Bi 1 .

K THUT C BN V PHA CH MI TRNG NUI CY

1. K THUT C BN
1.1 K thut v trng a. Dng c thu tinh Bng 1.1: Thi gian kh trng dng c thu tinh bng nhit v nhit kh trng Nhit (oC) Thi gian kh trng (pht) 160 170 180 190 45 18 7,5 1,5

b. Nt y Hp kh trng cng vi cc dng c thu tinh 1210C, 1atm. Khng kh trng kh, gy chy i vi nt bng v bin dng vi nt nha. c. Mi trng Bng 2: Thi gian kh trng dung dch v cc mi trng lng bng ni hp (autoclave) 1210C, 1atm. Th tch mi trng (ml) <50 75 250-500 1000 Thi gian hp kh trng (pht) 15 20 25 30

1.2 Kh trng m thc vt Bng 3: Ho cht kh trng, nng , thi gian x l v hiu qu thc nghim.

Tc nhn v trng Calci hypochlorit Natri hypochlorit Hydro peroxid Nc Brom HgCl2 Cht khng sinh

Nng % 9 10 2 10 12 1-2 0.1 1 4 50 mg/l

Thi gian x l ( pht) 5 30 5 30 5 15 2 10 2 10 30 60

Hiu qu Rt tt Rt tt Tt Rt tt Trung bnh Kh tt

Trong thi gian x l, m cy phi ngp hon ton trong dung dch dit khun. i vi cc b phn c nhiu bi ct, trc khi x l nn ra cn thn bng nc x phng bt v ra sch li bng nc my. Khi x l xong, m cy c ra nhiu ln bng nc ct v trng (ti thiu l 3 ln). 1.3 Kh trng ni thao tc cy v dng c cy - Sn v tng lau chi thng xuyn, trc khi a vo s dng, phng cy cn c x l hi formol. ng kn ca phng cy trong 24h, b formaldehyde i v kh hi formaldehyde cn tha bng dung dch NH3 25% cng trong 24h. - Kh trng trc t cy bng cch lau sch bng cn 700. Bt n UV kh trng phng cy 30 pht trc khi s dng. - Cc dng c mang vo phng cy u v trng trc: t o chong, m vi, khu trang ca ngi cy n dao, ko, kp (forceps), giy lc, bnh ng nc ct... - Trn bn cy thng xuyn c mt n cn s dng trong khi cy v mt cc ng cn 960 nhng cc dng c lm vic.

- Trc khi cy, ngi cy cn ra tay bng x phng v lau k n khu tay bng cn 960. - Cc dng c bng kim loi nh kp cy, dao m, que cy vng, kim mi nhn c th c kh trng bng cch t di ngn la n cn. Nhng dng c ny trc ht phi c nhng vo cn tuyt i ri mi t. Nh ro i cc git cn tha ri mi a vo ngn la n cn. - Khi m np chai hoc np ng nghim mi trng nui cy, th dng ngn tay k t v ngn tay t cm ly np gn, v khng chm tay vo b mt bn trong ca np gn cng nh khng th np gn xung bt c b mt no cho n khi gn n tr li chai mi trng.

2. PHA CH MI TRNG:
2.1 Thnh phn mi trng dinh dng Thnh phn mi trng Nc ct Cht hu c Cc cht iu ha sinh trng thc vt Cht v c Cc hp cht khng bit r thnh phn ng, Acid amin, Vitamin (B1, B6, H, PP) Auxin, Cytokinin,Gibberellin a lng N P K Ca Mg S Vi l ng Fe Zn B Co N Mn Cu Al Mo I Nc da, nc khoai ty, nc chui, casein,hydrolysalt, trypton, pepton

Thnh phn mui khong c bn ca mi trng MS (Murashige v Skoog, 1962) SKOOG I NH4NO3 KNO3 KH2PO4 MgSO4.7H2O CaCl2.2H2O 1650 mg/L 1900 mg/L 170 mg/L 370 mg/L 440 mg/L SKOOG II FeSO4.7H2O MnSO4.4H2O H3BO3 ZnSO4.7H2O 27,8 mg/L 22,3 mg/L 6,2 mg/L 8,6 mg/L KI Na2MoO4.2H2O CuSO4.5H2O CoCl2.6H2O SKOOG III 0,83 mg/L 0,25 mg/L 0,025 mg/L 0,025 mg/L

