nuoi_cay_te_bao

8/3/2019 Nuoi_cay_te_bao

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K thut nui cy t bo 2011

ng dng k thut nui cy trong phngth nghim.Tc Gi : L Hong DuyDanh mc :

Chng 1 : S ch cc cht cn thit cho nuicy.Chng 2 : Hng dn nui cy Vi khun.Chng 3 : Hng dn nui cy t bo thc vt.Chng 4 : Hng dn nui cy t bo ng vtv ng vt hu nh.

Li Ng :

Ti liu tham kho t 1 cun sch chuyn nghnh sinh hc xut bn nm 1972, ti liu thamkho thm cho sinh vin chuyn nghnh cng ngh sinh hc. Ti liu ny khng phi ti liu xtnghim ch tp trung vo kin thc nui cy t vo v vi sinh vt, kin thc c bn cho cngngh t bo gc sau ny, nhn chung cng ngh hin nay s dng my mc gim gnhnng cho cc nh nghin cu nhng cch thc nui cy vn khng khc g nhng nm u

ca cng ngh sinh hc.Chng 1 : S ch cc cht cn thit cho nui cy.Dung dch Hanks

m gp 10 ln.Cch pha :

Khotien.vn | Ti liu c vit bi L Hong Duy 1


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Pha 2 dung dch ring A v B ring r sau kt hp li :

Dung dch A :NaCl : 80g.

KCl : 40g.

7H2O + MgSO4 : 1g.

MaCl2. .6H2O : 1g.

CaCl2 : 1,4g

Nc ct 2 ln 500ml.

Dung dch B :Na2HPO4.2H2O : 0,75g

Na2HPO4.12H2O : 1,5g.

KH2PO4 : 0,6g.

Glucoz : 10g

Nc ct 2 ln 480 ml.

** Pha 2 dung dch mui nh trn sau dung dch A vo dung dch B mt cch t t va va khuy. Cui cng c mi lt dung dch cho thm 20 ml dung dch 1% phenol. Lc qualc giy, lm tit trng th c 2 cch :

+ Cch th 1 l cch tt nht l lc qua Seitz (lc ngn vi khun).

+ Cch 2 sy c 100 C trong 20 pht, sy 3 ln trong 3 ngy lin tip. Nhng trongtrng hp ny CaCl2 phi pha ring vo cho thm sau khi sy, trc khi dng.

Tit trng xong th ng vo l 500-1000 ml y kn bng nt cao su. Th v trngbng cch cy 0,5 ml dung dch vo mi trng canh thang tht pepton v thchSabouraud.

Bo qun dung dch m trong t lnh c th bo qun c n 6 thng. Trc khi

dng em pha long 10 ln vi nc ct 2 ln v trng. Dung dch lm vic em sy100 C, 10 pht vi p sut 0,7 at , iu chnh PH bngng dung dch bica 1,4% cPH = 7,2 7,4.

C 100 ml dung dch cho 2,5 3 ml.

Ch :+ Dung dch lm vic trong phng khng qu 1 thng.



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+ Cha pha bica c th s dng 6 thng.

Bica l tn vit tt : Bicacbonat Na, Bicacbonat Ca, Bicacbonat Mg....Dung dch Earle

Thnh phn dung dch chun c 10 ln trong 1 lt nh sau :

NaCl : 68g.

KCl : 4g.

CaCl2 : 2g.

MgSO4.7H2O : 2g.

NaH2PO4.7H2O : 1,25g.

Glucoz : 10g.

Phenol : 0,2g.

Ha tan cc thnh phn mi theo trt t k trn trong nc ct 2 ln. Ring CaCl2 thphi pha ring v cho vo sau va cho va trn lc qua lc Seitz. Kh v trng y ntcao su. Bo qun bung lnh. Trc khi pha long 10 ln vi nc ct 2 ln. Lc liln th 2. iu chnh PH c PH = 7,4 7,5 bng cch cho thm vo 4 4,4 ml dungdch 5% Bica vo 100 ml dung dch mui. Nu cn th cho gim PH th cho lun kh CO2i qua sau khi kh ny c i qua bnh cha axit H2SO4 hay i qua lc bng.

Dung dch m FotFatTy theo yu cu c th ca cng tc c th cn c th ca cng tc c th cn ndung dch m Fotfat cha cc ion Ca v Mg.

Cch pha 1 lt dung dch ny c 10 ln nh sau :

Dung dch A :NaCl : 80g

KCl : 2g

CaCl2 : 80g.KCl : 2g

CaCl2 : 1g

MgCl2.6H2O : 1g

Nc ct 2 ln 1000 ml.



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Dung dch B :

Na2HPO4.12H2O : 28,98g

KH2PO4 : 2g

Nc ct 2 ln : 10 ml

Trc khi dng c 100ml dung dch A cho thm 800 ml nc ct 2 ln sau cho thm100 ml dung dch B. Lc qua lc Seitz v gi t lnh.

Nu trong dung dch m Fotfat khng cn cc Ion Ca v Mg th cch pha nh sau :

NaCl : 8g.

KCl : 0,2g

Na2HPO4.12H2O : 2,9g

KH2PO4 : 0,2g

Nc ct 2 ln cho y 1 lt sy v trng 110 trong 30 pht c th gi c nhit phng.

