Tuhinga
Ngā kōwhiringa kē atu i te methylene blue mō te karuwhārahi: kōwhiria mā te mahi
Mō tētahi pani huakita māmā, ka taea te crystal violet te whakamahi hei whakakapi i te methylene blue mēnā ko te whāinga he kite i te āhua o ngā pūtau. Mō ngā pūtau ora, tērā pea ko te kōwhiringa pai ake ko te phase contrast kāore rawa he waikano. Kei te āhua o te tirohanga e hiahia ana koe te kōwhiringa.
Whakaarohia e rua ngā tāngata e tirotiro ana i te ahurea huakita kotahi. E hiahia ana tētahi ki te wehewehe i ngā pūtau tokotoko i ngā pūtau porowhita. E hiahia ana tētahi atu ki te mōhio mēnā kua whakamatea ngā huakita e tētahi maimoatanga. Ka taea e tētahi waikano te hoatu whakaahua mārama ki te tangata tuatahi, engari kāore pea e hoatu he whakautu whaihua ki te tangata tuarua.
I mua i te kōwhiri i tētahi whakakapi, whakaotia tēnei rerenga: “Me mātaki au i te ___ i roto i te ___, ā, me ora tonu / kāore e hiahiatia kia ora tonu ngā pūtau.” Mā taua rerenga e tautuhi te mahi me tutuki e te waikano.
Tīmata ki te tauira me te pātai
Tuatahi whakatauria mēnā me ora tonu ngā pūtau, kātahi ka tautuhia te āhuatanga e hiahia ana koe ki te mātaki:
- Me ora tonu ngā pūtau: mō te nekehanga, te āhua rānei, whakaarohia te phase contrast kāore he waikano, te DIC rānei; mō te DNA karihi, whakaarohia te Hoechst 33342 me te fluorescence; mō te noho tōtika o te kiriuhi, kōwhiria he whakamātautau kua whakamanahia pērā i te SYTO 9 me te propidium iodide.
- Kāore e hiahiatia kia ora tonu ngā pūtau: mō te morphology huakita, whakaarohia he waikano māmā crystal violet, he waikano kino nigrosin rānei; mō te tauhohenga Gram, whakamahia te tikanga Gram-stain katoa; mō te DNA karihi i ngā pūtau kua whakamaua, whakaarohia te DAPI fluorescence.
Whakamahia tēnei ripanga hei whakataurite i ngā herenga o ia ara. He kōwhiringa tikanga ēnei, ehara i te tohutohu kia whakawhitia noa ngā pounamu i roto i tētahi kawa e whakamahia kē ana.
| He aha tāu e hiahia ana ki te mātaki | Tauira me ngā taputapu | Kōwhiringa kē pea | Herenga matua |
|---|---|---|---|
| Te āhua me te whakaritenga o ngā huakita | Pani huakita kua whakaritea; brightfield | Waikano māmā crystal violet | Ka whakaatu i te morphology; kāore e whakapūmau i te tauhohenga Gram, i te oranga rānei. |
| Ngā tapa pūtau ki runga i tētahi papamuri pōuri | Whakaritenga bacteriological; brightfield | Waikano kino nigrosin | He mea nui te whakarite me te hanga ranunga; ehara te pūtau kāore i waikanotia i te pūtau ora i ngā wā katoa. |
| Tauhohenga Gram | Pani huakita kua whakaritea; brightfield | Tikanga Gram-stain katoa | Me whakamahi te raupapa reagent katoa me ngā mana whakahaere. |
| Te nekehanga, ngā panonitanga rānei i ngā pūtau ora | Whakaritenga mākū hototahi; phase contrast, DIC rānei | Kāore he waikano | Me whai optics e tika ana; kāore te contrast e tautuhi i tētahi rāpoi ngota. |
| DNA karihi | Ngā pūtau kua whakamaua; fluorescence | DAPI | Me whai whiriwhiringa excitation me emission hototahi. |
| DNA karihi i ngā pūtau ora | Ngā pūtau ora; fluorescence | Hoechst 33342 | Ko te urunga ki te pūtau kāore e whakapūmau he haumaru kore-pānga ngā tikanga whakaahua. |
| Te noho tōtika o te kiriuhi huakita | Ngā huakita kāore i whakamaua; fluorescence | SYTO 9 me te propidium iodide | He ine autaki te waikano kiriuhi mō te oranga. |
Mō tētahi pani māmā, whakakapia te tikanga me te tūpato
He kōwhiringa whaihua te crystal violet ina ko te mahi he tirohanga tae-kotahi ki te morphology huakita. Kei te Bacteriological Analytical Manual a te FDA e rua ngā hanganga crystal-violet e tika ana mō te waikano māmā. Whakamahia tētahi hanganga kua tuhia me tōna tikanga whakarite; kāore te ingoa o te waikano anake e whakamārama i tētahi reagent kua rite mō te whakamahi.
