Mayeso a yisiti ndi methylene blue: kuwerenga maselo amoyo poyerekeza ndi kuyeza nthawi ya kupuma

Machubu awiri ali patebulo la labotale. Onse ndi abuluu. Mu imodzi, maselo abuluu ooneka ndi maikulosikopu amatanthauza yisiti yakufa. Mu inayo, chubu chosanduka kuchoka ku buluu kukhala chopanda mtundu chimatanthauza kuti yisiti ndi yamoyo ndipo ikupuma. Utoto womwewo, koma matanthauzo otsutsana. Mukasokoneza mayeso awiriwa, tanthauzo lililonse la zotsatira limatembenuka.

Kusiyanitsa kumeneku ndi maziko a nkhani yonseyi. Methylene blue ndi utoto wa redox: ukakhala wooksidizidwa umakhala wabuluu, ndipo ukachepetsedwa umakhala leucomethylene blue yopanda mtundu. Kuwerenga maselo amoyo kumagwiritsa ntchito kusintha kwa mankhwala kumeneku pa kuyang’ana kwakanthawi kochepa ndi maikulosikopu. Kuyesa kupuma kwa maselo kumagwiritsa ntchito kusinthako m’chisakanizo chonse pamene nthawi ikuyezedwa. Mayeso aliwonse amafuna njira yake, zitsanzo zake zowongolera, ndi kusamala kwake pomasulira zotsatira.

Kuwerenga maselo amoyo: kupaka utoto kwakanthawi kochepa, n’kuwerenga mwamsanga

Njira yogwiritsidwa ntchito popanga mowa ndi yomwe ili ndi malangizo okhazikika kwambiri. Konzani methylene blue ya 0.01% w/v mu sodium citrate ya 2%, isakanizeni 1:1 ndi yisiti yomwazidwa m’madzi, ndipo yang’anani chisakanizocho patapita nthawi yochepa yokhazikika. Nthawi zotchulidwa m’mabuku zimasiyana kuchokera pa 1 mpaka 2 mphindi mu buku la Brewing Science Institute kufika pa pafupifupi 5 mphindi m’njira za kafukufuku, pamene njira ya OIV yofufuza tizilombo ta mu vinyo imafuna kuyang’ana pasanathe 10 mphindi. Utoto m’chisakanizo chowerengera umatha kukhala pafupifupi 0.005%. Ikani chisakanizocho m’chipinda chowerengera cha improved Neubauer, ndipo pa kukulitsa kwa pafupifupi 400x ikani maselo abuluu kwambiri m’gulu la osakhala amoyo ndi osapaka utoto m’gulu la amoyo. Maselo abuluu wopepuka amawerengedwa ngati amoyo mu njira ya OIV, zomwe zikusonyeza kale vuto la kusiyana kwa chiweruzo cha owerenga, lofotokozedwa pansipa.

Kuchuluka kwa maselo kumapezeka potengera miyeso ya chipinda chowerengera. Mukawerenga sikweya yonse yapakati ya 1 mm, maselo pa mL amafanana ndi N kuchulukitsa ndi mlingo wa kusungunula kuchulukitsa ndi 10^4, pomwe N ndi chiwerengero cha maselo m’deralo, monga momwe zalembedwera mu njira za ASBC za yisiti. Kuwerengera kuchuluka kwa maselo amoyo n’kosavuta kwambiri: maselo osapaka utoto kugawa ndi maselo onse, kuchulukitsa ndi 100. Werengani pafupifupi maselo 400 kuti muyerekezere kuchuluka kwa maselo amoyo, potsatira kafukufuku wa FEMS woyerekeza njira m’malo opangira mowa ndi mchitidwe wa OIV. Gwiritsani ntchito lamulo limodzi la malire mosasintha, mwachitsanzo kuwerengera maselo omwe ali pa mizere yapansi ndi yakumanzere ya gulidi, osawerengera omwe ali pa mizere yapamwamba ndi yakumanja.

Timaselo timene timaphukira pa maselo ena timafunika lamulo musanayambe kuwerenga. Lamulo lodziwika bwino limawerengera kamphukira ngati selo lapadera kakafika pafupifupi theka la kukula kwa selo limene kachokerako; kakakhala kakang’ono kuposa pamenepo, kamawerengedwa limodzi ndi selolo. Lamulo lililonse limene mwasankha, ligwiritseni ntchito pa sikweya iliyonse ndipo lilembeni papepala lowerengera kuti munthu wotsatira alibwereze.

