Phng php khuch tn qua ging thch (well diffusion agar)

Nguyn tc: Phng php ny da trn kh nng i khng ca bacteriocin

vi vi khun ch th ca vi khun acid lactic trn mi trng nui cy. Bacteriocin c kh nng khuch tn trong mi trng agar v tc ng ln vi khun ch th. Vi khun acid lactic sinh bacteriocin khng c vi khun ch th s xut hin vng khng khun xung quanh ging thch. Quy trnh thc hin: - Chun b mi trng cha dng ch th: + Dng ch th c nui cy trn a 24 gi, khun lc c cho vo nc ct v trng v tin hnh pha long dung dch vi khun xc nh mt s vi khun. + Chun b dch huyn ph ca dng vi khun ch th vi mt s 10 5 t bo/ml bng phng php m hng cu (Ph lc 2). + Tri 0,1ml dung dch cha B. subtilis vo mi trng c - peptone agar 2% vo a Petri (ng knh 90mm). + Sau khi mi trng ng li, to nhng ging nh c ng knh 9mm vi thanh kim loi v trng. - Chun b dch bacteriocin th: + Vi khun acid lactic c pht trin trong 2ml MRS broth di iu kin ym kh trnh hnh thnh H2O2, ti giai on pha tnh (khong 48 gi). + Dung dch c ly tm 8.000 vng/pht trong 15 pht 4oC. + Loi b phn cn cha xc t bo vi khun, ly phn nc trong ca dung dch sau ly tm, dng giy o pH iu chnh dung dch sau ly tm pH 6,5 bng NaOH 0,1N v tr lnh 4oC. Thu c dung dch bacteriocin th. - Ly 0,1ml dung dch bacteriocin th nh vo mi ging ca a thch cha dng ch th. - Tin hnh mu 4oC trong 15 pht cho dung dch trong ging khuch tn. - Sau , a c 37oC cho vi khun ch th pht trin.

i chng: ging cha 0,1ml mi trng MRS lng. Hot tnh khng khun ca nhng dng phn lp c tnh bng vng v khun quanh ming ging trn a. Thc hin o ng knh vng khng khun hnh thnh trn a v chn 5 dng vi khun Streptococcus spp. c hot tnh khng khun cao nht s dng cho cc th nghim tip theo. Xc nh v chn lc 5 dng Streptococcus spp. c hot tnh bacteriocin cao nht. - Hot tnh bacteriocin ca nhng dng phn lp c tnh bng vng khng khun quanh ming ging trn a. - Tnh khng khun c biu hin khi vng khng khun rng hn 2mm. + ng knh vng khng khun < 5mm: tnh khng yu. + ng knh vng khng khun t 5 n 10mm: tnh khng trung bnh. + ng knh vng khng khun > 10mm: tnh khng mnh. - So snh kh nng khng khun ca cc dng v chn lc ra 5 dng vi khun Streptococcus spp. c hot tnh bacteriocin cao bng phn mm thng k Statgraphics plus 4.0.