Dalilin amfani da methylene blue a matsayin rinin bango a gwajin acid-fast

Wani ma'aikacin fasaha ya duba shafar majina bayan kammala duk matakan rini. Ƙwayoyin sandar ja sun fito fili a kan bango mai shuɗi, kuma rahoton ya nuna cewa an ga bacilli masu riƙe rini duk da acid, wato acid-fast. Washegari, wani farantin gilashi dabam ya nuna bango mai shuɗi iri ɗaya ba tare da ƙwayoyin sandar ja ba, kuma aka bayar da rahoton cewa babu tarin fuka. Ɗaya daga cikin waɗannan rahotanni ya yi iƙirarin fiye da abin da farantin ya tabbatar, kuma ba na farkon ba ne.

Rashin fahimtar yana cikin ma'anar aikin. Ma'aikacin ya ɗauki rinin shuɗi a matsayin wani ɓangare na sinadaran gano ƙwayoyin cuta, kamar methylene blue yana taimakawa wajen gano mycobacteria. Ba haka ba ne. A lokacin da methylene blue ya taɓa farantin, gwajin ya riga ya kammala. Rinin yana ba bango launi ne kawai domin a iya karanta sakamakon. Wannan labarin yana bayyana manufar methylene blue a gwajin rini na acid-fast, dalilin zaɓin shuɗi maimakon wani launi, da abin da ganin ja a kan shuɗi yake ba ka damar kammalawa.

Amsa a taƙaice

Gwajin rini na acid-fast gwaji ne na bambancewa da aka gina a kan ra'ayoyi uku. Ana tilasta carbol fuchsin, rinin farko, shiga kowace ƙwayar halitta, har da mycobacteria masu bangon kakin zuma. Acid-alcohol, sinadarin cire launi, yana cire rinin ja daga ƙwayoyin halitta na yau da kullum amma ba ya iya cire shi daga masu acid-fast. Daga nan methylene blue, rinin bango, yana ba duk abin da sinadarin cire launi ya cire wa rini sabon launi. Saboda haka ƙwayoyin da ba su da acid-fast, ƙwayoyin halittar mai samfurin, da tarkace suna bayyana da shuɗi, yayin da bacilli masu acid-fast suke ci gaba da zama ja. Rinin bango yana samar da bambancin launi. Ba shi da wani aiki a tsarin sinadaran da ke sa ƙwayar halitta ta kasance acid-fast.

Abin da kowane mataki yake yi

Zanen da ke ƙasa yana nuna rabon ayyukan. Karanta shi daga hagu zuwa dama a matsayin tsarin aiki, sannan ka karanta jerin da ke ƙasa a matsayin tunatarwa cewa kowane rukuni na hanyoyin rini yana amsa wata tambaya dabam.

Shigar rini shi ne mataki na farko. Mycobacteria suna ƙin karɓar rinukan ruwa na yau da kullum domin lulluɓinsu yana ƙin ruwa sosai, don haka tsarin aikin yana tilasta rinin shiga. A hanyar Ziehl-Neelsen, dumamawa har tururi ya tashi tana tura carbol fuchsin mai phenol ta cikin bangon kakin zuma. A hanyar Kinyoun ta sanyi, ana maye gurbin zafi da haɗin rinin farko mai ƙarfi sosai, kuma wasu hanyoyin da aka gyara waɗanda ba sa amfani da zafi suna ƙara sinadaran rage tashin saman ruwa. Ƙa'idodin rinin acid-fast na American Society for Microbiology sun rubuta haɗin sinadaran hanyoyin biyu gefe da gefe, kuma bambancin yawan sinadarin a haɗin yana da girma: kusan 0.3 g na basic fuchsin a kowane 100 mL don hanyar dumamawa, idan aka kwatanta da kusan 4 g don hanyar sanyi.

