Inqaku
Ezinye iindlela endaweni ye-methylene blue kwimicroscopy: khetha ngokomsebenzi
Kwi-smear elula yeebhaktheriya, i-crystal violet inokuthatha indawo ye-methylene blue xa injongo ikukubona ukumila kweeseli. Kwiiseli eziphilayo, enye indlela engcono isenokuba yi-phase contrast ngaphandle kwesithambiso konke konke. Ukhetho luxhomekeke kwinto ekufuneka uyijongile.
Masithi abantu ababini bavavanya inkcubeko enye yeebhaktheriya. Omnye ufuna ukwahlula iiseli ezimile njengeentonga kwezingqukuva. Omnye ufuna ukwazi ukuba unyango luyibulele na ibhaktheriya. Isithambiso esinika umntu wokuqala umfanekiso ocacileyo sisenokungamniki umntu wesibini mpendulo isebenzisekayo.
Ngaphambi kokukhetha enye indawo, gqibezela esi sivakalisi: “Kufuneka ndijonge ___ kwi-___, kwaye iiseli kufuneka / akuyomfuneko ukuba zihlale ziphila.” Esi sivakalisi sichaza umsebenzi ekufuneka isithambiso siwenze.
Qala ngesampuli nangombuzo
Qala ngokugqiba ukuba iiseli kufuneka zihlale ziphila na, emva koko uchonge into ekufuneka uyijongile:
- Iiseli kufuneka zihlale ziphila: ukujonga intshukumo okanye ukumila, cinga nge-phase contrast engenasithambiso okanye i-DIC; kwi-DNA yenyukliyasi, cinga nge-Hoechst 33342 ene-fluorescence; ekupheleleni kwemembrane, khetha uvavanyo oluqinisekisiweyo olufana ne-SYTO 9 ene-propidium iodide.
- Akuyomfuneko ukuba iiseli zihlale ziphila: kwimorphology yeebhaktheriya, cinga ngesithambiso esilula se-crystal violet okanye i-negative stain ye-nigrosin; kwi-Gram reaction, sebenzisa indlela epheleleyo ye-Gram stain; kwi-DNA yenyukliyasi kwiiseli ezilungisiweyo, cinga nge-DAPI fluorescence. Sebenzisa le theyibhile ukuthelekisa imida yesebe ngalinye. Olu lukhetho lweendlela, hayi imiyalelo yokutshintshisa iibhotile ngaphakathi kweprotocol esele ikhona.
| Into ekufuneka uyijongile | Isampuli nezixhobo | Enye indlela enokukhethwa | Owona mda uphambili |
|---|---|---|---|
| Ukumila nokulungelelaniswa kweebhaktheriya | I-smear yeebhaktheriya elungisiweyo; brightfield | Isithambiso esilula se-crystal violet | Sibonisa imorphology; asiqinisekisi i-Gram reaction okanye ukuphila. |
| Imida yeeseli kwindawo engasemva emnyama | Ulungiselelo lwebacteriology; brightfield | I-negative stain ye-nigrosin | Ulungiselelo kunye nokwakheka kwesithambiso kubalulekile; iseli engathanjiswanga ayithethi ukuba iyaphila. |
| Gram reaction | I-smear yeebhaktheriya elungisiweyo; brightfield | Indlela epheleleyo ye-Gram stain | Ifuna ulandelelwano olupheleleyo lwee-reagent kunye nee-control. |
| Intshukumo okanye utshintsho kwiiseli eziphilayo | Ulungiselelo olumanzi oluhambelanayo; phase contrast okanye DIC | Akukho sithambiso | Ifuna ii-optics ezifanelekileyo; i-contrast ayichongi molecule. |
| DNA yenyukliyasi | Iiseli ezilungisiweyo; fluorescence | DAPI | Ifuna ii-filter zokuvuselela nokukhupha ukukhanya ezihambelanayo. |
| DNA yenyukliyasi kwiiseli eziphilayo | Iiseli eziphilayo; fluorescence | Hoechst 33342 | Ukungena kwiseli akuqinisekisi ukuba iimeko zokwenza umfanekiso azinabungozi. |
| Ukuphelela kwemembrane yeebhaktheriya | Iibhaktheriya ezingalungiswanga; fluorescence | SYTO 9 ene-propidium iodide | Ukuthanjiswa kwemembrane ngumlinganiselo ongathanga ngqo wokuphila. |
Kwi-smear elula, buyisela indlela ngononophelo
I-crystal violet ngumgqatswa osebenzayo xa umsebenzi kukufumana umfanekiso wombala omnye wemorphology yeebhaktheriya. I-Bacteriological Analytical Manual ye-FDA idwelisa iindlela ezimbini zokwakheka kwe-crystal violet ezifanelekileyo kwisithambiso esilula. Sebenzisa ukwakheka okubhaliweyo kunye nendlela yako yokulungiselela; igama ledayi lodwa alichazi i-reagent esebenzayo.