Dung dch TrypsinHa tan 1g Trypsin Difco trong 100ml dung dch p Fotfat. Lc qua lc giy hay lcbng sau lc qua lc Seitz th v trng bng cc cy vo canh thang tht Pepton vthch Sabouraud.

trng thi ng bng (-20 C) dung dch Trypsin c th bo qun c my thng + 4 C, ch gi c vi tun l. Trc khi dng em pha long 4 ln vi dung dchm Fotfat hay dung dch Hanks v trng c nng cui cng l 0,25%.

Dung dch Vecxen(Vecxen l mui ca axit etylen diamin tetra-axetic)

iu ch dung dch 0,02% ngi ta cy 0,5g Vecxen ha tan trong 2500ml dung dchm Fotfat khng cha Ion Ca v Mg rt vo l 50ml, sy v trng di p sut 1 at, 30pht. Bo qun 4 C trong thi gian 1 -2 thng trnh nh sng.

Dung dch Phenol 1%

Trc ht cha 100ml 1N NaOH bng cch trn 10ml dung dch NaOH bo ha(li tmdung dch NaOH bo ha b ht cn) vi 90 ml nc ct 2 ln. Sau c 10g Phenol ha tan trong cn cho thm 20ml dung dch 1N NaOH lc u lng my pht.Ht ht nc trong bn trn cho vo ng o lng 1 lt v cho thm vo ch cn 10ml 1N NaOH li lc v ht ly nc trong hp vo ng ong, c tip tc nh th chon khi thuc Phenol tan ht. Tuy nhin 1N NaOH dng ha tan 10g Phenol



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khng c qu 70ml v nu s lng NaOH nhiu qu th dung dch c mu thmkhng dng c vo nui cy t bo.

Dung dch trung tnhMun ch dung dch 0,1% trung tnh ngi ta ha tan 0,1g thuc vo 100ml cn Etyl

60 dung dch ny dng nhum ng t bo m v pht hin Plec .

Dung dch Cristal Violet(xem thm)Dung dch ny cng dng nhum sng t bo m trong khi lm Trypsin ha m.Pha 0,1% dung dch Cristal Violet trong 0,1N axit Nitric.

To Huyt Thanhng dng dng thm vo mi trng dinh dng 1 lng nht nh t 5 10% tbo d bm vo b mt thy tinh

iu ch : Ly mu ca ng vt thng l ca B, Nga & ca ngi.....

Cho vo l rng c trong t m 20 30 C cho mu ng, sau dng que tchmu ng ra khi l, t l vo t lnh +4 C trong 1 2 ngy.

Cht huyt thanh li tm 2000 vng/pht trong 20 pht lc ly huyt thanh qua lc Seitzrt vo l hay ng v y nm cao su, th v trng v bo qun t t 4 20 C.

Ty mc ch s dng c th dng ti hay un 50 C trn 30 pht trc khi dngcn th c tnh i vi t bo.

Cht lm nh haLin quan n cht bo chia nh cht bo...

Dung dch Glyxerin 15% trong H2O huyt thanh nga bnh thng khng cha cht boqun, dung dch Sacaroz 10% vi Gelatinh 1% c PH 6,8 7 hoc cc dung dch chaglucoz, lactoz c 10 30% sa.

Thy phn t inh btNguyn l : Dng(H2SO4 hoc HCl) vi p lc d. Dch thy phn thu c trung habng Na2CO3 hoc NaOH. Nu dng H2SO4 lm tc nhn thy phn th c th dng

CaCO3

hoc nc vi trung ha, sau lc qua lc p khung bn vi than hot tnh kh mu : Dch thy phn thng l glucoza, 1 lng nh aa, khon cht c th emlm c c li 60-70% s dng dn.

Dng enzim : Bt go, bt ng tch phi men amilaza hay Amilaza, Bt utng l 1 ngun Nit k thut cha 40% Protein 19% cht bo v cc Axit amin.

Cch lm cao ng Bp

http://en.wikipedia.org/wiki/Crystal_violethttp://en.wikipedia.org/wiki/Crystal_violet


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Cch lm cao ng thng c mu nu sm 150 kg ht khng mt khng mc, ra schngm vi 220l nc, my t l nc bng 1:1,3 thm 2 lt HCl 30% v 1,2 kg Na2SO3 lm dch ngm c PH = 3 v nng SO2 c 0,25% ngm 48 gi 50 55 C.

Sau khi kt thc dch ngm c gia nhit ti 75 80 C dng sa v lc sch, chnhPH 8 8,2 lng 30 pht cho protein kt ta. Lc qua lc p khung bn v ra b bngnc nng. Dch lc v nc ra c iu chnh PH bng HCl ti 5 v c chn khng 60 C ti 28 b.

Thnh phn lp to ph dng lm phng php plec :1. Dung dch Earcle c 10 ln khng cha 18 ml Phenol v Bica.

