Dzimwe Nzira dzekutsiva Methylene Blue paMicroscopy

Kune bacterial smear iri nyore, crystal violet inogona kutsiva methylene blue kana chinangwa chiri kuona chimiro chemasero. Kumasero mapenyu, imwe nzira iri nani inogona kuva phase contrast isina stain zvachose. Sarudzo inoenderana nezvaunoda kucherechedza.

Ngatitii vanhu vaviri vari kuongorora bacterial culture imwe chete. Mumwe anoda kusiyanisa masero akaita sematanda kubva kumasero akatenderera. Mumwe anoda kuziva kana treatment yauraya mabhakitiriya. Stain inopa munhu wekutanga mufananidzo wakajeka inogona kusapa munhu wechipiri mhinduro inoshandisika.

Usati wasarudza chinotsiva, pedzisa mutsara uyu: “Ndinofanira kucherechedza ___ mu ___, uye masero anofanira / haafaniri kuramba ari mapenyu.” Mutsara uyu unotsanangura basa rinofanira kuitwa nestain.

Tanga nespecimen nemubvunzo

Kutanga sarudza kana masero achifanira kuramba ari mapenyu, wobva waona chinhu chaunoda kucherechedza:

Masero anofanira kuramba ari mapenyu: pakufamba kana chimiro, funga nezve unstained phase contrast kana DIC; pa nuclear DNA, funga nezve Hoechst 33342 ine fluorescence; pakuvimbika kwemembrane, sarudza assay yakasimbiswa yakaita seSYTO 9 ine propidium iodide.
Masero haafaniri kuramba ari mapenyu: pabacterial morphology, funga nezve crystal-violet simple stain kana nigrosin negative stain; paGram reaction, shandisa nzira yakazara yeGram-stain; pa nuclear DNA mumasero akafixwa, funga nezve DAPI fluorescence. Shandisa tafura iyi kuenzanisa zvipingamupinyi zvebazi rimwe nerimwe. Idzi isarudzo dzenzira, kwete mirayiridzo yekuchinjanisa mabhodhoro mukati meprotocol yatovepo.

Zvaunoda kucherechedza	Specimen nemidziyo	Imwe nzira inogona kushandiswa	Chikanganiso chikuru
Chimiro nekurongeka kwemabhakitiriya	Bacterial smear yakagadzirwa; brightfield	Crystal violet simple stain	Inoratidza morphology; haisimbisi Gram reaction kana viability.
Miganhu yemasero pakatarisana nebackground yakasviba	Bacteriological preparation; brightfield	Nigrosin negative stain	Preparation neformulation zvakakosha; sero risina stain harirevi kuti riri benyu.
Gram reaction	Bacterial smear yakagadzirwa; brightfield	Nzira yakazara yeGram-stain	Inoda reagent sequence yakazara nema controls.
Kufamba kana shanduko mumasero mapenyu	Wet preparation inoenderana; phase contrast kana DIC	Hapana stain	Inoda optics dzakakodzera; contrast haitauri kuti molecule ipi iripo.
Nuclear DNA	Masero akafixwa; fluorescence	DAPI	Inoda excitation ne emission filters zvinoenderana.
Nuclear DNA mumasero mapenyu	Masero mapenyu; fluorescence	Hoechst 33342	Kupinda musero hakuratidzi kuti imaging conditions hadzikuvadzi.
Kuvimbika kwebacterial membrane	Mabhakitiriya asina kufixwa; fluorescence	SYTO 9 ine propidium iodide	Membrane staining chiyero chisina kunanga cheviability.

Pasimple smear, chinja nzira nokungwarira

Crystal violet inogona kushandiswa zvakanaka kana basa riri kuwana mufananidzo une ruvara rumwe chete webacterial morphology. FDA's Bacteriological Analytical Manual inonyora maformulation maviri ecrystal-violet akakodzera simple staining. Shandisa formulation yakanyorwa zvakanaka pamwe nenzira yayo yekugadzirira; zita redhayi chete haritsananguri reagent yakagadzirira kushanda.

Crystal violet inoshandiswawo muGram staining, asi izvozvo hazviiti kuti crystal-violet simple stain ive Gram stain. MuASM Gram-stain protocol, mhedzisiro yekusiyanisa inoenderana necrystal violet, iodine, decolorization, uye counterstaining zvichishanda zvakatevedzana. Kusiya mamwe ematanho iwayo kunochinja zvinogona kusimbiswa nemufananidzo. Ona simple staining ichienzaniswa neGram staining kuti unzwisise musiyano uyu.

Nigrosin inopa imwe nzira yekuratidza miganhu: inosvibisa background nepo bacterial cells achiramba akajeka kana achienzaniswa nayo. Pane ruzivo rwakakosha mumirayiridzo yeMerck yenigrosin runogona kushaikwa murondedzero yakajairika yezvinotsiva. Working formulation yayo yakatarwa ine formaldehyde. Saka haugoni kufungidzira kuti inoenderana nemasero mapenyu kubva pamashoko ekuti “negative stain,” kunyange masero pachawo achiratidzika seasina stain. Tarisa formulation yakazara nekugadzirirwa kwespecimen.

