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Cu 1: nh ngha khng sinh, n v & p/loi k/sinh? 1) nh ngha khng sinh: - C rt nhiu /ngha v k/sinh, song /ngha cht ch theo Waksmann (1942) c ngha kinh in nh sau: K/sinh l nhng sp trao i cht t nhin c cc vsv to ra, c t/dng c ch s ptr or tiu dit chn lc /vi cc vsv khc. Vd: k/sinh penicillin. - n nm 1972 c khong 4076 k/sinh c tm thy, ngy nay s k/sinh mi p/lp c hng nm l khong 100. 2) n v khng sinh: * L lg k/sinh ti thiu ha tan trong 1 th tch mi trng x/nh c t/dng c ch hay tiu dit vsv. * Mi k/sinh c /v h/tnh s/hc ring: + /v hot lc of penicillin t nht h/tan vo 50ml canh thang c t/dng c ch s ptr of Staphylococcus aureus 209p. + /v hot lc of streptomycin l lg streptomycin t nht h/tan trong 1ml canh thang c t/dng c ch s ptr of E.coli. 3) P/loi k/sinh khng sinh: * P/loi k/sinh nhm khi qut 1 cch h/thng danh mc cc k/sinh ngy cng nhiu thm. C th p/loi da vo p t/dng, c ch t/dng. p/loi theo ng/gc, con ng sinh tng hp hay c/trc ha hc. Nhng p/loi theo c/trc ha hc l khoa hc nht. * Gii thiu p/loi theo c/trc ha hc & v d: c 9 nhm k/sinh chinh. - Cc k/sinh cacbonhydrat. Vd: cc saccarid thun nht (nojirimycin); cc glycolipid (moenomycin), - Cc lacton macrocyclic 1

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Vd: cc k/sinh macrolid (erythromycin), cc anzamycin (rifamycin), - Cc k/sinh quinon & dn xut. Vd: Cc tetracycline (tetracyclin), cc benzoquinon (mitomycin), - Cc k/sinh peptid & aa. Vd: Cc dn xut aa (cycloserin), cc cromopeptid (actinomycin), - Cc k/sinh d vng cha nit. Vd: polyoxin - Cc K/sinh d vng cha O2. Vd: k/sinh polyete (monenzin). - Cc k/sinh mch vng no. Vd: Cc dn cht alcan (cyclohexinmid), k/sinh steroid (axit fuzidic). - Cc k/sinh mch thng: Vd: cc cht cha Phospho (phosphomycin). - Cc k/sinh cha nhn thm. Vd: cc dn cht benzene (chloramphenicol), cc cht nhn thm ngng t (griseofulvin), cc ete thm (novobiocin).

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Cu 2: Cc phng php p/lp vsv tng hp k/sinh? 1)Ng/l chung: T cc ngun c cht khc nhau(t, nc, cht thi, bn,..) tin hnh nghin nh, pha long mu & cy vo cc mtr nui cy chn lc ri cho ptr trn nhit thch hp, sau thun khit chng mi phn lp c, kt hp th k/nng sinh k/sinh ta thu c cc vss cn tm. Ty pp n/cu m ng dng ling hot cc pp phn lp, th khc nhau nhm thu ly kt qu mong mun. 2) Cc pp: a) Pp cy dch chit t ln b mt thch: Cn chnh xc 1 lg t (1-2g, t trng trt thg c ly su 10-15 cm) vo coois s ri cho vo 1 lg nc v trng, nghin nh. Dng nc v trng ri pha long thnh cc pha long thch hp(10-4 106 ). Nh dung dch pha long ln b/mt thch mtr dinh dng trong hp pitri ri gt nh cho phn tn u. trong t m nhit thch hp (28 320C) cho ptr 5-7 ngy, xut hin cc khun lc ring r. Cy tch khun lc trn thch nghing thun khit ha, sau nui cy ri th hot tnh k/sinh of cc chng mi c phn lp. b) Pp cy t trc tip ln b mt thch cha sn vsv kim nh: em reo cy trc tip cc ht t ln b mt thch trong hp Petri c mtr dinh dng cha sn vsv kim nh, t m 24-48h ri mang ra quan st. Nu xung quanh khun lc ptr t t xut hin vng v khun ta x/nh c trong ht t c vsv to ra k/sinh tiu dit c vsv kim nh. T khun lc tin hnh phn lp thu c vsv sinh tng hp k/sinh. c) Pp lm giu t: t trc khi c phn lp c cho vo ci thy tinh, gi m xc nh, thnh thong cho t vsv kim nh vo ri trn u. trong t m 1 t/gian ri em ra phn lp theo 1 3

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trong cc pp nu trn. Bng cch ny ta thu c vsv i khng vsv kim nh (d lc u vsv i khng c rt t trong t d) Pp b sung k/sinh vo mtr: phn lp x khun ta c th cho thm k/sinh chng nm & VK vo mtr phn lp vi nng t 5-25g/ml. Cc k/sinh thg dng nh: tetracylin, neomycin, penicillin,. Nng k/sinh thm vo cn phai c duy tr thch hp, nu k chnh s ptr of x khun mc tiu cng b c ch. e) P/lp vsv sinh k/sinh chng ung th: Cc k/sinh chng ung th va c t/dng tiu dit t/bo ung th, va c t/dng tiu dit t/bo.. Nn sng lc cc vsv to k/sinh chng ung th ta c th dng vsv lm test th, cng c th s/dng cc chng nm men b.

