TIU CHUN QUC GIA

TCVN 8129 : 2009

ISO 18593 : 2004VI SINH VT TRONG THC PHM V THC N CHN NUI - PHNG PHP LY MU B MT S DNG A TIP XC V LAU B MT

Microbiology of food and animal feeding stuffs - Horizontal methods for sampling techniques from surfaces using contact plates and swabs

Li ni u

TCVN 8129 : 2009 hon ton tng ng vi ISO 18593 : 2004;

TCVN 8129 : 2009 do Ban k thut tiu chun quc gia TCVN/TC/F13 Phng php phn tch v ly mu bin son, Tng cc Tiu chun o lng Cht lng ngh, B Khoa hc v Cng ngh cng b.

VI SINH VT TRONG THC PHM V THC N CHN NUI - PHNG PHP LY MU B MT S DNG A TIP XC V LAU B MT

Microbiology of food and animal feeding stuffs - Horizontal methods for sampling techniques from surfaces using contact plates and swabs

1. Phm vi p dng

Tiu chun ny quy nh cc k thut i vi cc phng php ly mu b mt s dng a hoc gc tip xc trn b mt trong mi trng cng ngh ch bin thc phm (v cc nh my ch bin thc phm), pht hin hoc nh lng vi sinh vt tn ti.

CH THCH: Thut ng "mi trng" ngha l bt k mt vt no tip xc vi sn phm thc phm hoc gn nh cho thy nhim bn hoc ngun ti nhim, v d: vt liu, nh xng, ngi vn hnh.

2. Ti liu vin dn

Cc ti liu vin dn sau rt cn thit cho vic p dng tiu chun ny. i vi cc ti liu vin dn ghi nm cng b th p dng phin bn c nu. i vi cc ti liu vin dn khng ghi nm cng b th p dng phin bn mi nht, bao gm c cc sa i, b sung (nu c).

TCVN 6404 (ISO 7218), Vi sinh vt trong thc phm v thc n chn nui - Nguyn tc chung v kim tra vi sinh vt.

TCVN 6507-1 (ISO 6887-1), Vi sinh vt trong thc phm v thc n chn nui - Chun b mu th, dung dch huyn ph ban u v cc dung dch pha long thp phn kim tra vi sinh vt - Phn 1: Cc nguyn tc chung v chun b huyn ph ban u v cc dung dch pha long thp phn.

3. Nguyn tc

3.1. V cc phng php ny khng xc nh c tin cy hoc ti lp, nn cc kt qu ch c s dng trong "phn tch xu hng".

3.2. a tip xc (hoc phin knh nhng) lm y bng mi trng thch thch hp c n vo b mt cn th nghim. Sau khi , c tnh s nhim bn b mt thu c bng cch m s lng khun lc pht trin.

3.3. S dng phng php lau b mt, th nh du vng b mt cn kim tra (v d: dng khun mu) ri dng tm bng lau vng nh du. Phn tm bng c b ri cho vo ng nghim hoc chai c cha dung dch lng v trng hoc cht lng trung tnh v lc bng tay trn.

Nu b mt c lau bng khn hoc bt bin v trng (t), th dng c ly mu c bo qun trong mt th tch xc nh ca dung dch lng (v d 100 ml cho 100 cm2).

Trong phng th nghim, s dng huyn ph ban u v cc dung dch pha long thp phn tip theo, nu cn, xc nh s lng vi sinh vt bng cc qui trnh nu trong cc phng php nh lng (nhm) cc vi sinh vt cn pht hin.

CH THCH: Thi gian v nhit ph thuc vo kiu vi sinh vt cn pht hin.

3.4. i vi mi trng chn lc, c th tin hnh cc php th khng nh thch hp. S lng n v hnh thnh khun lc ca cc vi sinh vt c th trn centimet vung hoc mi ming gc tnh c t s lng khun lc ( khng nh).

