Amagerageza y'umusemburo akoresha ubururu bwa metilene: kubara uturemangingo tuzima no gupima igihe cyo guhumeka

Udutembo tubiri turi ku meza ya laboratwari. Twombi turimo ubururu. Muri kamwe, uturemangingo tw'ubururu tubonwa muri mikorosikopi tugaragaza umusemburo wapfuye. Mu kandi, guhinduka kuva ku bururu kugera ku kutagira ibara bigaragaza ko umusemburo ari muzima kandi uhumeka. Irangi ni rimwe, ariko ibisobanuro by'ibibonwa biratandukanye rwose. Iyo witiranyije aya magerageza abiri, imyanzuro yose irahinduka.

Iryo tandukaniro ni ryo iyi nyandiko yose ishingiyeho. Ubururu bwa metilene ni irangi rya redox: iyo ryatakaje electron riba ubururu, iyo ryakiriye electron rikaba leucomethylene blue itagira ibara. Kubara uturemangingo tuzima bikoresha iyo miterere ya shimi mu kureba by'igihe gito muri mikorosikopi. Igerageza ryo guhumeka riyikoresha mu mpinduka ibera mu ruvange rwose, igapimwa uko igihe gihita. Buri gerageza rikenera uburyo bwaryo, amagerageza ngenzuzi yaryo n'ubwitonzi bwaryo mu gusobanura ibisubizo.

Kubara uturemangingo tuzima: gusiga irangi mu gihe gito, ukareba bidatinze

Uburyo bukoreshwa mu nzoga ni bwo bufite amabwiriza ahuriweho cyane. Tegura ubururu bwa metilene bwa 0.01% w/v muri sitirate ya sodiyumu ya 2%, ubuvange ku gipimo cya 1:1 n'uturemangingo tw'umusemburo tureremba mu mazi, maze urebe uruvange nyuma y'igihe gito cyagenwe. Ibihe byatangajwe biratandukanye: kuva kuri 1 kugeza kuri 2 iminota mu gitabo cya Brewing Science Institute, kugeza ku minota igera kuri 5 mu mabwiriza y'ubushakashatsi; uburyo bwa OIV bwo gusuzuma mikorobe zo muri divayi busaba kureba bitarenze iminota 10. Mu ruvange rubarirwamo uturemangingo, irangi riba hafi ya 0.005%. Shyira uruvange mu cyumba cyo kubariramo cya Neubauer cyavuguruwe, maze ku gukuba ishusho hafi 400x, ushyire uturemangingo tw'ubururu bwijimye mu cyiciro cy'utakiri tuzima, naho utatashwe n'irangi mu cyiciro cy'utuzima. Mu buryo bwa OIV, uturemangingo tw'ubururu bwerurutse tubarwa nk'utuzima; ibyo bitanga ikimenyetso cy'ikibazo cyo gushingira ku myumvire y'ureba, kivugwa hepfo.

Ubwinshi bw'uturemangingo mu ngano y'amazi bubarwa hashingiwe ku miterere n'ingero by'icyumba cyo kubariramo. Iyo ubaze kare yose yo hagati ifite uruhande rwa 1 mm, uturemangingo kuri mL tubarwa nka N ikubwe n'igipimo cyo koroshya uruvange, ikubwe na 10^4; N ni umubare w'uturemangingo two muri ako gace, nk'uko bisobanurwa mu buryo bwa ASBC bwo gusuzuma umusemburo. Ijanisha ry'uturemangingo tuzima ryoroshye kurushaho: uturemangingo tutatashwe n'irangi tugabanyijwe n'uturemangingo twose, bikubwe na 100. Bara uturemangingo tugera kuri 400 kugira ngo ubone ikigereranyo cy'uturemangingo tuzima, ukurikije ubushakashatsi bwa FEMS bugereranya uburyo bwo mu nganda z'inzoga n'imikorere ya OIV. Koresha itegeko rimwe ku turemangingo turi ku mbibi, urugero ukabariramo uturi ku mirongo yo hasi n'iy'ibumoso, ukareka uturi ku mirongo yo hejuru n'iy'iburyo.

Utubuto dushibuka ku turemangingo dukeneye itegeko mbere yo gutangira kubara. Itegeko rikunze gukoreshwa ribara agashibuka nk'akaremangingo kihariye iyo kageze hafi kuri kimwe cya kabiri cy'ubunini bw'akaremangingo kakabyaye; iyo kari munsi y'ubwo bunini, tubarirwa hamwe. Itegeko wahitamo ryose, rikoreshe kuri buri kare kandi uryandike ku rupapuro rwo kubaruriraho kugira ngo undi muntu azarikoreshe uko ryakoreshejwe.