Ka whakamahia hoki te crystal violet i te Gram staining, engari kāore tērā e huri i te waikano māmā crystal-violet hei Gram stain. I roto i te kawa Gram-stain a ASM, ka whakawhirinaki te hua wehewehe ki te crystal violet, iodine, decolorization, me te counterstaining e mahi raupapa ana. Mā te waiho i aua hikoinga ka rerekē ngā mea ka taea te whakapūmau e te whakaahua. Tirohia te waikano māmā me te Gram staining mō taua wehewehenga.
He ara rerekē tā te nigrosin hei whakaatu i ngā tapa: ka whakapōuri i te papamuri, ā, ka noho āhua māramarama ake ngā pūtau huakita. He taipitopito whaihua kei roto i ngā tohutohu nigrosin a Merck ka ngaro pea i tētahi rārangi whānui o ngā whakakapi. Kei roto i tana hanganga mahi kua tohua te formaldehyde. Nō reira, kāore e taea te whakatau he hototahi ki ngā pūtau ora i runga noa i ngā kupu “negative stain,” ahakoa kāore ngā pūtau tonu e kitea kua waikanotia. Tirohia te hanganga katoa me te whakariterite i te tauira.
Mō ngā tauira ora, whakaarohia te whakarerekē i ngā optics
Mēnā e hiahia ana koe ki te mātaki i ngā pūtau e neke ana, e wehewehe ana rānei, ka uaua ake pea te tirohanga mēnā ka tāpirihia he waikano anō. E whakamārama ana a Nikon i tana aratohu mō te contrast i ngā tauira pūataata, ka whakakitea ngā tauira e te phase contrast me te differential interference contrast, arā te DIC, mā ngā rerekētanga o ō rātou āhuatanga whatu. Ka taea e ēnei te whakaatu i ngā pūtau ora kāore i waikanotia, mēnā kei te karuwhārahi ngā wāhanga e hiahiatia ana.
Ka pupuri tēnei kōwhiringa i tētahi whakariterite kāore i waikanotia, engari ka whakarerekē i ngā mōhiohio e wātea ana. Ka taea e koe te whai i tētahi tapa, i tētahi nekehanga rānei me te kore e mōhio ko tēhea rāpoi ngota i whakaputa i te contrast. Mēnā ko te wāhi o tētahi rāpoi ngota te pātai, kōwhiria kē he tikanga tapanga e tika ana.
Mō te DNA karihi, ka wehewehe te whakataurite a Invitrogen i ngā waikano DAPI me Hoechst i te whakamahinga noa o te DAPI ki ngā pūtau kua whakamaua, me te Hoechst 33342 e māmā ake ana te kuhu ki te pūtau, ā, e manakohia ana mō te tapanga pūtau ora. Me whai taputapu fluorescence mō ēnei mea e rua. Kāore tētahi o ēnei i te whakakapi tika mō tētahi waikano brightfield whai tae.
Whakaarohia te pānga o te waikano me te whakamarama hei tikanga whakamātautau. Whakatauritea ngā whakariterite kua waikanotia me ngā mea kāore i waikanotia ina ko te whanonga o ngā pūtau tāu e ine ana.