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Cu 4: Trnh by c ch tc dng ca khng sinh? - Cc k/sinh t/dng c bn wa vic c ch cc pn ng tng hp rt khc nhau of t/bo vsv gy bnh. Chng lk vo cc v/tr chnh xc hay cc p/t ch of t/bo vsv m to ra cc pn ng trao i cht. Cc ch t/dng /trng cho tng nhm k/sinh, tuy nhin trong nhiu trng hp ng/ta vn cha bit chnh xc ht. - C 6 mc t/dng khc nhau /vi t/bo VK or nm: + T/dng ln thnh t./bo: tng hp murein (vancomycin, phosphomycin) + T/dng ln mng nguyn sinh cht: thay i c/trc (polymixin, amphotericin) lm mt chc nng of mng lm cho cc p/t c k/lng ln & cc ion b thot ra ngoi. + T/dng ln w/trnh tng hp AND: phn chia (antracyclin), dch m(anzamycin). + T/dng ln w/trnh tng hp Pr: ribosom(aminoglycosid gn vi receptor trn tiu phn 30S of ribosom lm cho w/trnh d/m ko chnh xc); + T/dng ln s trao i cht h hp: antimycin. + T/dng ln s trao i cht trung gian: sulfamid.

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Cu 5: Trnh by tnh khng thuc of vsv & nguyn tc s/dng khng sinh? 1) Tnh khng thuc of vsv: hiu v tnh khng thuc of vsv trc tin ta phi hiu t/no l khng thuc. Khng thuc l h/tng vsv mt di tnh nhy cm ban u of n trong 1 t/gian nht nh hay vnh vin vi t/dng of k/sinh hay ha tr liu. C 2 kiu khng thuc: Khng thuc t nhin & khng thuc mi nhn. +khng thuc t nhin: l c trng ca tng ni vsv nht nh i vi 1 ks nht nh no . Tnh cht ny c sn trc khi sd cc ks . Lin quan ti ph tc dng ca ks,c th c trng cho tng loi,cho tng nhm,tng sp. L thng tin di truyn c sn trong NST. V mt sinh hc c 2 c ch tc dng: tnh thm ca tb v s thiu vng phan t ch. +khng thuc mi nhn: xut hin trong chn lc t nhin cc chng khng ca qun th vsv nhy cm khi sd ks. a) C ch d/truyn hc: Xt trn phng din khng thuc mi nhn c th l do thay i gen NST(B NST), c th do tip nhn gen plasmid(d khng plasmid hay ngoi NST) * Khng thuc do B NST: Kiu khng thuc ny chim khong 10% tng s vsv khng thuc, x/hin ch yu /vi cc k/sinh nh: -lactam, aminosid,B kiu ny t n nh & t d/truyn. * Khng thuc plasmid: kiu khang thuc ny rt p/bin, chim khong 90% s vsv k/thuc. K/thuc ngoi NST l h/tng a khng thuc do nhn t R, nhn t R(gm 2 phn:phn x/dnh tnh k/thuc & phn truyn ti gen k/thuc) chnh l cc gen /khin tnh k/thuc. W/trnh truyn cht liu di truyn nhn t R theo 3 c ch: 6

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- G phi hp thit lp cu tip hp. - G bm v/liu d/truyn wa tip hp. - G ti t hp v/liu d/truyn t/bo nhn to thnh h v tnh k/thuc of vsv. b) C ch sinh ha cc k/thuc mi nhn: c 4 kiu chnh: * Thay i tnh thm thnh t/bo:k/sinh ko thm wa thnh t/bo c thi k pht huy t/dng(khng -lactam). * V hiu ha cc k/sinh=enzyme. * Thay i phn t ch Vd: thay i Pr ribosom lk vi streptomycin hay thay i ARNpolymeaza lk vi rifamicin. * Hot ha con ng trao i cht mi khc m hot cht k t/dng. 2) Nguyn tc s/dng khng sinh: Vic s/dng k/sinh cha hp l trong iu tr lm xut hin ngy cng nhiu vsv khng thuc. khc phc h/tng cn thc hin 1 s ng/tc sau: - Phn lp vsv gy bnh & th nhy cm of chng vi k/sinh. Vd:pp khoanh giy lc,.. - Ch s/dng k/sinh khi c nhim khun. - Phi hp k/sinh vi ch phm khc lm tng t/dng, gim phn ng ph, bnh nhn ko t dng thuc. - Chn la ng k/sinh. - S/dng k/sinh ng liu lng. - Nm vng chng ch nh of k/sinh. - Theo di hiu qu tr liu of k/sinh.

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Cu 6: Nu ng/l chung cc pp nh lng k/sinh, trnh by pp vsv nh lng khng sinh? 1) Ng/l chung cc pp nh lng k/sinh: - K/sinh c t/dng c ch s ptr of vsv kim nh, so snh vi t/dng of k/sinh chun bit s tnh c h/lng k/sinh of mu th - Da trn phn ng to mu of k/sinh or sp phn hy of chng ri o bng my quang ph t ngoi or bng my so mu ri tnh ra kt qu. - K/sinh khuych tn vo mtr thch cy vsv kim nh to ra cc vng v khun. o ng knh vng v khun k/sinh chun & k/sinh th ri tnh ra hay tra bng thu kt qu cn phn tch. 2) Pp vsv nh lng khng sinh: K/sinh c t/dng c ch s ptr of vsv kim nh, so snh vi t/dng of k/sinh chun bit s tnh c h/lng k/sinh of mu th. chnh xc of pp ny l 5-10% . a) Pp pha long l/tc: - Dng nh lg k/sinh trong dch chit hay dch l/men. Pha long k/sinh trong mtr canh thang trong sut trong cc ng nghim v trng thnh cc nng khc nhau. C th pha long theo: 1:2; 1:4; 1:8; (dy hnh hc), hay 10:10; 9:10; 8:10; (dy s hc),.. - Ti mi nng k/sinh cho vo 1 lg vsv kim nh, sau t vo t m 370C trong 18-24h ri em ra c kt qu, nu ng no trong sut ti nng pha long k/sinh tiu dit vsv kim nh. - t k/qu chnh xc cn ch : thnh phn mtr pha long phi n nh, lg VK kim nh cho vo cc ng nghim phi bng nhau & t/gian cho vsv kim nh phi n nh. 8

