13.9 Xc nh hot lc thuc khng sinh bng phng php th vi sinh vt

 

Hot lc khng sinh c th hin bng kh nng c ch mt loi chng ch th no , iu kin thch hp. S gim hot tnh sinh hc ca khng sinh c th hin trn chng ch th, rt kh pht hin bng phng php phn tch ho hc, v vy phng php vi sinh vt thng gi mt vai tr quan trng trong vic nh gi kh nng mt hot lc ca khng sinh, nht l nhng loi khng sinh c cu trc phc tp, hoc thnh phn gm mt hn hp khng sinh cng h m mc hot lc chnh lch nhau khng ln lm.

Phng php vi sinh vt xc nh hot lc ca mt khng sinh bng cch so snh tc ng c ch s pht trin ca mt loi vi sinh vt (c gi l chng ch th) bi nhng pha long bit ca khng sinh mang th (cha bit r hot lc) vi tc ng c ch bi nng bit ca khng sinh i chiu (cht chun bit r hot lc).

Th nghim phi dng phng php phn tch thng k nh gi nhng s liu thu c. Nu p dng m hnh ng thng song song, hai ng log liu - p ng ca cht chun v ch phm th phi song song vi nhau v phi tuyn tnh trong khong liu dng.

Hai phng php thng c s dng trong kim nghim l phng php khuch tn dng mi trng thch v phng php o c dng mi trng lng.

Phng php th nht da vo s khuch tn ca khng sinh t ch cha qua lp thch c hp Petri hoc khay vo khong pht trin ca vi sinh vt to ra mt vng c ch hon ton xung quanh ni cha (ng tr, l thch) dung dch khng sinh.

Phng php ng nghim da vo c ca canh khun, bit c s c ch pht trin ca vi khun gy ra bi mt lot dung dch khng sinh trong mi trng lng l mi trng thch hp i vi s pht trin ca vi sinh vt khi khng c khng sinh.

Phng tin v dng c

Tt c cc dng c phi c lm sch hon ton trc v sau mi ln th nghim. Nhng dng c thu tinh v nhng ng khc dng trong th nghim phi c loi ht vi khun v cht nh hng. Cc dng c dng gi v chuyn chng ch th cn lm sch cn thn v tit khun bng sc nng kh hoc hi nc iu kin thch hp.

iu kin nhit : Khi nui cy chng ch th v ch to nh dch cy truyn, cn thc hin trong mi trng m bo nhit qui nh. i vi phng php o c, cn kim tra cht ch bung iu khin nhit sao cho nhit quanh cc ng nghim phi ng u v phi t c nhit cn thit trong ng nghim. Nn dng iu nhit bng nc lun chuyn s tt hn iu nhit bng khng kh.

My o quang ph: c dng o s truyn qua hoc hp th trong khong sng kh hp, do cn c my o quang thch hp c th thay i c bc sng hoc dng dng knh lc 650 nm hoc 580 nm o nh dch nui cy pha ch v dng knh lc 530 nm o hp th nh sng phng php o c. Dng canh thang tin hnh nh mu nhng thm ngay dung dch formaldehyd chnh my cho hp th v im 0

Dng c th nghim:

- Phng php khuch tn: Dng cc khay hoc a Petri bng thu tinh hoc plastic, t tiu chun bng phng. a Petri c hai loi, c nh ng knh 100 mm, c ln ng knh ln hn 160 mm. Cc khay th nghim c tiu chun ho, bng knh hoc plastic vi kch thc 25 x 25 cm (loi vung) hoc 28,8 x 19,8 cm (loi ch nht). C th s dng nhng cht liu thch hp t tiu chun bng phng.

ng tr bng thp khng r, bng thu tinh, hoc bng s, c kch thc nh sau: ng knh ngoi 8,0 mm; ng knh trong 6,0 mm; cao 10,0 mm.

Vt liu dng lm ng tr phi l loi tr, ring i vi khng sinh nhy cm vi nh sng phi dng vt liu c pha mu nu. Khi dng phi lm sch cn thn, loi ht cn, kh khun. i khi phi ra bng acid nh dung dch acid nitric 2M, hoc acid sulfocromic.

