanyo ifuna inkqubo yayo, izilawuli zayo, nokuhlolwa ngononophelo kweziphumo zayo.

Ukubala iiseli eziphilayo: daya okwexeshana, uze ufunde iziphumo ngokukhawuleza

Inkqubo esetyenziswa ekwenzeni ibhiya yeyona ibekelwe imigangatho ecacileyo. Lungisa imethylene blue eyi-0.01% w/v kwisodium citrate eyi-2%, uyixube ngomlinganiselo we-1:1 nolwelo oluneeseli zegwele ezixhonyiweyo, uze uhlolisise lo mxube emva kwexesha elifutshane elimiselweyo. Amaxesha apapashiweyo ahluka ukusuka kwimizuzu eyi-1 ukuya kweyi-2 kwincwadi yesikhokelo yeBrewing Science Institute ukuya malunga nemizuzu eyi-5 kwiinkqubo zophando, ngoxa indlela ye-OIV yokuhlola iintsholongwane kwiwayini ifuna ukuba uhlolo lwenziwe kwisithuba semizuzu eyi-10. Idayi ekumxube wokubala iphelela kufutshane ne-0.005%. Faka umxube kwigumbi lokubala leNeubauer eliphuculweyo, uze xa ukhulise malunga ne-400x uhlele iiseli eziluhlaza okwesibhakabhaka obumnyama njengezingaphiliyo, neeseli ezingadaywanga njengeziphilayo. Kwindlela ye-OIV, iiseli eziluhlaza okwesibhakabhaka okhanyayo zibalwa njengeziphilayo, nto leyo esele ibonisa ingxaki yokuxhomekeka ekutolikeni komntu ekuxoxwa ngayo ngezantsi.

Uxinaniso lweeseli lubalwa kusetyenziswa imilinganiselo yegumbi lokubala. Xa kubalwa zonke iiseli ezikwisikwere esisembindini se-1 mm, iiseli nge-mL zilingana no-N ophindwe ngomlinganiso wokuxutywa ngolwelo, ophindwe nge-10^4, apho u-N linani leeseli ezibalwe kuloo ndawo, njengoko kuchaziwe kwiindlela ze-ASBC zokuhlola igwele. Ipesenti yokuphila kweeseli ilula ngakumbi: yahlula inani leeseli ezingadaywanga ngenani lazo zonke iiseli, uze uphindaphinde nge-100. Bala malunga neeseli eziyi-400 ukuze uqikelele ukuphila kwazo, ulandela uphononongo lwe-FEMS oluthelekisa iindlela kwindawo yokwenza ibhiya kunye nenkqubo ye-OIV. Sebenzisa umthetho omnye wemida ngokungaguquguqukiyo, umzekelo ubandakanye iiseli ezikwimigca yegridi engezantsi nasekhohlo, uze ungazibali ezikwimigca ephezulu nasekunene.

Amaqhuma amatsha akhula kwiiseli afuna umthetho ngaphambi kokuba kuqalwe ukubala. Umkhwa oqhelekileyo kukubala iqhuma njengeseli eyahlukileyo xa lifikelela malunga nesiqingatha sobukhulu beseli engumama; xa lingaphantsi koko, libalwa kunye neseli engumama. Nokuba ukhetha wuphi umthetho, wusebenzise kwisikwere ngasinye kwaye uwubhale kwiphepha lokubala ukuze umntu olandelayo enze okufanayo.

Iphepha lokubala (liprinte)

Eli phepha lokubala lenzelwe eli nqaku ngokukhethekileyo. Likhuphele kwincwadi yokubhalela okanye uprinte elinye kwisampulu nganye.

Ukuphila kweeseli (%) = 100 x ezingadaywanga / (ezingadaywanga + eziluhlaza okwesibhakabhaka obumnyama). Zonke iiseli nge-mL = N x umlinganiselo wokuxutywa ngolwelo x 10^4, apho u-N avela kwindawo echaziweyo yegumbi lokubala. Bhala ixesha lokudaya ecaleni kwesiphumo, kuba inani lokuphila kweeseli elingachazwanga ixesha lalo alinakuphindwa ngendlela ethembekileyo.