2. Nc ct v trng : 60 ml

3. Huyt thanh b hoc kh 3,6 ml

4. NaHCO2 (7,5%) : 5,4 ml

5. trung tnh (1:1000) : 3 ml

6. Penixilin : 18.000 n v

7. Streptomixin : 18.000 n v

Thch ph ny m bo t bo sng c 8 12 ngy

2% thch

Mi trng dinh dng dung dch Gey A c 10 ln 20%

Dung dch Gey B c 10 ln 10%

Tris 0,2 M, PH = 7,6 c 10 ln 10%

5% Lactoalbumin Hydrolisat 10%

Cha 10% huyt thanh b

trung tnh 1:1000

Nc ct 30%

Dung dch Gey BNaCl : 8g

KCl : 0,375g



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CaCl2 : 0,275g

MgCl2 : 0,21g

Na2HPO4 : 0,15g

KH2PO4 : 0,025g

Glucoza : 2g

NaHCO3 : 0,25g

Nc ct 2 ln 1000ml c thm Phenol lm cht ch th mu, bo qun nhit +4 C thi gian 1 thng.

Tch Enzim dng mui trung tnh kt ta l Sunfat amon [(NH4)2SO4] t l 50 60 % dng(Etanol, Izopropanol v axeton) chit nc nm men cho 3-4 th tch cn vo 1 th tchnc chit enzim trch gim hot tch enzim cn lnh xung 3 5 C khuy mnh, khi

enzim kt ta lng xung cn li tam cn ra n 2 3 ln ca cho vo bnh ht m hocmy sy chn kh6ong sn phm thu c dng bt.

Chng 2 : Nui cy vi khun.Tm tt ni dung chng 2 : bi ny hng dn nui cy nhng vi khun c li, v ng dngvo cuc sng, cch nui cy vi khun khc cng tng t cc bn tm hiu thm ti trnghoc hi nhng ngi c chuyn mn.

Bng vi khun v ng dng ca vi khun.Vi sinh vt sn sinh Bn cht sn phm trao i cht Hot tnh Dc hc

Aerobacter cloal Amanitamuscaria v cc nm khc.

Polisacarit Muscarin. Chng vim tng phgiao cm.

Bacillus anthracis. Dch lc th nc chit. Ging Eponefrin hoattnh tim mch.

Bacillus licheniforms. Nc chit th Ging ACTHBacillus subtilis. // //

Cc loi Claviceps. Ecgotalcaloit Phong b adreneric

kch thch c trn hottnh CNSCorticium caeruleum. Ruglovasin A & B H huyt p.Fusarium oxysporum. Axit Fusaric Caxit5-butylpicolinic H huyt p.Nhiu loi Fusarium. Nc chit th. Gy bun nn.

Gibbrella Zea Zlagenin Gy h protein, gyng dc.

Zearalenon Gy ng dc.Lactobacillus leichmannii Nc chit th. Ging ACTH.



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Lenzites trabea Rugulovasin A & B H huyt pLentinus Edodes Lentysin Gim lipit mu.Lenzites trabea Rugulovasin A & B H huyt p.

Naematoloma fasciculare Naemototin Gim ng mch vnhOospora-astringenes Oosponals oospo lacton, oospong

lycolCo c.

Oudemansiella radicata Oudenon H huyt p.Nhiu loi Panaeolus Serotonin 5 hydroxytriptophan Co mch.

Pellicularia filamentosa Rugulovasin A & B H huyt p.Penicillium convor ruglosum Rugulovasin A & B H huyt p.Penicillium corylophiloides Rugulovasin A & B H huyt p.Penicilium purpurogenum Rugulovasin A & B H huyt p

Penicilium Rugulosum // //Phialocepphala repens Phialoxin Chng ng.

Psilocybe mexicana v ccnm khc

Psiloxin Tng hot ng tmthn.

Rhizoc tonia leguminicola Slaframin Tng ph giao cm.

Salmoella enteriditis Nc chit th. Ging ACTH.Salmonella tiphi // //Serratia marcescens // //Nhiu loi Serratia Proteaza(TSP) Chng vim.

Streptomyces allbire ticuli Leupeptin Chng Plaxmin, chngvim c ch, cc men

Trypsin Papainmenchng ng mu v

cc men khc.Streptomyces argenteolus

Var.toyonakensisPepstatin c ch Pepxin.

Treptomyces cacaoi

Varasoensis

Proteinnaza trung tn. Chng vim.

Treptomyces caespitosus Pepstatin c ch pepsinTreptomyces chartreusis Leupeptin Nh stm.albireticuliStreptomyces grioseolus Proteinnaza trung tn Chng vim.

Streptomyces griseus proteaza Ging InsulinStreptomyces hygroc picus Chimostatin. c ch chimotripsinStreptomyces kinoluteus Kinonaza B1 Proteinnaza Chng vimStreptomyces lavendulae Chimostatin Leupeptin c ch chimotripsin

nh stm.albireticuliStreptomyces nigrifaciens

Var.FFD-101Nigrifactin Chng hixtamin

Streptomyces : noboritoensis Leupeptin Nh stm.albiraticuli

Streptomyces :roseochromogenes

// //

Streptomyces roseus Leupeptin Nh stm.albiraticuliStreptomyces testaceus Leupeptin c ch PeptinStreptomyces thioluteus Leupeptin Nh stm.albiraticuli

Streptomyces VerticillatusVar Zynogenes

Retikinonaza I & II Proteinaza trungtin.

Chng Vim.

Streptomyces EF-44-201 4 thnh phn S-PI c ch pepsin



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Cc loi streptomyces Enzim Gim nhm Protein A& B ca mu.