Dng c dng cho phng php o c: Dng cc ng nghim loi 16 x 125 mm hoc 18 x 150 mm bng thu tinh c cng chiu di, ng knh v dy, b mt phi khng c vt xc, khng dy bn. Khi dng, ng nghim phi sch hon ton, m bo khng cn vt khng sinh hoc vt cc cht ty ra v phi tit khun trc khi dng.

Mi trng, dung mi v cht pha long

Phi dng cc cht t t mc tinh khit ho hc tr ln pha ch.

Mi trng

Di y l cng thc mi trng cn thit dng cho kim nghim khng sinh. Cc loi mi trng ny c th c thay th bng mi trng kh tng ng hoc bng mt loi mi trng thc y vi sinh vt pht trin tt hn m cng cho mt ng p ng tng t. Pha mi trng bng cch ho tan thnh phn trong cng thc vo mt phn nc, sau thm nc va mt lt. Dng dung dch acid hydrocloric 1N hoc dung dch natri hydroxyd 1N iu chnh mi trng sao cho sau khi tit khun 121oC trong
15 pht c pH nh ghi trong cng thc. Thi gian cn cho vic tit khun c th ko di ti 30 pht i vi nhng mi trng c th tch ln hn 500 ml.

Mi trng s 1

Pepton kh

Casein pancreatic

Cao men bia

Dextrose monohydrat

Thch

Nc ct

pH: 6,0 n 7,0

6,0 g

4,0 g

3,0 g

1,0 g

20,0 g

1000 ml

 

Mi trng s 2:

Pepton kh

Cao men bia

Cao tht

Thch

Nc ct

pH: 6,0 n 7,0

6,0 g

3,0 g

1,5 g

20,0 g

1000 ml

Mi trng s 3:

Pepton kh

Cao men bia

Cao tht

Dextrose monohydrat

Dikali hydrophosphat (K2HPO4)

Kali dihydrophosphat (KH2PO4)

Natri clorid

Nc ct

pH: 6,0 n 7,0

5,0 g

1,5 g

1,5 g

1,0 g

3,58 g

1.32 g

3,5 g

1000 ml

Mi trng s 4:

Ging nh mi trng s 2 nhng c pH l 7,0 n 8,0.

Mi trng s 5:

Ging nh mi trng s 1, nhng c pH l 7,8 n 8,0.

Mi trng s 6:

Casein pancreatic

Bt u tng papaic

Natri clorid

Dextrose monohydrat

Dikali hydrophosphat (K2HPO4)

Thch

Polysorbat 80

Nc ct

pH : 7,0 n 7,3

17,0 g

3,0 g

5,0 g

2,5 g

2,5 g

15,0 g

10,0 ml

1000 ml

Ch : Polysorbat 80 c thm vo mt lng dung dch nng ca cc thnh phn khc trc khi thm nc va dung tch cn thit.

Mi trng s 7:

Pepton kh

Cao men bia

Cao tht

Dextrose monohydrat

Natri clorid

Thch

Nc ct

pH: 6,0 0,2

9,4 g

4,7 g

2,4 g

10,0 g

10,0 g

20,0 g

1000 ml

Dung mi v cht pha long

Dung mi v cht pha long dng trong th nghim phi khng nh hng n s pht trin ca vi sinh vt. Chng c dng pha cc dung dch theo yu cu trong tng th nghim. Cc loi dung mi nh nc v khun, acid hydrocloric, ethanol, dimethylformamid v cc dung dch m c di pH khc nhau hoc cc loi khc tu thuc vo tng khng sinh ring bit (bng 2).

Cc dung dch m phosphat c ch to bng cch ho tan lng dikali hydrophosphat v kali dihydrophosphat (theo bng 1) vo trong nc va c 1000 ml. Sau iu chnh vi acid phosphoric 18 N hoc natri hydroxyd 18 N n pH cn thit. Gi tr pH ch ra trong bng l pH ca dung dch sau khi tit khun.

Bng 1.