Pepala lowerengera (sindikizani ili)

Pepala lowerengera ili lapangidwa mwapadera pa nkhaniyi. Likopeni m’kope kapena sindikizani limodzi pa chitsanzo chilichonse.

Maselo amoyo (%) = 100 x osapaka utoto / (osapaka utoto + abuluu kwambiri). Maselo onse pa mL = N x mlingo wa kusungunula x 10^4, pomwe N imachokera ku dera lodziwika la chipinda chowerengera. Lembani nthawi yopaka utoto pafupi ndi zotsatira, chifukwa chiwerengero cha maselo amoyo chopanda nthawi yake sichingabwerezedwe modalirika.

Chifukwa chake ziwerengero za maselo amoyo zimasintha

Nthawi ndi chinthu choyamba chosokoneza, ndipo imakhudza zotsatira m’njira ziwiri. Maselo omwe akuchita ntchito zosinthira zinthu m’thupi amachepetsa utoto, motero kuwerenga msanga kungapeze maselo amoyo asanamalize kuchepetsa utotowo n’kuwawerengera molakwika ngati akufa. Koma kukhala mu utoto kwa nthawi yaitali kumawononga maselo, n’kupanga maselo abuluu omwe awonongekadi, zomwe zimapangitsa utoto kuoneka ngati ukusonyeza kufa pamene cholinga chinali kuyeza mmene maselo analili poyamba. Choncho njira yophunzitsira ya labotale imanena kuti maselo awerengedwe pasanathe 5 mphindi, monga zalembedwera mu njira yophunzitsira za yisiti ya Wisconsin. Mawu oyenera ndi akuti kuwerengera ndi methylene blue kumadalira nthawi, choncho sankhani nthawi imodzi yochepa ndipo isunge mofanana pa chitsanzo chilichonse m’gulu la mayeso.

Zitsanzo zokhala ndi maselo amoyo ochepa zimakhala ndi kupendekeka kwina kwa zotsatira. Njira yokhazikika imachenjeza kuti methylene blue ingayerekeze kuchuluka kwa maselo amoyo mopitirira kuchuluka kwenikweni kukakhala pansi pa pafupifupi 80%, chifukwa maselo omwe akutha kuchepetsa utoto sakutanthauza kuti akuthabe kuberekana. Mabuku opanga mowa amafotokoza kusadalirika komweku pamene kuchuluka kwa maselo amoyo kwatsika kwambiri. Gwiritsani ntchito methylene blue ngati njira yofulumira yopereka chiyerekezo, ndipo tsimikizirani zisankho zofunika zokhudza maselo amoyo ochepa, monga kugwiritsanso ntchito yisiti pa kupanga mowa, powerengera makoloni pama mbale okulitsira kapena pogwiritsa ntchito utoto wina wowonjezera mayesowa.

Zotsatira za zinthu zina m’chitsanzo ndi zosokoneza zachitatu. Njira ya OIV imagwira ntchito pafupi ndi pH 4.6 ndipo imachenjeza kuti zitsanzo za pH yotsika zomwe zimaletsa kwambiri kusintha kwa pH zimalepheretsa utoto kugwira ntchito moyenera. Imalangizanso kuti mayesowa asagwiritsidwe ntchito shuga akapitirira 100 g/L, chifukwa pamenepo maselo amaoneka abuluu wopepuka m’njira yosokeretsa. Kuyerekezera pH pa kupaka utoto m’mayesero kunapeza kusintha kwakukulu kwa kuthekera kozindikira maselo m’mitengo ya pH yomwe inayesedwa, mogwirizana ndi kafukufuku wa njira ya methylene blue yodalira pH. Zinyalala ndi msampha wina: tinthu topaka utoto m’chisakanizo chosayera cha zopangira mowa timafanana ndi maselo akufa ndipo timakulitsa kusiyana pakati pa owerenga.