Zygosporiu Masonii Zygosporin A Chng VimZygosporium Mycophilum Zygosporin A Chng VimPenicillium chrysogenum Penixylin iu tr cc bnh

nhim trng do vikhun, nm hoc

virus.Streptomyces Erythryus Tetraxiclin //Streptomyces Griseus Streptomyxin //Striptomyces erythreus Eritromixin //

Penixillium patulum Grizeofulvin //Clavileps purpurea Alcaloit ca nm ca g nh :

Ecgotamin, EcgotoxinTrong khoa sn (gipvo vic co t cung)v iu tr cc bnh

mch mu, bnh nhtu 1 bn.

Propionibacterium Shermanii Vitamin(B12) corinoit iu tr thiu mu ctnh.

Ca1v loi vi khun x khunv nm.

Steroit c cu trc c thay i 1cch c hiu nh vi sinh vt.

Ch phm hoocmonV d km hm srng trng iu trvim v thp khp.

Acetobacter subboxy dans L-socboza (Tin cht tng hpaxit ascorbic)

Tin cht ca VitaminC

Dextran Cht thay th huyt tng. Leuconostoemesenteroides

Aspergllus niger Enzim Chun on lm snha sinh.

Penixilium chryso genum Glucozooxydaza Chun on bnh ing.Escherichia coli Asparaginaza Kim hm khi u v

bnh bch cu.Streppiococcus spec Streptokinaza iu tr bnh mch

mu.Bacillus subtilis Penixilinaza Loi tr PenixilinRhizpus spec. Lipaza. Thuc tr tiu ha.

Mi trng nui dng khng sinhPenixi l in

Cc chng Penixi l in :Khotien.vn | Ti liu c vit bi L Hong Duy 9


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Penicyllum, Aspergllus Penicillum notatum, Pichrysogenum.

Mi trng nui cy :30g Lactoz k thut, 3g NaNO3, o,5g KH2PO4, 0,25g MgSO4

0,2g C6H5Ch2CooH, 0,044g ZnSO4

Nc ct va 1 lt dng NaOH iu chnh PH ti 5,5

Cy chm gam/l t :Cao ng : 2

Glucoz : 2

Lactoz : 0,5

Nitrat amol : 0,125

Sunfat Magie : 0,025

Sunfat Nitrat : 0,05

Kali Photohat monobazic : 0,2

CaCO3 : 0,5

TreptomixinMi trng :C glucoz , bt u tng, cao ng, ngoi ra cn c mui Amon, photphat v CaCO3 iuchnh PH.

Giberelin



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Mi trng :

Sacaroz : 40 60

Tatarat amon : 7

KH2PO4 : 2

MgSO4.7H2O : 0,2

Hn hp cha cc nguyn t vi lng PH = 5,5 thm 0,5 Cao ng.

Eremothecium a shbyii v Ashbya gossypii

Gii thiu chng vi khun to ra Ribolavin (hay cn gi l B12).



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E.ashbyii c khun ti dng si kch tht 2 4 x 60 80 Micromet. Khun ti c cu tophn nhnh. Ging ny sinh sn v tnh bng bo t, c hnh 1 u 1 u nh mi bo tnang cha 8 10 bo t ny mm pht trin thnh khun ti.

Trn mi trng thch E.ashbii cn non c mu trng dn sang mu vng gi hn c th bphn hy, c mu vng lc hunh quang (5 10 ngy cy 1 ln). Ashbya gosspii sng ksinh n bng, cafe, 1 s cy khc, cu to c hnh thoi 1 u kh di trnh nh nng mttri nhit pht trin 28 C.

Mi trng E.ashbyii :

B ma : 130 /gam.

Kh ht bng : 1000

Kh u tng : 2200

Kh lc : 3500.

Mm i mch : 6000

Mm thc : 5000

B u : 3000

C 5% ng vng, 3% dch thy phn protein cha ti Pepton 1% phi m, 0,3% cao tht,0,3 % cao tht, 0,3% KH2PO4, 0,25% NaCl v nc ct, PH = 6, 7 ngy cy 1 ln nhit =28 C.

TetraxyclinCn gi l Tetraxin, steclin, acromixin, polixiclin to bng phng php ha hcClotetraxylin hoc nui cy x khun Str.aureofaciens.

Mi trng nui cy :Sacacroza : 2%

Cao ng : 0,24

Bt u tng : 2

CaCO3 : 0,4

NaCl : 0,5

R ng : 0,2 .

Phng php chit ly cc cht t vi nm :Khotien.vn | Ti liu c vit bi L Hong Duy 12


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Phng php nui cy ring r cc khun lc trn mi trng rn l gelatin hoc thch(agar - agar)

Penixilin nh mc mu xanh lc Penicillium notatum.

i vi sn phm l cht ha tan trong dung dch nui cy th phi li tm tch ly phndch ri cho kt ta, lm sch, c th cho kt tinh tr li hoc sn phm nm trong tbo th phi ph v v t bo ri mi tin hnh tch v lm sch tip theo pectinazaphn hy pectin

S chuyn ha Steroit nh to thnh cortizol s oxi ha khng hon ton nh s tothnh axit axetic v sobboza sn xut axit ascorbic (Vitamin C), nhng thnh tu caqu trnh ny c ng dng trong sn xut Cortizon, axit asscorbic(Vitamin C), axitglucomic tin cht ca Aphidrin v cc cht iu tr cc chng vim khp.

