Gram staining yog txheej txheem nrawm thiab siv los saib pom muaj cov kab mob nyob hauv cov ntaub so ntswg thiab cais cov kab mob li Gram-positive lossis Gram-negative, raws li cov tshuaj thiab lub cev ntawm lawv cov phab ntsa ntawm tes. Gram stain yuav luag ib txwm siv ua thawj kauj ruam hauv kev kuaj mob kab mob.
Cov txheej txheem no yog hu ua tom qab Danish tus kws tshawb fawb Hans Christian Gram (1853 - 1938), uas tau tsim cov txheej txheem xyoo 1882 thiab luam tawm nws xyoo 1884 ua cov txheej txheem kom paub qhov txawv ntawm ob hom kab mob uas muaj cov tsos mob zoo sib xws: Streptococcus pneumoniae (tseem hu ua Streptococcus pneumoniae). Pneumococci) thiab kab mob Klebsiella pneumoniae.
Kauj ruam
Txoj Kev 1 ntawm 3: Npaj Lub Tshuab Ntsuas Xaub
Kauj Ruam 1. Npaj ua haujlwm hauv chav kuaj
Hnav hnab looj tes thiab khi cov plaub hau ntev los tiv thaiv kab mob kis ntawm cov qauv uas koj tab tom sim. Ntxuav qhov chaw ua haujlwm hauv qab lub fume hood, lossis lwm qhov chaw muaj cua nkag tau zoo. Txheeb xyuas Bunsen lub tshuab hlawv thiab xyuas kom lub tshuab tsom iav ua haujlwm zoo ua ntej koj pib.
Kauj Ruam 2. Sterilize lub iav tsom iav swb
Yog tias iav swb yog qias neeg, ntxuav nws nrog cov xab npum kom tshem cov roj thiab av. Ntxuav cov kab xev nrog ethanol, tshuaj ntxuav iav, lossis lwm txoj hauv kev siv los ntawm koj lub chaw kuaj ntshav.
Kauj Ruam 3. Muab cov qauv tso rau ntawm daim iav swb
Koj tuaj yeem siv cov txheej txheem Gram stain los pab txheeb xyuas cov kab mob hauv cov qauv tshuaj, lossis kab lis kev cai ntawm cov kab mob loj hlob hauv cov tais zaub mov. Kom tau txais txiaj ntsig zoo tshaj plaws, siv Gram stain ntawm cov kab mob me me ntawm tus qauv. Nws raug nquahu kom siv cov qauv uas muaj hnub nyoog tsawg dua 24 teev, vim tias cov kab mob qub tuaj yeem ua rau lawv cov phab ntsa ntawm tes puas thiab yuav tsis teb rau Gram staining.
- Yog tias siv cov qauv ntaub so ntswg, ntxiv 1-2 tee rau iav swb. Tshaj tawm tusyees ntawm tus swb kom tsim ib txheej nyias ntawm cov qauv txau, los ntawm zawv zawg nws siv ntug ntawm lwm qhov iav uas tsis muaj menyuam tsis huv. Cia nws qhuav ua ntej ua cov kauj ruam tom ntej.
- Yog tias koj tab tom noj cov kab mob los ntawm cov tais zaub mov, ua kom tsis huv lub voj inoculation hauv lub qhov cub Bunsen kom txog thaum nws ci, tom qab ntawd cia nws txias. Siv lub voj los tso dej kom tsis muaj menyuam mus rau qhov swb, tom qab ntawd ua kom tsis muaj menyuam thiab txias dua ua ntej siv nws los sau cov kab mob me me. Tom qab ntawd do maj mam.
- Cov kab mob tau npaj hauv cov kua zaub yuav tsum tau rov ua dua siv lub vortexer, tom qab ntawd nqa nrog lub voj inoculation li saum toj no, yam tsis ntxiv dej.
- Yog tias koj muaj tus qauv swab (feem ntau ua tiav nrog cov paj rwb me me tipped), kov thiab maj mam tig lub swab hla tus swb.
Kauj Ruam 4. Qhov kub me ntsis tuaj yeem ua rau pleev zoo
Cov cua sov yuav tuav cov kab mob nyob rau sab saum toj ntawm lub swb, yog li lawv tsis yaj yooj yim thaum txheej txheem txheej txheem. Coj mus rhaub tus swb sai li ob mus rau peb zaug hla lub Bunsen hlawv, lossis ua kom sov lub swb hla lub tshuab rhaub hluav taws xob. Tsis txhob overheat, cov qauv yuav raug puas tsuaj. Yog tias koj siv lub Bunsen hlawv, nws yuav tsum yog lub nplaim taws me me tab sis siv lub nplaim paj txiv kab ntxwv loj.