Kumaspecimen mapenyu, funga kuchinja optics

Kana uchida kuona masero achifamba kana kupatsanuka, kuwedzera imwe dhayi kunogona kuomesa kucherechedza. Sekutsanangura kunoita Nikon mugwara rayo recontrast mumaspecimen akajeka, phase contrast nedifferential interference contrast, kana DIC, zvinoita kuti specimen ioneke kuburikidza nemisiyano ye optical properties yayo. Zvinogona kuratidza masero mapenyu asina stain, kana microscope iine components dzinodiwa.

Sarudzo iyi inochengeta preparation isina stain, asi inoshandura ruzivo runowanikwa. Unogona kutevera muganhu kana kufamba usingazivi kuti molecule ipi yakakonzera contrast. Kana mubvunzo uri wemolecular localization, sarudza labeling method yakakodzera panzvimbo pezvo.

Pa nuclear DNA, kuenzanisa kweInvitrogen kweDAPI neHoechst stains kunosiyanisa kushandiswa kwakajairika kweDAPI nemasero akafixwa kubva kuHoechst 33342 inopinda mumasero zviri nyore, iyo inosarudzwa palive-cell labeling. Zvose zvinoda fluorescence equipment. Hapana pairi inotsiva zvakananga colored brightfield stain.

Bata dye exposure neillumination semamiriro ekuedza. Enzanisa preparations dzine stain nedzisina stain kana maitiro emasero ari iwo auri kuyera.

Sero dzvuku haringorevi kuti rakafa

SYTO 9 nepropidium iodide zvinowanzoshandiswa pamwe chete kuongorora kuvimbika kwebacterial membrane. BacLight manual inobatanidza membranes dzisina kukuvara negreen staining uye membranes dzakakuvadzwa nered staining. Kududzirwa ikoko kunoda kusimbiswa paspecimen iri kushandiswa.

A 2019 study yakaitwa naRosenberg nevamwe inoratidza dambudziko iri. Mu24-hour E. coli biofilms pagirazi mu phosphate-buffered saline, about 96% yemasero yakaratidzika sepropidium-iodide-positive in situ. Asi vanyori vakafungidzira kuti 82% aigona kurimwa mushure mekukohwa. Confocal images dzakaratidza masero ane green interiors pasi pezvinhu zvakanga zvastainwa red, zvichiratidza kuti extracellular nucleic acids dzaive nechikamu musignal inotsausa.

Mapesenti iwayo anotsanangura zviyero zvakasiyana papreparations dzechidzidzo, kwete universal correction factor. Chidzidzo chinobatsira ndechekuongorora chakagadzira signal yacho. Mubiofilm, nucleic-acid dye inogona kuona zvinhu zviri kunze kwemasero pamwe chete nezviri mukati memasero akakuvadzwa. Pakuita mhedziso yeviability, vatsvakurudzi vanokurudzira zvimwe zviyero zvinotsigirana nazvo, zvakadai sekurima kana metabolic activity, vachibvumawo kuti chimwe nechimwe chine zvipingamupinyi.

Tarisa kuti imwe nzira yacho ine chii chaizvo

Imwe staining method inogona kunge ichiri nemethylene blue. Giemsa muenzaniso: Merck inotsanangura product yayo seazure-eosin-methylene-blue solution, yakagadzirirwa kushandiswa kunosanganisira blood nebone-marrow smears. Inogona kuva imwe nzira yakasiyana yespecimen yakasiyana, asi haigutsi chinodiwa chekurega methylene blue.

Kana kubvisa compound iri chikamu chekuedza, tarisa reagent identity neformulation usati waenzanisa mifananidzo. Gwaro remethyl blue, methylene blue, nemadhayi ane mazita akafanana rinotsanangura misiyano yemazita.

Simbisa substitution usati washandisa mhedzisiro

Panzira itsva, ita kuenzanisa kudiki uchishandisa specimens dzinomiririra uye control yakakodzera. Chengeta microscope settings dzichienzanisika pazvinogoneka. Nyora reagent formulation, kugadzirirwa kwespecimen, exposure, uye imaging conditions kuitira kuti mufananidzo unotaridzika zviri nani uongororwe pane kungosarudzwa chete.

Ongorora kubudirira maererano nemubvunzo wepakutanga. Pamorphology, tarisa kana miganhu yemasero nekurongeka zvichiri kududzirika. Panuclear labeling, tarisa nzvimbo yesignal nebackground. Paviability, tsvaga humbowo hunopfuura kungopatsanuka kwemavara kunotaridzika zvakanaka. Gwaro rakakura remethylene blue staining nekushandiswa kwayo murabhoritari rinotsanangura kuti observation nepreparation zvinobatana sei.

Sarudza imwe nzira inochengetedza chiyero chaunoda. Ruvara chinhu chimwe chete pamufananidzo unobuda.