S dung dch m

Dikali hydrophosphat K2HPO4 (g)

Kali dihydrophosphat KH2PO4 (g)

pH

iu chnh n

1

2

3

4

5

6

2,0

16,73

0

20,0

35,0

13,6

8,0

0,523

13,61

80,0

0

4,0

6,0 0,1

8,0 0,1

4,5 0,1

6,0 0,1

10,5 0,1

7,0 0,2

Cht i chiu v n v

Cht i chiu c dng trong th nghim l nhng cht c hot lc c xc nh chnh xc so vi chun quc t hoc cht i chiu quc t tng ng.

Hot lc ca mt khng sinh c th hin bng n v quc t. Mt n v quc t l hot lc c bit cha trong mt lng (tnh theo cn nng) ca chun sinh hc quc t tng ng hoc ch phm i chiu sinh hc quc t. Tng thi k, Hi ng v chun sinh hc ca T chc y t th gii s c ch dn v qui nh chnh xc cho mi chun quc t . Trong mt vi trng hp, nh ngha s n v quc t c th bao gm mt tng lng ca mt vi nguyn liu v thc t kh cn mt lng nh d chnh xc ca cht chun sinh hc quc t hoc cht i chiu sinh hc quc t.

Hot lc ca mt vi cht khng sinh c th c biu th bng mg. mg hot lc khng nht thit tng ng vi khi lng ca cht khng sinh v n khng th bao hm ton b thc th ho hc tinh khit ring ca tng khng sinh .

Chng ch th v dch cy truyn

Cc chng ch th dng phi nhy cm, ph hp vi tng khng sinh, theo qui nh bng 2. C th dng chng ch th khc vi chng ch th gii thiu trong chuyn lun ny min sao chng phi nhy cm vi khng sinh cn nh lng.

Ch to dch cy truyn

Ch to nh dch vi khun khng c nha bo

Escherichia coli

Klebsiella pneumoniae (khng to v )

Micrococcus luteus

Saccharomyces cerevisiae

Staphylococcus aureus

Staphylococcus epidermidis

Streptococcus faecium

Dng chng ch th mi pht trin trn thch nghing sau 24 gi, cy truyn li ln mt mt ng thch nghing khc hoc mt b mt thch rng (nh trong bnh Roux) ca mi trng s 1 (Saccharomyces cerevicae dng mi trng s 8; Streptococcus faecium dng mi trng s 3) 32 - 350C trong 24 gi (ring vi Klebsiella pneumoniae v Streptococcus faecium 370C) vi Saccharomyces cerevisiae 300C. Thu vi khun pht trin trn b mt mi trng bng nc mui sinh l, ri ho long vi cng mt cht ho long ti c thch hp c p ng thch hp trong phng php o c, hoc to ra vng c ch r rng, sc nt thun li khi o trong phng php khuch tn.

Th d: Pha long hn dch vi khun ti nng sao cho khi o bc sng 650 nm, cc o dy 1cm, truyn qua T = 50%, bc sng 580 nm T = 25%, hay cho vng vo khun r, sc net c ng knh 13 mm.

Nh dch vi khun ch to nh trn em bo qun nhit di 40C c th s dng trong 2 tun.

Ch to nh dch nha bo

Bacillus cereus

Bacillus pumilus

Bacillus subtilis

Cho chng vi khun mc qua m trn ng thch nghing mi trng s 1 370C (ring vi Bacillus cereus nui 300C). Thu vi khun pht trin trn mt thch bng nc mui sinh l, ri chuyn sang mt b mt thch rng hn (nh trong bnh Roux) ca mi trng s 1, trong mi trng ny thm vo dung dch 0,001% (kl/tt) mangan sulfat (MnSO4) gip thc y s hnh thnh nha bo. Nh dch vi sinh vt c tri cho ph kn b mt mi trng bng cch cho vo b mt thch mt vi vin bi thu tinh v khun. em 370C (ring vi Bacillus cereus 300C) trong 7 ngy. Dng nc ct v khun ra vi khun mc (ch yu l nha bo) em ho long ti nng thch hp, th d khong 107 - 108 nha bo trong 1ml, un cch thy nh dch nha bo nhit 70oC trong 30 pht. Dng test phin knh kim tra kh nng sng ca nha bo. Xc nh lng nh dch nha bo cho vo 100 ml mi trng nh lng c th to c vng c ch r rng, sc nt. Nh dch nha bo c th gi lu nhit 40C.