I-crystal violet ikwasetyenziswa kwi-Gram staining, kodwa oko akuthethi ukuba i-crystal-violet simple stain yi-Gram stain. Kwi-protocol ye-ASM ye-Gram stain, isiphumo sokwahlula sixhomekeke kwi-crystal violet, iodine, decolorization, kunye ne-counterstaining ezisebenza ngokulandelelana. Ukushiya la manyathelo kutshintsha into umfanekiso onokuyiqinisekisa. Bona isithambiso esilula xa sithelekiswa ne-Gram staining ukuze ubone lo mahluko.
I-nigrosin inika indlela eyahlukileyo yokubonisa imida: yenza indawo engasemva ibe mnyama ngoxa iiseli zeebhaktheriya zihlala zikhanya xa zithelekiswa nayo. Kukho inkcukacha eluncedo kwi-miyalelo ye-Merck ye-nigrosin enokuphoswa luluhlu oluqhelekileyo lwezinye iindlela. Ukwakheka kwayo okuchaziweyo kokusebenza kubandakanya i-formaldehyde. Ngoko awunakugqiba ukuba iyahambelana neeseli eziphilayo ngenxa nje yamagama athi “negative stain,” nangona iiseli ngokwazo zibonakala zingathanjiswanga. Jonga ukwakheka okupheleleyo kunye nendlela yokulungiselela isampuli.
Kwiisampuli eziphilayo, cinga ngokutshintsha ii-optics
Ukuba ufuna ukubukela iiseli zihamba okanye zahlukana, ukongeza enye idayi kunokwenza uqwalaselo lube nzima ngakumbi. Njengoko i-Nikon ichaza kwisikhokelo sayo se-contrast kwiisampuli ezicacileyo, i-phase contrast kunye ne-differential interference contrast, okanye i-DIC, zenza iisampuli zibonakale ngenxa yomahluko kwiimpawu zazo ze-optical. Zingabonisa iiseli eziphilayo nezingathanjiswanga, ukuba imicroscope inamalungu afunekayo.
Olu khetho lugcina ulungiselelo lungenasithambiso, kodwa lutshintsha ulwazi olufumanekayo. Ungalandela umda okanye intshukumo ngaphandle kokwazi ukuba yeyiphi imolecule ebangele i-contrast. Ukuba umbuzo ngowendawo yemolecule, khetha indlela efanelekileyo yokuleyibhela endaweni yoko.
Kwi-DNA yenyukliyasi, uthelekiso lwe-Invitrogen lwe-DAPI kunye ne-Hoechst stains lwahlula ukusetyenziswa okuqhelekileyo kwe-DAPI kwiiseli ezilungisiweyo kwi-Hoechst 33342 engena lula ngakumbi kwiiseli, ekhethwayo ekuleyibhelweni kweeseli eziphilayo. Zombini zifuna izixhobo ze-fluorescence. Akukho nanye yazo ethatha indawo ngokuthe ngqo yesithambiso esinombala se-brightfield.
Phatha ukuvezwa kwidayi kunye nokukhanyiswa njengeemeko zovavanyo. Thelekisa amalungiselelo athanjisiweyo nalawo angathanjiswanga xa indlela iiseli eziziphatha ngayo ingumlinganiselo wakho.