Ib qho ntxiv, smear kuj tseem tuaj yeem ua nrog methanol, los ntawm kev ntxiv 1-2 tee ntawm methanol rau cov tshuaj pleev qhuav, kom qhuav cov methanol uas seem ntawm qhov swb los ntawm nws tawm hauv qhov cua qhib. Cov txheej txheem no txo qis kev puas tsuaj ntawm tes thiab muab cov duab huv huv tom qab
Kauj Ruam 5. Muab tus swb hla lub tais xim
Cov tais tais ua los ntawm cov hlau, iav, lossis cov yas yas ntiav nrog cov ntxaij mos muag sab hauv. Muab cov kab xaum tso rau saum cov nets, kom cov kua uas koj siv tau tuaj yeem tso rau hauv lub tais.
Yog tias koj tsis muaj lub tais xim, koj tuaj yeem tso tus swb rau ntawm lub tais yas los luam cov dej khov
Txoj Kev 2 ntawm 3: Txheej Txheem Gram Stain
Kauj Ruam 1. Ncuav cov kua ua kua violet tso rau ntawm lub qhov ncauj
Siv lub raj xa tshuaj tsuag cov kab mob ua piv txwv nrog ob peb tee dej ntawm cov xim siv lead ua violet, tseem hu ua gentian violet. Cia nws rau 30-60 vib nas this. Crystal violet (KV) sib cais hauv cov kua aqueous rau hauv KV+ thiab chloride (Cl-) ions. Cov ions no nkag mus rau phab ntsa ntawm tes thiab cov cell ntawm cov kab mob gram-positive thiab gram-negative. KV+ ion sib cuam tshuam nrog cov khoom tsis zoo ntawm cov kab mob hauv lub cev, muab cov cell rau xim liab.
Ntau lub chaw soj ntsuam siv "Hucker" siv lead ua violet, uas muaj ammonium oxalate los tiv thaiv nag lossis daus
Kauj Ruam 2. Yaug lub Crystal violet maj mam muab
Qaij tus swb thiab siv lub tshuab ntxhua khaub ncaws kom tsuag cov dej me me ntawm cov dej los yog cov kais dej hla tus swb. Dej yuav tsum ntws los ntawm qhov smear, thiab yuav tsum tsis txhob txau ncaj qha rau ntawm lub smear. Tsis txhob yaug ntau dhau. Nws tuaj yeem tshem tawm cov kab mob hauv cov kab mob Gram zoo.
Kauj Ruam 3. Yaug lub ntsej muag nrog iodine, tom qab ntawd yaug
Siv lub tee dej los yaug qhov ncauj nrog iodine. Tso nws tsawg kawg 60 vib nas this, tom qab ntawd yaug kom zoo zoo ib yam li saum toj no. Iodine, nyob rau hauv daim ntawv ntawm qhov tsis pom zoo ion, cuam tshuam nrog KV+ los tsim ib qho loj sib xyaw ua ke ntawm Crystal violet thiab iodine (CV-I compound complex)) hauv txheej sab hauv thiab sab nraud ntawm lub cell. Qhov sib xyaw ua ke no yuav khaws cov xim violet ntawm cov xim siv lead ua violet sab hauv ntawm lub xovtooj, ntawm qhov chaw tsis huv.
Iodine yog ib yam khoom uas muaj corrosive. Zam kev nqus pa, nqus los yog kov cov tawv nqaij
Kauj Ruam 4. Ntxiv cov tshuaj dawb, tom qab ntawd yaug tawm sai
A 1: 1 sib xyaw ntawm acetone thiab ethanol feem ntau yog siv rau cov kauj ruam tseem ceeb no, uas yuav tsum tau ua tib zoo ua raws sijhawm. Muab tus swb rau ntawm lub kaum ntse ntse, tom qab ntawd ntxiv cov tshuaj dawb kom txog thaum tsis muaj xim liab pom hauv cov dej siv los yaug. Qhov no feem ntau siv sijhawm tsawg dua 10 vib nas this, lossis txawm tias sijhawm tsawg dua yog tias cov tshuaj dawb muaj cov acetone siab. Nres cov kauj ruam no tam sim, txwv tsis pub cov zas xim tseem yuav ua rau siv lead ua violet los ntawm cov gram-positive thiab negative cells, thiab cov txheej txheem zas xim yuav tsum tau rov ua dua. Tam sim ntawd yaug tawm cov tshuaj dawb ntau dhau los siv cov txheej txheem yav dhau los.
