Gwaje-gwajen yisti da shuɗin methylene: ƙirga ƙwayoyin da ke raye da auna lokacin numfashi

Bututu biyu suna kan teburin gwaji. Dukansu shuɗi ne. A ɗaya, ƙwayoyin da suka yi shuɗi a ƙarƙashin madubin ƙara girma suna nufin yisti matacce. A ɗayan, bututun da ke sauyawa daga shuɗi zuwa marar launi yana nufin yistin yana raye kuma yana numfashi. Rini ɗaya, amma fassarar sakamakon ta saɓa. Idan aka rikita gwaje-gwajen biyu, kowace ƙarshe da aka cim ma za ta juya.

Wannan bambanci shi ne jigon labarin nan. Shuɗin methylene rini ne na redox: idan ya yi oxidation, shuɗi ne; idan aka rage shi, ya zama leucomethylene blue marar launi. Ƙirgar ƙwayoyin da ke raye tana amfani da wannan halayyar sinadarai a ɗan gajeren kallo ta madubin ƙara girma. Gwajin numfashi yana amfani da ita a wani martanin dukan gauraya da ake auna lokacinsa. Kowannensu yana buƙatar nasa tsarin aiki, nasa gwaje-gwajen kwatance, da nasa taka-tsantsan wajen fassara sakamako.

Ƙirgar ƙwayoyin da ke raye: ɗan gajeren rina, a karanta da wuri

Tsarin da masana'antun giya ke amfani da shi ne mafi daidaitaccen salo. Shirya shuɗin methylene 0.01% w/v a cikin sodium citrate 2%, gauraya shi 1:1 da ruwan da aka baza yisti a ciki, sannan a duba gaurayar bayan ɗan gajeren lokaci da aka kayyade. Lokutan da aka wallafa sun bambanta daga minti 1 zuwa 2 a cikin littafin jagorar Brewing Science Institute zuwa kusan minti 5 a hanyoyin bincike, yayin da hanyar OIV ta nazarin ƙananan halittu a ruwan inabi ke buƙatar a duba cikin minti 10. Ƙarfin rinin a gaurayar da za a ƙirga yana ƙarewa kusa da 0.005%. Cika ɗakin ƙirga na Neubauer da aka inganta, sannan a kusan 400x a ware ƙwayoyin shuɗi mai duhu a matsayin marasa rai, waɗanda ba su ɗauki rini ba kuma a matsayin masu rai. A hanyar OIV, ana ƙirga ƙwayoyin shuɗi mai haske a matsayin masu rai, abin da tuni ke nuni da matsalar bambancin hukuncin masu duba da za a tattauna a ƙasa.

Ana samun yawan ƙwayoyi a kowace ƙarar ruwa daga ma'aunin ɗakin ƙirga. Idan aka ƙirga dukan murabba'in tsakiya na 1 mm, yawan ƙwayoyi a kowace mL yana daidai da N sau ninkin tsarmawa sau 10^4, inda N yake adadin da aka ƙirga a wannan fili, kamar yadda aka bayyana a hanyoyin ASBC na yisti. Lissafin kaso na masu rai ya fi sauƙi: ƙwayoyin da ba su ɗauki rini ba a raba da jimillar ƙwayoyi, a ninka da 100. Ƙirga kusan ƙwayoyi 400 don kimanta kaso na masu rai, bisa binciken FEMS na kwatanta hanyoyin masana'antun giya da tsarin OIV. Yi amfani da ƙa'idar iyaka ɗaya a ko'ina, misali a haɗa ƙwayoyin da suke kan layukan ƙasa da hagu na ragar, a bar waɗanda suke kan layukan sama da dama.

Ana buƙatar ƙa'idar ƙirga toho kafin a fara ƙirga. Wata ƙa'ida da aka saba amfani da ita tana ƙirga toho a matsayin ƙwaya mai zaman kanta idan ya kai kusan rabin girman ƙwayar uwa; idan bai kai haka ba, ana ƙirga shi tare da uwar. Duk ƙa'idar da aka zaɓa, a yi amfani da ita a kowane murabba'i kuma a rubuta ta a takardar aiki don mai zuwa ya maimaita ta.