Cc loi th nghim

Th nghim hai liu v ba liu.

Phng php khuch tn

Pha dung dch chun v dung dch th: Dung dch chun nng bit v dung dch th c coi nh xp x nng dung dch chun. Cc dung dch chun v th ny ban u c pha vo dung mi thch hp c nhng dung dch gc. Sau pha long dung dch gc bng dung mi hoc dung dch m v khun n nng thch hp, c pH thch hp cho tng loi khng sinh ( xem bng 2). xc nh hiu lc ca khng sinh, dng ba liu khc nhau ca chun (S1, S2, S3) mang so snh vi ba liu khc nhau ca th (U1, U2, U3) c hot lc c coi nh dung dch chun. Nng c pha theo cp s nhn, th d pha mt lot pha long theo t l 2 : 1 hoc 4 : 1. Nu quan h gia logarit nng khng sinh v ng knh vng c ch ch ra gn nh thng vi h s dng th c th dng nh lng thng xuyn bng cch ch dng hai nng chun (S1, S2) v hai nng th (U1, U2) - Th nghim hai liu. Trong trng hp c tranh chp bt buc phi s dng nh lng ba liu.

Chun bޠ cy truyn

Cc th nghim c th tin hnh trn nhng a Petri hoc khay bng phng. vo hp Petri hoc khay mi trng thch hp cy truyn chng ch th thch hp cho tng khng sinh. Lp mi trng ny thng c dy 3 - 4 mm (ring vi amphotericin B nystatin dy l 1 - 2 mm, i vi bleomycin, mithramycin dy 2 - 3 mm). Thch dinh dng c th c thnh mt lp hoc hai lp (gm lp nn khng c vi khun v lp gieo cy), nn chn nng nh dch cy vo mi trng sao cho to c vng c ch sc nt nht tng ng vi cc liu lng ca khng sinh. Dng nh dch chng ch th ch to nh phn trn, thng cy vo mi trng vi nng 1 ml chng cho 100 ml mi trng l thch hp. Khi nh dch chng l loi khng c nha bo, thng phi cy vo mi trng thch un chy hon ton v ngui n 45 - 500C. Trng hp nh dch chng l nha bo, nhit cho php khi cy ti a l 65 - 700C. Dng mt lng thch hp mi trng cy chng ch th, rt mt cch v khun vo a petri hoc khay knh (trn a thch hoc khay c thch nn hoc khng tu theo yu cu) c mt lp mi trng c dy thch hp, dn tri nhanh v y np ngay. Trong khi i thch ng c, t khay knh hoc hp petri trn mt tm knh to mt mt phng gip cho mi trng trn khay hoc hp c dy ng nht. Nhng mi trng chun b c kh nhit phng 30 pht trc khi s dng (ring i vi colistin v colistin sulfomethat natri, a petri hoc khay knh cy truyn phi t lnh 40C trong vi gi).

Tin hnh th:

i vi th nghim 3 liu, dng 6 ng tr v khun t ln mt thch cy truyn trong mt hp petri (vi th nghim 2 liu dng 4 ng tr cho mi hp). C th thay cc ng tr bng cch dng dng c c l tit khun, c vo mi trng thch to ra nhng l thch ng knh 6 - 8 mm (hoc thay bng nhng khoanh giy tm khng sinh). Phn phi cc dung dch th nghim ca chun v mu th trn mi hp Petri hay khay vung sao cho ph hp vi kiu b tr th nghim chn (xem ph lc 13.10). Vic b tr ng tr, l khoan, hoc khoanh giy trn mt thch phi sao cho cc vng c ch khng b trm ln nhau.