Urupapuro rwo kubaruriraho (rucapishe)

Uru rupapuro rwateguwe by'umwihariko ku bw'iyi nyandiko. Rwandukure mu ikaye cyangwa ucapishe urupapuro rumwe kuri buri cyitegererezo.

Viability (%) = 100 x unstained / (unstained + dark blue). Total cells per mL = N x dilution factor x 10^4, aho N iva mu gace kagenwe k'icyumba cyo kubariramo. Muri izi ngero, Viability ni ijanisha ry'uturemangingo tuzima, unstained ni uturemangingo tutatashwe n'irangi, dark blue ni utw'ubururu bwijimye, Total cells per mL ni uturemangingo twose kuri mL, naho dilution factor ni igipimo cyo koroshya uruvange. Andika igihe cyo gusiga irangi iruhande rw'igisubizo, kuko umubare w'uturemangingo tuzima udafite igihe wapimiweho udashobora kongera kuboneka mu buryo bumwe.

Impamvu imibare y'uturemangingo tuzima ihindagurika

Igihe ni cyo kibangamira igipimo mbere y'ibindi, kandi gikora mu byerekezo byombi. Uturemangingo dukora ibikorwa byo guhindura ibinyabutabire twakira electron mu irangi; bityo, kureba hakiri kare bishobora gutuma ubona uturemangingo tuzima tutararangiza guhindura irangi, ukatwibeshyaho nk'utwapfuye. Ariko kumara igihe kirekire mu irangi ubwabyo biratwangiza, bigatuma habaho uturemangingo tw'ubururu twangiritse koko, bikagaragaza urupfu rutari ruhari mbere yo gusiga irangi. Ni yo mpamvu amabwiriza yo kwigisha muri laboratwari asaba gushyira uturemangingo mu byiciro mu minota 5, nk'uko byanditse mu mabwiriza ya Wisconsin yo kwigisha ibijyanye n'umusemburo. Imvugo yitonze ni uko gushyira uturemangingo mu byiciro hakoreshejwe ubururu bwa metilene biterwa n'igihe; bityo gena igihe kimwe kigufi kandi ugikoreshe ku byitegererezo byose byo mu ruhererekane.

Ibyitegererezo birimo uturemangingo tuzima duke bifite ikindi kibogamisha igipimo. Uburyo bufite amabwiriza ahuriweho buburira ko ubururu bwa metilene bushobora kugaragaza ijanisha ry'uturemangingo tuzima riruta iririho koko iyo riri munsi ya hafi 80%, kuko uturemangingo tugishobora guhindura irangi tutaba byanze bikunze tugifite ubushobozi bwo kororoka. Inyandiko zo mu rwego rwo gukora inzoga zisobanura uko kutizerwa na zo iyo ubushobozi bw'uturemangingo bwo gukomeza kubaho bwagabanutse cyane. Fata ubururu bwa metilene nk'uburyo bwihuse butanga ikigereranyo, kandi wemeze ibyemezo bikomeye bishingiye ku turemangingo tuzima duke, nko kongera gukoresha umusemburo mu kindi cyiciro cyo gukora inzoga, ukoresheje kubara koloni ku masahani y'ubuhinzi bwa mikorobe cyangwa irindi rangi ryunganira.

Ingaruka z'ibindi bigize icyitegererezo ziza ku mwanya wa gatatu. Uburyo bwa OIV bukorerwa hafi ya pH 4.6 kandi buburira ko ibyitegererezo bifite pH iri hasi kandi bigoye kuyihindura bibuza irangi gukora neza; bunagira inama yo kudakoresha iri gerageza iyo isukari irenze 100 g/L, kuko uturemangingo tugaragara mu bururu bwerurutse bushobora kuyobya usuzuma. Igerageza ryagereranyije pH ikoreshwa mu gusiga irangi ryabonye ko ubushobozi bwo gutahura uturemangingo bwahindutse cyane mu ntera ya pH yapimwe, bihuje n'ubushakashatsi ku buryo bw'ubururu bwa metilene buterwa na pH. Ibisigazwa ni undi mutego: uduce twatashwe n'irangi mu mvange z'ibinyampeke zirimo umwanda dusa n'uturemangingo twapfuye, bikongera itandukaniro ry'ibisubizo hagati y'ababipima.

