C TNH MT S MI TRNG PHN LP V CHN ON VI KHUN GY BNH

Trong phng th nghim y t, phn lp v nh danh vi khun gy bnh cn phi s dng n nhiu loi mi trng nui cy v ho cht thch hp cho mt s th nghim, c bit l th nghim sinh ho. Bn cnh , vic nh gi cht lng pha ch mi trng ng quy cch cng l mt trong nhng khu quan trng trong cng tc xt nghim vi khun. Cho n nay, c nhiu loi mi trng khc nhau c dng cho phn lp v nh danh vi khun, v thng c xp thnh 3 loi ch yu l: mi trng nui cy c bn, mi trng tng sinh vi khun (c giu cht dinh dng) v mi trng nui cy chn lc. Mi loi mi trng s c s dng vi nhng mc ch khc nhau trong nui cy, phn lp v nh danh vi sinh vt c trong mu xt nghim ca bnh nhn. 1. MT S MI TRNG NUI CY THNG THNG 1.1. Mi trng Chocolate agar Mi trng thch chocolate l mi trng giu dinh dng v thng c dng cho vi khun Haemophilus, S.pneumoniae v cc loi vi khun kh pht trin khc. y l mi trng thch mu cu, khi thm mu cu vo mi trng c bn trong iu kin nhit gii phng t bo hng cu v nicotinamid - adenin dinucleotid (NAD). Mi trng a c bo qun 40C. a mi trng cy bnh phm c m nhit 350C trong kh trng c 5% CO2, thi gian 18 24 gi, quan st s pht trin ca vi khun. 1.2. Mi trng Simmons citrate Mi trng thch Cimmons citrate c s dng cho cc trc khun ng rut gram m khc nhau. Ging nh nguyn l s dng mi trng acetat ca vi khun, phn bit cc loi vi khun ngi ta da vo s khc bit do kh nng s dng citrate nh l ngun carbon duy nht ca chng phn lp. Mi trng c pH khong 6,9. ng mi trng sau khi cy huyn dch vi khun ln mt nghing, t m 35oC, quan st s thay i trong 4 ngy. 1.3. Mi trng Deoxychocolate citrate

Thch Deoxychocolate citrate l mi trng chn lc, mi trng khc nhau dng cho phn lp trc tip cc cn nguyn gy bnh ng rut t phn v bnh phm nc tiu hoc gin tip t canh thang giu dinh dng nh selenite-F. Cc thnh phn chn lc c trong mi trng nh citrate natri v deoxycholate natri phi hp vi nhau c ch cc trc khun ng rut khc m khng phi l cn nguyn gy bnh nguy him. Thnh phn khc nhau y l lactose, cn nguyn gy bnh ng rut khng ln men ng s xut hin cc khun lc khng mu. Nu ln men ng lactose, xut hin nhng khun lc chuyn t mu hng sang mu , l kt qu ca s thay i pH trong qu trnh ln men ng lactose. Cy khun lc ln mi trng a, m 350C/48 gi. Cht nui cy m nhiu nh mu phn, nc tiu, hoc cc cht khc ca c th th c th cy trc tip vo mi trng, ngc li nu cht nui cy t th nn lm thm mt bc l cy vo mi trng tng sinh trc ri mi cy vo mi trng chn lc. 1.4. Mi trng Kligler Iron Agar (KIA) Mi trng thch Kligler Iron Agar c s dng nui cy trc tip cho trc khun gram m ln men ng glucose, y l c im c bn trong phn loi bc u ca cc trc khun gram m. Mi trng ny cng ng thi th tnh ln men ng lactose, sinh hi trong qu trnh ln men carbohydrat v sinh H2S. Tt c nhng c im c s dng cho cc trc khun gram m thuc h vi khun ng rut. Mi trng KIA gm glucose v lactose (c th ln men carbohydrat), phenol (ch th pH), pepton (ngun carbon/nitrogen) v mui st cng vi thiosulfate natri. C 3 hnh thi ln men carbohydrat c th xy ra l: Acid (mu vng) phn gc v kim (mu ) phn nghing ca mi trng, khi cho thy vi sinh vt ln men ng glucose nhng khng ln men ng lactose. Acid (mu vng) phn gc v acid (mu vng) phn nghing ca mi trng, khi cho thy vi sinh vt ln men c ng glucose v ng lactose. Kim (mu ) phn gc v kim (mu ) phn nghing mi trng, khi cho thy vi sinh vt khng th ln men ng glucose v ng lactose. Cc sn phm sinh ra khng phi l sn phm acid. Nu sinh hi, c hi phn gc ca ng mi trng, mi trng b chia ct hoc y mi trng ln khi y ng. Nu thy mu en phn gc ng, vi sinh vt

 sinh ra hydrogen sulfid gas t thiosulfid. Phn ng c c sau khi nui cy t 18 24 gi, nu mi trng c c qu sm, vi sinh vt ch c th ln men ng glucose. Nu c qua mun, cc cht gy men lactose c th dng ht lactose v bt u d ho pepton, phn nghing ca mi trng tr li mu .