Kutheni amanani okuphila kweeseli etshintsha

Ixesha yinto yokuqala ephazamisayo, kwaye linokuphazamisa kumacala omabini. Iiseli ezisebenzayo ngokwemetabolism ziyayinciphisa idayi, ngoko ukufunda iziphumo kwangethuba kunokufumana iiseli eziphilayo zingekagqibi ukuyinciphisa, zize zihlelwe ngempazamo njengezifileyo. Kodwa ukuhlala kwidayi ixesha elide nako kuyityhefu kwiiseli kwaye kudala iiseli eziluhlaza okwesibhakabhaka ezonakeleyo ngokwenene, nto leyo enokunika umfanekiso ongachanekanga wokufa kwazo. Ngoko inkqubo yokufundisa yaselabhoratri icacisa ukuba iiseli mazihlelwe kwisithuba semizuzu eyi-5, njengoko kubhalwe kwinkqubo yaseWisconsin yokufundisa ngegwele. Indlela echanekileyo yokuyichaza kukuba ukuhlelwa kweeseli ngemethylene blue kuxhomekeke kwixesha, ngoko misela ixesha elinye elifutshane uze uligcine lifana kuzo zonke iisampulu ezikuluhlu.

Iisampulu ezineeseli eziphilayo ezimbalwa zinenye ingxaki yokuthambekela kweziphumo. Indlela ebekelwe umgangatho ilumkisa ukuba imethylene blue inokunika uqikelelo oluphezulu kunokuphila kokwenene xa oko kungaphantsi malunga ne-80%, kuba iiseli ezisakwazi ukunciphisa idayi azisoloko zisakwazi ukuzala. Uncwadi lokwenziwa kwebhiya luchaza ukungathembeki okufanayo xa ukuphila kweeseli kwehle kakhulu. Yithathe imethylene blue njengendlela ekhawulezayo yokufumana uqikelelo nje, uze uqinisekise izigqibo ezibalulekileyo xa ukuphila kweeseli kusezantsi, ezifana nokuphinda usebenzise igwele elikhuliswe ngaphambili, ngokubala kwiipleyiti zokukhulisa okanye ngenye idayi encedisayo.

Iziphumo ezibangelwa lulwakhiwo lwesampulu ziza kwindawo yesithathu. Indlela ye-OIV isebenza kufutshane ne-pH 4.6 kwaye ilumkisa ukuba iisampulu ezine-pH ephantsi egcinwa ingatshintshi zizithinteli ezinamandla zithintela ukuziphatha okufanelekileyo kwedayi. Ikwacebisa ukuba olu vavanyo lungasetyenziswa xa iswekile ingaphezu kwe-100 g/L, apho iiseli zibonakala ziluhlaza okwesibhakabhaka okhanyayo ngendlela elahlekisayo. Uthelekiso lovavanyo lwe-pH ngexesha lokudaya lwafumanisa utshintsho olukhulu kubuntununtunu bovavanyo kuluhlu oluvavanyiweyo, nto leyo ehambelana nophononongo lwendlela yemethylene blue exhomekeke kwi-pH. Inkunkuma yenye ingozi eyahlukileyo: amasuntswana adayiweyo kwimixube emdaka yokwenza ibhiya afana neeseli ezifileyo kwaye andisa umahluko phakathi kweziphumo zabantu abenza uvavanyo.

Lo mahluko ulinganisiwe, awusekelwanga kumabali nje. Kuvavanyo lweenyanga ezintandathu kwindawo yokwenza ibhiya oluchazwe kuthelekiso lwe-FEMS lweendlela zokuhlola igwele, imilinganiselo yemethylene blue eyenziwe kabini yayinokuphambuka okuqhelekileyo okungumndilili we-6.1%, xa kuthelekiswa ne-0.2% yokubala iiseli ngokuhamba kolwelo nge-oxonol. Abantu ababini abenza uvavanyo babenokwahluka ukuya kuthi ga kumanqaku epesenti ayi-13 kwisampulu enye. Uthelekiso lwamanani lweendlela zokulinganisa ukuphila kweeseli lwango-2023 lwafumanisa ukuba imikroskopu ye-fluorescence nokubala iiseli ngokuhamba kolwelo zinika iziphumo ezichane ngakumbi, ngoxa lubonisa nokuba ukusebenza kuxhomekeke kuhlobo lwegwele nakulwakhiwo lwesampulu. Inkcazelo eyaziwayo yale ndlela eyenziwa ngu-Painting noKirsop (PMID 24430080) iseyeyona ndawo yokubhekisa, kwaye uphononongo olubanzi lweemvavanyo zokuphila nokusebenza kweeseli zegwele (PMID 25154541) lubeka imethylene blue phakathi kweemvavanyo ezincedisanayo, kungekhona ezinikela isigqibo esiqinisekileyo. Itrypan blue ayisosiphuculo esizenzekelayo apha: ibonisa ukulahleka kokuthembeka kwenwebu yeseli, ngoxa imethylene blue ibonisa ukufunxwa kwedayi kunye nomsebenzi wokuyinciphisa, ngoko iiseli eziphantsi koxinzelelo zinokuhlelwa ngokwahlukileyo ngezi dayi zimbini.