3.5. Sau khi ly mu, b mt c lm sch v ty trng, nu cn, sau khi ly mu khng li vt dinh dng trn b mt c ly mu.

4. Mi trng nui cy v cht pha long

CH THCH: c thm thng tin, xem cc tiu chun c lin quan v cc vi sinh vt ch cn pht hin cn nh lng.4.1. Cht lng trung tnh

Nhn chung, thnh phn c bn i vi cht lng trung tnh l nc pepton m, hoc mui pepton hoc bt k cht pha long thch hp no khc (nh dung dch Ringer nng mt phn t, cht m phosphat pH 7,5, dung dch pepton 1 g/l).

Trng hp khi c cc d lng cht kh trng, th cn b sung cc cht trung ha thch hp vo dch pha long v vo mi trng c s dng trn cc a tip xc trnh mi nh hng c ch cht ty trng n s pht trin ca vi sinh vt.

5. Thit b v dng c thy tinh

V cc yu cu chung, xem TCVN 6404 (ISO 7218).

C th dng dng c s dng mt ln thay th cho dng c thy tinh s dng nhiu ln, nu c cc qui nh thch hp.

S dng cc thit b, dng c ca phng th nghim vi sinh thng thng v c th nh sau:

5.1. a tip xc, bng cht do c ng knh 65mm, c lm y mt lng xc nh mi trng thch (chn theo vi sinh vt ch), c bit l dng ly mu b mt.

Cc a c ng knh hoc din tch khc nhau, ty theo kiu b mt cn ly mu. Thch phi to thnh mt khum trn a.

CH THCH: Cng c th s dng bt k dng c no khc (mi trng dinh dng trong vt cha cng hoc do) m c th tip xc vi b mt cn ly mu, nh phin knh nhng (5.2).5.2. Phin knh nhng, phin knh tng hp (7cm2 n 10cm2), mt hoc hai mt c ph mt lp mi trng c (c chn theo cc vi sinh vt ch).

CH THCH: Sn c cc mi trng pht trin khc nhau theo cc vi sinh vt cn tm.

5.3. Tm bng c th b gy c hoc gc bng vt liu tng hp (nh algimat hoc rayon) ng trong ng hoc trong bao.Gc c kh trng v c bao gi ring, bao gi khng c cha cc cht c ch.

CH THCH: i vi mt s kiu b mt, bng cn st li c th lm nhim bn cc phn bn trong ca b mt ny sau khi ly mu.

5.4. Vi, m t, vt liu an dt, khng cha cc cht khng sinh, c ng gi ring r trong cc bao gi bng cht do v trng, dng ly mu cc b mt ln ( 100 cm2).

5.5. Bt bin, m t, phng, vung, khng cha cc cht khng sinh, c ng gi ring r trong cc bao gi bng cht do v trng, dng ly mu cc b mt ln ( 100 cm2).

5.6. Vt cha, nh chai, ng nghim hoc bnh cu, thch hp kh trng v bo qun mi trng nui cy.

5.7. Hp lm mt, cch nhit, c th duy tr mu nhit thp trong qu trnh vn chuyn n phng th nghim.

5.8. Pipep chia , c ming rng v dung tch danh nh 1 ml, c chia cc vch 0,1 ml hoc pipet t ng c th phn phi 100(l v 1000(l.5.9. B trn, dng trn cc cht lng trong cc ng nui cy.

5.10. B ng ha kiu nhu ng hoc b ng ha c lc theo phng nm ngang, c cc ti cht do v trng chun b huyn ph ban u bng chuyn ng xoay (b trn nhu ng) hoc chuyn ng rung (b ng ha c lc theo phng nm ngang).

5.11. a Petri, bng cht do, c ng knh 65mm.

5.12. My o pH, c th c chnh xc n 0,01 n v pH 250C 10C, c th o chnh xc n 0,1 n v pH.

5.13. Khun mu, bng vt liu khng n mn (v d: khung bng thp khng g ng khung vi din tch t 20 n 100 cm2), d dng lm sch v c th kh trng c.