Bambancewa shi ne mataki na biyu. Acid-alcohol, wanda galibi ake haɗawa da 3 mL na hydrochloric acid mai ƙarfi a cikin 97 mL na ethanol mai 95 percent, yana wanke rinin ja daga kowace ƙwayar halitta da bangonta ba ya nuna juriya ta musamman. Ƙwayoyin halitta masu cikakken lulluɓin lipid na mycobacteria suna riƙe rinin. Wannan shi ne matakin da duk hanyar ta dogara a kansa, kuma shi ne wanda ya fi saurin shafar sakamako idan an yi kuskuren dabara, kamar yadda za a tattauna a ƙasa.

Bambancin launi shi ne mataki na ƙarshe. Yanzu farantin yana ɗauke da ƙwayoyin acid-fast masu ja a kan bango kusan marar launi na ƙwayoyin da aka cire wa rini. Methylene blue mai 0.3 percent w/v yana sake sa a iya ganin wannan bango da launin da ya bambanta sosai da ja. Yin rinin bango yana ɗaukar ɗan lokaci kaɗan, yawanci 20 zuwa 30 seconds a hanyar dumamawa, kuma har zuwa 'yan mintoci a wasu nau'ikan hanyar sanyi. Lokacin yana dogara ne da ƙa'idar aiki, ba wata siffa ta rinin kansa ba.

Me ya sa wannan rini, kuma me ya sa shuɗi?

Methylene blue rini ne na asali mai cation, wato ion ɗinsa mai launi yana ɗauke da cajin tabbatacce kuma yana manne wa saman bakteriya da aka cire wa launi, ƙwayoyin halittar mai samfurin, da tarkace masu cajin korau. OpenStax Microbiology yana bayyana wannan tsarin caji a matsayin ƙa'idar gama-gari ta rinukan asali. Amma methylene blue ba shi kaɗai ba ne ke da wannan aikin sinadarai. Crystal violet da safranin su ma rinukan asali ne. Zaɓin ya shafi yadda ido yake ganin launi ne, ba sinadarai ba.

Ƙwayoyin sandar ja suna buƙatar bango mai launin da ya bambanta da nasu, kuma shuɗi yana ba da bambanci da ja ta hanyar da ta fi amfani a madubin ƙara girma mai bango mai haske. Safranin ba zai zama rinin bango mai kyau ba domin launin ruwan hoda-ja da yake bayarwa yana kusa sosai da launin carbol fuchsin da aka riƙe, yana rushe bambancin da tsarin aikin ya samar. Crystal violet zai ba da bango mai duhun shunayya da zai yi gogayya da siraran bacilli ja wajen jan hankali. Hujjar da ta fi ƙarfi cewa aikin shi ne bambancin launi maimakon sinadarai ita ce yiwuwar musanyawa: ana amfani da rinukan bango masu kore kamar malachite green da brilliant green a wasu ƙa'idodin aikin dabbobi da wasu da aka gyara. Gwajin yana ci gaba da aiki domin kowane launi da ya bambanta a fili yana yin aikin ɗaya.

Wannan yana haifar da sakamako biyu masu amfani a aikace. Na farko, haɗin sinadaran da aka saba amfani da shi bai tanadi gyaran pH ba; kada ka rikita wannan narkakken rinin ruwa na 0.3 percent da haɗe-haɗen alkaline kamar methylene blue na Loeffler, waɗanda suke cikin wata dabarar rini dabam. Na biyu, amfani da rini fiye da kima yana kawo matsala. Rubuce-rubucen ilimin nazarin kyallen halitta sun yi gargaɗi musamman cewa bango mai shuɗi mai kauri sosai zai iya ɓoye bacilli kaɗan. Saboda haka barin rini a kan farantin fiye da lokacin da ƙa'idar aiki ta amince yana rage sauƙin karanta sakamakon ba tare da wata fa'ida ba.

