Madadin Methylene Blue don Microscopy

Ga shafaffen ƙwayoyin bakteriya mai sauƙi, crystal violet na iya maye gurbin methylene blue idan manufar ita ce ganin siffar ƙwayoyin. Ga ƙwayoyin da suke da rai, madadin da ya fi dacewa na iya zama phase contrast ba tare da wani tabo ba. Zaɓin ya dogara da abin da ake buƙatar lura da shi.

A ce mutane biyu suna nazarin al'adar bakteriya iri ɗaya. Ɗaya yana son bambance ƙwayoyi masu siffar sanda da ƙwayoyi masu zagaye. Ɗayan kuma yana son sanin ko wani magani ya kashe bakteriyar. Tabo da ke ba mutum na farko hoto mai haske na iya kasa ba mutum na biyu amsa mai amfani.

Kafin zaɓar abin maye, kammala wannan jimla: “Ina buƙatar lura da ___ a cikin ___, kuma dole / ba dole ba ne ƙwayoyin su ci gaba da kasancewa da rai.” Wannan jimlar tana ayyana aikin da tabon dole ya yi.

Fara da samfurin da tambayar

Da farko ka yanke shawara ko dole ne ƙwayoyin su ci gaba da kasancewa da rai, sannan ka tantance abin da kake buƙatar lura da shi:

Dole ne ƙwayoyin su ci gaba da kasancewa da rai: don motsi ko siffa, yi la'akari da phase contrast ko DIC ba tare da tabo ba; don DNA na nucleus, yi la'akari da Hoechst 33342 tare da fluorescence; don mutuncin membrane, zaɓi gwajin da aka tabbatar da ingancinsa kamar SYTO 9 tare da propidium iodide.
Ba dole ba ne ƙwayoyin su ci gaba da kasancewa da rai: don morphology na bakteriya, yi la'akari da simple stain na crystal violet ko negative stain na nigrosin; don Gram reaction, yi amfani da cikakkiyar hanyar Gram-stain; don DNA na nucleus a cikin ƙwayoyin da aka gyara, yi la'akari da fluorescence na DAPI.

Yi amfani da wannan teburin don kwatanta iyakokin kowace hanya. Waɗannan zaɓuɓɓukan hanyoyi ne, ba umarnin musanya kwalabe a cikin wata hanyar gwaji da ake amfani da ita ba.

Abin da kake buƙatar lura da shi	Samfuri da kayan aiki	Madadin da za a iya amfani da shi	Babban iyaka
Siffar bakteriya da yadda suke jere	Shafaffen bakteriya da aka shirya; brightfield	Simple stain na crystal violet	Yana nuna morphology; ba ya tabbatar da Gram reaction ko viability.
Iyakokin ƙwayoyi a kan duhun bango	Shirye-shiryen bacteriological; brightfield	Negative stain na nigrosin	Shirye-shirye da formulation suna da muhimmanci; ƙwayar da ba ta sami tabo ba ba lallai ba ne tana da rai.
Gram reaction	Shafaffen bakteriya da aka shirya; brightfield	Cikakkiyar hanyar Gram-stain	Yana buƙatar cikakken jerin reagents da controls.
Motsi ko sauye-sauye a cikin ƙwayoyin da ke da rai	Wet preparation mai dacewa; phase contrast ko DIC	Babu tabo	Yana buƙatar optics masu dacewa; contrast ba ya gano wani molecule.
DNA na nucleus	Ƙwayoyin da aka gyara; fluorescence	DAPI	Yana buƙatar excitation da emission filters masu dacewa.
DNA na nucleus a cikin ƙwayoyin da ke da rai	Ƙwayoyin da ke da rai; fluorescence	Hoechst 33342	Shigar dye cikin ƙwaya ba ya tabbatar da cewa yanayin ɗaukar hoton ba ya cutarwa.
Mutuncin membrane na bakteriya	Bakteriya da ba a gyara ba; fluorescence	SYTO 9 tare da propidium iodide	Tabo na membrane hanya ce kai tsaye ba ta auna viability ba.

Ga simple smear, maye gurbin hanyar cikin taka-tsantsan

Crystal violet zaɓi ne mai amfani idan aikin shi ne samun hoto mai launi guda na morphology na bakteriya. Bacteriological Analytical Manual na FDA ya lissafa formulations biyu na crystal violet da suka dace da simple staining. Yi amfani da formulation da aka rubuta tare da hanyar shirya shi; sunan dye kaɗai ba ya fayyace reagent mai aiki.

Crystal violet kuma yana cikin Gram staining, amma wannan ba ya sa simple stain na crystal violet ya zama Gram stain. A cikin ASM Gram-stain protocol, sakamakon bambancewa ya dogara da crystal violet, iodine, decolorization, da counterstaining da suke aiki a jere. Barin waɗannan matakan yana sauya abin da hoton zai iya tabbatarwa. Duba simple staining da Gram staining don wannan bambanci.

Nigrosin yana ba da wata hanya ta daban don bayyana iyakoki: yana duhun bango yayin da ƙwayoyin bakteriya suke kasancewa masu haske idan aka kwatanta da shi. Akwai muhimmin bayani a cikin umarnin nigrosin na Merck wanda jerin madadin na gama-gari zai iya rasa. Formulation ɗin aiki da aka fayyace yana ɗauke da formaldehyde. Saboda haka ba za ka iya ɗauka cewa ya dace da ƙwayoyin da ke da rai kawai saboda kalmomin “negative stain” ba, ko da yake ƙwayoyin kansu suna bayyana kamar ba su sami tabo ba. Duba cikakken formulation da yadda aka shirya samfurin.