6. K thut ly mu

6.1. Yu cu chung

iu quan trng l phng th nghim nhn c ng mu i din ca b mt th nghim v khng b bin i trong sut qu trnh vn chuyn v bo qun, hoc do d lng cht kh trng.

Cc cht ty trng thng c cho thi gian tip xc kh trng t 5 min n 15 min. Thi gian i theo qui nh ca kh trng trc khi kim tra b mt bng a tip xc hoc gc nh gi hiu nng lm sch v chng trnh kh trng (hoc theo quy nh kh trng).

Khng th qui nh mt cht trung ha dng cho tt c mi tnh hung. Nhn chung, sorbitan monooleat (30g/l) v lexitin (3g/l) l c th c dng trung ha cc lng d ca cc cht ty trng hp th (v d: cc hp cht amoni bc bn, amphotericid). Natri thiosulfat (5g/l) l cht trung ha tt i vi cc sn phm cha halogen. Trong trng hp cc cht ty trng cha cc peroxit, th c th dng cc catalaza hoc peroxidaza lm cht trung ha. Mt n v ca cc enzym ny xc tc phn hy 1(mol hydro peroxit trong mt pht 250C v pH 7,0 0,2. Mt s cc cht trung ha cc cht ty trng c khuyn co trong EN 1276 [1], EN 1650 [2], EN 13697 [3] v EN 13704 [4].Cc thnh phn ca cht trung ha c th c s dng trong hu ht cc tnh hung c nu trong Bng 1. Thnh phn trong dung dch c pepton (1 g/l) v natri clorua (8,5g/l), c phn phi vo cc ng hoc chai v c kh trng 15min 1210C.

Bng 1 - Cht trung ha c th s dng trong hu ht cc tnh hung

Thnh phnNng

Sorbitan monooleat (Polysorbat 80)30 g/l

Lexitin3 g/l

Natri thiosulfat5 g/l

L-Histidin1 g/l

Saponin30 g/l

6.2. Phng php a tip xc

Sau khi ly ra khi hp vn chuyn, n b mt thch ca a tip xc hoc ca phin knh nhng tht cht v khng b dch chuyn sang cc bn ca b mt th nghim. Theo ti liu cho bit rng cc kt qu ti u i vi cc a tip xc thu c vi thi gian tip xc 10 s v lc nn c khi lng 500 g. y cc a tip xc hoc phin knh ngay sau khi cy v t tr li vo hp vn chuyn.

6.3. Phng php dng gc hoc vi/bt bin

6.3.1. Phng php dng gc

Ly gc ra khi bao gi v trng v lm m u mt bng cch nhng vo ng nghim c cha dch pha long. n u mt ca ming gc vo thnh ng b phn nc d. t u mt ming gc ln b mt cn kim tra v vch trn mt vng rng 20 cm2 n 100 cm2, trong khi quay ming gc gia ngn tay tr v ngn tay ci theo hai hng t cc gc phi sang tri.B hoc ct que cm mt cch v trng v t ming gc vo ng nghim ng cht lng.

6.3.2. Phng php dng bt bin/vi

M ti hoc hp bng cht do ng ming vi hoc ming bt bin.

Dng kp v trng hoc mang gng tay v trng ly ming vi hoc bt bin ra. Lm m bt bin hoc ming vi bng mt lng va ca dch pha long. i vi cc b mt m th khng cn.

t li ming vi hoc ming bt bin vo ti ng bng cht do v y tht kn.

Ly mu b mt chn theo hai hng vung gc, thay b mt ca ming vi hoc bt bin. t ming vi hoc bt bin ny vo hp ng v trng, b sung dch pha long v y np. Thm mt lng va xc nh ca dch pha long sao cho ming vi hoc bt bin vn m cho n khi phn tch.