1 s loi Vi khun c li cho cng mi trng :

Vi khun dng du m lm thc n ng dng dng phn hy du mhay cc vt du loang .. . .Achromabater, Alkigenes,Bacillus, Bacterium, Corynerbacterium,Flavobacterium,Micrococcus, Pseudomonas, Vibria....

Chng nm men, nm mc :Acremonium, Aspergilus, Candida, Cyfromyces, Debaryomyces, Endomyces, Hansenulamonilia, Penicillium, Scopuluriosis, Torulopsis.

Khun Ty x khun :Micromonospora , Mycobacterium....

Cc chng s dng Hydrocacbua :Candida lipolytica, Cointermedias, C.Tropicalis, C.Parasilops, S.guilliermondii.....

Tn cc loi vi khun gy bnh:Ph cu : Pneumococcus.

Lu cu : Conococcus.

Mn no cu : Miningococcus.

Bruxela (Brucella).

Micobacterium bn vi axit .

Trc khun than : Bacillus anthracis.



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Vi khun him kh gy bnh trc khun ng rut.

Cc vi khun influenxa

Strepto v Staphylococcus

T cu khun : Staphylococcus.

...............................

1 s cch t bin c bn :

Treptomixin l x khun Streptomyces griseus cn gi l Actinomyces Streptomixin tbin nh tia t ngoi.

Polysacarit ca sinh vt sinh ra ta thy c dextran trng hp t cc gc glucoza, Levantrng hp t Fructoza nh vi khun Aerobacter Levanicum v Pulalan trng hp t Maltozanh loi nm tng t nm men l Pullularia Pullulans.

Dextran l Plysacarit do vi khun lactic c phn t lng l 15000 50000000 lin kt giacc gc glucoza vi nhau bng mi 1,6 glucorit vi khun sinh ra Leuconostocmesenteroides.

t bin : Hp cht cha Nit nh Nitrozomety guanidin metyldicloroety lamin, nitrix,Hydroxylamin hay EtylametanSunfotnat. Inozit ru mch vng 6 nguyn t.

Chng 3 : Hng dn nui cy t bo thc vt.V chng ny cng nh v VN cch vin sinh hc VN u lm c ht nn ti cng chnhc li i nt v hoocmon Thc vt.

Hoocmon thc vt gm c : Auxin, Giberilin, Xitokin kch thch m non phn ha thnh cycon.

Cch thc : Trc ht dng 1 t hp ph mn xenlulo bng Trypxin thnh t bo trn sau nui trong mi trng dinh dng tip n x l hoocmon thnh cy con.

Hin nay trn nt c rt nhiu ti liu v hoocmon thc vt

GIi thiu cc cht trong thnh phn : Giberelin c tm thy trong vic ph ng cc ht ging c tc dng kch thch ny

mm hnh thnh bng con ng ln men (lt b v ht cho ln men) c phn lp tr cn la Von v 1 s khc, ging vi khun Fusariummonilifome, Fusarium OxySprumc tt c 38 loi :

+ A1 : C19H24O6



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+ A2 : C19H26O6

+ A3 : C19H22O6

+ A4 : C19H24O5

.......

A12 : C20H28O4........ A38 : C20H26O6.

Giberelinthc qu xanh chng chn v cm ng ra hoa cy da

Trn chng nui vi khun c gii thiu cu trc.

Auxin l hp cht ca -indolaxetic cha trong b phn sinh sn ca cy ci tham giavo qu trnh sinh trng ny mm ca ht ging hnh nh minh ha :

Chc nng : Auxin c ch ht ny mm v kch thch s rng l. Xitokin (Cytokin ) l cht c chit sut trong t bo bch cu ca ng vt hay

nha cy thc vt t Limphokin



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Chc nng : Kch thch ra r ca cnh gim (chit) v kch thch thu tinh to ht

Etilen : Nui cy t bo v m thc vt (nhn ging v tnh) v kch thch sinh trng

ca chi non

Axit Abscisic Pha ng cho mm ht, c khoai ty v to qu khng ht

Chng 4 : Hng dn nui cy t bo ng vtv ng vt hu nh.Tinh ch Phi :

Tinh ch m phi c dng rng ri nht cho thm vo mi trng nui t bo

+ Phi g c trng p 9-11 ngy ly phi ct b mt, ,m, chn, cnh, ra bng dung dchhanks trong 1 ci cc, ri dng ko ct tht nh hay nghin nh cho vo ng ri cho thmvo 1 th tch bng y dung dch hanks cha 100 n v trong 1ml ca Penixilin vSterptomyxin. Li tm 30 pht, ht ly nc trong trn l tinh ch phi g 50% th v trngnh thng l, bo qun 12-20 C trong ng y nt cao su, trc khi dng phi lmtan chy li tm 30 pht, tc 2000 3000 vng/ pht, ht ly nc trong.

i vi phi ngi hay cc ng vt khc cng tin hnh tng t ch khc l ngm m nghin trong dung dch hanks 30 pht trong t m 37 trc khi li tm.

Mi trng dinh dng5% Lactoalbumin hydrolysat. Gi tt l LH

y l mi trng, ph cp nht dng nui t bo thnh phn gm c :

- Lactoalbumin Hydrolysat : 5g



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- NaCl : 0,85g

- Nc ct 2 ln : 1000ml

Trc tin l ha tan NaCl vo nc ct. Sau cho Lactoal bumin Hydrolysat vo lc qualc Seitz rt vo l, y nt cao su hn Parafin (l sp nn hay sp n cy), th v trng

bng cch cy vo canh than tht Pepton v thch Saboureaud.