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b) Pp khuych tn trn thch: * Nguyn l: K/sinh khuych tn vo mtr thch cy vsv kim nh to ra cc vng v khun. o ng knh vng v khun k/sinh chun & k/sinh th ri tnh ra hay tra bng thu kt qu cn phn tch. * Pp khuych tn trn thch thg c s/dng nh lg k/sinh, cn ch ti dy lp thch, pH mtr & t0C nui cy. Nhng k/sinh polypeptide kh khuych tn trn thch nn thg cho vo mtr nh lg cc cht lm tng khuych tn of k/sinh(CaCl2 lm tng khuych tn of k/sinh gramicindin C), or t hp Petri c VK kim nh vo t lnh (40C) trong 2-6h k/sinh khuych tn vo mtr thch ri mi em t vo trong t m cho vsv ptr. Trong pp ny ta p/bit 3 bin thin khc nhau: pp tr thp, pp c l thch & pp khoanh giy lc.

PP. ng tr thp

PP. khoanh giy lc

Cu 7: Trnh by cc k/nng ng dng ngoi y hc.

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Cc nh n/cu & k/t kt hp vi cht ch vi nhau nhm pht huy ti a tnh hiu wa of k/sinh. a) Khng sinh trong chn nui: K/sinh c dung nh cht k/thch tnagw trng gia sc, gia cm, gim chi ph thc n, k/thch tng sn lng trn g vt. Cc k/sinh: enduracidin,mikamicin,virginiamicin(peptid), tilozin, - K/sinh t/dng ln h sv ng rut lm tng s/lng vsv c ch trong rut, tng cng tng hp VTM, tng cng ti hp th thc n,k/sinh lm gim cc vsv c hi tit ra cht c or s/dng VTM, lm gim pH rut, Cc t/dng trn gip v tng cng trao i cht nhanh. - Mt cch gin tip tng cng iu tit hormone /bit l hormone s/trng gip c th ln nhanh. b) K/sinh trong trng trt: Vic chn k/sinh dit vsv gy bnh cy trng ko ch ch n t/dng k/sinh m cn phi w/tm n h/qu k/t. * K/sinh dng u tranh vi bnh thc vt phi tha mn cc yo cu sau:- Hot tnh k/sinh mnh /vi mm bnh.- D thm vo cc t/bo cy.- Liu iu tr k hi n cy.- K/sinh phi bn vng trong 1 t/gian d b mt hay thm su vo trong cy. * u im of k/sinh trong b/v TV:- Liu rt thp nhng vn c t/dng.- D b cc vsv t phn hy.- Ko c or rt t c /vi V mu nng & cc cn trng c ch. c) K/sinh trong cng nghip thc phm: Bo qun thc phm ti & cc thc phm ng hp l vn rt w/trng. Cc k/sinh c s/dng trong bo qun thc phm ph thuc vo cc quy nh lut php of nh nc. vd: 1 s cht bo qun nh: Nizin cht l tng b/v phormat, c chua, u xanh, bp ci
Cu 8: Trnh by cc ng/liu cht dinh dng trong sx k/sinh?

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Mi trng dinh dng nui vsv phi cha all cc ng/t cn thit cho t/bo s/trng & sinh tng hp di dng d hp th & tip nhn. Cc ng/liu phi tha mn yu cu l d kim, chuyn tr thun tin & c sch cn thit. a) Cc ngun cacbon: Cc loi cacbonhydrat l ng/liu truyn thg of cg nghip k/sinh & VTM. S/dng cc loi ng sch lm mtr: sacaroza(lmen sx Fumagillin), glucoza, lactoza(sx penicillin), T/bt & Dextrin l cc ng/liu lmen k/sinh tt /vi cc chng vsv c k/nng to ra cc enzyme amylaza. C th s/dng t/bt vi cc ngun gc khc nhau: ng. Khoai\, sn, Cc loi du thc vt(du u tng, du c,)c th dng nh ngun cacbon h tr. b) Cc ngun Nit: C th s/dng cc mui amoni [(NH4)2SO4, NH4NO3], mui nitrat[NaNO3, KNO3], lm ngun nito ty thuc vo chng vsv. Cao ng: l ngun nito d hp th (cn thg s/dng 50% cht kh) l w/trnh ph trong sx t/bt ng. Cao n/men: l ngun m c nhiu loi vsv a thch. Cao n/men c sx t nm men bnh m l chnh bng pp t phn (ahttolysis) 50-550C, hay bng plasmolysis vi nng cao or bng thy phn acid nh (pH=5-6 vi HCl). Cao n/men cha cc acid amin, cc peptid, cc VTM tan trong nc & cc loi cacbonhydrat. Bt u tng: c s/.dng nhiu trong l/men k/sinh do hp th tg i chm & ko xy ra cc c ch /khin trao i cht. c) Cc cht khong, vi lng & k/thch: /bo cho vsv ptr b/thng cn phi /bo cc cht khong nh: NaCl, K2HPO4,Bn cnh cc cht khong phi b/sung 1 lng nh cc ion vi lng cho vsv. Tuy nhin nng cn thp, nhng k v th m xem nh v/tr of chng. i khi cn b/sung them vo mtr dinh dng 1 s cht k/thch ptr nh: VTM,

