Ubundi buryo busimbura Methylene Blue muri Mikorosikopi

Ku bacterial smear yoroshye, crystal violet ishobora gusimbura methylene blue igihe intego ari ukubona imiterere ya selile. Ku selile nzima, ubundi buryo bwiza bushobora kuba phase contrast idakoresha irangi na rimwe. Guhitamo biterwa n’icyo ukeneye kwitegereza.

Tekereza abantu babiri basuzuma bacterial culture imwe. Umwe ashaka gutandukanya selile zimeze nk’uduti n’izizungurutse. Undi ashaka kumenya niba treatment yarishe bacteria. Irangi riha uwa mbere ishusho isobanutse rishobora kudaha uwa kabiri igisubizo ashobora gukoresha.

Mbere yo guhitamo igisimbura, uzuza iyi nteruro: “Nkeneye kwitegereza ___ muri ___, kandi selile zigomba / ntizigomba kuguma ari nzima.” Iyo nteruro isobanura umurimo irangi rigomba gukora.

Tangirira kuri specimen no ku kibazo

Banza umenye niba selile zigomba kuguma ari nzima, hanyuma umenye ikintu ukeneye kwitegereza:

Selile zigomba kuguma ari nzima: ku kugenda cyangwa imiterere, tekereza kuri phase contrast cyangwa DIC bidakoresha irangi; kuri nuclear DNA, tekereza kuri Hoechst 33342 hamwe na fluorescence; ku busugire bwa membrane, hitamo assay yemejwe nka SYTO 9 hamwe na propidium iodide.
Selile ntizigomba kuguma ari nzima: ku miterere ya bacteria, tekereza kuri simple stain ya crystal violet cyangwa negative stain ya nigrosin; kuri Gram reaction, koresha uburyo bwuzuye bwa Gram-stain; kuri nuclear DNA yo muri selile zafikijwe, tekereza kuri DAPI fluorescence.

Koresha iyi mbonerahamwe kugira ngo ugereranye imbogamizi za buri nzira. Ibi ni amahitamo y’uburyo, si amabwiriza yo gusimburanya amacupa muri protocol isanzwe.

Icyo ukeneye kwitegereza	Specimen n’ibikoresho	Ubundi buryo bushoboka	Imbogamizi nyamukuru
Imiterere n’imitunganyirize ya bacteria	Bacterial smear yateguwe; brightfield	Crystal violet simple stain	Yerekana morphology; ntigaragaza Gram reaction cyangwa viability.
Imbibi za selile ku background yijimye	Bacteriological preparation; brightfield	Nigrosin negative stain	Uburyo bwo gutegura na formulation bifite akamaro; selile idafite irangi si ngombwa ko iba iri nzima.
Gram reaction	Bacterial smear yateguwe; brightfield	Uburyo bwuzuye bwa Gram-stain	Busaba reagent sequence yose hamwe na controls.
Kugenda cyangwa impinduka muri selile nzima	Wet preparation iboneye; phase contrast cyangwa DIC	Nta rangi	Bisaba optics ziboneye; contrast ntigaragaza molecule runaka.
Nuclear DNA	Selile zafikijwe; fluorescence	DAPI	Bisaba excitation na emission filters bihuye.
Nuclear DNA muri selile nzima	Selile nzima; fluorescence	Hoechst 33342	Kwinjira muri selile ntibigaragaza ko imaging conditions nta ngaruka bifite.
Ubusugire bwa bacterial membrane	Bacteria zidafikijwe; fluorescence	SYTO 9 hamwe na propidium iodide	Gusiga membrane ni igipimo kiziguye cya viability.

Ku simple smear, hindura uburyo witonze

Crystal violet ni uburyo bufatika igihe umurimo ari ukubona morphology ya bacteria mu ibara rimwe. Bacteriological Analytical Manual ya FDA igaragaza formulations ebyiri za crystal violet ziboneye kuri simple staining. Koresha formulation yanditswe neza n’uburyo bwayo bwo gutegura; izina ry’irangi ryonyine ntirisobanura reagent yiteguye gukoreshwa.

Crystal violet kandi ikoreshwa muri Gram staining, ariko ibyo ntibivuze ko crystal-violet simple stain ari Gram stain. Muri ASM Gram-stain protocol, igisubizo gitandukanya gishingira kuri crystal violet, iodine, decolorization, na counterstaining bikurikirana. Gusiba izo ntambwe bihindura ibyo ishusho ishobora kugaragaza. Reba itandukaniro hagati ya simple staining na Gram staining kugira ngo ubone iryo tandukaniro.