Kusiyana kumeneku kwayezedwa, sikungokhala nkhani zongofotokozedwa. M’mayesero a miyezi isanu ndi umodzi pamalo opangira mowa omwe anafotokozedwa mu kuyerekezera kwa FEMS kwa njira za yisiti, miyeso iwiri yobwerezedwa ya methylene blue inali ndi kupatuka kokhazikika kwa pafupifupi 6.1%, poyerekeza ndi 0.2% ya kuyeza maselo oyenda pogwiritsa ntchito oxonol. Owerenga awiri anasiyana ndi mpaka mfundo 13 za peresenti pa chitsanzo chomwecho. Kuyerekezera kwa mu 2023 kwa njira zoyezera maselo amoyo pogwiritsa ntchito ziwerengero kunapeza kuti maikulosikopu ya fluorescence ndi kuyeza maselo oyenda zimapereka miyeso yolondola kwambiri, ndipo kunasonyeza kuti mmene njira imagwirira ntchito kumadalira mtundu wa yisiti ndi zinthu zina m’chitsanzo. Kufotokoza kodziwika kwa njira imeneyi kochokera kwa Painting ndi Kirsop (PMID 24430080) kukadali maziko ofunika, ndipo ndemanga yotakata ya mayeso a kukhala ndi moyo ndi mphamvu yogwira ntchito za yisiti (PMID 25154541) imaika methylene blue pakati pa mayeso othandizana ndi ena, osati opereka chigamulo chomaliza paokha. Trypan blue si njira yabwinoko nthawi zonse pano: imasonyeza kuwonongeka kwa nembanemba ya selo, pamene methylene blue imasonyeza kulowa kwa utoto ndi ntchito yochepetsa utotowo. Choncho maselo omwe ali pa mavuto angagawidwe m’magulu osiyana pogwiritsa ntchito mitundu iwiri ya utotoyi.

Kuyesa kupuma kwa maselo: yezani nthawi imene buluu ukutha

Mayeso achiwiriwa sagwiritsa ntchito maikulosikopu. Yisiti, glucose, ndi methylene blue yosungunuka kwambiri zimayikidwa m’chubu chotsekedwa, ndipo wotchi imayeza nthawi imene buluu umatenga kuti uthe. Pamene maselo akusintha zinthu m’thupi mwawo, amapanga zinthu zopereka maelekitironi zomwe zimasintha utoto wabuluu kukhala wopanda mtundu, pamene oxygen yosungunuka m’madzimo imauoksidizanso. Maselo akachepetsa oxygen, kuchepetsedwa kwa utoto kumaposa kuoksidizidwanso kwake ndipo mtundu umatha m’chubu. Choncho mapeto a mayesowa amasonyeza kugwirizana kwa kupuma kwa maselo, kuchuluka kwa utoto, ndi kupezeka kwa oxygen. Ayenera kulembedwa ngati nthawi yochepetsa methylene blue kapena nthawi yotha kwa mtundu, osati ngati mlingo wachindunji wa kugwiritsa ntchito mamolekyulu a oxygen.

Gwiritsani ntchito utoto wosungunuka kwambiri kuposa umene mumagwiritsa ntchito powerengera maselo amoyo, kuti nthawi ikhale yabwino kuyezera. Buku la AQA la mayesero ofunikira a A level limapereka malangizo okhazikika bwino pa izi: glucose ya 1 g pa madzi a 100 mL otenthedwa kufika pafupifupi 30 C, yisiti youma ya 5 g yowonjezedwa musanayambe kugwiritsa ntchito, ndi utoto wogwiritsira ntchito wa pafupifupi 0.001%, wokonzedwa posungunula 0.1 mL ya utoto woyambira wa 1% kufika pa 100 mL. Sakanizani 2 mL ya yisiti ndi glucose zomwazidwa m’madzi ndi 2 mL ya utoto wogwiritsira ntchito, siyani mpaka zifike pa kutentha kwa mayeso, gwedezani kamodzi kwa masekondi 10, yambitsani wotchi, ndipo musagwedezenso. Kugwedeza kumalowetsanso oxygen n’kubwezeretsa buluu m’chubu, ndipo uku ndi kusokoneza kwakukulu kwambiri komwe kumachitika chifukwa cha mmene njira yagwiritsidwira ntchito. AQA imanena kuti chisakanizocho nthawi zambiri chimayenera kutaya mtundu pasanathe pafupifupi 5 mphindi pa 35 C, ndipo nthawi zapakati m’mayesero zinali 275 s pa 20 C, 128 s pa 35 C, ndi 145 s pa 45 C. Izi ndi zitsanzo zochokera ku dongosolo limodzi la mayeso, osati miyezo yogwira ntchito kulikonse. Kugwirizana kwa mlingo wa kuchepetsedwa kwa utoto ndi yisiti yomwe ikuchita ntchito zosinthira zinthu m’thupi pansi pa mikhalidwe yoyendetsedwa kumathandizidwa ndi kafukufuku wapadera wa kuchepetsedwa kwa utoto, kuphatikizapo ntchito yolembedwa ngati PMID 25773544 ndi kafukufuku wa mmene kusinthaku kumachitikira mu Yeast (2014).