Iseli ebomvu ayithethi ngokuzenzekelayo ukuba ifile
I-SYTO 9 kunye ne-propidium iodide zidla ngokusetyenziswa kunye ukuvavanya ukuphelela kwemembrane yeebhaktheriya. Incwadana ye-BacLight inxulumanisa iimembrane ezipheleleyo nokuthanjiswa okuluhlaza kunye neemembrane ezonakeleyo nokuthanjiswa okubomvu. Loo ndlela yokutolika kufuneka iqinisekiswe kwisampuli.
Uphononongo luka-2019 lukaRosenberg noogxa bakhe lubonisa le ngxaki. Kwi-biofilm ye-E. coli yeeyure ezingama-24 kwiglasi kwi-phosphate-buffered saline, malunga ne-96% yeeseli zabonakala njenge-propidium-iodide-positive in situ. Kodwa ababhali baqikelela ukuba i-82% yayinokukhuliswa emva kokuqokelelwa. Imifanekiso ye-confocal yabonisa iiseli ezinamacala angaphakathi aluhlaza phantsi kwezinto ezithanjiswe bomvu, nto leyo eyalatha kwii-nucleic acid ezingaphandle kweeseli njengonobangela womqondiso olahlekisayo.
Ezo pesenti zichaza imilinganiselo eyahlukeneyo kumalungiselelo ophononongo, hayi into enokusetyenziswa njenge-correction factor kuzo zonke iimeko. Isifundo esiluncedo kukujonga ukuba yintoni evelise umqondiso. Kwi-biofilm, idayi ye-nucleic acid inokufumanisa izinto ezingaphandle kweeseli kwakunye nangaphakathi kwiiseli ezonakeleyo. Ukuze kufikelelwe kwisigqibo malunga nokuphila, abaphandi bacebisa imilinganiselo eyongezelelweyo, efana nokukhulisa okanye umsebenzi we-metabolic, ngoxa bevuma ukuba nganye inemida yayo.
Jonga ukuba enye indlela eneneni iqulethe ntoni
Enye indlela yokuthambisa isenokuyiqulatha i-methylene blue. IGiemsa ngumzekelo: i-Merck ichaza imveliso yayo njenge-azure-eosin-methylene-blue solution, eyenzelwe ukusetyenziswa okubandakanya ii-smear zegazi kunye nomongo wethambo. Inokuba yindlela eyahlukileyo yesampuli eyahlukileyo, kodwa ayihlangabezani nemfuneko yokukhuphela ngaphandle i-methylene blue.
Ukuba ukukhuphela ngaphandle ikhompawundi yinxalenye yovavanyo, jonga isazisi se-reagent kunye nokwakheka kwayo ngaphambi kokuthelekisa imifanekiso. Isikhokelo se-methyl blue, methylene blue, kunye needayi ezinamagama afanayo sicacisa umahluko wamagama.
Qinisekisa ukutshintshwa ngaphambi kokusebenzisa isiphumo
Kwindlela entsha, yenza uthelekiso oluncinci usebenzisa iisampuli ezimele uvavanyo kunye ne-control efanelekileyo. Gcina useto lwemicroscope luthelekiseka apho kunokwenzeka. Rekhoda ukwakheka kwe-reagent, ukulungiselelwa kwesampuli, ukuvezwa, kunye neemeko zokwenza umfanekiso ukuze umfanekiso okhangeleka ungcono uphandwe kunokuba ukhethwe nje.
Vavanya impumelelo ngokusekelwe kumbuzo wokuqala. Kwimorphology, vavanya ukuba imida yeeseli kunye nokulungelelaniswa kwazo kusatolikwa na. Ekuleyibhelweni kwenyukliyasi, vavanya indawo yomqondiso kunye nemvelaphi. Ekuvavanyeni ukuphila, funa ubungqina obungaphezulu kokwahlulwa kwemibala okubukekayo. Isikhokelo esibanzi se-methylene blue staining kunye nokusetyenziswa kwayo elabhoratri sicacisa indlela uqwalaselo nolungiselelo oludibana ngayo.
Khetha enye indlela egcina umlinganiselo owudingayo. Umbala yenye kuphela yeempawu zomfanekiso ophumayo.