Ga samfuran da ke da rai, yi la'akari da sauya optics

Idan kana son kallon ƙwayoyi suna motsi ko rarrabuwa, ƙara wani dye na iya rikitar da abin da ake lura da shi. Kamar yadda Nikon ya bayyana a jagoransa kan contrast a cikin samfura masu bayyana haske, phase contrast da differential interference contrast, ko DIC, suna sa samfura su bayyana ta bambance-bambancen halayensu na optical. Za su iya bayyana ƙwayoyin da suke da rai kuma ba a yi musu tabo ba, muddin microscope ɗin yana da abubuwan da ake buƙata.

Wannan zaɓin yana barin preparation ɗin ba tare da tabo ba, amma yana sauya irin bayanin da ake samu. Za ka iya bin iyaka ko motsi ba tare da sanin wane molecule ne ya haifar da contrast ɗin ba. Idan tambayar ita ce inda molecule yake, zaɓi hanyar labeling da ta dace maimakon haka.

Don DNA na nucleus, kwatancen DAPI da Hoechst stains na Invitrogen yana bambanta yawan amfani da DAPI ga ƙwayoyin da aka gyara da Hoechst 33342, wanda ya fi iya shiga ƙwaya kuma ake fifita shi don labeling na ƙwayoyin da ke da rai. Dukansu suna buƙatar kayan fluorescence. Babu ɗayansu da yake maye gurbin tabo mai launi na brightfield kai tsaye.

Ɗauki fallasa ga dye da illumination a matsayin yanayin gwaji. Kwatanta preparations masu tabo da marasa tabo idan halayen ƙwayoyin ne ma'aunin da kake nazari.

Jan ƙwaya ba yana nufin matacciyar ƙwaya kai tsaye ba

Ana yawan haɗa SYTO 9 da propidium iodide don tantance mutuncin membrane na bakteriya. BacLight manual yana danganta membranes marasa lalacewa da kore staining, da membranes da suka lalace da ja staining. Wannan fassarar tana buƙatar tabbatarwa ga samfurin da ake gwadawa.

Wani bincike na 2019 da Rosenberg da abokan aikinsa suka yi ya nuna matsalar. A cikin biofilms na E. coli masu awanni 24 a kan gilashi cikin phosphate-buffered saline, kusan 96% na ƙwayoyin sun bayyana propidium-iodide-positive a wurinsu. Duk da haka marubutan sun ƙiyasta cewa 82% za a iya noma su bayan tattara su. Hotunan confocal sun nuna ƙwayoyi masu koren ciki ƙarƙashin kayan da suka sami jan tabo, wanda ya nuna cewa extracellular nucleic acids ne suka taimaka wajen haifar da siginar da ke iya ruɗarwa.

Waɗannan kaso suna bayyana ma'aunai daban-daban a kan preparations na binciken, ba universal correction factor ba ne. Muhimmin darasi shi ne a bincika abin da ya haifar da siginar. A cikin biofilm, nucleic-acid dye na iya gano kayan da ke wajen ƙwayoyin da kuma cikin ƙwayoyin da membranes ɗinsu suka lalace. Don yanke hukunci kan viability, masu binciken suna ba da shawarar ma'aunai masu ƙarin juna, kamar cultivation ko metabolic activity, tare da amincewa cewa kowannensu yana da iyakoki.

Duba abin da madadin yake ƙunshe da shi a zahiri

Wata madadin hanyar staining na iya har yanzu ɗauke da methylene blue. Giemsa misali ne: Merck ya bayyana samfurinsa a matsayin azure-eosin-methylene-blue solution, wanda aka yi niyya don amfani da suka haɗa da shafaffen jini da bone-marrow. Zai iya zama wata hanya daban don wani samfurin daban, amma ba ya cika buƙatar cire methylene blue.

Idan cire wani compound wani ɓangare ne na gwajin, duba ainihin reagent da formulation kafin kwatanta hotuna. Jagorar kan methyl blue, methylene blue, da dyes masu sunaye masu kama da juna tana bayani kan bambance-bambancen suna.

Tabbatar da musanyawar kafin amfani da sakamakon

Ga sabuwar hanya, yi ƙaramin kwatance ta amfani da samfura masu wakiltar abin da ake nazari da control da ya dace. Ka kiyaye settings na microscope suna da kusanci da juna inda zai yiwu. Rubuta formulation na reagent, shirya samfurin, exposure, da yanayin imaging domin a iya bincika dalilin da ya sa wani hoto ya fi kyau maimakon kawai a fifita shi.

Auna nasara bisa tambayar asali. Don morphology, tantance ko iyakokin ƙwayoyi da tsarin jerinsu har yanzu suna da sauƙin fassara. Don nuclear labeling, tantance wurin sigina da background. Don viability, buƙaci hujja fiye da kyakkyawan rabuwar launuka. Babbar jagorar kan methylene blue staining da aikace-aikacen dakin gwaje-gwaje tana bayyana yadda abin da ake lura da shi da preparation suke haɗuwa.

Zaɓi madadin da yake kiyaye ma'aunin da kake buƙata. Launi ɗaya ne kawai daga cikin halayen hoton da ake samu.