- Acetone ntshiab (95%+) tuaj yeem siv los hloov pauv tau. Qhov ntau koj siv acetone, cov tshuaj dawb sai yuav ua haujlwm yog li koj yuav tsum tau ua tib zoo saib xyuas lub sijhawm.
- Yog tias koj tab tom muaj teeb meem taug qab lub sijhawm rau cov kauj ruam no, ntxiv cov xim dawb tso los ntawm qhov poob.
Kauj Ruam 5. Sprinkle cov xim ziab khaub ncaws hla lub smear, tom qab ntawd yaug
Lub txee xim, feem ntau yog safranin lossis fuchsin, yog siv los nce qhov sib piv ntawm cov kab mob gram-negative thiab gram-positive, los ntawm kev ua kom cov kab mob decolorized (decolorized), piv txwv li gram-negative bacteria, liab dawb lossis liab. ces yaug.
Fuchsin yuav ua kom hnyav ntau ntau cov kab mob tsis zoo, xws li Haemophilus spp thiab Legionella spp. Txoj kev no zoo rau cov pib tshiab
Kauj Ruam 6. Qhuav qhov swb
Koj tuaj yeem ziab lawv hauv qhov qhib cua kom qhuav, lossis ua kom qhuav lawv siv cov ntawv bibulous muag rau lub hom phiaj no. Cov txheej txheem Gram staining tiav.
Txoj Kev 3 ntawm 3: Txheeb Xyuas Cov Xim
Kauj Ruam 1. Npaj lub tsom iav raj
Muab tus swb hauv qab lub tshuab tsom iav. Cov kab mob sib txawv hauv qhov loj me, yog li qhov xav tau kev nthuav dav yuav txawv ntawm 400x txog 1000x. Lub hom phiaj tsom iav nrog tso roj tso rau pom zoo kom pom cov duab zoo dua. Tso cov roj tso rau hauv qhov swb, zam kev txav chaw thaum ntws los tiv thaiv kom tsis txhob muaj npuas.
Kev tso roj tuaj yeem tsuas yog siv tshwj xeeb tsom iav, tsis yog lo ntsiab muag "qhuav"
Kauj Ruam 2. Sim txheeb xyuas cov kab mob gram zoo thiab gram tsis zoo
Txheeb xyuas tus swb hauv lub tsom iav qhov muag. Cov kab mob gram-positive tshwm rau xim liab, vim tias cov xim siv lead ua violet raug daig hauv cov phab ntsa tuab. Cov kab mob gram-tsis zoo yuav yog xim liab lossis xim liab, vim tias cov xim siv lead ua violet tau yaug tawm los ntawm cov phab ntsa ntawm cov cell nyias, qhov uas cov xim paj yeeb rau hauv.
- Yog tias cov qauv hnyav dhau, qhov tshwm sim tuaj yeem yog qhov tsis zoo. Khaws ib qho qauv tshiab yog tias nws qhia tag nrho cov kab mob gram zoo, kom ntseeg tau tias qhov txiaj ntsig raug.
- Yog tias koj siv tshuaj dawb ntau dhau, qhov tshwm sim tuaj yeem yog qhov tsis raug cuav. Stain tus qauv tshiab yog tias nws qhia tag nrho cov kab mob gram tsis zoo, kom ntseeg tau tias qhov txiaj ntsig raug.
Kauj Ruam 3. Sib piv cov txiaj ntsig koj pom nrog daim duab siv
Yog tias koj tsis paub cov kab mob zoo li cas, kawm sau cov duab siv, feem ntau txheeb los ntawm cov duab thiab Gram stain. Koj tseem tuaj yeem saib cov ntaub ntawv online ntawm [National Microbial Pathogen Database, Kab mob hauv Duab, thiab ntau lwm qhov chaw online. Txhawm rau pab txheeb xyuas tus kheej, hauv qab no yog piv txwv ntawm cov kab mob uas feem ntau ntsib lossis tseem ceeb rau kev kuaj mob, cais raws li lawv li xwm txheej Gram thiab lub cev.
Kauj Ruam 4. Txheeb xyuas kab mob gram-positive raws li lawv lub cev
Cov kab mob tau muab cais tawm ntxiv raws li lawv cov duab hauv lub tsom iav, feem ntau yog cocci (kheej kheej) lossis pas nrig (cylindrical). Nov yog qee qhov piv txwv ntawm cov kab mob gram-positive (ntshav xim) raws li lawv lub cev:
- Gram positive coj feem ntau yog staphylococci (lub ntsiab lus pab pawg cocci) lossis streptococci (lub ntsiab lus saw cocci).
- 'Gram zoo rods xws li Bacillus, Clostridium, Corynebacterium, thiab Listeria. Cov kab mob hu ua Actinomyces spp. feem ntau muaj cov ceg lossis cov ceg ntoo.