1.5. Mi trng MacConkey Agar Thch MacConkey l mi trng chn lc cho cc vi khun thuc h vi khun ng rut v cc trc khun gram m khc c trong mt qun th hn tp v phn bit chng c ln men lactose hay khng ln men lactose. Mui mt v tinh th mu tm c ch hu ht cc vi sinh vt gram dng nhng cho php cc trc khun gram m mc c. ng lactose nh mt ngun cung cp carbohydrat duy nht. Trc khun gram m, nu ln men lactose s sinh ra khun lc mu hng hoc . Ngc li, nu khng ln men lactose th khun lc khng c mu hoc khun lc c mu trong. Sau khi cy vi khun ln a mi phn lp, t vo t m 350C, thi gian t 18 24 gi. Vi khun c th ln men lactose mnh hoc yu, sau 24 gi nui cy sinh ra nhng khun lc c mu hoc xut hin mu hng nh sau 24 48 gi. Khng nui cy ko di qu 48 gi, nh th c th lm o ln kt qu. Thch MacConkey Sorbitol, c cha cc thnh phn ging nh thch MacConkey thng thng khc nhng c thm D-sorbitol. Mi trng ny c dng cho phn lp E.coli O157:H7. 1.6. Mi trng Mueller-Hinton Agar Mi trng thch Mueller-Hinton Agar l mi trng trong, dng cho th nghim tnh nhy cm ca vi sinh vt vi khng sinh. Mi trng cng thng c dng th nghim s thu phn tinh bt. Thch Mueller-Hinton c cha hn dch ng vt, casamino acid v tinh bt gip cho hu ht cc vi sinh vt pht trin. Hn na, c th cho thm mu cu vo cng thc c bn thc hin th nghim tnh nhy cm ca chng Streptococci. Khi un nng mu cu to thnh chocolate vi thch Mueller-Hinton th c th th nghim vi cc vi sinh vt kh mc, nh Heamophilus v Neisseria. 1.7. Mi trng dinh dng (Nutrient Agar)

Mi trng thch dinh dng c s dng phn bit gia mt s t loi Neisseria d mc vi cc loi Neisseria khc, nh N.gonorrhoeae v N.meningitidis. S t loi Neisseriae d mc th pht trin c trn mi trng thch dinh dng, ngc li c nhiu loi khng pht trin c. Thch dinh dng cng thng c dng lm mi trng lu gi chng. Thch dinh dng c cha cc cht dinh dng ng vt v c bit l hm lng protein thp. Chng phn lp no pht trin c trn mi trng ny th cng c ngha l chng rt d mc v khng i hi bt c thnh phn c bit no.

1.8. Mi trng Oxydative-Fermentative (OF) Mi trng thch Oxydative-Fermentative hoc OF l mi trng lun c dng xc nh cch s dng carbonhydrat ca vi sinh vt l kiu oxy ho, ln men hoc tr. y l mi trng rt quan trng nh danh cc trc khun gram m khng ln men ng glucose nh: Pseudomonas, Acinetobacter spp . Mi trng c cha mt lng nh pepton ngn cn s to thnh sn phm kim, v cc sn phm ny c th trung ho mt lng nh sn phm acid qua qu trnh oxy ho. Lng ln carbonhydrat trong mi trng lm tng s lng acid m n c th to thnh. Mi trng cng c lng nh thch to cho mi trng khng rn cng khng lng, to iu kin cho php acid to thnh trn b mt v khuch tn qua mi trng. Mu xanh bromthymol nh ch th mu pH pht hin acid. 1.9. Mi trng th nghim tnh di ng Mc ch ca mi trng th tnh di ng l xc nh mt vi khun c th di ng c hay khng. y l th nghim c bit dng cho nh danh cc thnh vin ca h vi khun ng rut, thng cho 2 loi Shigella v Klebsiella lun khng di ng, v cc loi Yersinia chc chn di ng nhit phng nhng khng di ng 350C. Cc vi khun khng di ng l do khng c lng roi, ch pht trin trong ng cy v xung quanh mi trng trong. Cc vi sinh vt di ng, s dng lng roi di chuyn ra ngoi ca ng cy v xut hin m khi trong mi trng. Lng thch trong mi trng thp lm cho mi trng khng lng, khng cng cho php pht hin di ng tt nht.

