CCH PHN LP V XC NH NM MEN RU TRONG DY THC HNH SINH HC PGS.TS.

Bin Vn Minh Trng HSP - i hc Hu Nm men (Yeasts), l nhng nm c cu to n bo, nhn c mng nhn, sinh sn ch yu theo hnh thc ny chi. T bo nm men c nhiu hnh dng khc nhau, c th hnh cu, hnh bu dc, hnh elip, hnh ng... Kch thc t bo thay i t 1,5-12m (micromet). Nu t bo dng si th chiu di c th ti 20m hay hn na, thng l si nm gi gm nhiu t bo dnh li vi nhau theo chiu di hoc dng cnh cy mt cch lng lo. Nm men c mt khp mi ni, chng phn b nhiu trong t vn, trong bnh men ru, trn b mt v qu cng nh cc dch qu p da, l, to, nho, m... Trong bi vit ny chng ti mun gii thiu v cch phn lp v xc nh nm men ru trong dy thc hnh sinh hc. 1. Chun b a. Dng c, thit b Bung cy v trng; cn k thut 1 ci; gi ng nghim 2 ci, mi gi c 10 ng nghim; a thy tinh; que trang 1 ci; que cy 1 ci; n cn 1 ci; bp in 1 ci; soong 2 lt 1 ci; besher loi 250 ml 1 ci; pipette loi 1ml 2 ci; ng nghim 50 ng; chai tam gic loi 250 ml 3 chai; ko ct giy 1ci; a Petri 25 hp; khn lau 2 ci; bt la 1 ci; ng nh git 1 ci. b. Nguyn liu v mu vt - Bng khng thm nc 50g; nc ct 2 lt; cn t 100 ml; bng thm nc 20g; giy thm 1 hp; giy gi a Petri; dy buc bng nilon - t vn, tri cy chn, mi loi 30-50g. Cc ng ging nm men chun. * Mi trng Hansen c thnh phn: ng glucose 50g; KH2PO4 3g; MgSO4.7H2O 3g; peptone 10g; thch (agar-agar) 20g; pH=6,0; kh trng 0,8 -1 atm/30 pht. * Hoc mi trng mch nha c thnh phn: mch nha 150g, thch 20g; pH=7,0; kh trng 1 atm/30 pht. 2. Cc bc tin hnh a. Chun b mi trng Hansen thch a, thch nghing. Cc a mi trng Hansen hoc mch nha c cha 0,03g/l streptomycin hay rosebengal (1/3000) 100ml/l. b. Thu mu, x l mu v pha long - Thu mu: bnh men ru, hoc t vn, hoc qu chn nho, to, m, mn... - Cn 1-2g mu trn thm 1-2g ng saccharose thm 2ml nc ct, trn u 3-5 ngy. - Ly 1ml dch huyn ph trn pha long t 10-1 n 10-10 ln. c. Phn lp nm men ru - Cy dch huyn ph ln mi trng c trong hp Petri + Dng pipet v trng nh 1ml dch huyn ph pha long nng nht nh ln b mt ca mi trng trong hp Petri.
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+ Dng que trang dn u git dch huyn ph trn ton b b mt mi trng hp thch. + y hp Petri, gi li v t vo t m nhit 28 - 300C trong 48 - 72h. - Trn dch mu pha long trong cc mi trng thch nng chy Dng pipet v trng nh 1ml dch huyn ph pha long nng nht nh vo mi trng thch nng chy v ngui n 45 - 500C trong bnh nn, lc u ri ra cc hp Petri mi hp c 15ml. Gi cc hp Petri li v t vo t m nhit 28 300C trong 48 - 72h. - Nui nm men mc thnh khun lc. p ngc cc hp Petri ri t vo t m nhit 28 - 300C trong 48 - 72h. Sau thi gian trn b mt thch a s c cc khun lc nm men mc tch bit mt thng quan st c. - Thu nhn chng nm men ru thun khit + Cy nm men t khun lc mc tch ri vo ng mi trng thch nghing. + Nui nm men nhit v thi gian thch hp (nh trn). + Loi b cc ng b nhim, chn ra cc ng c cc chng thun khit. - Phng php kim tra thun khit ca mt chng nm men mi phn lp C nhiu cch kim tra: 1) Kim tra thun khit ca cc khun lc: mu sc, b mt, hnh dng c trng ging nh khun lc chng mu. 2) Quan st nm men: lm tiu bn t dch ln men, nhum n bng xanhmethylen, ri quan st vi vt knh x100 (vt knh dng du). T bo nm men S.cerevisiae c hnh cu hay van, c mt s ang giai on ny chi.

