13.7 Th v khun

Qui nh chung

K thut ny p dng th v khun nhm pht hin s c mt ca vi khun, nm mc v nm men trong cc nguyn liu, ch phm v dng c m theo tiu chun ring cn phi v khun. Th v khun phi c tin hnh trong iu kin v khun nh trong bung thi kh v khun hoc trong bung sch mu th khng b nhim. Trong qu trnh th, mu khng c tip xc vi cc tc nhn c nh hng n vi khun, nm mc, nm men (nh: tia t ngoi, cht st khun, nhit cao ...). Cc dng c, dung mi, mi trng nui cy phi c dit khun trc khi dng.

Phng php th

Nguyn tc

Nu vi khun, nm mc, nm men c cy vo mi trng c cht dinh dng v nc, c gi iu kin nhit thch hp th chng s pht trin. S c mt ca vi sinh vt lm cho mi trng bin i trng thi t trong sang c, hoc c cn lng y mi trng, hoc thay i mu sc mi trng.

Chn mt trong hai phng php th thng dng l phng php mng lc hoc phng php cy trc tip. Khi tin hnh th phi lm sch b ngoi ca ng (hoc chai, l, bnh ...) ng ch phm bng mt cht st khun thch hp. Sau ly mt lng ch phm dng theo qui nh, cy trc tip vo mi trng (nu theo phng php cy trc tip) hoc theo phng php mng lc: em ch phm sau khi c ho long vi dung mi thch hp lc qua mng lc, ri ct cc mng lc thnh ming nh em nhng vo mi trng, mi trng cy ch phm hoc cy mng lc trong thi gian qui nh.

Chun b mi trng v dung mi

Mi trng pht hin vi khun hiu kh v k kh

Mi trng thioglycolat c thch (dng cho th nghim nhng ch phm lng v trong).

Mi trng thioglycolat khng c thch (dng cho th nghim nhng ch phm c hoc c sn st dng cao).

Mi trng Soybean - casein ( pht hin vi khun hiu kh v nm mc)

C hai loi mi trng trn phi c pha ch v kim tra cht lng theo ng qui nh mc "Cch pha ch v kim tra cht lng cc mi trng" trc khi dng.

Dung mi dng ho long

Khi nhng mu th khng dng dung dch lng, cn ho long th nghim. Ch c dng lm dung mi ho long l mt cht lng bt k khng c tnh khng khun v khng tc ng n xp ca mng lc. Thng dng cc dung mi sau y:

Dung mi A: Ho tan 1 g pepton vo nc cho va 1 lt. Lc (hoc ly tm) cho trong. iu chnh pH bng 7,1 0,2. ng vo nhiu bnh, mi bnh khong 100 ml. Hp 1210 C trong 18 - 20 pht.

Dung mi B: Nh dung mi A, nhng thm 1 ml polysorbat 80 cho 1 lt dung mi, dng ho long nhng ch phm th c lecithin.

Dung mi C: isopropyl myristat

Dng pha long nhng ch phm dng du hay dung dch du.

 

Tin hnh th theo phng php mng lc

Dng c

Cc dng c dng trong th nghim phi m bo v khun gm:

- Cc ng nghim hoc cc bnh ng mi trng.

- B lc gm c: Phu lc, gi mng lc, bnh hng, v cc ph kin ghp ni; h thng ht chn khng.

- Mng lc: Thng dng loi c dng knh l lc khong 0,2 - 0,45 mm. Loi mng Cellulose nitrat c dng cho cc ch phm dng nc, dng du v cc dung dch c lng cn thp . Loi mng lc dng cellulose acetat c dng cho cc ch phm dng c nng cn cao. c bit i vi cc ch phm khng sinh cn phi chn loi mng lc thch hp m bo khng cn d lng khng sinh trn mng sau khi lc.

- Ko ct mng lc.

Lng mu th cn dng cho mt ln th nghim

Tu theo tng loi mu th, ly s lng thch hp theo qui nh bng 1.

 

Bng 1.

 

STT

Loi mu th

Lng ti thiu ch phm cn ly

1

 

 

 

 

 

 

 

2

 

 

 

3

 

Dung dch thuc tim c cha trong

mt n v ng gi:

- Di 1 ml

- C t 1 n t hn 4 ml

- C t 4 n t hn 20 ml

- C t 20 n 100 ml

- Ln hn 100 ml

 

Dung dch thuc nh mt v cc ch phm lng khng tim

 

 

Nhng mu th l cht rn, dch treo, nh dch, kem, hoc thuc m.