Na2EDTA

37,3 mg/L

Thnh phn vitamin ca Morel (MorelsVitamin)

Pirydoxine (B6) 1 mg/ L

Biotin (H) 0,01 mg/L

Mesoinositol 100 mg/L

Nicotinic acid Thiamin HCl (P.P) 1 mg/L (B1) 1 mg/L

Pantotate Calci 1 mg/L

2.2 Cch pha cc dung dch m (stock) * Stock a lng MS : SKOOG I (x10) (Pha thnh 1Lvi nng s dng khi pha 1L mi trng MS l 100ml/L)

Thnh phn NH4NO3 KNO3 KH2PO4 MgSO4.7H2O CaCl2.2H2O

Khi lng 16500 mg 19000 mg 1700 mg 3700 mg 4400 mg

Cn v dng nc ct ho tan ln lt tng cht trong bcher cho tan hon ton. Dng ng ong 1L iu chnh cho 1 lt.

* Stock st MS: SKOOG II (x100) (Pha thnh 200ml vi nng s dng khi pha 1lt mi trng MS l 2ml/l)

Thnh phn Na2EDTA FeSO4.7H2O

Khi lng 3730 mg 2780 mg

Cn v ho tan tng cht bng 100mL nc ct trong mi bcher ring.

t 2 bcher dung dch ln bp v gia nhit cho dung dch m l n va c hi nng bc ln l c. Khuy u v dung dch Na2EDTA vo ng ong 200 ml, ri cho t t dung dch FeSO4 vo, va cho va khuy u. ngui ri cho vo bnh ti bo qun trong t lnh.

* Stock vi lng MS: SKOOG III (x100) Trc tin cn chun b cc stock con: Thnh phn KI Na2MoO4.2H2O CuSO4.5H2O CoCl2.6H2O Khi lng 8300mg 2500mg 250mg 250 mg Th tch dd 100ml 100ml 100ml 100ml

SKOOG III (x100): pha thnh 500ml vi nng s dng khi pha 1lt mi trng MS l 5ml/l Thnh phn MnSO4.4H2O H3BO3 ZnSO4.7H2O KI Na2MoO4.2H2O CuSO4.5H2O CoCl2.6H Khi lng 2230 mg 620 mg 860 mg 83 mg/1 ml 25 mg/1 ml 2,5 mg/1 ml 2,5 mg/1 ml

Cn 3 cht u tin v ho tan vi nc ct trong tng becher ring.Cho tng dung dch theo th t vo ng ong 500ml. Sau ht 1ml dung dch trong stock con ca 4 cht tip theo cho vo ng ong, va cho va khuy u v thm nc ct cho 500ml.

Thnh phn vitamin Pha cc stock con Dung dch Pirydoxine (B6) (10mg/ml) Biotin (H) (1mg/ml) Nicotinic Acid (P.P) (10mg/ml) Phng php Cn 1000mg B6 ha tan vi nc ct cho 100ml Cn 100mg biotin ha tan vi nc ct cho 100 ml Cn 1000 mg nicotinic acid ha tan vi nc ct cho 100ml Thiamin-HCl (B1) (10mg/ml) Pantotate-Ca (10mg/ml) Cn 1000 mg B1 ho tan vi nc ct cho 100 ml Cn 1000 mg Pantotate-Ca ha tan vi nc ct cho 100ml

Bng thnh phn vitamin Morel (x 100)

Thnh phn Meso-inosito Pirydoxine (B6) Biotin (H) Nicotinic acid(P.P) Thiamin HCl(B1) Pantotate Calci

Khi lng th tch 100 g 10ml 1 ml 10ml 10ml 10 ml

Cn meso-inositol ri ha tan vi 100ml nc ct. Cho th t tng cht vo ng ong c cha nc ct, va cho va khuy u v thm nc cho 200ml.

* Thnh phn cc cht iu ha sinh trng thc vt (tnh theo mg) Dung dch cc cht Dung dch BAP (1mg/ml) Phng php thc hin Cn 100 mg Benzylaminopurine (BAP) ho tan trong 5ml NaOH 1N.