Sinadaran bangon da suke sa gwajin ya yiwu

Dalilin da ya sa acid-alcohol yake iya bambance ƙwayoyin halitta yana cikin lulluɓin mycobacteria. Tushen tsarinsa shi ne haɗin mycolyl-arabinogalactan-peptidoglycan: peptidoglycan da ke haɗe da arabinogalactan, wanda ƙarshen ɓangarorinsa na waje suke ɗauke da mycolic acids masu sarƙoƙi masu tsawo sosai. Wannan yana samar da mycomembrane na waje mai yalwar lipid wanda abubuwa ba sa ratsa shi cikin sauƙi. Bitar fatar waje ta mycobacteria ta bayyana wannan tsari dalla-dalla. Wannan shinge yana aiki ta ɓangarorin biyu. Yana hana rini shiga, wanda ya sa shigar rinin yake buƙatar zafi ko rinin farko mai ƙarfi, kuma yana hana cire rini, wanda ya sa acid-alcohol ba ya iya cire carbol fuchsin bayan ya shiga ciki. Bakteriya na yau da kullum ba su da wannan lulluɓi kuma suna rasa rinin. Don haka an fi fahimtar acid-fast a matsayin sakamakon shingen lipid, ba a matsayin fuchsin yana haɗuwa da mycolic acid ta hanyar sinadarai ba.

Wannan tsarin sinadaran kuma yana bayyana wani abin mamaki da ake yawan gani: mycobacteria ba sa karɓar rini sosai ko a kai a kai ta hanyar Gram, wani lokaci suna bayyana a matsayin siffofi marasa kaifin launi masu ɗigo kamar ƙullaye ko siffofi marasa rini. Bangonsu mai ƙin ruwa yana iyakance shigar crystal violet. Rinin acid-fast ba gyaran rinin Gram ba ne. Wata ƙa'idar bambancewa ce dabam, da aka gina a kan wata siffar bango dabam, kamar yadda kwatancen rini mai sauƙi da rinin Gram yake nuna wa sauran hanyoyin biyu.

Hanyoyi uku, tambayoyi uku

Don ƙarin bayani kan rukuni mafi faɗi na hanyoyin rini ɗaya na bakteriya, duba kundin hanyoyin rini na madubin ƙara girma.

Abin da ja a kan shuɗi ba zai iya tabbatarwa ba

Ƙwayoyin sandar ja suna nufin ƙwayoyin sun riƙe carbol fuchsin duk da amfani da acid-alcohol. Wannan abin da aka gani ya dace da Mycobacterium tuberculosis, amma jagorar CDC kan tantance tarin fuka ta bayyana a sarari cewa sauran mycobacteria su ma suna da acid-fast. Saboda haka tantance nau'in ƙwayar cuta yana buƙatar noman ƙwayoyin cuta da gwajin nucleic acid, ba launi ba.

Kuskuren dabara ya fi taruwa a matakan wankewa biyu. Rashin cire launi yadda ya kamata yana barin kayan bango da ja kuma yana iya sa a karanta sakamako tabbatacce na ƙarya. Cire launi fiye da kima yana cire rinin farko da ba a riƙe sosai ba kuma yana taimakawa wajen haifar da sakamako marar tabbatacce na ƙarya. Rinin bango da aka yi da yawa yana binne bacilli kaɗan a cikin shuɗi. Shafafe masu kauri, sinadaran gwaji da suka yi daskararren ɗanɗano, da gurɓataccen ruwan kurkurewa suna ƙara nasu alamomin da ba na ainihin samfurin ba.

Iya gano ƙwayoyin cuta yana da iyaka da yawansu, komai ingancin dabarar aikin. Duban shafar samfurin da madubin ƙara girma na yau da kullum yana buƙatar kusan 5,000 zuwa 10,000 bacilli a kowane milliliter na majina. Tsofaffin bitocin shaidu na CDC sun sanya iya gano ƙwayoyin ta wannan hanya a kusan 45 zuwa 80 percent a cikin masu cutar huhu da noman ƙwayoyin cuta ya tabbatar. Saboda haka shafar da ba ta nuna ƙwayoyin ba ba ta taɓa kawar da yiwuwar tarin fuka. Jagorar CDC ta yanzu tana riƙe noman ƙwayoyin cuta tare da kowace shafa, kuma tana ba da shawarar yin gwajin ƙara yawan nucleic acid a aƙalla samfurin hanyar numfashi ɗaya idan ana zargin tarin fukar huhu kuma sakamakon zai canza yadda za a kula da mara lafiya.

