Isihloko
Ezinye izindlela ze-methylene blue zokuhlola nge-microscope: khetha ngokomsebenzi
Ku-smear elula yamabhaktheriya, i-crystal violet ingasetshenziswa esikhundleni se-methylene blue uma inhloso kuwukubona ukuma kwamaseli. Kumaseli aphilayo, enye indlela engcono ingaba yi-phase contrast ngaphandle kokusebenzisa umbala nhlobo. Ukukhetha kuncike kulokho odinga ukukubona.
Ake sithi abantu ababili bahlola isiko elifanayo lamabhaktheriya. Omunye ufuna ukuhlukanisa amaseli ayizinti kwawayindilinga. Omunye ufuna ukwazi ukuthi ukwelashwa kuwabulalile yini amabhaktheriya. Umbala onikeza umuntu wokuqala isithombe esicacile ungase unganikezi owesibili impendulo esebenzisekayo.
Ngaphambi kokukhetha enye indlela, qedela lo musho: “Ngidinga ukubona ___ ku-___, futhi amaseli kufanele / akudingeki aqhubeke ephila.” Lo musho uchaza umsebenzi umbala okufanele uwenze.
Qala ngesampula nombuzo
Qala ngokunquma ukuthi amaseli kufanele aqhubeke ephila yini, bese uhlonza isici odinga ukusibona:
- Amaseli kufanele aqhubeke ephila: uma ufuna ukubona ukunyakaza noma ukuma, cabanga nge-phase contrast engenambala noma i-DIC; nge-DNA yenucleus, cabanga nge-Hoechst 33342 kanye ne-fluorescence; ngobuqotho be-membrane, khetha ukuhlola okuqinisekisiwe njenge-SYTO 9 kanye ne-propidium iodide.
- Amaseli akudingeki aqhubeke ephila: nge-morphology yamabhaktheriya, cabanga nge-crystal-violet simple stain noma i-nigrosin negative stain; nge-Gram reaction, sebenzisa indlela ephelele ye-Gram stain; nge-DNA yenucleus kumaseli afixed, cabanga nge-DAPI fluorescence.
Sebenzisa leli thebula ukuqhathanisa imikhawulo yegatsha ngalinye. Lezi yizinqumo zendlela, hhayi imiyalelo yokushintshana ngamabhodlela ngaphakathi kwephrothokholi ekhona.
| Okudingeka ukubone | Isampula nemishini | Enye indlela engacatshangelwa | Umkhawulo omkhulu |
|---|---|---|---|
| Ukuma nokuhleleka kwamabhaktheriya | I-smear yamabhaktheriya elungisiwe; brightfield | Crystal violet simple stain | Ibonisa i-morphology; ayiqinisekisi i-Gram reaction noma ukuphila kwamaseli. |
| Imingcele yamaseli ibonakala ngemuva elimnyama | Ukulungiselelwa kwe-bacteriological; brightfield | Nigrosin negative stain | Indlela yokulungiselela nokwakheka kwereagent kubalulekile; iseli elingafakwanga umbala akusho ukuthi liyaphila. |
| Gram reaction | I-smear yamabhaktheriya elungisiwe; brightfield | Indlela ephelele ye-Gram stain | Idinga ukulandelana okuphelele kwama-reagent nama-control. |
| Ukunyakaza noma izinguquko kumaseli aphilayo | Ukulungiselelwa okumanzi okuhambisanayo; phase contrast noma DIC | Akukho stain | Kudinga i-optics efanele; i-contrast ayikhombi i-molecule ethile. |
| DNA yenucleus | Amaseli afixed; fluorescence | DAPI | Kudinga amafilter ahambisana ne-excitation ne-emission. |
| DNA yenucleus kumaseli aphilayo | Amaseli aphilayo; fluorescence | Hoechst 33342 | Ukungena eselini akuqinisekisi ukuthi izimo zokuthwebula azilimazi. |
| Ubuqotho be-membrane yamabhaktheriya | Amabhaktheriya angafixed; fluorescence | SYTO 9 with propidium iodide | Ukufakwa umbala ku-membrane kuyisilinganiso esingaqondile sokuphila kwamaseli. |
Ku-smear elula, shintsha indlela ngokucophelela
I-crystal violet iyindlela esebenzayo uma umsebenzi kuwukuthola ukubonakala kombala owodwa kwe-morphology yamabhaktheriya. I-Bacteriological Analytical Manual ye-FDA ibala izinhlanganisela ezimbili ze-crystal-violet ezifanele i-simple staining. Sebenzisa ukwakheka okubhalwe phansi kanye nendlela yako yokulungiselela; igama lombala lodwa alichazi i-reagent esebenzayo.