Cho thm nc ct cho 100 ml. Mi ml dung dch c cha 1mg BAP. Dung dch IAA (1mg/ml) Cn 100mg Indolacetic acid (IAA) ha tan trong 5ml NaOH 1N. Cho thm nc ct cho 100ml. Mi ml dung dch c cha 1mg IAA. Dung dch IBA (1mg/ml) Cn 100mg Indolebutyric acid (IBA) ha tan trong 5ml NaOH 1N. Cho thm nc ct cho 100ml. Mi ml dung dch c cha 1mg IBA Dung dch Kinetin (1mg/ml) Cn 100mg Kinetin (KIN) ha tan trong 5ml NaOH 1N. Cho thm nc ct cho 100ml. Mi ml dung dch c cha 1mg KIN. Dung dch NAA (1mg/ml) Cn 100mg Naphthaleneacetic acid (NAA) ha tan trong 5ml NaOH 1N. Cho thm nc ct cho 100ml. Mi ml dung dch c cha 1mg NAA. Dung dch 2,4-D (1mg/ml) Cn 100mg 2,4-D (2,4-dichlorophenoxyacetic acid) ha tan trong 50ml ethanol 50%. Cho thm nc ct cho 100ml. Mi ml dung dch c cha 1mg 2,4-D.

* Cc dung dch cht iu ha sinh trng thc vt (tnh bng mol/l) BAP c trng lng phn t (MW) l 225.26 - Ho tan 225.26gr BAP trong 1l nc ct tc ta c 1l dung dch BAP1M hay 1mol/l - Cn 225.26 mg BAP ho tan trong 1l nc ct tc ta c 1l dung dch BAP 1mM hay 1mmol/l

- Cn 225.26x10-3 mg BAP ho tan trong 1lt nc ct tc ta c 1l dung dch BAP 1M hay 1mol/l Dung dch BAP ( MW 225.26) (10mM) Cn 225.26 mg BAP ho tan trong 5 ml NaOH 1N. Cho thm nc ct cho 100 ml, tc ta c 100 ml dung dch BAP 10mM V d: Cho 1ml dung dch BAP 10mM vo mi trng MS pha c 1lt mi trng. Nh vy ta c 1l mi trng MS c cha 10M BAP. Mun pha 1lt mi trng MS c cha 1M BAP, ta s dng 0.1mL dung dch BAP 10 mM. Tng t nh vy pha cho cc loi kch thch t cn li, bit: - IAA (MW 175.19) - IBA (MW 203.24) - Kinetin (MW 215.22) - NAA (MW 186.21) - 2,4-D (MW 221.04) Thc hin pha cc mi trng nui cy sau: - Mi trng nui cy phi cam chanh. - Mi trng nui cy nh sinh trng lan Dendrobium. - Mi trng kho st s bit ha c quan t l Saintpaulia. - Mi trng tng sinh (m so).

Bi 2. NUI CY NH SINH TRNG LAN DENDROBIUM

1. MC TIU CA BI:
- Thc hin thao tc tch nh chi t chi bn Dendrobium. - Theo di s thnh lp cm chi v nhn cm chi. - Tch chi con v nui cy cy con hon chnh

2. DNG C, HA CHT V MI TRNG NUI CY NH SINH TRNG

2.1. Dng c Bnh tam gic Becher ng nghim + mi trng Kp inox a petri v trng Dao cy, n cn Pipet thy tinh Bnh nh mc, ng ong Qu bp cao su Giy cy v trng

2.2. Ha cht v mi trng Khong a lng Knudson 100ml Khong vi lng Heller 5ml Skoog II MS : 2ml Vitamin Morel: 2ml Glycerin :2ml Inositol : 5ml
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Vitamin B1: 5ml ng: 30g Nc da: 150ml Khoai ty: 60g Than hot tnh: 0,25g Agar: 6g pH: 5,5 * Khong a lng ca KnudsonC (Morel,G.M.,1965) NH4NO3: 500mg/l NH4(SO4)2: 500mg/l Ca(NO3)2: 241,3mg/l KCl : 250 mg/l KH2PO4: 250 mg/l MgSO4: 122,15mg/l * Khong vi lng ca Heller (1953) AlCl3.6H2O : 0,054 mg/l CuSO4.5H2O : 0,03 mg/l H3BO3: 6,2 mg/l KI : 0,01 mg/l MnSO4.H2O : 0,08 mg/l NiCl2.6H2O : 0,03 mg/l ZnSO4.7H2O :1,0 mg/l