Lacoalbumin Hydrolisat thng c dng nng 0,5% nn trc khi dng phi phalong dung dch m ra 10 ln vi dung dch hanks hay dung dch Earle.

Mi trng Parker cn gi l (199)y l mi trng tng hp thng c dng dng t bo sau khi gy nhim virus

iu ch mi trng Parker c 10 ln :- Chun b cc dung dch ring r :

+ Dung dch 1 t nh bng gam cho 1 l t mi trngL - acginin : 0,7g

L - histidin : 0,2g

L Lizin : 0,7g

Dl Triptophan : 0,2g

Dl phenilalanin : 0,5g

Dl metionin : 0,3g

Dl axit glutamic : 1,5g

Dl serin : 0,5g

Dl treonin : 0,6g

Dl lxin : 1,2g

Dl izolxin : 0,4g

Dl valin : 0,5g

L Hydroxyprolin : 0,1g

Glyxin : 0,5g

Dl axit asparaginic : 0,6g

Dl alfa alanin : 0,5g (dl - alanin)



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L prodin : 0,4g

L glutamin : 1g

Sodium axetat : 0,5g

Cc axit amin ny cng vi sodium axetat c c sn v hn hp dng bt v scho vo mi trng theo trt t s ni sau .

+ Dung dch 2 :Ha 400mg L lirozin v 200mg L-xistin trong 200mg 0,075N HCl un nng ln ti 85 90 C. Lc mnh khng si, sau dung dch vo ni un cch thy nhit 37 C thnh thong lc cho tan hon ton.

+ Dung dch 3 :Axit Nicotinic : 25mg

Amit axit nicotic : 25mg

Piridioxin Hydroclorit : 25mg

Tiamin Hydroclorit : 10mg

Ribolavin : 10mg

Calcium Pantotenat : 10 mg

L Inozit : 50mg

Axit para aminobenzoic : 50 mg

Cholin Clorit : 500mg

Ha tan tt c trong 175 ml nc ct 2 ln, lc mnh v khuy bng que thy tinh chonc ct 2 ln thm cho 200ml. Rt ra mi ng 2ml v bo qun - 20 C.

+ Dung dch 4 :S b ch 2 dung dch vi 2 thnh phn nh sau

A :L glutation : 10 mg

L Sytin Hydroclorit : 20 mg

Axit Ascocbic : 10 mg

Ha tan trong 100 ml nc ct 2 ln.



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B :Vitamin A : 20 mg

Cn etyl (95%) 2 ml

Tween 80 (dung dch 5% trong 20ml nc )Vitamin A ha tan trong cn etyl sau cho thm tween 80 (axit oleic)

Trn 2 dung dch A v B cho nc ct 2 ln thm vo cho 200 ml v cho thm vocho 200ml v cho thm 1g Adeno Zintrifotfat Sodium (ATP)

Ch xong rt dung dch vo ng 10ml v bo qun -20 C

+ Dung dch 5 :Ha 100mg biotin trong 75ml, nc ct 2 ln cho thm 1ml dung dch 1N HCl bo

qun c bn hn cho nc ct 2 ln vo cho 100ml rt ra ng 1ml v bo qun-20 C

+ Dung dch 6 :Ha 10mg axit folic (tinh th) trong 100ml dung dch Hanks, rt ra ng 1ml v bo qun - 20 C

+ Dung dch 7 :Ha 20mg Vitamin D2 (Calciferol) , trong 40ml cn etyl 90% cho thm 40mg Cholesterin.

lm tan Cholesterin phi lc mnh v khuy bng que. Sau cho thm 60ml dung

dch 5% Tween 80 rt ra ng tng 5ml mt v bo qun -20 C.

+ Dung dch 8 :10 mg Vitamin E (alfatocaferol fotfat)

Ha tan trong 100ml nc ct 2 ln rt ra 2 ng tng 1 ml v bo qun - 20 C.

+ Dung dch 9 :10 mg Vitamin K ha trong 100ml nc ct 2 ln, lc mnh v t v ni cch ty 37 cho n khi tan hon ton. Rot ra tng 1 ml v bo qun - 20 C.

+ Dung dch 12

Ha 18 mg Guanin (Chlorit) trong 167 ml nc ct 2 ln cho thm 0,5 ml NH4OH mc v cho nc ct 2 ln thm vo cho 180 ml. un si ngui v li cho thmnc ct cho 180ml.

+ Dung dch 13 :Khotien.vn | Ti liu c vit bi L Hong Duy 19


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Ha 100 mg d-riboza v 100mg dezoxyriboza trong 100 ml nc ct 2 ln. Rt tng5 ml 1 vo ng v bo qun - 20 C

+ Dung dch 14 :Ha 100 mg nc ct 2 ln ha tan 40 mg axit adenilic : Rt ra ng tng 5ml mt v

bo qun -20 C.

+ Dung dch 15 :Ha tan 10 mg Fe(NO3)3.9H2O trong 100 ml nc ct 2 ln. Cho thm 1 git HNO3m c. Rt ra ng tng 2ml mt v bo qun -20 C.