I-crystal violet nayo iyasetshenziswa ku-Gram staining, kodwa lokho akwenzi i-crystal-violet simple stain ibe yi-Gram stain. Ku-ASM Gram-stain protocol, umphumela wokuhlukanisa uncike ekutheni i-crystal violet, iodine, decolorization, kanye ne-counterstaining kusebenze ngokulandelana. Ukushiya lezo zinyathelo kushintsha lokho isithombe esingakuqinisekisa. Bheka i-simple staining uma iqhathaniswa ne-Gram staining ukuze ubone lowo mehluko.
I-nigrosin inikeza enye indlela yokwenza imingcele ibonakale: yenza ingemuva libe mnyama kuyilapho amaseli amabhaktheriya ehlala ekhanya uma kuqhathaniswa. Kunemininingwane ebalulekile ku-Merck's nigrosin instructions engase ingabonakali ohlwini olujwayelekile lwezinye izindlela. Ukwakheka kwayo okuchaziwe okusebenzayo kuqukethe i-formaldehyde. Ngakho-ke awukwazi ukuphetha ngokuthi iyahambisana namaseli aphilayo ngokusekelwe kuphela emagameni athi “negative stain,” nakuba amaseli ngokwawo ebonakala engafakwanga umbala. Hlola ukwakheka okuphelele kanye nokulungiselelwa kwesampula.
Kumasampula aphilayo, cabanga ngokushintsha i-optics
Uma ufuna ukubuka amaseli ehamba noma ehlukana, ukufaka omunye umbala kungase kwenze ukubhekwa kube nzima. Njengoba i-Nikon ichaza kumhlahlandlela wayo nge-contrast in transparent specimens, i-phase contrast kanye ne-differential interference contrast, noma i-DIC, yenza amasampula abonakale ngokusebenzisa umehluko ezicini zawo ze-optical. Lezi zindlela zingaveza amaseli aphilayo angafakwanga umbala, inqobo nje uma i-microscope inezingxenye ezidingekayo.
Lokhu kukhetha kugcina ukulungiselelwa kungafakwanga umbala, kodwa kushintsha ulwazi olutholakalayo. Ungalandela umngcele noma ukunyakaza ngaphandle kokwazi ukuthi iyiphi i-molecule ebangele i-contrast. Uma umbuzo umayelana nokuthi i-molecule itholakala kuphi, khetha indlela efanele yokuyilebula.
Nge-DNA yenucleus, ukuqhathanisa kwe-DAPI ne-Hoechst stains kwe-Invitrogen kuhlukanisa ukusetshenziswa okuvamile kwe-DAPI kumaseli afixed ne-Hoechst 33342 engena kalula kakhulu kumaseli, evame ukukhethwa ekulebuleni amaseli aphilayo. Kokubili kudinga imishini ye-fluorescence. Akukho kokubili okungena esikhundleni ngokuqondile se-stain enemibala esetshenziswa ku-brightfield.
Phatha ukuchayeka kudayi nokukhanya njengezimo zokuhlola. Qhathanisa ukulungiselelwa okufakwe umbala nokungafakwanga umbala lapho ukuziphatha kwamaseli kuyisilinganiso sakho.