3 CC BC TIN HNH:
+ Nguyn vt liu th nghim: Cc on chi con Dendrobium cao 10cm - Ra sch chi Dendrobium di vi nc chy. Dng dao m lt ht cc l non bao xung quanh chi cho n khi l ra cc chi bn v chi ngn
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- Ngm chi trong nc x phng long v dng gn lau nh ln thn chi. Ra cho sch x phng di vi nc chy - Lc chi trong cn 700 trong 1 pht. Sau x l vi dung dch javel thng phm theo t l 1:5 trong vng 25-30 pht - Trong t cy, dng kp v trng cho chi vo cc bcher c cha nc ct v trng. Lc ra 3-5 ln cho ht mi javel. - t cc chi ln giy v trng. Dng dao ct ring tng chi bn v chi ngn ra khi chi m ban u. Nh ct b ht cc m cht b cht kh trng lm trng phn gc chi. Cy tng chi vo ng nghim c cha mi trng chun b. - Nui trong phng sng - Ghi r tn nhm, ngy cy, ging cy vo tt c cc ng nghim

4. BO CO THC HNH
- Thc hin thao tc tch v nui cy v trng nh chi lan Dendrobium. - Thu c cm chi v cy con v trng trong ng nghim. - Quan st v ghi nhn thi gian tng trng ca cy con hon chnh.

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Bi 3. NUI CY PHI CAM CHANH

1. MC TIU CA BI
Gip sinh vin lm quen vi thao tc v trng mu cy v k thut cy v trng i vi mu ht. Thc hin pha mi trng gieo ht Thc hin cc thao tc v trng trong nui cy v k thut gieo ht

2. DNG C, THIT B V HA CHT

2.1 Dng c: Bnh tam gic Becher ng nghim + mi trng Kp inox a petri v trng Dao cy, n cn Pipet thy tinh Bnh nh mc, ng ong Qu bp cao su Giy cy v trng

2.2 Thit b Autoclave T sy T cy (laminar) T lnh Cn k thut

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My ct nc 2 ln My khuy t pH k Bp t

2.3 Ho cht

Dung dch Skoog I, II v III ca mi trng MS Stock vitamin Morel Cn 90%, 70% Stock glycine ng saccharose X phng bt Dung dch hypochloride calcium 10% Nc ct v trng Agar

3. CC BC TIN HNH
3.1 Chun b mi trng (thnh phn cho 1l mi trng) - Skoog I (MS):100ml - Skoog II (MS): 2ml - Skoog III (MS): 5ml - Vitamin Morel: 2ml - Glycine: 2ml - Sucrose: 30g - pH :5,8 - Agar:7g 3. 2 Nguyn liu thc vt:
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- Cc ht cam chanh cn nguyn vn, khng b h hi 3.3 Cc bc thc hin - Cho ht vo bcher c cha nc x phng long, ra ht cho sch cht nht bm xung quanh ht - Ra ht cho sch x phng bng nc ct. Ngm ht 2pht trong cn 70% - Ra ht sch etanol bng nc ct. Sau ngm ht 10pht trong dung dch hypochloride calcium 10% - Trong t cy v trng, ra ht cho sch cht kh trng bng cch lc ht trong nc ct v trng (3 ln) - Tin hnh tch o ht bng kp v dao m v trng. Cho cc ht va tch b v o vo a petri v trng c cha giy thm v trng v lm m giy thm bng mt t nc ct v trng. - Gieo ht: gieo 4-5 ht vo cc bnh tam gic cha mi trng nui cy c hp kh trng. - y np bnh cy v ghi r h tn, ngy cy, s nhm, tn ging v mi trng.

4. BO CO THC HNH
- Tm tt phng php thc hin nui cy m ht cam chanh. - Thu c cc cy con v trng trong ng nghim - Quan st v ghi nhn thi gian tng trng ca cy con hon chnh.

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Bi 4. KHO ST S BIT HA C QUAN T M L SAINTPAULIA (AFRICAN VIOLET)

1. MC TIU CA BI:
Chng minh nguyn tc vi nhn ging v kh nng ti to c quan hon chnh t mu cy thc vt. Theo di s bit ha phi sinh dng, chi, m so v r trn cc mu cy l Saintpaulia.