 

 

Ton b mt n v ng gi

1/2 n v ng gi

2 ml

2 - 10 ml

1/2 n v ng gi

 

5 - 10 ml ca dung dch ho long n 10 ln (tt/tt) bng dung mi thch hp nu (mc c)

 

0,5 - 1 g mu th c ho long thnh dung dch vi t l 1% (kl/tt) bng dung mi thch hp (nu mc dung mi dng ho long)

 

 

K thut th

Dng dung mi A va thm t mng lc. Rt ln mng lc mt lng mu cn th nh qui nh bng 1. Dng my ht chn khng rt ngn thi gian lc. Ra mng lc t nht 3 ln, mi ln dng 100 ml dung mi thch hp, v khun. Ly mng lc ra khi gi trong phu lc. Cy mng lc vo mi trng bng nhng chm vo loi mi trng pht hin nm hoc vi khun (mc chun b mi trng v dung mi ).

Khi mu th c tnh khng khun, ra mng lc t nht ba ln bng cch lc qua mng dung mi tit trng chn dng cho th nghim.Lm th nghim kim tra bit chc chn khng cn tc dng khng khun ca mu th hp th trn mng. Chuyn ton b mng lc vo mi trng nui cy thch hp pht hin vi khun, nm mc.

Nu khng c ch dn trong chuyn lun ring, mi trng thyoglycolat 300C n 350C v mi trng soybean - casein 200C n 250C t nht 14 ngy.

Nu mu th l dng c (b dy truyn, ti lc mu...) th dng 100 ml dung mi B cho chy qua dng c. Hng ly dung mi, rt ln mng lc c thm t bng dung mi A, ri lm tip theo k thut ghi mc k thut th.

Tin hnh th theo phng php cy trc tip

Dng c:

Cc dng c dng cho th nghim u phi m bo v khun mi c s dng vo th nghim. Dng c dng cho phng php cy trc tip gm: Bm tim, pipet, cc ng nghim hoc cc bnh ng mi trng.

Lng ch phm cn dng th nghim

Tu theo tng loi mu th, ly lng ch phm thch hp theo qui nh bng 2.

K thut th

Dng bm tim hoc pipet ly mu th cy vo hai loi mi trng theo s liu bng 2. Nu mu th l cht rn th cy trc tip vo mi trng v ch lc mu th phn tn u trong mi trng. Nu khng c ch dn chuyn lun ring th mi trng pht hin vi khun 300C - 350C ; mi trng pht hin nm mc 200C - 250C, thi gian t nht 14 ngy; thng xuyn theo di cc mi trng cy. Nu ch phm lm c mi trng, c th d dng quan st s mc ca vi khun, sau khi mi trng 3 - 7 ngy nn chuyn mt phn nh mi trng ny sang mt lot mi trng mi cng loi. Tip tc cc ng mi trng cy li t nht 7 ngy.

Trng hp mu th l bng, gc, ch khu phu thut: Nu kch thc v hnh dng cho php, nhng ton b mu th vo 100 ml mi trng tng ng. Khi mu th l dng c (dy truyn, ng lc mu, ...) th dng dung mi A hoc B (mc chun b mi trng v dung mi) cho chy qua thu c t nht 15 ml dch ra, cy vo t nht 100 ml mi loi mi trng nui cy. v c kt qu nh ghi trn.

Bng 2

STT

 

Loi mu th

Lng ti thiu
ch phm cn ly

Th tch ti thiu mi trng cn ly

 

Ghi ch

1

 

 

 

 

 

 

 

Mu th l cht lng cha trong mt n v ng gi loi:

Di 1 ml

T 1 ml n t hn 5 ml

T 5 ml n t hn 20 ml

T 20 ml n t hn 50 ml

T 50 ml n 100 ml

 

 

 

Ton b n v ng gi

1/2 n v ng gi

2 ml

5 ml

10 ml

 

 

 

15 ml

15 ml

20 ml

40 ml

80 ml

 

Nu mu th l du hay dung dch du phi thm vo mi trng 1% polyethoxy- ethanol hoc 1% polysorbat nh ho.

 

2

 

Mu th l cht rn, thuc m hay kem, cha trong mt n v ng gi loi:

Di 50 mg

 

T 50 mg n 200 mg

 

Ln hn 200 mg

 

 

 

 

Ton b 1 n v ng gi

 

1/2 khi lng ca
1 n v ng gi

100 mg

 

 

 

 

40 ml

 

80 ml

 

80 ml

 

Nu mu th l thuc m hay kem th cn ho long vi dung mi B theo t l 1/10 (kt/tt) trc khi cy vo mi trng.