Takardar aikin ƙirga (a buga wannan)

An ƙirƙiri wannan takardar aiki musamman don wannan labarin. Kwafe ta cikin littafin rubutu ko a buga ɗaya ga kowane samfur.

Viability (%) = 100 x unstained / (unstained + dark blue). Total cells per mL = N x dilution factor x 10^4, inda N yake adadin da aka ƙirga a filin ɗakin ƙirga da aka kayyade. Rubuta lokacin rina a gefen sakamakon, domin ba za a iya maimaita adadin kaso na masu rai ba idan ba a san lokacin da aka yi amfani da shi ba.

Dalilin da adadin ƙwayoyin masu rai ke sauyawa

Lokaci shi ne abu na farko da ke rikita sakamako, kuma yana yin hakan ta hanyoyi biyu. Ƙwayoyin da ayyukan metabolism ɗinsu ke gudana suna rage rinin, don haka karantawa da wuri na iya kama ƙwayoyi masu rai kafin su gama rage shi, a yi kuskuren ware su a matsayin matattu. Amma tsawaita lokacin da suke cikin rinin shi ma yana da guba kuma yana haifar da ƙwayoyin shuɗi da suka lalace da gaske, yana ba da alamar mutuwa ta ƙarya. Saboda haka, wata hanyar koyarwa ta dakin gwaje-gwaje ta kayyade a tantance cikin minti 5, kamar yadda aka rubuta a tsarin koyar da gwajin yisti na Wisconsin. Abin da ya dace a ce shi ne tantancewa da shuɗin methylene ya dogara da lokaci, don haka a kayyade ɗan gajeren lokaci ɗaya kuma a kiyaye shi ga kowane samfur a jerin gwaje-gwajen.

Samfuran da ke da ƙarancin ƙwayoyi masu rai suna da wata karkatar sakamako ta biyu. Hanyar da aka daidaita tana gargaɗin cewa shuɗin methylene na iya nuna kaso na masu rai fiye da na gaskiya idan ya yi ƙasa da kusan 80%, domin ƙwayoyin da har yanzu suke rage rini ba lallai ne su riƙe ikon haihuwa ba. Rubuce-rubucen aikin giya suna bayyana irin wannan rashin tabbas idan kaso na masu rai ya ragu sosai. Ɗauki shuɗin methylene a matsayin hanya mai sauri ta samun kimantawa kawai, kuma a tabbatar da muhimman shawarwarin da suka shafi ƙarancin masu rai, kamar sake amfani da yistin da aka reno wajen sabon zagayen giya, ta ƙirga a farantin reno ko wani rini na taimako.

Tasirin sauran abubuwan da ke cikin samfur shi ne na uku. Hanyar OIV tana aiki kusa da pH 4.6 kuma tana gargaɗin cewa samfuran ƙarancin pH da ke da ƙarfin hana sauyin pH suna hana rinin yin aikin da ya dace, tare da ba da shawarar kada a yi gwajin idan sukari ya wuce 100 g/L, inda ƙwayoyin ke nuna shuɗi mai haske da zai ruɗi mai karantawa. Wani gwajin kwatanta pH na rina ya gano babban sauyi a ikon gano bambance-bambance a faɗin matakan da aka gwada, wanda ya yi daidai da binciken hanyar shuɗin methylene mai dogaro da pH. Tarkace wata matsala ce dabam: barbashin da ya ɗauki rini a gaurayar hatsi marar tsabta yana kama da matattun ƙwayoyi kuma yana ƙara bambancin sakamakon masu duba.