Iseli elibomvu alisho ngokuzenzakalela ukuthi lifile
I-SYTO 9 ne-propidium iodide kuvame ukusetshenziswa ndawonye ukuhlola ubuqotho be-membrane yamabhaktheriya. I-BacLight manual ihlobanisa ama-membrane aqinile nokufakwa umbala oluhlaza kanye nama-membrane alimele nokufakwa umbala obomvu. Lokho kuchazwa kudinga ukuqinisekiswa ngesampula ethile.
Ucwaningo luka-2019 olwenziwa nguRosenberg nozakwabo lubonisa inkinga. Kuma-biofilm e-E. coli anamahora angu-24 ayesengilazini ku-phosphate-buffered saline, cishe ama-96% amaseli abonakala ene-propidium-iodide-positive lapho ehlolwa endaweni yawo. Kodwa ababhali balinganisela ukuthi ama-82% ayekwazi ukukhuliswa ngemva kokuvunwa. Izithombe ze-confocal zabonisa amaseli anezingaphakathi eziluhlaza ngaphansi kwezinto ezifakwe umbala obomvu, okukhomba ama-nucleic acid angaphandle kwamaseli njengembangela yesignali edidayo.
Lawo maphesenti achaza izilinganiso ezihlukene ezenziwe ekulungiseleleni kwalolo cwaningo, hhayi isici sokulungisa esisebenza yonke indawo. Isifundo esibalulekile ukuhlola ukuthi yini eyakhiqiza isignali. Ku-biofilm, udayi we-nucleic acid ungathola izinto ezingaphandle kwamaseli kanye nangaphakathi kwamaseli alimele. Ukuze kufinyelelwe esiphethweni ngokuphila kwamaseli, abacwaningi batusa izilinganiso ezengeziwe, njengokukhulisa amaseli noma umsebenzi we-metabolic, kuyilapho bevuma ukuthi indlela ngayinye inemikhawulo.
Hlola ukuthi enye indlela iqukethe ini ngempela
Enye indlela yokufaka umbala ingase isalokhu iqukethe i-methylene blue. I-Giemsa iyisibonelo: i-Merck ichaza umkhiqizo wayo njenge-azure-eosin-methylene-blue solution, ohloselwe ukusetshenziswa okuhlanganisa ama-smear egazi nawe-bone marrow. Kungaba indlela ehlukile yesampula ehlukile, kodwa ayihlangabezani nemfuneko yokungafaki i-methylene blue.
Uma ukukhipha i-compound ethile kuyingxenye yokuhlola, hlola ubuwena be-reagent nokwakheka kwayo ngaphambi kokuqhathanisa izithombe. Umhlahlandlela othi methyl blue, methylene blue, and similarly named dyes uchaza umehluko wamagama.
Qinisekisa ukushintshwa ngaphambi kokusebenzisa umphumela
Ngendlela entsha, yenza ukuqhathanisa okuncane usebenzisa amasampula amele kahle kanye ne-control efanele. Gcina amasethingi e-microscope eqhathaniseka lapho kungenzeka. Bhala phansi ukwakheka kwe-reagent, ukulungiselelwa kwesampula, ukuchayeka, nezimo zokuthwebula ukuze isithombe esibukeka singcono sikwazi ukuphenywa kunokuba nje sikhethwe.
Hlola impumelelo ngokombuzo wokuqala. Nge-morphology, hlola ukuthi imingcele nokuhleleka kwamaseli kusabonakala kahle yini. Ngokulebula i-nucleus, hlola indawo yesignali nengemuva. Ngokuphila kwamaseli, funa ubufakazi obungaphezu kokuhlukaniswa kwemibala okubukeka kahle. Umhlahlandlela obanzi nge-methylene blue staining and laboratory applications uchaza ukuthi ukubhekwa nokulungiselelwa kuhambisana kanjani.
Khetha enye indlela egcina isilinganiso osidingayo. Umbala uyisici esisodwa kuphela sesithombe esiphumayo.