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Cu 9: Cc pp ci to ging(CLTN, B nhn to) & trnh by cc k/nng trong ci to ging? 1)Cc pp ci to ging: a)b t nhin: cc vsv b b theo tun xut khc nhau, nn coong vic ci to ging y l cn phi tuyn chn ly cc c th c hot tnh cao nht n/cu tip theo. y l pp chn cc chng c h/tnh cao, tuy nhin bng pp sng lc cc c th b t nhin c h/tnh cao ch n/cu ban u, k c gi tr p dng vo sx. b) b nhn to: Cc chng vsv chu t/dng of tc nhn gy b mnh nh tia X, UV hay cc tc nhn ha hc nh: etylenimin, dimetylsulfat,..phn ln cc vsv b git cht, cc c th cn sng xut hin nhiu dng b or gim (mt) kh nng sinh k/sinh (b m tnh) or lm tng k/nng sinh tng hp k/sinh(b dng tnh). 2) Cc k/nng trong ci to ging: - T chng vsv sinh ra 1 loi cht c c/trc tg t ng/ta to b & phn lp cc b ch to ra 1sp cn thit lm tng cao sn lng & lm cho qu trnh tinh ch k/t hn. - Lm gia tng sn lng hot cht m/tiu nh ng dng 1 cch linh hot cc pp b & sng lc hay ti t hp, sang lc da vo kinh nghim hay h/ng c thc bng c ch cc con ng sinh tng hp nht nh hay bng vic thay i h/thng /khin. - a vo s/dng cc chng b c tip or tin phn ng thay i to ra cc k/sinh ng ng, c c/trc thay i so vi k/sinh bit. Adramycin l cht c t/dng chng ung th mnh hn, l k/sinh antitumor c ng dng rng ri hin nay. 12

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Cu 10: Cc tc nhn b & cc kiu b trong ci to ging vsv sn xut k/sinh? a)Cc tc nhn gy b: * Tc nhn vt l: Tia Rnghen(tia X), tia cc tm bc song 250280 nm c t/dng gy cht mnh, t/dng gy b khi x/l cc chng vsv. Ngoi ra cn c th s/dng tia nowtron,, ngy nay tia cc tm bc song di (=380 nm) kt hp vi ha cht cng thng c s/dng. * Tc nhn ha hc: + N-mustar: tris-(-clor-etyl)-amin or Metyl-bis-( -clor-etyl)amin CH2 CH2 - Cl CH3 N < CH2 CH2 Cl + Etylenimin: tietylerimelamin: (cng thc) + Dimetylsunfat, HNO3, HN = C NH NO2 O = N N CH3 b) Cc kiu b: ci to ging ta x/l cc chng vsv vi cc tc nhn b, ri kt hp vi sng lc phn lp cc chng c h/lc sx h/cht tng ln tt nht so vi chng ban u. Sau khi x/l vi cc tc nhn b c th xy ra cc kiu b sau: * b kiu tng gen (genom): kiu b ny lm thay i s/lng NST trong t/bo, c ngha trong ci to ging thc vt. * B kiu gen: trong ci to ging vsv ch yu l cc b im c ngha rt w/trng vi 1 s dng: b i cp bazo, B trt khung 13

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Cu 11: Trnh by pp ti u ha b, tuyn chn (sng lc) b ci to ging. 1)Phng php ti u ha b: Cc chng b m trong b dn n s thay i cc tnh trng ph thuc vo rt nhiu yu t, bn cnh cc nhn t /bit /trng cho tng chng th k mtr x/l cng /hng rt mnh ti b l : pH mtr, h m, nng (cng ) tc nhn b, t/gian x/l hay trng thi ptr of vsv => to c k/qu mong mun all cc nhn t ny u phi c ti u ha. Phng php hu hiu nht l lp ng cong liu t/dung kt hp vi xy dng biu tn sut. Tuy nhin cch ny i hi rt nhiu cng sc, nhng li l con ng duy nht em li kt qu. 2) Tuyn chn b: (ch n/cu chn lc ngu nhin) * S tng trng to ln of cc vsv trong cng nghip k/sinh a s u t c bng cch sng lc ny, tc l tuyn chn sau b. * Ng/l chung: Sauk hi x/l vi t/nhn b & nui cy ptr, trong s cc c th (clon) sng st c la chn ngu nhin ta tin hnh kho st k/nng to h/cht of chng. * Pp l/men m hnh c ng dng khi m cc tham s l/men (mtr dinh dng, cc k ngoi cnh) c duy tr ging k cng nghip. Cc chng tt nht thu c bc ny li em b & sng lc tip. Sau 1 lot cc bc b & sng lc phn lp sx s tng ln dn dn. Pp ny cn gi l tuyn chn bc thang hay b bc thang. * Trong ci to ging cng nghip khi kho st cht lg to h/cht of cc c th (clon) sau cc bc b mnh xut hin 3 tnh th sau: - Cc clon c sn lg cao hn of b m mc va phi xut hin vi tn sut cao hn & d xc nh hn. 14

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- Cc clon c sn lg cao hn of b m nhiu xut hin vi tn s thp, do vy tm kim chng rt mt thi gian. - Do phi s/dng tc nhn b liu lg mnh thg xut hin b nhiu tng lm hiu sut k tng tip c na. trnh mt thi gian tm kim chng c hiu sut cao nht ta ly 5-10 chng c mc tng trng va phi lm chng b m & tip tc b tip(trnh c b nhiu tng). * Tn s xut hin cc tng trng sx ph thuc vo chng vsv, k b cng nh vo q/trnh sinh tng hp & /khin of h/cht cn ti u. Thng thng theo chu k b phi kho st t vi trm n vi ngn c th.Cng sut sng lc s quyt nh vn tc tin ln pha trc of ci to ging. tng cng sut sang lc ng/ta xy dng cc h/thng t ng kiu Glaser Dumb-Waiter hon ton t ng.

