Njira zina m'malo mwa methylene blue pa microscopy: sankhani malinga ndi ntchito

Pa smear yosavuta ya mabakiteriya, crystal violet ingalowe m'malo mwa methylene blue ngati cholinga ndi kuona mawonekedwe a maselo. Pa maselo amoyo, njira yabwinoko ingakhale phase contrast popanda utoto uliwonse. Kusankha kumadalira zomwe muyenera kuona.

Tiyerekeze kuti anthu awiri akuwunika bacterial culture yomweyo. Wina akufuna kusiyanitsa maselo ooneka ngati ndodo ndi maselo ozungulira. Winayo akufuna kudziwa ngati treatment yapha mabakiteriyawo. Utoto womwe umapatsa munthu woyamba chithunzi chomveka ukhoza kusapatsa munthu wachiwiri yankho lothandiza.

Musanankhe cholowa m'malo, malizitsani chiganizo ichi: “Ndikufunika kuona ___ mu ___, ndipo maselo ayenera / sayenera kukhalabe amoyo.” Chiganizochi chimatanthauzira ntchito yomwe utotowo uyenera kuchita.

Yambani ndi specimen ndi funso

Choyamba dziwani ngati maselo ayenera kukhalabe amoyo, kenako pezani chinthu chimene mukufuna kuona:

• Maselo ayenera kukhalabe amoyo: pa kuyenda kapena mawonekedwe, ganizirani unstained phase contrast kapena DIC; pa nuclear DNA, ganizirani Hoechst 33342 ndi fluorescence; pa membrane integrity, sankhani assay yotsimikiziridwa monga SYTO 9 yokhala ndi propidium iodide.
• Maselo sayenera kukhalabe amoyo: pa bacterial morphology, ganizirani crystal-violet simple stain kapena nigrosin negative stain; pa Gram reaction, gwiritsani ntchito njira yonse ya Gram-stain; pa nuclear DNA m'maselo okhazikitsidwa, ganizirani DAPI fluorescence.

Gwiritsani ntchito tebuloli poyerekezera malire a njira iliyonse. Izi ndi zosankha za njira, osati malangizo osinthanitsa mabotolo mkati mwa protocol yomwe ilipo kale.

Pa smear yosavuta, sinthani njirayo mosamala

Crystal violet ndi njira yothandiza ngati ntchito yake ndi kuona bacterial morphology ndi mtundu umodzi. FDA's Bacteriological Analytical Manual imatchula ma formulation awiri a crystal violet oyenera simple staining. Gwiritsani ntchito formulation yolembedwa bwino ndi njira yake yokonzekera; dzina la utotowo lokha silikufotokoza reagent yomwe yakonzeka kugwiritsidwa ntchito.

Crystal violet imagwiritsidwanso ntchito mu Gram staining, koma zimenezi sizitanthauza kuti crystal-violet simple stain ndi Gram stain. Mu ASM Gram-stain protocol, zotsatira zosiyanitsa zimadalira crystal violet, iodine, decolorization, ndi counterstaining zomwe zimagwira ntchito motsatizana. Kusiya masitepe amenewo kumasintha zomwe chithunzicho chingatsimikizire. Onani simple staining poyerekeza ndi Gram staining kuti mumvetse kusiyanako.

Nigrosin imapereka njira ina yoonetsera malire: imadetsa background pamene bacterial cells amakhalabe owala poyerekezera. Pali mfundo yofunika mu malangizo a Merck a nigrosin yomwe mndandanda wamba wa njira zina ungaphonye. Working formulation yake yomwe yafotokozedwa ili ndi formaldehyde. Choncho simungaganize kuti ndi yogwirizana ndi maselo amoyo chifukwa chongoti imatchedwa “negative stain,” ngakhale maselowo akuwoneka ngati sanapakidwe utoto. Yang'anani formulation yonse ndi momwe specimen yakonzedwera.

Pa specimens zamoyo, ganizirani kusintha optics

Ngati mukufuna kuona maselo akuyenda kapena kugawikana, kuwonjezera utoto wina kungapangitse observation kukhala kovuta. Monga Nikon ikufotokozera mu kalozera wake wa contrast mu transparent specimens, phase contrast ndi differential interference contrast, kapena DIC, zimapangitsa specimens kuoneka chifukwa cha kusiyana kwa optical properties zawo. Zingasonyeze maselo amoyo osapakidwa utoto, ngati microscope ili ndi zigawo zofunikira.