An auna wannan bambancin; ba zance ne kawai ba. A wani gwajin masana'antar giya na watanni shida da aka ruwaito a kwatancen yisti na FEMS, ma'aunai biyu-biyu na shuɗin methylene sun ba da matsakaicin karkacewar ma'auni na 6.1%, idan aka kwatanta da 0.2% na ƙirgar ƙwayoyi ta kwarara da oxonol. Sakamakon masu duba biyu a samfur ɗaya ya bambanta da har maki 13 na kaso. Wani kwatancen hanyoyin tantance masu rai ta ma'auni na 2023 ya gano cewa kallon ƙwayoyi ta madubin hasken fluorescence da ƙirgar ƙwayoyi ta kwarara sun fi bayar da sakamako mai daidaito sosai, tare da nuna cewa ingancin aiki ya dogara da nau'in yisti da sauran abubuwan da ke cikin samfur. Bayanin hanyar na asali daga Painting da Kirsop (PMID 24430080) ya kasance tushen komawa, yayin da wani bita mai faɗi kan gwaje-gwajen rayuwa da kuzarin yisti (PMID 25154541) ya sanya shuɗin methylene cikin gwaje-gwajen taimako maimakon waɗanda ke ba da hukunci na ƙarshe. Shuɗin trypan ba shi ne ingantaccen madadi kai tsaye a nan ba: yana nuna lalacewar cikakken shingen ƙwaya, yayin da shuɗin methylene ke nuna shigar rini da aikin rage shi. Don haka, ƙwayoyin da ke fuskantar matsin yanayi na iya shiga rukuni daban-daban a ƙarƙashin rinin biyu.

Gwajin numfashi: auna lokacin gushewar shuɗi

Gwaji na biyu ba ya amfani da madubin ƙara girma sam. Ana sanya yisti da glucose da shuɗin methylene mai rauni a cikin rufaffen bututu, sannan agogo ya auna tsawon lokacin da shuɗin ke ɗauka kafin ya gushe. Yayin da ƙwayoyi suke gudanar da metabolism, suna samar da abubuwan da ke ba da ƙarfin ragewa, waɗanda ke sauya shuɗin rini zuwa sifarsa marar launi, yayin da oxygen da ya narke a cikin ruwan ke sake yin oxidation ya mayar da shi shuɗi. Da zarar ƙwayoyin sun rage oxygen ɗin, aikin ragewa ya rinjaya kuma launin bututun ya gushe. Saboda haka, ƙarshen gwajin yana nuna daidaiton numfashi, ƙarfin rini, da samuwar oxygen. Ya kamata a bayyana sakamakon a matsayin lokacin rage shuɗin methylene ko gushewar launinsa, ba a matsayin ma'aunin kai tsaye na saurin amfani da oxygen na ƙwayoyin molecule ba.

Yi amfani da rini da aka tsarma fiye da wanda ake amfani da shi wajen ƙirgar masu rai, domin lokacin ya faɗa cikin zangon da ya dace. Littafin jagorar gwaje-gwajen wajibi na AQA A level ya daidaita wannan sosai: glucose 1 g a kowace ruwa 100 mL da aka ɗumama zuwa kusan 30 C, a ƙara busasshen yisti 5 g nan da nan kafin amfani, da rinin aiki kusa da 0.001% da aka shirya ta tsarma 0.1 mL na ruwan rini na asali 1% zuwa 100 mL. Gauraya 2 mL na ruwan yisti da glucose da 2 mL na rinin aiki, a bar shi ya kai zafin gwajin, a girgiza sau ɗaya na sakan 10, a fara agogon auna lokaci, sannan kada a sake girgizawa. Girgizawa tana sake shigar da oxygen kuma tana mayar da bututun shuɗi; wannan shi ne babban abin da tsarin aiwatarwa ke haifarwa wanda ke karkatar da sakamako. AQA ta lura cewa yawanci launin gaurayar ya kamata ya gushe cikin kusan minti 5 a 35 C, tare da matsakaicin lokutan gwaji na 275 s a 20 C, 128 s a 35 C, da 145 s a 45 C. Waɗannan misalai ne daga tsarin gwaji guda, ba ma'aunan kwatance na ko'ina ba. Bincike na musamman kan ragewa ya goyi bayan alaƙar ma'auni tsakanin saurin ragewa da yistin da metabolism ɗinsa ke gudana a ƙarƙashin yanayin da aka sarrafa, ciki har da aikin da aka lissafa a matsayin PMID 25773544 da binciken yadda wannan ke aiki a Yeast (2014).