 

 

 

Theo di v nh gi kt qu

Trong sut thi gian , hng ngy phi quan st cc mi trng cy mng lc hoc mu th . Nu khng thy c vi khun, nm mc mc trong sut thi gian qui nh, mu th c coi l t tiu chun v khun.

Nu c t 1 tr ln trong s cc mi trng nui cy, c vi khun hoc nm mc mc, cn phi:

R sot li qui trnh th.

Th li cc mu th nghi ng.

Gi li cc mi trng c vi khun, nm mc mc.

Tin hnh phn lp, so snh vi khun hoc nm trn mi trng, cy li vi vi khun hoc nm mc mi trng c.

Nu trn tiu bn phn lp, vi khun (hoc nm mc) ging vi tiu bn lm ln u, mu th c coi l khng t tiu chun v khun.

Nu trn tiu bn phn lp, vi khun (hoc nm mc) khc bit so vi ln th nghim u tin, cn thc hin php th lp li vi s lng mu gp i. Nu khng pht hin thy vi khun (hoc nm mc), mu th c coi l t tiu chun v khun. Nu vn thy vi khun (hoc nm) mc ln lp li ny, mu th c coi l khng t tiu chun v khun.

Cch pha ch v kim tra cht lng cc mi trng

Mi trng pht hin cc vi khun hiu kh v k kh

Mi trng thioglycolat:

Dng cho th nghim nhng ch phm lng v trong

L - Cystin

Natri clorid

Dextrose (C6H12O6.H2O)

Thch bt (c m nh hn 15%)

Cao men bia (c kh nng tan trong nc)

Casein pancreatic

Natri thioglycolat

(hoc acid thioglycolic 0,3 g )

Resazurin (dung dch 0,1% mi pha)

Nc ct

pH sau khi tit khun: 7,1 0,2

0,50 g

2,50 g

5,50 g

0,75 g

5,00 g

15,00 g

0,50 g

 

1,0 ml

1000 ml

Trn tt c cc thnh phn theo th t ghi trn (tr resazurin v thioglycolat) trong ci nghin, thm vo mt t nc nng, trn k, chuyn sang dng c thch hp. Thm s nc cn li, un hn hp cch thu si n khi to thnh dung dch trong. Thm natri thioglycolat, dng dung dch natri hydroxyd 1 N iu chnh sao cho mi trng sau khi tit khun c pH7,1 0,2. un nng li dung dch (trnh un si). Lc (nu cn) qua giy lc thm t, ri thm dung dch resazurin trn u. ng vo cc ng nghim (hoc bnh) thch hp, hp v khun 1210C trong 18 - 20 pht. Ly ra lm ngui nhanh ti 250C, tip tc bo qun nhit 20 - 300C, trnh nh sng. Nu 1/3 th tch pha trn ca ng (hoc bnh) mi trng c mu hng, mi trng khng thch hp th nghim. C th phc hi li mi trng bng cch un cch thu cho mt mu ri lm lnh t ngt. Nhng ch s dng mi trng phc hi ny mt ln, khng dng mi trng phc hi ln 2. Cc mi trng thioglycolat lng ch dng trong khong 3 tun.

Mi trng thioglycolat khng c thch

Dng cho th nghim nhng ch phm c hoc c sn st dng cao.

L. Cystin

Natri clorid

Dextrose (C6H12O6.H2O)

Cao men bia (c kh nng tan trong nc)

Casein pancreatic

Natri thioglycolat

(hoc acid thioglycolic 0,3 g )

Resazurin (dung dch 1% mi pha)

Nc ct

pH sau khi tit khun: 7,1 0,2

0,50 g

2,50 g

5,50 g

5,00 g

15,0 g

0,50 g

 

10 ml

1000 ml

Cho tt c cc thnh phn trn vo mt dng c thch hp, un cch thu, khuy u n khi thnh dung dch, dng dung dch natri hydroxyd 1N iu chnh cho pH sau khi tit khun t 7,1 0,2. C th lc (nu cn). Phn chia vo cc ng nghim (hoc bnh) thch hp. Hp tit khun 1210C trong khong 18 - 20 pht. Sau khi tit khun em lm ngui nhanh ti 250C, mi trng khng c un nng tr li.