Iryo tandukaniro ryarapimwe; si ibyo abantu bavuga gusa. Mu igerageza ry'amezi atandatu mu ruganda rw'inzoga ryatangajwe mu igereranya rya FEMS ku musemburo, ibipimo bibiri by'ubururu bwa metilene byasubiwemo byagize impuzandengo y'ugutandukira bisanzwe ya 6.1%, ugereranyije na 0.2% mu kubara uturemangingo tunyura mu gikoresho hifashishijwe oxonol; abapimyi babiri batandukanyaga kugeza ku ngingo 13 z'ijanisha ku cyitegererezo kimwe. Igereranya ryo mu 2023 rishingiye ku mibare ry'uburyo bwo gupima uturemangingo tuzima ryasanze mikorosikopi ya floresansi no kubara uturemangingo tunyura mu gikoresho bitanga ibipimo bihamye cyane kurushaho, ariko ryerekana ko imikorere iterwa n'ubwoko bw'umusemburo n'ibindi bigize icyitegererezo. Ibisobanuro by'uburyo busanzwe byatanzwe na Painting na Kirsop (PMID 24430080) biracyari ishingiro ryifashishwa, kandi isuzuma ryagutse ry'amagerageza y'ubushobozi bw'umusemburo bwo kubaho n'imbaraga z'ibikorwa byawo (PMID 25154541) rishyira ubururu bwa metilene mu bipimo byunganira aho kuba ibitanga umwanzuro wa nyuma. Ubururu bwa trypan si bwo buryo buhita buruta ubu: bugaragaza kwangirika k'agahu k'akaremangingo, naho ubururu bwa metilene bukagaragaza kwinjira kw'irangi hamwe n'ubushobozi bwo kuriha electron; bityo, uturemangingo twahuye n'ibidushegesha dushobora gushyirwa mu byiciro bitandukanye muri ayo marangi abiri.

Igerageza ryo guhumeka: pima igihe ubururu bumara bushira

Igerageza rya kabiri ntirikoresha mikorosikopi na rimwe. Umusemburo, gilikoze n'ubururu bwa metilene bworoshywe bishyirwa mu gatembo gafunze, maze isaha igapima igihe ubururu bumara bushira. Mu gihe uturemangingo duhindura ibinyabutabire, dutanga ibinyabutabire biha electron irangi ry'ubururu, bikarihindura rikabura ibara, naho ogisijeni yashonze mu mazi ikongera kurikuramo electron, ikarisubiza ubururu. Iyo uturemangingo tumaze kugabanya ogisijeni, iyakira rya electron riruta itakaza ryazo, maze ibara ryo mu gatembo rigashira. Bityo, igihe igerageza rirangirira kigaragaza uko guhumeka, ubwinshi bw'irangi n'ogisijeni ihari biringanira; kigomba gutangazwa nk'igihe ubururu bwa metilene bumara bwakira electron cyangwa bushirwaho n'ibara, aho kuba igipimo kiziguye cy'umuvuduko wo gukoresha ogisijeni yo mu buryo bwa molekile.

Koresha irangi ryoroshywe cyane kurusha iryo mu kubara uturemangingo tuzima, kugira ngo igihe ripimwa kibe mu ntera yoroshye gukoreramo. Igitabo cya AQA cy'amagerageza ategetswe ku rwego rwa A level gitanga amabwiriza ahuriweho neza: 1 g ya gilikoze kuri 100 mL z'amazi ashyushye kugera hafi 30 C, 5 g z'umusemburo wumishijwe zongerwamo ako kanya mbere yo gukoresha, n'irangi ryo gukoresha rya hafi 0.001%, ritegurwa mu koroshya 0.1 mL y'igisubizo kibitswe cya 1% kugeza kuri 100 mL. Vanga 2 mL z'uturemangingo tw'umusemburo tureremba mu gisubizo cya gilikoze na 2 mL z'irangi ryo gukoresha, utegereze ko bigera ku bushyuhe bw'igerageza, unyeganyeze rimwe mu masegonda 10, utangize isaha, kandi ntuzongere kunyeganyeza. Kunyeganyeza byongera kwinjiza ogisijeni kandi bigasubiza ubururu mu gatembo; iki ni cyo gitera ikosa rikomeye kurusha ibindi mu buryo bwo gukora. AQA ivuga ko uruvange rusanzwe rugomba gushirwaho n'ibara mu minota igera kuri 5 kuri 35 C, ikagaragaza impuzandengo zo mu magerageza za 275 s kuri 20 C, 128 s kuri 35 C na 145 s kuri 45 C. Izo ni ingero zavuye mu buryo bumwe bwo gutegura igerageza, si ibipimo rusange byo kugereranyaho. Isano ibarika hagati y'umuvuduko wo kwakira electron n'umusemburo ukora ibikorwa byo guhindura ibinyabutabire mu mimerere igenzurwa ishyigikirwa n'ubushakashatsi bwihariye kuri iyo mpinduka, burimo ubushakashatsi bubaruwe nka PMID 25773544 n'ubushakashatsi ku mikorere bwatangajwe muri Yeast (2014).