Dng u que cy chc thng vo mi trng, thn trong di chuyn u que cy trn 1 ng, khng chc que cy xung tn y tupe. mi trng nui cy 350C. Bn cht lng roi l protein, do vy khng to thnh tt nhit cao, mt s nh sinh vt hc thch nhit t 18 200C. 1. 10. Mi trng thch Salmonella- Shigella (SS Agar) Thch Salmonella- Shigella thng dng chn lc Salmonella v mt vi chng Shigella t mu xt nghim phn. Nhng vi sinh vt ny c phn bit trn mi trng thch SS bng c im khun lc trn mi trng. Thnh phn mi trng SS c cha mui mt, natri citrate v xanh brillian, c ch cc trc khun gram dng v rt nhiu trc khun ln men ng lactose thng thng khc tm thy trong phn. Nu mt vi sinh vt pht trin trn mi trng v ln men lactose, th n s sinh ra acid v thay i thnh mu - hng. Nu thiosulfate natri tham gia vo nh mt ngun sulfur cho qu trnh sinh ra hydrogen sulfid. Nu hydrogen sulfid hin din trong mi trng th s to thnh cht kt ta mu en gia khun lc. Khun lc Shigella xut hin khng mu trn mi trng SS, v nhng vi sinh vt ny khng ln men ng lactose hoc sinh hydrogen sulfid. Khun lc Salmonella khng mu v c im en gia khun lc, v vi khun ny to ln hydrogen sulfid nhng khng ln men ng lactose. Khun lc chuyn t mu hng n mu cho thy sinh vt ln men ng lactose, nu c mu en gia th cng sinh ra hydrogen sulfid. Nu Proteus pht trin trn mi trng ny th kh nng to thnh nhng m s b hn ch. 1. 11. Mi trng Thiosulfate Citrate Bile Salts Sucrose (TCBS) Mi trng thch Thiosulfate Citrate Bile Salts Sucrose l mi trng chn lc dng phn lp cc loi Vibrio t mu xt nghim phn c ln vi vi khun ch khc. Cc loi Vibrio c phn bit bng c im do khun lc sinh ra trn mi trng. Cc kiu khun lc khc nhau c sinh ra t cc loi Vibrio khc nhau, th hin qua s ln men ca sucrose. V d: V.cholerae v V. alginolyticus sinh ra cc khun lc mu vng, v chng ln men ng sucrose, ngc li V. parahaemolyticus v V.vulniticus lun sinh ra khun lc mu xanh do thiu ln men sucrose. Vi sinh vt Vibrio khng sinh ra hydrogen sulfid. Khi dng thch TCBS, chng ta nn dng cht nui cy c, nhng loi Vibrio d cht t ngt v mi trng ny c ngn chn c iu . Mu xt nghim

ti l tt nht, v vi sinh vt ny nhy cm vi iu kin kh, nh sng v pH acid. Nu x l chm th nn s dng mi trng Cary-Blair vn chuyn bnh phm tt hn l mi trng vn chuyn c m glycerol. a mi trng nui cy nn nhit 350C, thi gian t 18 24 gi, di nht l 48 gi. Khng ly khun lc mi trng TCBS lm th nghim oxydase. 1.12. Mi trng Esculin Agar Mi trng thch esculin l mi trng phn bit kh nng thy phn esculin ca vi khun. Nhng sn phm thu phn t phn ng esculin vi mui st c trong mi trng gy ra kt ta cc thnh phn iron, v sn phm ca s kt ta lm i mu ca mi trng t mu xm ti mu en. Cy vi khun ln phn nghing ca mi trng, 350C trong iu kin hiu kh v quan st s pht trin, mi trng c mu en cho thy c s thy phn esculin.