Uvavanyo lokuphefumla kweeseli: linganisa ixesha lokunyamalala kombala oluhlaza okwesibhakabhaka

Uvavanyo lwesibini alusebenzisi mikroskopu konke konke. Igwele kunye neglucose kunye nemethylene blue exutywe kakhulu ngolwelo zifakwa kwityhubhu evaliweyo, kuze kulinganiswe ixesha elithathwa ngumbala oluhlaza okwesibhakabhaka ukuba unyamalale. Njengoko iiseli zisenza imetabolism, zivelisa izinto ezidlulisela ii-elektroni eziguqula idayi eluhlaza okwesibhakabhaka ibe kwimo engenambala, ngoxa ioksijini enyibilikileyo iyibuyisela kwimo eneoksideshini. Xa iiseli zehlise inani leoksijini, ukuncitshiswa kwedayi kuyongamela kwaye ityhubhu iphelelwe ngumbala. Ngoko indawo yokuphela kovavanyo ibonisa ulungelelwano phakathi kokuphefumla kweeseli, uxinaniso lwedayi, nokufumaneka kweoksijini. Ifanele ichazwe njengexesha lokuncitshiswa okanye lokuphela kombala wemethylene blue, kungekhona njengomlinganiselo othe ngqo wesantya sokusetyenziswa kweoksijini yeemolekyuli.

Sebenzisa idayi exutywe ngolwelo kakhulu kunaleyo isetyenziswa ekubaleni iiseli eziphilayo ukuze ixesha lokuphela kombala libe kuluhlu olulungele ukulinganiswa. Incwadi yesikhokelo ye-AQA A level yomsebenzi owenziwa ngezandla oyimfuneko iyimisela kakuhle le nkqubo: i-1 g yeglucose kwi-100 mL yamanzi afudunyezwe afikelela malunga ne-30 C, kongezwe i-5 g yegwele elomisiweyo kanye ngaphambi kokusetyenziswa, kunye nedayi yokusebenza ekufutshane ne-0.001% elungiswa ngokuxuba i-0.1 mL yesisombululo esisisiseko se-1% ngolwelo ide ibe yi-100 mL. Xuba i-2 mL yolwelo oluneeseli zegwele neglucose kunye ne-2 mL yedayi yokusebenza, vumela umxube ufikelele kubushushu bovavanyo, wushukumise kube kanye kangangemizuzwana eyi-10, uqalise isilinganisi-xesha, uze ungaphindi uwushukumise. Ukushukumisa kubuyisela ioksijini kuze kubuyisele umbala oluhlaza okwesibhakabhaka kwityhubhu, nto leyo eyeyona mpazamo inkulu ibangelwa yinkqubo. I-AQA iqaphela ukuba umxube olungisiweyo ufanele ngokuqhelekileyo uphelelwe ngumbala kwisithuba esimalunga nemizuzu eyi-5 kwi-35 C, kwaye kuvavanyo amaxesha angumndilili ayeyi-275 s kwi-20 C, i-128 s kwi-35 C, ne-145 s kwi-45 C. Le yimizekelo yenkqubo enye, ayiyomigangatho esebenza kuzo zonke iimeko. Unxibelelwano olulinganisekayo phakathi kwesantya sokuncitshiswa kwedayi negwele elisebenzayo ngokwemetabolism phantsi kweemeko ezilawulwayo luxhaswa luphando olujolise ekuncitshisweni kwedayi, kuquka umsebenzi odweliswe njenge-PMID 25773544 kunye nophononongo lwendlela le nkqubo esebenza ngayo kwi-Yeast (2014).