Aikin zahiri yana ƙarƙashin ƙa'idodin da aka amince da su

Bayanai na koyarwa, har da wannan labarin, ba sa ba da izinin aikin asibiti. Don aikin bencin dakin gwaje-gwaje, bi ingantacciyar ƙa'idar aikin dakin gwaje-gwajen da aka tabbatar da sahihancinta, wadda ta dace da takardun CDC, APHL, ko CLSI na yanzu; takardar CDC ta rinin acid-fast da ke cikin ma'ajiyarta ta Stacks tana nuna yadda irin waɗannan ƙa'idodi suke fayyace sinadaran gwaji da yadda ake karanta sakamako. Aikin samfurin tarin fuka yana buƙatar matakan hana yaɗuwar ƙwayoyin cuta: ana shirya shafafe a cikin akwatin kariyar halittu domin shirya su yana samar da ƙananan ɗigon da ke shawagi a iska da fantsama. Jagorar tsaron dakin gwaje-gwaje ta CDC ta bayyana cewa ko shafafen da aka kayyade da zafi suna iya ɗauke da bacilli na tarin fuka masu rai, kuma har yanzu dole ne a kula da su a matsayin gurɓatattu. Bayanin CDC Public Health Image Library kan shafar carbol-fuchsin da methylene blue da aka duba don M. leprae tunatarwa ce mai amfani cewa hanyar ba ta takaitu ga tarin fuka ba: bambancin ja a kan shuɗi yana aiki duk inda ake neman ƙwayoyin acid-fast, kuma karatun da aka yi a hankali wanda bai nuna ƙwayoyin ba shi kansa sakamako ne da ya cancanci rubutawa.

Karanta farantin da aka gama

Rinin bango yana nuna amfaninsa a lokacin dubawa. Mai amfani da madubin ƙara girma yana fara bincike da ƙaramin ƙarfin ƙara girma, yana amfani da bangon shuɗi wajen tantance ingancin shafar: rarrabuwar ƙwayoyin halitta a daidaita, cikakkun ƙwayoyin halittar mai samfurin don daidaita kaifin gani, da rashin tarin daskararren sinadari mai yawa. Daga nan abubuwan ja da ake zargi suna jan ido musamman domin babu wani abu dabam a farantin da yake ja. Ana tabbatarwa ta hanyar amfani da man dubawa, inda bacilli na gaske suke nuna siraran siffofin sanda da ake tsammani, wani lokaci masu ɗan lanƙwasa ko masu ɗigo kamar ƙullaye, maimakon tarkacen rini mai siffa iri ɗaya.

Rinin bango kuma yana tsara kalmomin rahoton. Farantin da ke da bacilli ja ana bayar da rahotonsa da cewa an ga bacilli masu acid-fast, sau da yawa tare da darajar kimanta yawansu bisa ma'aunin dakin gwaje-gwajen. Farantin da ba shi da ƙwayoyin ja ana bayar da rahotonsa da cewa ba a ga bacilli masu acid-fast ba, ba cewa babu tarin fuka ba. Bambancin yana da muhimmanci domin bangon shuɗi yana tabbatar da cewa rinin ya yi aiki, ƙwayoyin halitta sun karɓi rinin bango, kuma tsarin ganin madubin ya wadatar, amma ba ya faɗin komai game da ƙwayoyin da yawansu yake ƙasa da iyakar ganowa. Bango mai shuɗi da babu ƙwayoyin ja sakamako ne ingantacce game da farantin, ba hukunci game da mara lafiya ba.

Ka tuna rabon ayyukan, kuma rubuta rahotanni zai zama mai sauƙi. Carbol fuchsin yana nuna ƙwayoyin da ake zargi. Acid-alcohol yana aiwatar da gwajin. Methylene blue yana sa a iya karanta amsar ne kawai: ja ya riƙe rinin duk da acid, shuɗi ya rasa shi. Duk sauran abubuwa—nau'in ƙwayar cuta, yawanta, da ma'anar sakamakon a asibiti—suna fitowa daga aikin da ya wuce duban farantin.