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Cu 13: Trnh by cc pp gi ging vsv? - Gi ging nhm gi gn k/nng sng st & cc tnh cht c gi tr k/t k thut of cc chng vsv sx l ht sc w/trng nhng li tn nhiu cng sc & kh khn. - Gi ging trng thi tim sinh gip duy tr c cc t/cht ban u of vsv vi s chm sc ti u. - La chn pp gi ging trong mi trng hp ph thuc vo k vt cht, k thut of phng th nghim, sc sng of vsv, tnh bn vng dt hc & yo cu s/dng sau nay. a) Gi ging trn mtr thch: L pp c ng dng kh rng ri trong thc t, mc d k phi l k/t & ng tin cy nht. Sau 1 chu k t/gian nht nh phi cy truyn chng vsv sang mtr mi, cho ptr ri li ct gi. Vic ny i hi tn cng sc & a n nguy c thoi ha chng dn n gim thiu of mt i nhng t/cht c bn of chng. Trong gi ging /bit trn mtr giu cht hu c c th xut hin phagolysts t pht chng vsv. Nhim phage c th lm gim or mt h/tnh of chng. lm gim kh b/mt thch c th trng paraffin nt or bc mng nilon hay mng polyetylen, mt khc c th dung mtr 1% du thc vt. Gi ging trn thch c th duy tr nhit -20 -220C . Trong thc t ng ta gi bo t or t/bo s/dng 48h tui trong mtr glycerin 15%. Phng php ny n gin, nhng li nm trong k/nng cht of vsv t cc t/b ng lnh. b) Gi ging trn ct v trng: Ly ct song ra trong dng nc chy ti khi ht bn, sy kh ri un nng trn ry st, sau ry wa sang nh ri cho vo ng nghim hay ampul, ng nt li ri kh trng 1200C trong 1h trong ni hp. 16

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T 1 lg ging c th s/dng lm nhiu ln trong t/gian tg i di. c) Gi ging trn t v trng: Ly 2g t vn, nghin nh, cho vo ng nghim ri them 2-3 git nc, lm nt & kh trng 1200C trong 1h, sau m 300C trong 24h ri kh trng nh trn 1 ln na. Sau khi th v trng, cc ng t c cy vsv & c bo qun. T 1 lg ging c th s/dng lm nhiu ln trong t/gian tg i di. d) Gi ging trn ht ng cc: T ht k c vng cn ly 1kg cho vo bnh, them vo 800ml nc si, un si trong t/gian nht nh cho k no nc, sau y lm nt & ng 30 ri em ra mt bn lau cn etylic cho ngui. Cn ly ra 15g vo bnh nn 250ml ri kh trng 1150C/40 . Th v trng, bnh k v trng c ly cy 2ml hn dch bo t trong Tween hay 3ml si dinh dng. Ni cy trong 2 ngy m trn my lc, ty tng chng m k c cho ptr 1 tun 280C or phi lc ph v khi k hay long si. Vsv ptr n/vy c em sy chn k 5-10 mmHg trong 3 ngy nhit 250C cho n khi khi lg cc bnh k k thay i na, m ht k k thay i 8% . Cc bnh cn li c trng paraffin nt & ct gi nhit phng & ni kho ro. C th s/dng nhiu ln sx k/sinh. e) Gi ging bng ng kh: ng kh c s/dng rng ri gi ging vsv t/gian di. Pp ng kh da trn thng hoa nc di chn k t cht liu ng lnh. Vi m/dch ny phi chun b hn dch bo t m c trong huyt thanh nga k c cht bo qun or trong dung dch cha 10% sacaroza & 1% gelatin, pH=6,8 7,0. Phn 0,2 ml hn hp dch bo t vo mi ng ampul(l thy tinh trung tnh c ch tht) bng pipet. Ampul c lm lnh xung bng hn hp CO2 bng & etanol ( 17

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70 -800C) trong 5 & xp vo my chn k lm kh p sut 100mmHg. T/gian lm kh ph thuc vo chn k, b/mt bc hi & th tch cn lm kh. m cn li k vt wa 3-5%. Ly ampul ra khi my chn k, c ct gi nhit 50C thun li hn. S/dng pp ny s: Chng c bi nhim,duy tr c cc /tnh tt of chng & tit kim c v/tr. f) Duy tr h/tnh k/sinh of chng sx: duy tr h/tnh of k/sinh bban u of chng sx th k th c gii php ton din ch bng cc pp bo qun, tn tr ging, m phi p dng cc pp phc hp bao gm: la chn pp tt nht, nui cy chng sx trn mtr gi ging thch hp nht kt hp vi vic a vo ng dng cc pp tuyn chn l/tc cc dng c h/tnh cao nht.