Nigrosin itanga ubundi buryo bwo kugaragaza imbibi: yijimisha background mu gihe bacterial cells ziguma zisa n’izifite umucyo kurushaho. Hari ikintu cy’ingirakamaro kiri mu mabwiriza ya Merck kuri nigrosin urutonde rusanzwe rw’ibisimbura rushobora kwirengagiza. Working formulation yayo yasobanuwe irimo formaldehyde. Bityo rero, ntushobora gufata ko ihuye na live-cell use kubera gusa amagambo “negative stain,” nubwo selile ubwazo zigaragara nk’izidasizwe irangi. Reba formulation yose n’uburyo specimen yateguwe.

Ku specimens nzima, tekereza guhindura optics

Niba ushaka kureba selile zigenda cyangwa zigabanyika, kongeramo irindi rangi bishobora kugora observation. Nk’uko Nikon ibisobanura mu gitabo cyayo kuri contrast muri transparent specimens, phase contrast na differential interference contrast, cyangwa DIC, bituma specimens zigaragara binyuze mu itandukaniro ry’imiterere yazo ya optical. Bishobora kugaragaza selile nzima kandi zidasizwe irangi, igihe microscope ifite components zikenewe.

Iri hitamo rigumana preparation idasizwe irangi, ariko rihindura amakuru ashobora kuboneka. Ushobora gukurikirana boundary cyangwa movement utazi molecule yateje contrast. Niba ikibazo ari molecular localization, hitamo uburyo buboneye bwo labeling.

Ku nuclear DNA, igereranya rya DAPI na Hoechst stains rya Invitrogen ritandukanya ikoreshwa risanzwe rya DAPI kuri selile zafikijwe na Hoechst 33342 yinjira muri selile kurushaho, ikunda gukoreshwa kuri live-cell labeling. Byombi bisaba fluorescence equipment. Nta na kimwe gisimbura mu buryo butaziguye colored brightfield stain.

Fata dye exposure na illumination nk’experimental conditions. Gereranya preparations zisizwe irangi n’izidasizwe igihe imyitwarire ya selile ari yo measurement yawe.

Selile itukura si ngombwa ko iba yapfuye

SYTO 9 na propidium iodide bikunze gukoreshwa hamwe mu gusuzuma ubusugire bwa bacterial membrane. BacLight manual ihuza membranes zidasenyutse na green staining, naho damaged membranes ikazihuza na red staining. Iyo interpretation igomba kwemezwa kuri specimen.

Ubushakashatsi bwo muri 2019 bwa Rosenberg na bagenzi be bugaragaza icyo kibazo. Muri biofilms za E. coli zari zimaze 24-hour ku kirahure muri phosphate-buffered saline, hafi 96% bya selile byagaragaye ko ari propidium-iodide-positive in situ. Nyamara abanditsi bagereranyije ko 82% zari cultivable nyuma yo kuzikusanya. Confocal images zerekanye selile zifite green interiors munsi y’ibintu byasizwe red, bikerekana ko extracellular nucleic acids zashoboraga kugira uruhare muri signal iyobya.

Izo percentages zisobanura measurements zitandukanye zakozwe kuri preparations zo muri ubwo bushakashatsi, si universal correction factor. Isomo ry’ingirakamaro ni ukureba icyateye signal. Muri biofilm, nucleic-acid dye ishobora detecta material iri hanze ya selile kimwe n’iri imbere muri damaged cells. Ku mwanzuro wa viability, abashakashatsi basaba complementary measurements, nko cultivation cyangwa metabolic activity, banemera ko buri kimwe gifite limitations.

Reba neza icyo ubundi buryo burimo

Ubundi buryo bwa staining bushobora kuba bugikubiyemo methylene blue. Giemsa ni urugero: Merck isobanura product yayo nka azure-eosin-methylene-blue solution, igenewe applications zirimo blood na bone-marrow smears. Ishobora kuba uburyo butandukanye ku specimen itandukanye, ariko ntiyujuje requirement yo gukuramo methylene blue.

Niba gukuramo compound runaka biri mu bigize experiment, banza urebe reagent identity na formulation mbere yo kugereranya images. Igitabo kuri methyl blue, methylene blue, n’amarangi afite amazina asa gisobanura itandukaniro ry’amazina.

Emeza substitution mbere yo gukoresha igisubizo

Ku buryo bushya, kora igereranya rito ukoresheje representative specimens na control iboneye. Gumana microscope settings zigereranywa aho bishoboka. Andika reagent formulation, specimen preparation, exposure, na imaging conditions kugira ngo ishusho isa neza kurushaho ishobore gusuzumwa aho kuyikunda gusa.

Suzuma intsinzi ukurikije ikibazo cy’ibanze. Kuri morphology, reba niba cell boundaries na arrangement bigikomeza gusobanuka. Kuri nuclear labeling, reba signal location na background. Kuri viability, saba ibimenyetso birenze color separation isa neza. Igitabo cyagutse kuri methylene blue staining n’imikoreshereze yayo muri laboratory gisobanura uko observation na preparation bihuzwa.

Hitamo ubundi buryo bugumana measurement ukeneye. Ibara ni kimwe gusa mu biranga ishusho iboneka.