Cch khc, m ti cht do cha ming vi hoc bt bin. kp cht ming bt bin cui ti, ko ti ngc theo tay. Dng ming bt bin ly mu nh m t trn v chuyn ming vi hoc bt bin vo ti cht do v trng. y tht kn np.

7. Vn chuyn

Vn chuyn mu ly c bng phng php dng gc, tt nht l trong khong 4 h trong hp lnh 10C n 40C. Tin hnh kim tra trong phng th nghim cng sm cng tt, khng qu 24 h, nh m t trong iu 8.

Chuyn a tip xc v/hoc phin knh nhng, tt nht trong khong 4h trnh nhim bn.

8. Cch tin hnh

8.1. Phng php dng a tip xc

cc a tip xc hoc phin knh nhng ty theo kiu loi vi sinh vt cn m (xem Ch thch ca iu 4).

Khng s dng phng php dng a tip xc pht hin c th cc vi sinh vt gy bnh.

8.2. Phng php dng gc

Trn k lng cha trong cc ng c cha gc, dng b trn (5.9) trong 30s, chnh tc sao cho thnh ng c lm t n khong 2cm n 3cm tnh t nh ng.Thm mt lng dch pha long hoc cht trung ha (4.1), ty thuc vo din tch cn kim tra (v d: 100ml cho 100cm2), vo ti cht do ng ming vi hoc bt bin. Sau x l lng cha trong ti trong b trn nhu ng (5.10) trong 1 min, hoc bng b trn lc ngang (5.10) trong 30s, thu c huyn ph ban u.

Nu d kin c lng ln vi sinh vt th chun b cc dung dch pha long thp phn tip theo trong dch pha long nc pepton thu c s khun lc c th m c [xem TCVN 6507-1 (ISO 6887-1)].

Ty theo phng php nh lng c s dng (xem cc tiu chun tng ng), cy huyn ph ban u vo cc a kp ng mi trng, s dng 1 ml dch cy trn mi a rt v 0,1 ml dch cy cho k thut dn a. X l cc dung dch pha long tip theo theo cng cch. Lt ngc cc a v trong khong thi gian v nhit thch hp cho tng loi mi trng.

thay th cho phng php a ni trn, c th s dng phng php a nh git. Bt u vi dung dch pha long nht, dng pipet v trng chuyn cc lng 0,05 ml cc huyn ph ban u (gc, vi hoc bt bin) v cc dung dch pha long tip theo sang cc phn thch hp ca mi trng cy c nh du trn y a thch (cc a kp, s dng cng dung dch pha long i vi cc a thch khc nhau). Mi a thch lm kh trc c th c dng nh git cho khng nhiu hn su dung dch pha long khc nhau. S dng k thut pipet ht ngc bng cch n hn pittong xung khi ht lng 0,05 ml v cy vo cc a bng cch n pittong ch vo im dng th nht. Gi cc a thch t th nm ngang (np hng ln trn) cho n b mt thch kh.Nu s dng phng php dng gc kim tra s c mt ca cc vi sinh vt c th (v d: Listeria monocytogens hoc Salmonella spp.), th din tch cn kim tra t nht phi l 100 cm2 v tt nht l 1000cm2. Chuyn gc, vi hoc bt bin vo canh thang tng sinh thch hp v trn k.

8.3. Pht hin v m khun lc

8.3.1. Phng php a tip xc

m s lng cc khun lc in hnh trn cc a tip xc hoc phin knh nhng trc tip sau thi gian qui nh v khng nh theo cc vi sinh vt cn tm, nu cn.

8.3.2. Phng php dng gc (gm vi hoc bt bin)

m s lng khun lc in hnh trn mi a v khng nh theo cc vi sinh vt cn tm, nu cn.

8.3.3. Phng php dng a nh git

m s lng cc khun lc trn mi a ti cc dung dch pha long cho 5 n 50 khun lc.

8.3.4. Qui trnh tng sinh

Sau khi tng sinh s b, tin hnh theo hng dn ca nh sn xut i vi cc vi sinh vt cn tm.