1.13. Mi trng Methyl Red-Voges-Proskauer (MR-VP) Mi trng canh thang MRVP dng cho cc th nghim methyl v VogesProskauer. Quy trnh ny dng phn bit cc thnh vin thuc h

Enterobacteriaceae (h vi khun ng rut), v d: E.coli, methyl dng v Voges-Proskauer m; Enterobacter aerogene v Klebsiella pneumoniae cho cc phn ng ngc li. Cc thnh vin thuc h vi khun ng rut c chia thnh 2 nhm da trn cch chuyn ho ng glucose. Mt nhm sn sinh ra mt lng ln hn hp acid (lactic, formic, succinic, acetic). Khi cho thuc th methyl vo mt trong cc vi khun thuc nhm ny, mu xut hin do pH c tnh acid. Nhm th 2 sn sinh ch yu cc sn phm trung tnh, acetoin hoc acetyl-methylcarbinol. Th nghim methyl, canh khun phi c m 48 gi. Ly vo mi ng nghim 0,5 n 1 ml canh khun, 350C, thi gian 18 24 gi l c th thc hin c th nghim. Trong qu trnh th nghim, lc ng nghim canh khun hp th oxy v tng thm phn ng. 1.14. Mi trng Blood Agar Mi trng thch mu (Blood Agar) l mi trng thng xuyn dng nui cy cc vi khun khng kh mc lm. Mi trng c pha ch hoc thch triptic soy

c bn c lm giu thm bng cch thm 5 10% mu cu, mu th hoc mu ngi ph v si fibrin. S kt hp ca mu khng ch cung cp cht dinh dng cho vi khun pht trin m cn cho php pht hin c tnh tan mu ca vi khun. 1.15. Mi trng Mannitol Sald Agar Mi trng thch Mannitol l mi trng nui cy chn lc c bn dng tng sinh v nh danh Staphylococci t mu xt nghim c ln vi vi khun ch khc. m mui cao (7,5%) c tc dng c ch hu ht vi khun gram m v gram dng tr Staphylococcus spp. Vi khun Staphylococcus aureus c kh nng ln men ng mannitol, sinh ra sn phm acid lm pH ca mi trng gim, phenol t mu chuyn sang mu vng, khun lc in hnh ca Staphylococcus aureus xut hin mu vng. Nhng loi Staphylococcus v Micrococcus khc thng khng ln men ng mannitol, do sinh ra khun lc mu hi . Sau khi cy vi khun ln a mi trng, m 350C trong kh trng khng c CO2, thi gian 24 48 gi. Mt s chng Staphylococcus aureus c th ln men ng mannitol chm, do vy khng nn loi b mi trng nui cy trong vng 48 gi. Cc chng ngh n l S.aureus cn c lm thm cc th nghim nh coagulase hoc bng mt thng quy chun khc.

1.16. Mi trng Selenite F Canh thang Selenite F l mi trng tng sinh cho s t thnh vin ca Salmonella v mt vi chng Shigella t phn v mu xt nghim cha s lng ln vi sinh vt hp. Mi trng c Selenite natri c ch rt nhiu trc khun gram m v cu khun ng rut (Enterococci) nhng cho php tng sinh Salmonella v mt vi loi Shigella. Selenite hiu qu nht l trung ho pH, trong qu trnh pht trin ca vi khun lm gim Selenite, cc sn phm kim ho c sinh ra, cho nn ng lactose cng c trong mi trng ny. Cc cht ln men ng lactose sinh ra acid, v cc sn phm kim c trung ho v tr li mi trng trung ho pH. 1.17. Mi trng Thayer-Martin, Modified (MTM) Thch Modified Thayer-Martin l mi trng giu dinh dng dng tng sinh Neisseria gonorrhoeae v N. meningitidis t mu xt nghim c cc vi khun ch hp. Mi trng thch Modified Thayer-Martin giu dinh dng, do vy nhiu loi Neisseria