Kusankha kumeneku kumasunga preparation yopanda utoto, koma kumasintha zomwe mungadziwe. Mutha kutsatira malire kapena kuyenda kwa selo popanda kudziwa molecule yomwe yapanga contrastyo. Ngati funso ndi molecular localization, sankhani labeling method yoyenera m'malo mwake.

Pa nuclear DNA, kuyerekezera kwa Invitrogen kwa DAPI ndi Hoechst stains kumasiyanitsa kugwiritsidwa ntchito kofala kwa DAPI pa fixed cells ndi Hoechst 33342 yomwe imalowa m'maselo mosavuta kwambiri ndipo imakondedwa pa live-cell labeling. Zonsezi zimafuna fluorescence equipment. Palibe imodzi yomwe imalowa m'malo mwachindunji mwa colored brightfield stain.

Onani dye exposure ndi illumination ngati experimental conditions. Yerekezani preparations zopakidwa utoto ndi zosapakidwa utoto ngati khalidwe la maselo ndilo measurement yanu.

Selo lofiira silitanthauza nthawi zonse kuti lafa

SYTO 9 ndi propidium iodide nthawi zambiri zimagwiritsidwa ntchito limodzi poyesa bacterial membrane integrity. BacLight manual imagwirizanitsa intact membranes ndi green staining, ndipo damaged membranes ndi red staining. Kutanthauzira kumeneku kuyenera kutsimikiziridwa pa specimen yomwe ikugwiritsidwa ntchito.

Kafukufuku wa 2019 wa Rosenberg ndi anzake akusonyeza vutoli. Mu 24-hour E. coli biofilms pa galasi mu phosphate-buffered saline, pafupifupi 96% ya maselo anaoneka propidium-iodide-positive in situ. Komabe olemba kafukufukuyo anayerekezera kuti 82% anali cultivable atakololedwa. Confocal images anaonetsa maselo okhala ndi green interiors pansi pa red-stained material, zomwe zinasonyeza kuti extracellular nucleic acids zinkathandiza kupanga signal yosokeretsa.

Maperesenti amenewo amafotokoza measurements zosiyanasiyana pa preparations za kafukufukuyo, osati universal correction factor. Phunziro lofunika ndi kufufuza chomwe chinapanga signalyo. Mu biofilm, nucleic-acid dye ingazindikire zinthu zomwe zili kunja kwa maselo komanso zomwe zili mkati mwa maselo owonongeka. Kuti afike pa viability conclusion, ofufuzawo amalimbikitsa complementary measurements, monga cultivation kapena metabolic activity, pamene akuvomereza kuti iliyonse ili ndi malire ake.

Onetsetsani zomwe njira inayo ili nazo kwenikweni

Njira ina ya staining ikhoza kukhalabe ndi methylene blue. Giemsa ndi chitsanzo: Merck imafotokoza product yake ngati azure-eosin-methylene-blue solution, yomwe cholinga chake chimaphatikizapo blood ndi bone-marrow smears. Itha kukhala njira yosiyana ya specimen yosiyana, koma siyikwaniritsa kufunika kochotsa methylene blue.

Ngati kuchotsa compound inayake ndi gawo la experiment, yang'anani reagent identity ndi formulation musanayerekezere zithunzi. Kalozera wa methyl blue, methylene blue, ndi utoto wa mayina ofanana amafotokoza kusiyana kwa mayina.

Tsimikizirani kusinthako musanagwiritse ntchito zotsatira

Pa njira yatsopano, pangani kuyerekezera kochepa pogwiritsa ntchito representative specimens ndi control yoyenera. Sungani microscope settings zofanana momwe zingathere. Lembani reagent formulation, specimen preparation, exposure, ndi imaging conditions kuti chithunzi chowoneka bwino kwambiri chithe kufufuzidwa m'malo mongosankhidwa chifukwa cha maonekedwe ake.

Weruzani kupambana molingana ndi funso loyambirira. Pa morphology, onani ngati malire ndi dongosolo la maselo zikadali zomveka. Pa nuclear labeling, onani signal location ndi background. Pa viability, funani umboni woposa kungokhala ndi kusiyana kokongola kwa mitundu. Kalozera wokulirapo wa methylene blue staining ndi ntchito zake mu laboratory amafotokoza momwe observation ndi preparation zimagwirizanirana.

Sankhani njira ina yomwe imasunga measurement yomwe mukufuna. Mtundu ndi property imodzi yokha ya chithunzi chomwe chimatuluka.