Umboniso wokutshintsha ekuhambeni kwexesha (owenzelwe eli nqaku)

Itheyibhile engezantsi ngumboniso owenzelwe eli nqaku kusetyenziswa inkqubo efana neye-AQA echazwe ngasentla: i-2 mL yomxube wegwele neglucose kunye ne-2 mL yedayi yokusebenza ekufutshane ne-0.001%, ukushukumisa kube kanye kangangemizuzwana eyi-10, kuze kulandelwe kukugcina umxube kwiimeko zobushushu ezimiselweyo ungaphazanyiswa. Amanani akuyo yimizekelo yeziphumo ngendlela iqela labafundi elifanele ukuzibhala ngayo, apho isalathisi sesantya sichazwa njenge-1 eyahlulwe ngexesha lokuphela kombala elilinganiswe ngemizuzwana. Yenza uvavanyo ngezi tyhubhu zifanayo uze utshintshe la manani ufake amaxabiso owalinganisileyo.

Zoba igrafu yesalathisi sesantya xa sithelekiswa nobushushu kwiityhubhu A, B, no-C. Igophe liyenyuka lize lizinze okanye lehle, okuyindlela elindelekileyo yomsebenzi oqhutywa zii-enzyme, kungekhona umgca othe tye. Iityhubhu D, E, no-F azingomanqaku edatha kwelo gophe. Zizilawuli ezenza kube lula ukutolika igophe.

Izilawuli ezenza uvavanyo ngalunye lube nentsingiselo

Uvavanyo ngalunye lokuphefumla kweeseli lufuna izilawuli ezintathu ezihamba nalo. Igwele elibilisiweyo okanye elibuleweyo kunye neglucose kunye nedayi kufanele lihlale liluhlaza okwesibhakabhaka, libonise ukuba ukuncitshiswa okukhawulezayo kwedayi kuxhomekeke kwimetabolism ephilayo. Igwele eliphilayo kunye nedayi ngaphandle kokongezwa kweglucose sisilawuli sento esetyenziswa ziiseli, kodwa lindela ukuphela kombala okucothayo nangona kunjalo, kuba igwele lisebenzisa oovimba balo bangaphakathi. Iglucose kunye nedayi ngaphandle kwegwele yisampulu esisiseko yemichiza, ebalulekileyo kuba iglucose ekwiimeko zealkali inokunciphisa imethylene blue kungekho naziphi na iiseli.

Ukubala iiseli eziphilayo kufuna uhlobo olwahlukileyo lolawulo: ukungaguquguquki kwenkqubo. Sebenzisa isisombululo esisisiseko sedayi esifanayo, umlinganiselo wokuxuba ofanayo, ixesha lokudaya elifanayo, indawo efanayo yegumbi lokubala, nemithetho efanayo yamaqhuma amatsha neyemida, kubalwe ngaphandle kokwazi iinkcukacha zesampulu apho kunokwenzeka. Xa iziphumo zabantu ababini abenza uvavanyo zahluka ngamanqaku alishumi, iphepha lokubala lidla ngokutyhila unobangela: amaxesha ahlukileyo, imida eyahlukileyo yokugweba ubunzulu bombala oluhlaza okwesibhakabhaka, okanye imithetho eyahlukileyo yamaqhuma amatsha. Ubuchule bokulungisa nokujonga iiseli ezidayiweyo bunxulumene neendlela ezilula zokudaya iibhaktheriya, ngoxa uphononongo olubanzi lwezinto eziphawulwa yile dayi kwiisampulu ezahlukeneyo lukwi-atlasi yokudaya kwimicroscopy. Ngokuphathelele ifiziksi yokulinganisa nabuphi na ubunzulu bombala oluhlaza okwesibhakabhaka, isalathiso sokufunxwa kokukhanya esineemeko ezichaziweyo sichaza ukuba kutheni ikhofishiyenti nganye kufuneka ihambisane neemeko zesinyibilikisi sayo, i-pH, noxinaniso.

Khetha uvavanyo ngokombuzo. Umbuzo wokuba zingaphi iiseli eziphilayo ngoku ngumbuzo wokubala, ophendulwa kwimicroscope kusetyenziswa iphepha lokubala. Umbuzo wokuba igwele elikhulisiweyo liphefumla ngamandla kangakanani ngumbuzo wesantya senkqubo, ophendulwa ngesilinganisi-xesha kunye neetyhubhu ezilawulwayo. Imethylene blue isebenza kuzo zombini, kodwa ayikhe izenze zombini kwityhubhu enye ngaxeshanye.