Ch mi trng 199 c 10 ln trong dung dch Hanksa. Ha tan 80 g NaCl trong 200 ml dung dch 2 cho thm 30 ml dung dch 12 v 30 ml

dung dch 13. Ha tan vo trong hn hp ny 4g HCl v sau cho 2gMgSO4.7H2O

b. Trong 1 bnh khc ha tan 0,6g KH2PO4 trong 55 ml nc ct 2 ln. Ha tan thmv 0,6g Na2HPO4 (khng ngm nc) hay 1,52g Na2HPO4.12H2O v cho thmnc va 111ml. Dung dich va mi ch ny em trn vi dung dch ch ima.

c. Ha tan ring bit 10g Glucoza (Dextroz khng ngm nc) trong 55 ml nc ct2 ln, cho thm vo y 20ml dung dch 1% phenol v trn vi hn hp va chbn kia.

d. Trong 1 bnh ring ha tan ha tan 1,4g CaCl2 khng ngm H2O trong 60ml 2 lndung dch ny em rt t t vo hn hp trn, va rt va lc mnh phngkt ta Ca3(PO4)2 khng tan.

e. Cho hn hp va ch vo nhng dung dch ch sn di y :

Dung dch 3 2mlDung dch 5 1mlDung dch 6 1mlDung dch 7 5mlDung dch 8 1ml

Dung dch 9 1mlDung dch 13 5mlDung dch 14 5mlDung dch 15 2mlNu nhng dung dch ny rt sn vo ng vi nhng khi lng ni trn th saukhi rt vo hn hp phi trn k ng.



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f. Cho dung dch 10 vo hn hp v cho thm nc ct va 945ml

g. Cho vo hn hp ny nhng axit amin v Natri axetat vi khi lng ni trongn ca dung dch 1 trn cn thn cho n khi cc tinh th tan gn ht.

h. Cho thm 5ml hn hp Penixillin v Streptomixin cha mi th 20000 n v trong1ml

i. Hn hp va ch trn t lnh 1 m.

j. Cho thm 10ml dung dch 4 v nc ct 2 ln va 1000ml v trn cn thn.

Mi trng c 10 ln va ch trn em lc Seits vi p sut dng, th v trng, rtvo chai v bo qun 4 C.

Khi s dng pha long 10 ln vi nc ct 2 ln.

Mi trng Eagle (Ign)Sau khi nghin cu iu kin nui cy t bo ng vt b sa Eagle i n kt lun l a sloi t bo cn 13 axit amin, 8 sinh t (vitamin) Glucoz v 5 ion (Na, K, Ca, Mg, Fotfat,Clorat )v ngh 1 mi trng t phc tp hn so vi mi trng 199.

Cch ch : thng ch ring tng dung dch v ch kt hp li trc khi dng. Dung dch 1 : Dng dung dch hanks khng cha Glucoz v Bicacbonat Natri thiu 2

cht ny c th lm v trng bng Sy.

ch 1000 ml mi trng nui cy cha 10% huyt thanh nga. Trc khi dngngi ta trn 84ml dung dch 1 vi 6,1 ml dung dch m v 10 ml huyt thanh nga sau cho thm dung dch 7 (Natri Bicacbonat) a PH = 7,2 7,4, Mi trng ch xongc th bo qun my ngy trong t lnh, tuy nhin 1 s lin kt khng bn c th b phhy nhanh hn.

Nui cy t bo lng biMi trng : Eagle cha 0,5% Lactoalbumin , khi t bo bt u mc th thay mitrng mi.

Phng php tch ADNCy t bo ln mi trng thch Hottinger. Sau 18 gi chuyn sang bnh cu cha 200ml mitrng Spizizen v canh thang Hottiger vi t l 2:1 . bnh ny trong bung m 37 C l lclin tc, sau 18 gi tui cy truyn sang 16 bnh cu khc c cha 200ml mi trng trn.Nhng bnh ny cng nui trong 19h. Sau c c t bo bng cch li tm 2,5 300 vng /pht trong 30 pht v ra 2 ln bng dung dch sinh l (0,14M NaCl + 0,014 Xitrat Na).



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Bt u tch ADN bng s ph hy t bo bng lizozim nguyn sinh cht ph hy bngDodesysunfat 1%, dng phenol tch Protein, lc mnh v trong 1,5 gi, sau em litm v dng Pipet ht ly lp trn. Cho thm v ete vi th tch tng ng.

Lc mnh v li tm 5 pht tc 2000 vng / pht, ADN nm lp di bng cch y ta tch ADN khi phenol v Protein lp trn b i v cho vo cn Etylic kt ta ADN dnga thy tinh trn ADN.

Ph hy ARN bng ARN AZA (50 /ml) trc em un ARN aza trong ni cch thy 90 C ph hy ADN- aza ha tan trong 0,15 M NaCl, vi ARN- aza trong 30 pht nhit 37 C sau x l ADN bng 80% Phenol nhit 37 C trong 30 pht v li tm 10 pht.

Hai ln x l bng etylic, ADN bo qun trong cn 70 t lnh.

Nng ADN

ADN (/ml) = ((E270 E290).10,1a )/0,19

E : quang hc.

a: long.

Bo qun sinh vt bng phng php ng lnh -15 C : 20 C.