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Cu 14: Qu trnh l/men gin on, cc pha c th & cc tham s /trng (tg, x, ) ? 1) Qu trnh l/men gin on: Hay cn gi l ln men m, l/men chu k & h/ng nh 1 h/thng. Ti thi im t=0h, ta cy ging vsv to hot cht vo mtr dinh dng trong bnh l/men 1 cch v trng, ri tin hnh l/men trong /kin kh nn ti u. Ngoi cp O2, cht ph bt, cht iu chnh pH th trong thi gian l/men ta k t/ng, bsung g thm vo h/thng. V vy q/trnh l/men thnh phn of h sinh khi vsv, nng cc sp trao i cht bin i k ngng. theo st cc din bin of qu trnh l/men ng/ta tin hnh phn tch cc mu dch l/men mi ly x/nh tham s chnh. 2) Cc pha c th & cc tham s c trng : 4 pha c th trong l/men gin on. * Pha lag: y l pha ptr tim tang vsv thch nghi vi mtr mi. Trong vic hnh thnh pha lag ny trng thi sinh l of ging c ngha quyt nh. Cm ng to h/cht trong vsv k ph thuc vo bn cht ngn hay di of pha lag. * Pha log: Cui pha lag l pha log, pha ny ptr nng ng nht = 1/x * dx/dt * Pha dng hay pha n nh: Sau khi ng ha cc cht dinh dng hay sau khi tch ly cc sp trao i cht, s/trng of vsv gim xung hay h/ton ngng li.Do s t phn of 1 s t/bo, 1 s cht dinh dng mi (cacbonhydrat, Pr) c gii phng, iu ny a li s ptr chm of 1 s t/bo sng st. C rt nhiu sp trao i cht c gi tr c to thnh. * Pha suy tn: Cc cht dinh dng b cn kit, nng lg of t/bo gim n ti thiu. t/bo cht i nh trong s/trng trong thi k s/trng. 19

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/trng of pha ny l ph thuc tng loi vsv & vo cc pp nui cy c ng dng. Trong 1 s trng hp vic sinh tng hp h/cht tuy cha dng hn, nhng chm li & vic duy tr tip tc qu trnh l/men k cn k/t na v th cng cn kt thc l/men.

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Cu 15: Qu trnh l/men bsung: ng/l, cc cng thc chnh & ng dng? Q/trnh l/men y cng bt u l/men 1 cch gin on, sau o ti 1 thi im nht nh bt u bsung. T/gian l/men do th tch t/b & tc bsung xc nh. Thg khi bt u ch s/dng 0,5 0,6V tng th tch, sau bng k thut fed batch tng ln 0,7 0,85 V th kt thc. Pp c bsung khi bt u l/men cc t/phn nguy hi of mtr c ng dng vi nng thp hn so vi pp l/men m rt nhiu(thp hn nng cho tng loi vsv). Cc cht ny trong q/trnh l/men c bsung l/tc vo mtr l/men thnh tng phn nh. Nu trong q/trnh l/men k th o c 1 cch trc tip & l/tc hm lg cht dinh dg th cn phi tm cc tham s gin tip l/wan & wa o of n tnh ra h/lng cn bsung. Pp c ng dng trong sx penicillin, acid glutamic, ng dng trong cc trng hp: nng c cht kh thp nhng li cn n nh(l/men nm,), nng c cht kh cao & k thay i (l/men acid xitric), bsung tin cht lin tc (penicillin, indol,) Cc cng thc chnh:

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Cu 16: L/men lin tc chemostat: ng/l h/ng, hnh nh qua th, cc cng thc /trng?

* Ng/l: khi iu khin Chemostat trng thi cn bng (dng, steady state) ng sinh trng cc t bo vi sinh vt c duy tr bng thay i nng ca cht dinh dng ang c ch no . lm c vic ny bt k cht dinh dng no (cacbon, ngun Nito, mui, O2,...) * Hnh nh: X3 S0 S03 X Sjx S.x X2 S02 S Dmj D X1 S01

Bin thin cc tham s c trng ln Chemostat theo D * ng dng: - Sn lg cao hn. - Nhiu k/nng o & iu khin. - Trong trng thi k i vsv ptr cn i( sinh khi ng nht). - Chemostat ng dng ch yu trong l/men SCP, nm men bnh m. - Chemosta ng dng trong sx etanol, bia.

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Cu 17: L/men lin tc turbidostat, l/men bn l/tc & ng dng? a) L/men l/tc turbidostat: Khi l/men dch l/men c 1 my bm chu k ngoi vng wa c quang hc (OD = Opitical Densty) of dch l/men o c l/tc. Qu trnh l/men c bt u gin on.

X X max X min t Hnh nh ln men turbidostat t

Do phi o l/tc quang hc m pp ny ch c th s/dng vsv 1 t/bo & cc dch l/men tg i long. K thut turbidostat ch yu n/cu: ti u ha, ko di tng trng ly tha.

t Nguyn cu ti u ha vi turbidostat 23

t1 S2

t2 S3

t3

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b) L /men bn l/tc & ng dng : y l bin tng l/men khi vic rt bt dch l/men & bsung them mtr dinh dng k xy ra l/tc m ch nh k trong 1 s thi im nht nh. L qu trnh c li v tng c lg dch l/men cha h/cht mc tiu (nu vic bsug cht dinh dg k lm gim hiu sut to sp). Tuy nhin thi im bsung & ly bt dch phi da trn tnh ton cng nghip hp l.

Xmax

Xmin

t t

Hnh nh ln men bn lin tc

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Cu 19: Trnh by cc h/thng bnh l/men: ng/l h/ng, ng dng my tnh in t trong l/men sx k/sinh? a) Cp kh vi cc b phn vn ng: y l loi bnh l/men sc(cp) ko kh kt hp vi khuy trn, c ng dng rng ri nht & cng ptr mnh m nht.

C khuy

khuy ng dn

khuy t ht

Trong thc t bnh l/men cp kh c khuy trn l thng dng nht. b) Cp kh vi my bm vng ngoi:

L loi l/men ct bt kh, hi lu tia hay tia chm cng kh thng dng.

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T/dng cp kh of p sut kh c t/dng chuyn ng dng cht lng nh my bm tng cng thm nn h/qu t c cng kh cao. c) Cp kh vi p sut kh & pha kh ng: L loi hnh l/men kh thng dng trong cng nghip, nhng ng dng loi hnh ny trong nhiu trng hp phi ci to c cc bin chng ging cho nht dch l/men thp. Nh vy m gim c chi ph nng lg, nng cao c tnh cnh tranh cao of cng ngh.