Dng pipet nh vo ng tr, hoc l thch mt lng bng nhau cc dung dch khng sinh. Nu dng cc khoanh giy phi tiu chun ho trc th tch chnh xc cht lng cho mi khoanh giy. Gi hp petri hoc khay nhit phng trong khong 1 - 4 gi tu theo c tnh ca mi khng sinh. i vi nhng khng sinh nhy cm vi nhit , phi c gi nhit 40C s khuch tn ca khng sinh vo mi trng xy ra chm v lm gim n mc ti thiu hiu qu ca s bin i theo thi gian gia cc dung dch khc nhau. i vi colistin v colistin sulfomethat natri nn 2 - 4 gi nhit phng trc khi . Cc hp Petri hoc khay sau thi gian khuch tn, mang nhit thch hp cho tng khng sinh ring (bng 2). Nhit phi c kim tra khong nhit chnh lch so vi quy nh ch trong khong 0,50C. Thi gian thng ko di 16 - 18 gi. Dng dng c o c chnh xc ti thiu 0,1mm, o ng knh vng c ch to bi cc nng khc nhau ca chun v th.

Kim tra tnh c gi tr ca nh lng, tnh hot lc v gii hn tin cy ca hot lc khng sinh theo ph lc 13.10

Phng php o c

Pha cc dung dch th nghim ca chun v mu th

Dung dch dch th nghim ca chun v mu th c pha trong dung mi thch hp v c pha long bng dung dch m phosphat c pH thch hp cho tng khng sinh theo ch dn bng 2. m bo gi tr hiu lc ca php th, lm th nghim 3 liu l thch hp. Pha song song nhng ho long gm 3 liu chun (S1, S2, S3) v 3 liu th (U1, U2, U3).

Ch to canh thang cy truyn

c c mt c thch hp dng c ngay dch cy truyn, pha dch cy truyn nh m t phn ch to nh dch cy truyn ri dng ngay. Chng ch thޠ v iu kin th nghim ca tng khng sinh s dng trong phng php ny c ghi trong bng 2.

Tin hnh

Cc ng nghim c xp thnh tng nhm, mi nhm 3 - 5 ng, thm vo mi ng 1 ml dung dch ca tng pha long ca dung dch chun hoc mu th. t cc ng nghim vo gi ng nghim theo quy tc ngu nhin theo khi hoc theo hnh vung latinh. Trong mi gi c 2 ng dng kim tra khng thm khng sinh, mt ng cha 1 ml dch pha long, cn ng kia cha 1 ml dch pha long v 0,5 ml dung dch formaldehyd 12% (tt/tt).

Thm vo mi ng 9 ml canh thang cy truyn. t gi chun b xong ngay vo trong t m hoc cch thu, gi nhit thch hp, khng ch nhit mc sai s 0,50C so vi nhit cn (theo bng 2) trong 3 - 4 gi. Sau khi , lm ngng s pht trin ca vi khun bng cch thm vo mi ng 0,5 ml dung dch formaldehyd 12% (tt/tt) tr ng kim tra thm formaldehyd 12% trc hoc nhng gi c cc ng nghim vo nc si . Ly tng gi ng nghim khi ni , xc nh truyn qua hoc hp th ca mi dung dch bng my o quang 530 nm.

Kim tra tnh c gi tr ca nh lng, tnh hot lc v gii hn tin cy ca hot lc khng sinh theo ph lc 13.10

Phng php ng chun

Ho tan mt lng cn chnh xc cht i chiu hoc cht chun sinh hc ca mt khng sinh cho trong dung mi thch hp, c th phi lm kh trc. Pha long vi dung mi qui nh c dy chun c 5 nng . T l tng gia cc nng nn l 1: 1,25. (xem hng dn bng 2)

Tin hnh

Khi tin hnh xy dng ng chun, kho st trn t nht 12 hp Petri. Khi xc nh hm lng ca mt khng sinh dng t nht 3 hp Petri. vo mi hp Petri 21 ml mi trng un chy, i thch ng cng li to thnh lp c dy ng u v mt b mt bng phng lm nn. (Ring vi amphotericin B, nystatin th khng dng lp nn). i vi bleomycin, cycloserin, mithramycin hoc mytomycin dng 10 ml lm lp nn. Lp nui cy dng 4 ml cho mi hp, c v lng u sau khi lp nn ng cng, t ln mt thch trong a 6 ng tr bng thp khng r hoc nhng ng bng cht liu khc nh quy nh phn dng c. Cc ng tr to thnh mt hnh lc gic u cch tm khong 2,8 cm. Chia 12 hp Petri xy dng ng chun thnh 4 nhm, mi nhm 3 hp. Dung dch c nng trung gian c ly lm nng kim tra dng hiu chnh cc gi tr o c mi nhm. y dung dch kim tra vo 3 ng tr, b tr xen k trong mi hp Petri. Cc ng tr cn li c y dung dch c nng ca mt nhm. Sau khi cc hp nhit thch hp 32 - 350C (theo ch dn bng 2) khong 16 - 18 gi s o c 9 gi tr ng knh vng v khun ca nng kim tra theo nhm. Tng s c 4 nhm s o c 36 gi tr ng knh vng v khun nng kim tra. chnh xc ca mi kt qu o l 0,1 mm.