9. Tnh v biu th kt qu

CNH BO - V phng php dng a tip xc v phng php dng vi/gc khng cho cc kt qu ging nhau, do cn phi nu r trong kt qu phng php s dng.

9.1. Phng php dng a tip xc

Chia s lng cc khun lc c trng cho din tch b mt ca a. Ghi li s m l s lng n v hnh thnh khun lc (CFU) trn centimet vung b mt.9.2. Phng php dng gc (k c vi v bt bin)

9.2.1. Tnh s n v hnh thnh khun lc (CFU) trn mililit huyn ph ban u, theo tiu chun c lin quan.

9.2.2. Tnh s n v hnh thnh khun lc (CFU) trn centimet vung b mt cn kim tra, Ns, theo cng thc sau y:

Ns =

trong :

N l s lng CFU trong 1 ml dung dch pha long (hoc cht lng trung ha);

F l dung dch pha long (hoc cht lng trung ha) trong ng hoc trong ti ng ha, tnh bng mililit (ml);

A l b mt cn kim tra, tnh bng centimet vung (cm2);

D l s nghch o ca pha long dng.

9.2.3. i vi phng php a nh git, tnh s lng N, CFU trn mililit dung dch huyn ph, theo cng thc sau:

trong

l s lng khun lc m c trn tt c cc git c gi li t hai dung dch pha long lin tip, trong t nht mt git cha 5 khun lc;

V l th tch cht cy dng trong mi git (trong trng hp ny l 50 (l)

n1 l s git c gi li pha long th nht;

n2 l s git c gi li pha long th hai;

d l h s pha long ng vi pha long th nht c gi li.

thu c s m trn centimet vung b mt, s dng cng thc:

Trong

F l dung dch pha long (hoc cht lng trung ha) trong ng hoc trong ti ng ha, tnh bng mililit (ml).A l din tch lau, tnh bng centimet vung (cm2)

9.2.4. Nu khng xc nh c din tch lau, th tnh s lng CFU trn mi ming gc, Nsw, theo cng thc sau:

Khi s dng cc phng php bn nh lng, th cc kt qu thu c theo CFU trn centimet vung b mt c th c gi l im v sinh. V cc im ny c th thay i nhiu ty thuc vo cc b mt cn kim tra, do nn cn c cc bn quan tm xc nh v i n thng nht.

9.2.5. Trong trng hp cc quy trnh tng sinh, th bo co vi sinh vt ch l pht hin c hoc khng pht hin c trong din tch lau, hoc trn mi ming gc nu khng bit din tch.TH MC TI LIU THAM KHO[1] EN 1276:1997, Chemical disinfectants and antiseptics - Quantitative suspension tests for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the food, industrial domestic and institutional areas - Test methods and requirements (phase 2, step 1)

[2] EN 1650:1997, Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal activity of chemical disinfectants and antiseptics in food, industrial, domestic, and industrial areas - Test method and requirements (phase 2 step 1)

[3] EN 13697:2001, Chemical disinfectants and antiseptics - Quantitative non-porous surface test for the evaluation of bactericidal and/or fungicidal activity of chemical disinfectants used in food, industrial, domestic and industrial areas - Test method and requirements without mechanical action (phase 2/step 2)

[4] EN 13704:2002, Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of spohcidal activity of chemical disinfectants used in food, industrial, domestic and industrial areas - Test and requirements (phase 2. step 1)

[5] TCVN 6263 (ISO 8261) Sa v sn phm sa - Hng dn chung v chun b mu th, huyn ph ban u v dung dch pha long thp phn kim tra vi sinh vt[6] TCVN 8128-1: 2009 (ISO/TS 11133-1:2009), Vi sinh vt trong thc phm v thc n chn nui - Hng dn chun b v sn xut mi trng nui cy - Phn 1: Hng dn chung v m bo cht lng i vi vic chun b mi trng nui cy trong phng th nghim.

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