Misalin sauyi da lokaci (abin da aka ƙirƙira musamman)

Teburin da ke ƙasa misali ne da aka ƙirƙira musamman don wannan labarin, ta amfani da tsarin AQA da aka bayyana a sama: gaurayar yisti da glucose 2 mL da rinin aiki 2 mL kusa da 0.001%, girgizawa sau ɗaya na sakan 10, sannan a bar shi a yanayin gwaji ba tare da motsawa ba. Abubuwan da ke ciki misalan karatu ne a tsarin da ƙungiyar ɗalibai za ta rubuta, inda ma'aunin sauri yake 1 a raba da lokacin gushewar launi a sakan. Yi gwajin waɗannan bututun iri ɗaya, sannan a maye gurbin waɗannan bayanai da ƙimomin da aka auna.

Zana jadawalin ma'aunin sauri da zafin jiki na bututun A, B, da C. Layin jadawalin yana tashi sannan ya lafa ko ya sauka, wanda shi ne tsarin da ake sa ran enzymes za su haifar, ba madaidaicin layi ba. Bututun D, E, da F ba maki ne na bayanai a wannan layin ba. Su ne gwaje-gwajen kwatance da ke sa a iya fassara layin.

Gwaje-gwajen kwatance da ke sa kowane gwaji ya kasance mai ma'ana

Kowane gwajin numfashi yana buƙatar abokan kwatance uku. Dafaffen yisti ko wanda aka kashe, tare da glucose da rini, ya kamata ya kasance shuɗi, yana tabbatar da cewa ragewa mai sauri ta dogara da metabolism na ƙwayoyi masu rai. Yisti mai rai da rini ba tare da ƙarin glucose ba gwajin kwatance ne na abin da ƙwayoyin ke amfani da shi, amma a sa ran gushewar launi a hankali duk da haka, domin yisti yana amfani da ajiyar abincinsa ta ciki. Glucose da rini ba tare da yisti ba shi ne gwajin tushe na sinadarai, wanda yake da muhimmanci domin glucose a yanayin alkaline na iya rage shuɗin methylene ba tare da wata ƙwaya ba.

Ƙirgar ƙwayoyin masu rai tana buƙatar wani nau'in kwatance dabam: daidaiton tsarin aiki. Ruwan rini na asali ɗaya, rabon gaurayawa ɗaya, lokacin rina ɗaya, filin ɗakin ƙirga ɗaya, ƙa'idodin toho da iyaka iri ɗaya, tare da ƙirga ba tare da sanin ainihin samfur ba idan zai yiwu. Idan sakamakon masu duba biyu ya bambanta da maki goma, takardar aiki yawanci tana bayyana dalili: bambancin lokaci, bambancin matakin shuɗi da ake ɗauka a matsayin alama, ko bambancin ƙa'idar toho. Dabarun shirya da kallon ƙwayoyin da aka rina suna da alaƙa da hanyoyin rina bakteriya masu sauƙi, yayin da bita mafi faɗi kan abin da rinin ke nuna wa a samfura daban-daban yake cikin atlas na rina samfura don kallon madubin ƙara girma. Game da ka'idodin kimiyyar lissafi da ke bayan auna ƙarfin kowane launin shuɗi, bayanin ma'aunin kwatance na shan haske da aka yi wa lakabin yanayin gwaji yana bayyana dalilin da ya sa dole ne kowace ƙimar coefficient ta zo tare da bayanan sinadarin narkarwa, pH, da ƙarfin ruwanta.

Zaɓi gwajin bisa tambayar da ake son amsawa. Yawan ƙwayoyin da suke raye a yanzu tambayar ƙirga ce, wadda ake amsawa da madubin ƙara girma da takardar aiki. Yadda yistin da aka reno yake numfashi da ƙarfi tambayar saurin aiki ce, wadda ake amsawa da agogon auna lokaci da bututun da aka sarrafa yanayinsu. Shuɗin methylene yana aiki ga duka biyun, amma ba a cikin bututu ɗaya a lokaci ɗaya ba.