-20 C : - 30 C ......

tng kh nng sng st ca cn thm nhng cht nh ha cht keo bo v hn dch nh :sa, huyt thanh, lng trng trng, Pepton muxin hay cc loi ng, cht lm nh ha l dungdch glyxerin 15% trong nc huyt thanh nga thanh nga bnh thng khng cha cht bo

qun, dung dch Saccaroza 10% vi Gelatin 1% c PH 6,8 7 hoc cc dung dch chaGlucoza , Lactoza c 10 30% sa...

13 axit amin : Glutamin, Lxin, Izolxin, Valin, Fenillamin, Alginin, Lizin, Hystidin, Triptophan,Metionin, Treonin, Xistin, Tirozin.

Sinh t B (Vitamin)Tiamin, Calci Umpatotenat, axit Nicotinic, Biotin, cholin, axit-folic, Piridoxal.

T bo s mc tt trong sinh t B di dng Coenzin : Adenozindifotfat (ADP), Adnozintrifotfat(ATP), Coenzim A.....

Dung dch bo qun m phiCho mu m vo bnh v trng ng mi trng gm 0,5% Hydrolisat lactoalbumin,, 5% huytthanh b cc khng sinh B (100 g/ml, erytromixin 100 g/ml), Tetraxilin (15 g/ml Micostatin100 g/ml) nc mui m Hanks a n phng th nghim tch t bo.

Cch tnh thng dngKhotien.vn | Ti liu c vit bi L Hong Duy 22


23/25


: khi lng t bo vi khun kh to t 100g Glucoza

G : tc pht trin tnh bng pht

Milimol oxy/pht = ((3333)/ ).41,8G

Nu mun sn xut c 40g/l t bo kh ca vi khun him kh cha cacbon bng 50% khilng ca n v c th bin i c 50% cacbon c trong c cht th c cht phi cha

((40 x 50 )/100) x (100/50) = 40g (cacbon)

Nu dng cacbon c cung cp di dng Glucoza cng thc mi trng l

40 x (180/72) = 100g (Glucoza).

B sung thm 1 s thng t in cn thiu trong cc dung dch :Tween : 40 Polyoxyetilen socbitan monopalmitat.

Tween : 60 polyoxy etylen - socbitan MonoStrearat.

Ax amino axetic : cn gi l Glyxin hay glycocol.

Ion kim loi : Ca(HCO3)2 + 2NaR = CaR2 + 2NaHCO3

Formol : 2CnH2n+1OH + O2 2Cn-1H2n-1CHO + 2H2O

Ax amino glutaric (ax glutamic).

Biotin : vitamin H.

Lizin :Axit , dimironic.



24/25


CaSO4 + 2H2O : Thch cao sng.

CaSO4 : thch cao khan.

Hn the (borax) : Na2B4O7

R ng nhiu st thm K4[Fe(CN)6]

4Fe+++ + 3[Fe(CN)6] Fe4[Fe(CN)6]3.

Note : lm ng ht t bo cc bn kim 1 ng ht nh ging rut vit bt bi, sau canh t gigi nhit t va nng v bn t t ko 2 u ra cho n khi ta c 1 ng ht c nh th cth dng tch, ht t bo.

V khoa hc hin nay con ngi ang i vo im ct v i ln qun cha c khm ph g mi,cc bn hc mn sinh chc nghe qua phng php sinh sn v tnh n cng ch khc g vini cy m phi l ta c 2 t bo(trng) sau ht 2 nhn ca 1 t bo ny chuyn qua nhnca t bo kia v em nui cy cho khi ph trin.

Khi ti cn nh c nghe qua 1 nh khoa hc phng ty khm ph ra 1 phng phpnui tim g vn gi nhp p trong phng th nghim ti 12 nm, v khi ng ta cht ngi pht ca ng ta c tnh ph ton b phng th nghim ca ng ta ton b cng trnh nghincu v tri tim g b thiu tri.... lm cho c gii khoa hc luyn tic.

Vi khoa hc hin nay ch c th gi c 1 tri tim ngi hoc ng vt trong 6 -24 gi thi.

Ti rt mong cc bn yu thch mn sinh VN c th khm ph ra nhiu iu k b m giikhoa hc cha khm ph ra, v hy chia s kin thc nc VN ta pht trin mnh v milnh vc cng ngh.

Tng kt: ni dung nui cy t bo : cn phi tit trng tt c cc l ng nu khng c mymc hin i, n tia cc tm th dng phng php th cng : ngm cc chai l chun b chonui cy trong dung dch axit long un si, sau cho vo dung ni cha nc ct un sitip. Cn i vi dung dch nui cy t bo th khng c qu chua hoc qu kim gi PH nnh trong 7,2 7,4. V phi lc u trch cc t bo lng xung y m cht.

Li Kt : ti xin gii thiu cc bn 1 phng php lm trong sut(v hnh) vt th(Xc ngvt) gi l tiu bn

Cch lm Tiu bn (lm xc cht trong sut trong dung dch) c cng chit sut sau khingm xc cht ng vt vo ete-metylic v axit Salixilic th vt cht s tr nn trong sut doSpantegonxer (Ngi c th nghim), lu l khi ta em xc con vt ra n vn y c m khngb tan hay b ra nha, n ch trong sut khi ta t xc con vt vo 2 dung dch trn. V hin nayngi ta to ra c o tn hnh nh cng ngh nano tn nh sng, ti hy vng nhng aic ebook ny s tr thnh ngi thnh cng v c nhiu thnh tu.



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L Hong Duy .Ti liu c chia s min ph ti din n Khotien.vn.

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