Cch tnh

Gi tr trung bnh cc ng knh vng v khun mi nng l a, b, d, e; gi tr trung bnh cc ng knh vng v khun nng kim tra ca mi nhm 3 hp (9 kt qu) l Ca, Cb, Cd, Ce v gi tr trung bnh cc ng knh vng v khun ca nng kim tra tt c 12 hp (k hiu l C), gm 36 kt qu .

So snh gi tr trung bnh ca cc ng knh vng v khun nng kim tra ca mi nhm vi gi tr trung bnh ca cc ng knh vng v khun nng kim tra ca 12 hp(C). Hiu ca chng (C - Ca; v. v... ) l s hiu chnh dng iu chnh gi tr trung bnh ca cc ng knh vng v khun nng mi nhm. Cng i s gi tr hiu chnh t c vo gi tr trung bnh ca ng knh vng v khun nng ca mi nhm ta c cc gi tr trung bnh ca cc ng knh vng v khun hiu chnh ca mi nng l a, b, d, e.

Th d: Gi tr trung bnh ca ng knh 36 vng v khun nng kim tra tnh c l 19,2 mm; gi tr trung bnh ca ng knh vng v khun nng kim tra ca nhm nng a l Ca = 19,0 mm. S hiu chnh l 19,2 - 19,0 = 0,2 mm. Gi tr trung bnh ca ng knh vng v khun nng ca nhm l 17,9 mm. Vy gi tr trung bnh ca ng knh vng v khun nng ca nhm sau khi hiu chnh l a = 17,9 + 0,2 = 18,1 mm.

Trn hai chu k giy bn logarit v ng chun qua cc im gi tr trung bnh hiu chnh. Trn trc tung (chia theo thang logarit) ly nng khng sinh bng g hoc n v hot lc trong ml. Trn trc honh (chia theo thang s hc) ly gi tr ng knh vng c ch. Nh vy ng chun c v qua im tng ng vi gi tr ng knh vng c ch cao nht v thp nht tnh c bng phng trnh sau:

 

 

L =

3a + 2b + C - e

5

 

H =

3e + 2d + C - a

5

L: ng knh vng v khun tnh cho nng thp nht ca ng chun.

H: ng knh vng v khun tnh cho nng cao nht ca ng chun.

C: ng knh trung bnh ca 36 kt qu ca nng kim tra.

a, b, d, v e: Nhng gi tr ng knh trung bnh hiu chnh cho tng dung dch cn li xp theo th t t nng thp nht ti nng cao nht.

Ba hp Petri dng cho mu th nghim cng c tin hnh ng thi khi xy dng ng chun. Trong ba hp ny nh xen k gia nng trung gian ca dung dch chun vi nng ca mu th pha long vi dung dch m thch hp c nng gi nh tng ng nng trung gian ca chun.

Sau khi o ng knh cc vng v khun ca mu th, tnh trung bnh hiu chnh theo gi tr ng knh nng trung gian s c ng knh ca ch phm th. Trn trc honh ng vi gi tr ng knh va tm c ca mu th, k ng vung gc vi trc honh ct ng ng chun va v c ti mt im v t im ny trn ng chun h vung gc vi trc tung s tm c gi tr nng ng vi ng knh ca mu th.

Hot lc ca ch phm c tnh ra phn trm so vi chun theo cng thc sau:

 

 

R% =

Hot lc tm thy ca ch phm 100

Hot lc tm thy nng trung gian