Kauj Ruam 5. Txheeb xyuas kab mob gram tsis zoo
Cov kab mob gram-negative (xim liab) feem ntau muab cais ua peb pawg. Cocci yog kheej kheej, cov pas nrig ntev thiab nyias, thaum coccoid pas nrig nyob nruab nrab.
- Gram tsis zoo cocci Feem ntau yog Neisseria spp.
- Gram-negative rods xws li E. coli, Enterobacter, Klebsiella, Citrobacter, Serratia, Proteus, Salmonella, Shigella, Pseudomonas, thiab ntau yam ntxiv. Vibrio cholerae tuaj yeem pom ua qia tsis tu ncua lossis qia qag.
- Gram-negative "coccoid" (lossis "coccobacilli") cov kab mob qws xws li Bordetella, Brucella, Haemophilus thiab Pasteurella
Kauj Ruam 6. Txheeb xyuas kom zoo yog tias cov txiaj ntsig tau sib xyaw
Qee cov kab mob nyuaj rau xim kom raug, vim yog qhov nkig nkuav lossis cov av nplaum ntawm lawv cov phab ntsa ntawm tes. Cov kab mob no tuaj yeem pom sib xyaw xim liab lossis paj yeeb hauv tib lub cell, lossis nruab nrab ntawm cov cell sib txawv hauv tib lub smear. Kev kuaj cov kab mob loj dua 24 teev tuaj yeem qhia qhov teeb meem no, tab sis kuj tseem muaj qee hom kab mob uas tseem nyuaj rau xim rau txhua lub hnub nyoog piv txwv. Cov kab mob no xav tau kev tshuaj xyuas tshwj xeeb ntxiv rau kev txheeb xyuas, xws li ua kom cov kua qaub-nrawm, soj ntsuam hauv kab lis kev cai kab mob, TSI kab lis kev cai tshaj tawm, lossis kuaj kab mob.
- Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, thiab Propionibacterium spp. txhua yam yog cov kab mob gram-positive, tab sis feem ntau tsis pom meej meej.
- Cov kab mob me me, xws li Treponema, Chlamydia, thiab Rickettsia spp. nyuaj rau stain los ntawm cov txheej txheem Gram.
Kauj Ruam 7. Muab cov khoom siv thiab cov cuab yeej siv pov tseg
Cov txheej txheem pov tseg no txawv ntawm cov chaw kuaj mob thiab nyob ntawm cov khoom siv. Feem ntau, cov kua hauv lub tais uas tsis zoo yog pov tseg hauv cov fwj uas sau tias yog cov khoom pov tseg. Tsau swb hauv 10% tshuaj dawb, tom qab ntawd muab pov rau hauv lub thawv ntim khoom.
Lub tswv yim
- Nco ntsoov tias Gram stain cov txiaj ntsig yuav tsuas yog qhov zoo yog tias cov qauv zoo. Nws yog ib qho tseem ceeb los qhia rau cov neeg mob kom lawv tuaj yeem muab cov hnoos qeev zoo (piv txwv li, qhov sib txawv ntawm kev hnoos thiab hnoos hnoos kom tau txais cov hnoos qeev).
- Raws li cov tshuaj dawb, cov tshuaj ethanol reacts qeeb dua li acetone.
- Ua raws li cov txheej txheem kuaj kev sim kom ntseeg tau kev nyab xeeb.
- Siv lub puab tsaig swab los xyaum, vim nws muaj cov kab mob gram-positive thiab gram-negative. Yog tias koj pom tsuas yog ib hom kab mob, koj tuaj yeem siv tshuaj dawb tsawg lossis ntau dhau.
- Koj tuaj yeem siv cov ntoo txuas kom ruaj ntseg swb.
Ceeb toom
- Acetone thiab ethanol yog cov tshuaj tua tau yooj yim. Cov acetone yuav tshem tawm cov roj los ntawm koj txhais tes, ua kom yooj yim rau koj cov tawv nqaij kom nqus tau lwm yam tshuaj. Hnav cov hnab looj tes thiab siv lawv nrog kev saib xyuas.
- Tsis txhob tso cov tshuaj pleev kom qhuav ua ntej koj yaug tawm cov Gram stain lossis counterstain.
Koj xav tau dab tsi
- Qauv Network
- Iav swb
- Pipette
- Cov hluav taws me me, lossis lub tshuab rhaub dej kub, lossis tshuaj methanol
- Dej
- Crystal liab
- Iodine
- Xim tshuaj dawb, xws li cawv lossis acetone
- Safranin