Chionetsero cha kusintha pakapita nthawi (chopangidwa mwapadera)

Tebulo ili pansipa ndi chionetsero chopangidwa mwapadera pa nkhaniyi pogwiritsa ntchito dongosolo la mtundu wa AQA lofotokozedwa pamwambapa: 2 mL ya chisakanizo cha yisiti ndi glucose kuphatikiza 2 mL ya utoto wogwiritsira ntchito wa pafupifupi 0.001%, kugwedeza kamodzi kwa masekondi 10, kenako kusunga pa kutentha komweko popanda kusokoneza. Zolembedwazi ndi zitsanzo za miyeso m’maonekedwe amene gulu la ophunzira liyenera kulemba. Chizindikiro cha liwiro chimatanthauzidwa ngati 1 kugawa ndi nthawi yotha kwa mtundu m’masekondi. Yesani machubu omwewa ndipo sinthani zolembedwazi ndi miyeso yomwe mwapeza.

Jambulani tchati cha chizindikiro cha liwiro poyerekeza ndi kutentha kwa machubu A, B, ndi C. Mzerewo umakwera kenako umakhala wopingasa kapena kutsika, zomwe ndi mmene ntchito yotsogozedwa ndi ma enzyme imayembekezeredwa kukhala, osati mzere wowongoka. Machubu D, E, ndi F si mfundo za miyeso pa mzerewo. Ndi zitsanzo zowongolera zomwe zimathandiza kumasulira mzerewo.

Zitsanzo zowongolera zomwe zimapangitsa mayeso aliwonse kukhala ndi tanthauzo

Mayeso aliwonse a kupuma kwa maselo amafuna zitsanzo zina zitatu. Yisiti yowiritsidwa kapena yophedwa, glucose, ndi utoto ziyenera kukhalabe zabuluu, posonyeza kuti kuchepetsedwa kwachangu kwa utoto kumadalira ntchito za maselo amoyo. Yisiti yamoyo ndi utoto popanda glucose wowonjezedwa ndi chitsanzo chowongolera cha zinthu zomwe maselo amagwiritsa ntchito, koma yembekezerani kuti mtundu utha pang’onopang’ono ngakhale zili choncho, chifukwa yisiti imagwiritsa ntchito zosungidwa m’maselo ake. Glucose ndi utoto popanda yisiti ndi chitsanzo chowongolera kusintha kwa mankhwala popanda maselo. Ichi n’chofunika chifukwa glucose m’mikhalidwe ya alkaline imatha kuchepetsa methylene blue popanda maselo alionse.

Kuwerenga maselo amoyo kumafuna kuwongolera kwa mtundu wina: kusasinthasintha kwa njira. Gwiritsani ntchito utoto woyambira womwewo, chiŵerengero chosakanizira chomwecho, nthawi yopaka utoto yomweyo, dera la chipinda chowerengera lomwelo, ndi malamulo a timaselo tophukira ndi malire omwewo. Werengani popanda kudziwa chitsanzocho kumene zingatheke. Owerenga awiri akasiyana ndi mfundo khumi za peresenti, pepala lowerengera nthawi zambiri limasonyeza chifukwa chake: nthawi zosiyana, malire osiyana osankhira buluu wopepuka ndi buluu kwambiri, kapena malamulo osiyana a timaselo tophukira. Njira zokonzera ndi kuona maselo opaka utoto zimagwirizana ndi njira zosavuta zopakira mabakiteriya utoto, pamene kufotokoza kwakukulu kwa zomwe utotowu umaonetsa m’zitsanzo zosiyanasiyana kuli mu buku lazithunzi za kupaka utoto poona ndi maikulosikopu. Pa mfundo za fiziki zomwe zimathandiza kuyeza mphamvu ya buluu ndi ziwerengero, mfundo yofotokozera kuyamwa kwa kuwala yomwe ili ndi mikhalidwe yake yotchulidwa imalongosola chifukwa chake nambala iliyonse yoyezera iyenera kuperekedwa limodzi ndi zosungunulira zake, pH, ndi kuchuluka kwa zinthu zomwe zinagwiritsidwa ntchito.

Sankhani mayeso potengera funso. Funso lakuti ndi maselo angati omwe ali amoyo panopa ndi funso lowerengera, loyankhidwa pa maikulosikopu pogwiritsa ntchito pepala lowerengera. Funso lakuti yisiti imeneyi ikupuma mwamphamvu motani ndi funso la liwiro la kusintha, loyankhidwa ndi wotchi yoyezera nthawi ndi machubu omwe mikhalidwe yawo ikuwongoleredwa. Methylene blue imagwira ntchito pa zonsezi, koma osati m’chubu chomwecho panthawi yomweyo.