Urugero rw'impinduka uko igihe gihita (rwateguwe by'umwihariko)

Imbonerahamwe ikurikira ni urugero rwateguwe by'umwihariko ku bw'iyi nyandiko, hakoreshejwe uburyo bwa AQA bwasobanuwe hejuru: 2 mL z'uruvange rw'umusemburo na gilikoze hamwe na 2 mL z'irangi ryo gukoresha rya hafi 0.001%, kunyeganyeza rimwe mu masegonda 10, hanyuma kubireka mu bushyuhe bwagenwe nta kongera kubikoraho. Ibyanditse ni ingero z'ibipimo, mu buryo itsinda ry'abanyeshuri rikwiye kubyandikamo; igipimo cy'umuvuduko gisobanurwa nka 1 igabanyijwe n'igihe ibara rimara rishira mu masegonda. Kora igerageza ku dutembo nk'utu maze usimbuze ibyanditsemo ibipimo wabonye.

Shushanya igipimo cy'umuvuduko ugereranyije n'ubushyuhe ku dutembo A, B na C. Umurongo urazamuka, nyuma ukaringanira cyangwa ukamanuka; iyo ni yo shusho itegerejwe mu bikorwa biterwa na enzime, si umurongo ugororotse. Udutembo D, E na F si ingingo z'ibipimo zo kuri uwo murongo. Ni amagerageza ngenzuzi atuma uwo murongo ushobora gusobanurwa.

Amagerageza ngenzuzi atuma buri gipimo kigira icyo gisobanura

Buri gerageza ryo guhumeka rikenera andi atatu ariherekeza. Umusemburo watetswe cyangwa wishwe, hamwe na gilikoze n'irangi, ugomba kugumana ubururu, ukagaragaza ko kwakira electron vuba biterwa n'ibikorwa by'uturemangingo tuzima. Umusemburo muzima hamwe n'irangi, nta gilikoze yongewemo, ni igerageza ngenzuzi ry'ibinyabutabire bikorerwaho; ariko utegereze ko ibara rishira buhoro n'ubwo nta gilikoze irimo, kuko umusemburo ukoresha ibiwutunga bibitswe imbere mu turemangingo. Gilikoze hamwe n'irangi, nta musemburo urimo, ni igerageza ngenzuzi rya shimi; ni ngombwa kuko gilikoze iri mu mimerere ya alikali ishobora guha electron ubururu bwa metilene nta turemangingo na duke turimo.

Kubara uturemangingo tuzima bikenera ubundi bwoko bw'igenzura: kudahindura uburyo bwo gukora. Igisubizo cy'irangi kibitswe kimwe, igipimo cyo kuvanga kimwe, igihe cyo gusiga irangi kimwe, agace k'icyumba cyo kubariramo kamwe, amategeko amwe ku dushibuka no ku mbibi, no kubara utazi icyitegererezo icyo ari cyo aho bishoboka. Iyo abapimyi babiri batandukanyijeho ingingo icumi z'ijanisha, urupapuro rwo kubaruriraho akenshi rugaragaza impamvu: ibihe bitandukanye, imipaka itandukanye yo kwemeza ubururu cyangwa amategeko atandukanye ku dushibuka. Uburyo bwo gutegura no kureba uturemangingo twasizwe irangi bufitanye isano n'uburyo bworoshye bwo gusiga irangi bagiteri, naho isuzuma ryagutse ry'ibyo irangi rigaragaza mu byitegererezo bitandukanye riri mu gitabo cy'amashusho yo gusiga irangi mu bushakashatsi bwa mikorosikopi. Ku bijyanye na fiziki yo gupima ubukana bw'ubururu ubwo ari bwo bwose mu mibare, inyandiko yifashishwa ku iyinjizwa ry'urumuri, igaragaza imimerere yakoreshejwe, isobanura impamvu buri gihamya kigomba guherekezwa n'amakuru y'umushongi, pH n'ubwinshi bw'irangi byakoreshejwe.

Hitamo igerageza ukurikije ikibazo. Kumenya umubare w'uturemangingo tuzima ubu ni ikibazo cyo kubara, gisubirizwa kuri mikorosikopi hifashishijwe urupapuro rwo kubaruriraho. Kumenya uko umusemburo worowe uhumeka cyane ni ikibazo cy'umuvuduko w'impinduka, gisubirizwa hifashishijwe isaha ipima igihe n'udutembo turi mu mimerere igenzurwa. Ubururu bwa metilene bukoreshwa muri byombi, ariko ntibukora byombi mu gatembo kamwe mu gihe kimwe.