kh mc cng c th pht trin c. Mi trng c cc yu t pht trin nh hemoglobin, vitamin, cocarboxylase, diphosphopyridin nucleotid v glutamin. Cng thc ca Modified cha nhiu thch ngn chn s lan to ca proteus. Thch Modified Thayer-Martin c cha mt s khng th, c ch vi khun ch thng thng v ngn chn s pht trin ca hu ht cc vi khun nh vancomycin c ch cu khun gram dng pht trin, colistin c ch trc khun gram m v nistatin ngn chn s pht trin ca nm. a mi trng MTM sau khi cy vi khun, c m trong kh trng c CO2 hoc bnh nn trong vi ngy. 1.18. Mi trng Peptone-Yeast Extract-Glucose (PYG) Canh thang Peptone-Yeast Extract-Glucose l mi trng c dng cho nui cy vi khun. C th dng thng quy ghi sc k lng pht hin cc sn phm chuyn ho cui cng trong canh cy PYG phn lp vi khun k kh. Canh thang PYG c cha mt s cht dinh dng v thnh phn b sung kch thch vi khun k kh pht trin. Thnh phn dinh dng gm vitamin K (cho sinh sc t Prevotella v Porphyromonas), cao men, hemin v glucose. Cht Resazurin p ng nh cht ch th vi khun k kh. Khi mi trng xut hin mu hng ngha l c hin din ca oxy. 1.19. Mi trng Lowenstein-Jensen (LJ) Mi trng Lowenstein-Jensen c dng nui cy Mycobacterium spp. Trong mi trng hu ht c cha thnh phn c th c ch s pht trin ca Mycobacteria. Bt khoai ty, trng v glycerol c trong mi trng LJ c tc dng gip kh c mi trng ny, ng thi cung cp cht dinh dng cho vi khun ny pht trin. Mi trng LJ m bo tt trong 1 thng nu vn cht np, ni kh ro v nhit t 4 60C. Mi trng LJ cng phi c gi ni ti, v xanh malachite rt nhy vi nh sng. Bnh phm c nui cy ln mi trng, m trong kh trng CO2 (5 10%), t 6 10 tun. Mt iu quan trng l phi ni lng np cho thay i khng kh bn trong. 2. CC TH NGHIM TRN MI TRNG THNG THNG Mi trng nui cy c th c chun b t nhng thnh phn ring hoc chun b t mi trng di dng bt kh c thng mi ho. Ngy nay, mi trng dng bt kh c thng mi ho tr ln ph bin ti cc phng th

nghim vi khun lm sng. Mt s im quan trng kim sot cht lng ca mi trng nh sau: Khng dng mi trng ht hn s dng; Hp mi trng cn c vn cht bng np trnh m; Gi mi trng vo ni ti, thong kh, mt; Khng s dng mi trng dng bt kh b ng mu hoc vn cc. Thng xuyn o mi trng d tr theo nguyn tc: ngoi vo, trong ra. Khi chun b mi trng cn phi tun th nghim ngt ch dn ca nh sn xut. Khng mi trng c pha ch ngoi nh sng v nhit phng. Kim tra tit trng v pH ca mi trng chun b.

Bng 1. Cc th nghim trn mt s mi trng thng dng Mi trng Thch mu (Blood Agar) 24h, CO2 S.pneumoniae Thch Chocolate 24h, CO2 H.influenzae E.coli Thch MacConkey vi crystal violet 24h P.mirabilis E.faecalis Methyl red/VogesProskauer E.coli 48h K.pneumoniae E.coli ATCC 25922 24h Agar P.aeruginosa ATCC 27853 ln ca vng v khun (-)/(+) ln ca vng v khun Pht trin v tan mu khng hon ton Pht trin Khun lc c mu Khun lc khng mu Khng pht trin (+)/(-) T m Sinh vt kim tra S. aureus Kt qu mong i Pht trin v tan mu hon ton

Mueller-Hinton

Peptone water (indole)

E.coli 24h K.pneumoniae E.coli

(+) (-) Khng pht trin Pht trin v sinh sc t sanh nc bin

Thch Simmons citrate

48h K.pneumoniae

Thch Thiosulfate citrate bile salt (TCBS) 24h

Vibrio spp.( Khng dnh) N.meningitidis

Khun lc mu vng

Pht trin Pht trin Khng pht trin Khng pht trin

Thayer Martin Agar

24h, CO2

N.gonorrhoeae Staphylococci E.coli

Bng 2. Gim st cht lng ca cc th nghim thng dng Quy trnh Vi sinh vt S. aureus Catalase Streptococcus spp. Coagulase S. aureus E. coli Indole E. aerogenes E. coli Methyl red E.. aerogenes P.aeruginosa Oxidase E. coli + Khng i mu Mu tm trong vng 20 giy Khng mu trong vng 20 giy Mu + Vng mu vng trn b mt Lp tc c mu + + Khng si bt To thnh vn cc sau 4 g Vng mu trn b mt + Phn ng mong i Phn ng si bt

Voges

E. aerogenes

Proskauer a khng

E. coli

Khng i mu C vng c ch C vng c ch C vng c ch Khng c vng c ch Mu tm trong vng 30 giy Khng i mu

sinh Streptococcus group A + E. faecalis sinh S. pneumoniae S. viridans P.aeruginosa + +

Bacitracin

khng

Optochin

a Oxidase E. coli

Lu : cc th nghim kim tra cht lng phi c thc hin cho mi ln pha dung dch. mi