Ehara te pūtau whero i te pūtau mate i ngā wā katoa
He maha ngā wā ka whakamahia ngātahitia te SYTO 9 me te propidium iodide hei aromatawai i te noho tōtika o te kiriuhi huakita. Ka hono te pukapuka BacLight i ngā kiriuhi tōtika ki te waikano kākāriki, ā, i ngā kiriuhi kua pakaru ki te waikano whero. Me whakamana taua whakamāramatanga mō te tauira e whakamahia ana.
Ka whakaatu tētahi rangahau 2019 nā Rosenberg me ōna hoa i te raruraru. I ngā biofilm E. coli 24-hour i runga i te karāhe i roto i te phosphate-buffered saline, tata ki te 96% o ngā pūtau i kitea he propidium-iodide-positive i tō rātou wāhi tonu. Heoi anō, i whakatau tata ngā kaituhi he 82% ka taea tonu te whakatipu i muri i te hauhaketanga. I whakaatu ngā whakaahua confocal i ngā pūtau he kākāriki o roto i raro i ngā rawa kua waikanotia whero, e tohu ana ko ngā nucleic acid kei waho o ngā pūtau tētahi take o te tohu whakapohehe.
E whakaahua ana aua ōrau i ngā inenga rerekē i runga i ngā whakariterite o taua rangahau, ehara i tētahi tau whakatikatika e tika ana mō ngā horopaki katoa. Ko te akoranga whaihua ko te tirotiro he aha tonu i whakaputa i te tohu. I roto i tētahi biofilm, ka taea e tētahi waikano nucleic-acid te kite i ngā rawa kei waho o ngā pūtau, me ngā mea kei roto i ngā pūtau kua pakaru. Mō tētahi whakatau mō te oranga, e taunaki ana ngā kairangahau kia whakamahia ētahi atu inenga tautoko, pērā i te whakatipu, i te metabolic activity rānei, me te mōhio he herenga anō tō ia tikanga.
Tirohia he aha tonu kei roto i te kōwhiringa kē
Tērā pea kei roto tonu i tētahi tikanga waikano kē te methylene blue. He tauira te Giemsa: e whakaahua ana a Merck i tana hua hei azure-eosin-methylene-blue solution, mō ngā whakamahinga tae atu ki ngā pani toto me te wheua-wheua. Ka taea pea tēnei hei tikanga rerekē mō tētahi tauira rerekē, engari kāore e tutuki i a ia tētahi whakaritenga kia kaua rawa te methylene blue e uru.
Mēnā ko te whakakore i tētahi pūhui tētahi wāhanga o te whakamātautau, tirohia te tuakiri reagent me te hanganga i mua i te whakataurite whakaahua. Kei te aratohu mō te methyl blue, te methylene blue, me ngā waikano he ōrite ngā ingoa ngā whakamārama mō ngā rerekētanga ingoa.
Whakamana i te whakakapinga i mua i te whakamahi i te hua
Mō tētahi tikanga hou, mahia tētahi whakataurite iti mā ngā tauira māngai me tētahi mana whakahaere e tika ana. Kia rite tonu ngā tautuhinga karuwhārahi i ngā wā e taea ana. Tuhia te hanganga reagent, te whakariterite tauira, te exposure, me ngā tikanga whakaahua, kia taea ai te tirotiro he aha i pai ake ai te āhua o tētahi whakaahua, kaua ko te kōwhiri noa i taua whakaahua nā te mea he pai ake te titiro.
Aromatawaitia te angitu ki te pātai taketake. Mō te morphology, aromatawaitia mēnā ka taea tonu te whakamārama tika i ngā tapa me te whakaritenga o ngā pūtau. Mō te tapanga karihi, aromatawaitia te wāhi o te tohu me te papamuri. Mō te oranga, me whai taunakitanga i tua atu i tētahi wehewehenga tae ātaahua. Kei te aratohu whānui mō te methylene blue staining me ngā whakamahinga taiwhanga te whakamārama me pēhea te hono o te tirohanga me te whakariterite.
Kōwhiria te kōwhiringa kē e pupuri ana i te inenga e hiahiatia ana e koe. He āhuatanga kotahi noa te tae o te whakaahua ka puta.