Ct bt kh

airlift

Cp kh vi pha kh ng l h/thng /trng cho l/men b/mt khi dung kh nn hay qut gi cp kh cho bung l/men. Re = NRe = Di2 . N . P Di: g knh my sy (cnh khuy) (cm). N: vn tc my sy ( v/s, s-1). P: khi lg ring (g/cm3). : nht ng (g/cm.s). d) m bo v trng: Phi /bo 1 s quan im v kt cu: bnh l/men ch c t ca m, l nhng ca ti cn thit, c b dung vng m, ca ln dng dong lt.

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Nn trc l h/thng dong trt (Simmering), kh trng bng hi & sau dng nc ngng ngng hay dng glycerin gi. Qu trnh l/men v mt ng/tc kha hi phi loi h/ng l/tc nhy m.

Hi vo

Hi ra Kha hi v hot ng

Cu 21: Cng sut my khuy of bnh l/men c cp kh? * Do sc ko kh vo bnh l/men m hnh thnh h kh-lng lm gim khi lg ring, do o lm gim yu cu cng sut. Cng thc(3.62) [Michel & Miller](t vit) 27

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Thng s: Pg cng sut my khuy c cp kh. K - Hng s (kt cu) (0,706 / V). P1 cng sut my khuy k sc ko kh. N vn tc my khuy. D1 ng knh my khuy (cm, dm.m). Q - vn tc cp kh (VVM, L / L.min, m3/m3.min). * Trong thc t cng nghip khi th tch bnh l/men tng, yu cu v cng sut my khuy gim. Th tch (m3) Cng sut (Kwh/m3) 0.1 11 - 13 15 47 15 - 20 13 *Nu ko c my khuy ta co cng thc: t vit Thng s: F dng cp kh (). P p sut ko kh . Vo tc kh ti b chia. Pc - p sut kh ti b chia. 0.06

Cu 24: Trnh by qu trnh kh l/tc mtr l/men. a) Kh trng l/tc dch l/men: * Mtr dinh dg sau khi s un nng c a vo b phn un nng tc thi vi hi c bm trc tip nng nhit ln 130-1400C, dch

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dinh dg c chy trong rut xon b phn duy tr t 2 3, qua van gin n x tc thi sang b phn lm lnh h nhit xung 800C & c lm lnh xung nhit l/men nh mtr lnh trong b trao i nhit thng.
Mt v trng
Lm lnh s b

hi

Lm lnh

ung nng

Nc lnh

un Mi nng trng s b 20 S 2-3 pht 20 S 1400

t(min)
B kh trng lin tc trao i nhit tm * U im of pp: - D /khin & t ng ha. - Trn nhit kh trng cao hn, thi gian kh trng ngn hn, b/m an ton hn & du nh.

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- Qu trnh l/tc cho dinh dg ng nht hn ( tng cng hiu sut l/men). Pp kh trng ang ngy cng c ng dng rng ri trong cng nghip.
hi

Chn ko M trng

Phn mt Mt v trng Kh trng mt lin tc-h bm hi

b) Kh trng ko kh: - Cc ht bi l lng trong ko kh & s vsv giao ng rt mnh ph thuc vo v/tr, chuyn ng cng nh vic x/l trc. Ngoi tri c khong 10.000 ht/m3 , trong khong 2.000 vsv/m3 , 50% s vsv l bo t nm, 40% l VK. - Cc bnh l/men c vn hnh vi vn tc cp kh 0.5 1.0 VVM (m3/m3.min). Nu th tch bnh l/men l 50m3 & vn tc cp kh l 1.0 VVM th phi cp 3.00m3/h k kh v trng. - Trong cc pp hnay c cng nghip ng dng pp lc v trng.

Cu 26:trnh by cu trc ho hc ca cc ks penixillin tn nhin,sinh tng hp,bn tng hp. Khung c bn ca pen l 6-APA (6- amino pen axit) c nhn thiazolidin v vng -lactam 30

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Trong sx nu k thm cc tin cht vo trong dch ln men thi cc ks t nhin c hnh thnh. Vit 1 cch tng qut R=OOC-CH(NH3)-(CH2)3-COV nu thm cc tin cht vo dch ln men thi c cc ks tng hp. Sd cc phn ng ho hc gn 6- APA vs dx R th c ks bn tng hp. tn Gc R1 Cc khng sinh t nhin 2-pentenyl-pennixilin(pen CH3-CH2-CH=CH-CH2F) COn-amyl-pen(dihydro-pen CH3-(CH2)4-COF) CH3-(CH2)6-COn-heptyl-pen(pen K) p-hdroxy-benzyl-pen(pen X) -OOC-CH(NH3+)(CH2)3-COpen N(D) iso-pen N(L) benzyl-pen(pen G) cc khang sinh tng hp phenoxy-metyl-pen(pen V) CH2=CH-CH-S-CH3COallyl=mercapto-metyl-pen cc khang sinh bn tng hp propicillin(bn ax,khng -lactam) 31

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ampicillin,dit G-,bn ax,nhy cm penicillinaza

cu 38: trnh by sx vtm B12 nh Ps.denitrificans v qu trnh tch chit tinh ch vtm B12 a-ln men sx vtm B12 sx vtm B12 chng nh Ps. L pp rt kinh t. S sx vtm B12 nh chng Ps. 32