(a)

(b)

(c)

Hnh 1: (a) Khun lc ; (b) Hnh dng t bo nm men S.cerevisiae di knh hin vi quang hc; (c) S cu to t bo nm men (1.Thnh t bo; 2.Th bin; 3.Mng sinh cht; 4.Nhn t bo; 5.Hch nhn; 6.Mng nhn; 7.Khng bo; 8. Mng li ni cht; 9.Ht d tr; 10.Ti th; 11.Cht t bo).

3. Lm mt s th nghim xc nh nm men ru a. Chun b dch ln men ru Rt 150ml dung dch 10% glucose vo bnh tam gic dung tch 250 ml v trng ri cy khong 1,5-5ml dch nm men (ging hot ha 24 gi). t vo t m 300C trong 1- 2 gi.
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1) o th tch kh CO2 nh gi kh nng ln men ru c th xc nh cc sn phm hnh thnh nh CO2, acetaldehyte hoc sn phm cui cng l ethanol. Trong cch o th tch CO2 c hnh thnh l n gin hn c. Gi kh CO2 trong ng phao hoc bnh Smith (xem hnh di). Mi trng Hansen dch th v trng c phn vo ng nghim c ng phao 1/2 chiu cao hay bnh Smith khong 1/2 bu trn bng pipet v trng. Cy ging nm men khong 1ml hot ha 24 gi, nt cht ming bnh ri t vo t c nhit thch hp. Kh CO2 sinh ra s y ct mi trng tt xung t t. Tnh th tch kh c hnh thnh trong mt n v thi gian.

(a)

(b)

(c)

(d)

(e)

Hnh 2: (a, b) Cch gi kh CO2 trong ng phao v (c,d,e) trong bnh ln men Smith 2) Phn ng vi Ba(OH)2: (Xc nh s c mt ca CO2 trong dch ln men) - Cho vo ng nghim 5ml dch ln men, thm 1ml dung dch Ba(OH)2 10%. Dng kp g cp ng nghim v un nh trn ngn la n cn. lng. Nu c mt ca CO2 s to thnh kt ta mu trng do s to thnh BaCO3 theo phn ng : CO2 + Ba(OH)2 BaCO3 + H2O 3) Phn ng vi kali bicromat : (Xc nh s c mt ca ru ethylic trong dch ln men) - Cho vo ng nghim 2ml dch ln men ru, thm 2 ml H2SO4 m c, thm vi tinh th K2Cr2O7 lc u cho n khi xut hin mu xanh lc. Phng trnh phn ng din ra nh sau: 2 K2Cr2O7 + 8 H2SO4 + 3CH3CH2OH 4KCr(SO4)2 + 3CH3COOH + 11 H2O ng i chng: Lm tng t nh trn, song thay dch ln men bng dch cha ln men. So snh s i mu hai ng th nghim v i chng. 4. Thu nhn nhanh nm men ru trong thc t Bn cnh phng php phn lp nh trn, ty iu kin tng trng chng ta c th t thu nhn nhanh cc chng nm men lm i tng dy thc hnh sinh hc nh sau: a. Hot ha men n bt m: 50 ml dung dch ng glucose 10% thm 1g NaCl un si, ngui thm 1g men bnh m dng bt. Chun b trc khi th nghim 2-3h. b. Pha long dch chit t cm men ru sau 2-3 ngy.
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c. Pha long men p lm n bt m (0,1g men p ha long trong 9ml nc ct v trng). d. Pha long dch tri cy (nho, to, chui, sim...) ngm ng (10g qu chn + 10g ng + 5ml nc un si ngui) sau 5-7 ngy ri ly 1ml dch qu ngm ng trn pha long trong 9ml nc ct v trng. * Cu hi 1) Trnh by phng php phn lp nm men ru thun khit. 2) Th no l chng nm men ru thun khit (chng sch)? 3) V sao khi phn lp nm men ru ngi ta thng thm streptomycin hay rosebengal vo trong mi trng. Hy gii thch. 5. Nhng im cn lu Khi phn lp nm men ru cn lu : cc hp thch mi trng phi v trng c b mt kh nc v c rn chc, khng g gh, gip cc khun lc nm men mc ri nhau ra. phng s nhim tp do khng kh bn, cc thao tc phi tuyt i v trng. Cc chng sau khi lm sch cn c kim tra li bng cch quan st hnh dng t bo di knh hin vi cng nh xc nh li cc phn ng nh tnh nh kh nng sinh kh CO2, kh nng ln men sinh ru ethylic. Vic to ra mt chng nm men thun khit cng nh bo qun khi b tp nhim l vic lm c ngha rt ln trong hc tp, nghin cu cng nh sn xut. Ti liu tham kho N.X., (1983). Thc tp Vi sinh vt, NXB Mir, Maxcva. Nguyn Ln Dng dch, NXB H&THCN H Ni. [2] John William Henry Eyre, (2009). A Laboratory Guide for Medical, Dental, and Technical Students, http://www.gutenberg.org/
[1] Egorov

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