Mi trng pht hin vi khun hiu kh v nm

Mi trng Soybean - casein

Casein pancreatic

Bt papaic soybean

Natri clorid

Dikali hydrophosphat (K2HPO4)

Dextrose (C6H12O6.H2O)

Nc ct

pH sau khi tit khun: 7,3 0,2

17,0 g

3,0 g

5,0 g

2,5 g

2,5 g

1000 ml

 

Ho tan tt c cc cht rn trong nc, un nng nh cho tan hon ton. ngui nhit phng. Dng dung dch natri hydroxyd 0,1N iu chnh (nu cn) cho pH sau khi tit khun khong 7,1 n 7,5. Lc (nu cn) cho mi trng trong. Phn chia vo nhng dng c thch hp, hp tit khun 1210C trong khong 20 pht.

Kim tra cht lng mi trng

a) v khun:

Ly ngu nhin mt vi ng (hoc bnh) mi trng mi sn xut, em nhit 30 - 350C trong 4 ngy i vi nhng loi mi trng dng nui cy vi khun hiu kh v k kh; nhit 20 - 250C trong t nht 7 ngy i vi nhng loi mi trng dng nui cy vi khun, nm mc. Cc loi mi trng phi khng c c vi khun, nm mc mc.

b) Kh nng dinh dng:

Cy vo mi trng dng th nghim khong 100 t bo sng ca nhng loi vi khun sau:

Loi hiu kh dng Staphylococcus aureus ATCC 6538.

Loi vi khun hiu kh c nha bo dng Bacillus subtilis ATCC 6633.

Loi vi khun k kh dng Clostridium sporogenes ATCC 9404.

Loi nm dng Candida albicans ATCC 10231.

Aspergillus niger ATCC 16404

Mi loi chng ch th c cy vo loi mi trng tng ng, ri mang nhit thch hp cho tng loi t nht 3 ngy i vi vi khun v t nht 5 ngy i vi nm mc. Trn mi loi mi trng, sau thi gian u phi thy vi khun mc tt.

 

c) Kim tra tc dng c ch mu mang th:

Mt s ch phm do yu cu ca sn xut hoc nhng ch phm mi ngi ta cho thm cht bo qun. Loi ch phm ny c nh hng n s pht trin ca vi khun, nm mc, nn cn phi kim tra tc dng c ch ca chng i vi vi khun nm mc.

i vi vi khun k kh

Dng 4 ng mi trng pht hin vi khun k kh. Hai ng dng lm chng ch cy vo mi ng chng ny 100 nha bo vi khun k kh Clostridium sporogenes ATCC 9404 (khong 0,1 ml nh dch nha bo pha long thch hp). Hai ng cn li cy vo mi ng mt lng bng nhau ch phm cn kim tra, ri cng cy vo mi ng 100 nha bo Clostridium sporogenes ATCC 9404. em tt c cc ng 30 - 350C t nht 7 ngy.

i vi vi khun hiu kh

Dng 4 ng mi trng pht hin vi khun hiu kh. Hai ng dng lm chng ch cy vo mi ng chng ny 100 t bo vi khun hiu kh Staphylococcus aureus ATCC 6538 (khong 0,1 ml nh dch t bo pha long thch hp). Hai ng cn li cy vo mi ng mt lng bng nhau ch phm cn kim tra, v cng cy vo mi ng 100 t bo Staphylococcus aureus ATCC 6538. em tt c cc ng 30 - 350C t nht 7 ngy.

i vi nm mc

Dng 4 ng mi trng pht hin nm, mc. Hai ng dng lm chng ch cy vo mi ng chng ny 100 t bo nm Candida albicans ATCC 10231. Hai ng dng kim tra, cy vo mi ng 100 t bo nm Candida albicans ATCC 10231 v mt lng ch phm bng nhau. em tt c cc ng 20 - 250C t nht 7 ngy.

nh gi kt qu:

Nu trong thi gian , vi khun, nm mc mc tt, (d dng v phong ph), i vi tng loi, mc ging nhau gia ng kim tra v ng chng: Mu th khng c tc dng c ch.

Nu trn cc ng mi trng c cy mu th, vi khun, nm, mc mc yu, chm pht trin hoc khng pht trin so vi ng chng vn mc tt: Mu th c tc dng c ch. Phi loi b tc dng c ch.

Tc dng c ch ca mu th c th loi b c mt cch c kt qu bng cch cy truyn nhc li trn mt lot mi trng mi hoc lc qua mng lc vi khun.