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Ging ct gi=>mt A(hot ho ging 4 ngy,28C)=>(bnh nn 2l;0,6l mi trng;my lc)ging cp 1, 28C,48h(mt E)=>(2550l/40-48l,vkh=200rpm)ging cp 2,32C,25-30h;0,5VVM(mt E)=>(300l mt/500l tng,cp kh:0,5 VVM,khuy:150200rpm)ln men,32C,140-160h(mt P) Trong qu trnh ln men cn quan tm ti ho tan ca oxy,trong g u(20-30h) cn duy tr ho tan cao,n sang g sau li cn oxy thp(xp x 0) s c li cho sx. b-qu trnh tch chit tinh ch vtm B12 c nhiu bin thin trong qu trnh tch chit v tinh ch vtm B12 dch ln men Ps. + un nng 30 120C,lm lnh chnh pH v 8.5 +thm KCN,khuy 16h,25C +thm ZnCl2 200g +chnh pH 8 +thm cht tr lc 200g,khuy lc

Dch lc chit 3 ln vi 350ml hn hp cresol:CCl4(t l 1/2) Dch chit hu c 1 butanol 33 thm 500ml nChit 3 ln vi

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100ml nc Dch chit nc hp cresol: CCl4 t l 1/2 chit 3 ln vi hn

Dch chit hu c 2 thm 200ml axeton v 100ml ete, vtm B12 th ta xung VTM B12 th metanol nhm hot ho 4g Phn hp ph vs a.axetic 2% trong metanol,ta ete VTM B 12 kt tinh ho trong 10ml Sc k trn oxit

Cu 37:cng thc cu to v tc dng ca VTM B12,ln men sx vtm B12 nh P.shermannii Cng thc cu to: -cha khung tetrapyrrol.trong cobalanmin ti C1 ca ribose lk vs 5,6-dimetyl-benzinmidazol. Bn cnh CN- cha CO + trung

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tm. Thay CN- = cc nhm khc nhau,ta c cc dx khc nhau. Cc ng ng vtm B12 vs cc bazo d vng khc(cc purin,benzimidazol th) c cc vsv to ra t pht hay khi cho thm cc cht y vo mt. Tc dng ca vtm B12 C vai tr quan trng trong ti to tb,tb thn kinh, phc hi cc nhu m gan,tham gia to hng cu,vtm B12 tham gia tng hp DNA v 1 s cht khc: cholin metionin,c trong gan ,sa trng. Thiu vtm B12 c th b suy nhc ton b Vtm B12 hu nh k c c tnh. Trong iu tr c th dng vtm B12 : -iu trn mu c tnh: c th dng phi hp vs cc thuc khc(ax folic, ax ascorbic) -iu tr cc bnh v vim thn kinh.

Cu 32: ln men sx ,tinh ch ,tch chit streptomycin -ln men sx streptomycin: Trong LM sx streptomycin ging S,griseus c nui cy 2628C/48h. Ging cp 2,3 c nui trong 24h pH 6,5-7

35

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i vs chng S.gieus DTH -2 mt nhn ging v LM c cc thnh phn sau: Mt nhn ging: Glucoza 40g, dextrin 2g,bt u tng 20g,(NH4)2 SO4 5,5g,CaCO3 6,5g, NaCl 2g,KH2PO4 50mg,pH t nhin Mt ln men: Glucoza 50g,dextrin 10g,bt u tng 30g,(NH4)2SO4 9g,NaCl 2g,CaCO3 9g,cao ng 4g, KH2PO4 25mg,du u tng 7mg/l pH =7,2 Sau khi time ging chuyn Sng mt ln men v ln men 2628C,Ph 6,8-7,cp kh 1 VVM,khuy lin tc 4-7 ngy m. Tch chit: Sau khi ln men kt thc,tin hnh lc loi sinh khi. Thm ax oxalic,loi ion Ca2+,chnh pH v trung tnh ri em lc loi Ca(C2O4)2. S dng nha trao i ion cationit yu ang chu k Na+ hp ph streptomycin theo phn ng: R-COONa+ +(streptomycin) => R3(strep) + 3Na+ Nha trao i ion c th l Amberlite IRC 50, Woffatit CP300....sau khi ct bo ho dng ax HCl 1N phn hp ph streptomycin ra khi nha theo phn ng: R3(STREP) + 3 H+ => 3ROO-H + strep Phn on chnh c c chn k.dch c c c cho thm metanol laoij NaCl i dng ta ri m lc. Thm CaCl2 vo cha metanol v mui kp str.clohydratcanxiclorid kt tinh s c em lc. T mui kp cho Streptomycin c cho tc dng vs H2SO4 to mui sunfat. Sau khi tu mu v c li n nhit cn thit th tin hnh lc v trng. Dch lc v trng c em ng kh. Sp thu c t tiu chun dc im.

Cu 25: ng dng my tnh in t trong ln men sx khng sinh. 36

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Trong LM sx ks sd my tnh in t trong 2 linh vc: +ti u ho vs s tr gip ca my tnh:tnh ton cc m hnh kt hp x l cc kt qu,x td cu cc tham s ln qt LM +kim sot,iu khin qt LM :trong cng ngh LM cc nc tin tin kt ni my tnh iin t on-line LM tr thnh hin thc

CU 27 trnh by con ng sinh tng hp v iu khin sx pen trong P.chrysogennum

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Vng- lactam-thiazilidin ca pen c xd t L-cistein v L-valin. Con ng sinh tonhr hp bt u t ngoi ribosome t L--AAA to dipeptid vs L-xistein ri qua tripeptid vs L-valin m tip tc, sau 1 s bc kt hp ng vng c th tch c isopen N. Pen G c to thnh khi L--AAA i sang nhm phenyl axetic. Cn 6-APA k pai l sp trung gian m ch do thiu tin cht m cha c loi ra.

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