2. DNG C, HA CHT V MI TRNG NUI CY.

2.1 Dng c: Bnh tam gic Becher ng nghim + mi trng Kp inox a petri v trng Dao cy, n cn Pipet thy tinh Bnh nh mc, ng ong Qu bp cao su Giy cy v trng

2.2 Ho cht v mi trng - Dung dch Skoog I, II v III ca mi trng MS - Stock vitamin Morel - Stock glycine - ng saccharose
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- Agar - Nc ct v trng - Cn 960, 700 - Dung dch hypochloride calcium 10% - X phng bt Chun b 1lt mi trng nui cy - Cho 500ml nc ct vo bnh 2000ml - Ln lt cho nh sau: + 10ml cc mui khong MS + 10ml dung dch m thiamine (40mg/l) + 30g ng + 20ml dung dch m IAA (10mg/100ml) + 20ml dung dch m kinetin (10mg/100ml) + 10ml dung dch m NaH2PO4.H2O (17g/l) + 8ml dung dch m adenine sulfate (17g/l) - Thm nc ct 1000ml. Chnh pH 5,7 - Cho 8g agar. Nu tan - Chia 25ml vo mi ng nghim. y kn. - Hp kh trng 30 pht, 1210C, 1atm. - thch nghing. 2.3. Nguyn vt liu th nghim L Saintpaulia

3. CC BC THC HIN:
Ct nhng l kho, non t cy m. ghi li tn ging mi cy. Ra mu l bng nc x phng m, ra sch. Cn thn v m l d b dp. t vo cc 250ml, y li bng a petri Kh trng bng thuc ty 19% trong 15 pht Ra sch bng nc ct v trng 3 ln
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Ct mu l vi kch thc 1-2 cm2, mu cung 1cm. t nhng mu ny ln mi trng Nui cy trn k Nui cy v kho st s thnh lp c quan trn cc mi trng c cc hormon khc nhau. Nui cy chi c ti sinh t l. To cm chi v nhn cm chi. Tch chi con ra khi cm chi v to cy con hon chnh.

4. BO CO THC HNH
- Thc hin thao tc cy v trng m l Saintpaulia. - Quan st v ghi nhn kt qu v s ti to c quan v thi gian thnh lp. - Ghi nhn nh hng ca cc kt hp hormon thc vt ln s bit ha ca m l.

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Bi 5: NHN GING, TO M SO TRN DENDROBIUM

1. MC TIU
Thc hin thao tc nhn ging in-vitro lan dendrobium bng m so Kho st s thnh lp m so t cy non in-vitro

2. DNG C, HA CHT V MI TRNG NUI CY.

2.1 Dng c: Bnh tam gic Becher ng nghim + mi trng Kp inox a petri v trng Dao cy, n cn Pipet thy tinh Bnh nh mc, ng ong Qu bp cao su Giy cy v trng 2.2. Mi trng v ha cht nui cy - Mi trng MS (20g/l ng) c b sung 0.1M 2,4-D v 1M 2,4-D. - Cn 960 v 700 - Nc ct v trng - Dung dch hypochloride calcium 10% - X phng bt 2.3. Nguyn liu thc vt Cy con lan dendrobium in- vitro

3. CC BC THC HIN

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Cn thn gp cy con in-vitro ra khi bnh nui cy. Trnh kp qu mnh lm dp mu cy. - Dng dao cy ct on r, thn v l chuyn qua mt da cy khc x l. - L: ct thnh nhiu mnh nh vi kch thc 0,8 -1mm x 8 10mm. t cc mnh l ny nui trn cc a petri cha mi trng MS + 1M 2,4-D BC. - Thn: ct lt mng 0,05 0,1mm bng li dao tht sc. Cc lt ct c t nm trn cc a petri cha mi trng MS + 1M 2,4-D - R: Ra sch agar bng nc ct v trng, ct nh thnh tng on 1-1,5mm t ln cc a petri c cha mi trng MS + 0.1M 2,4-D - Dng nha nylon cun quanh mp a petri m bo s v tr ng trong thi gian nui cy. - Ghi r s nhm, tn mu cy, tn mi trng v ngy cy. - t nui trong ti Yu cu: Thao tc x l mu cy tt, lt ct dt khot cng mng cng tt; trnh dp mu

4. BO CO THC HNH
Ghi nhn v so snh thi gian pht sinh so, hnh dng v mu sc khi m so, v tr pht sinh so t cc mu cy khc nhau.

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Bi 6: NUI CY M HOA HNG

1. MC TIU CA BI:
Theo di s tng trng v nhn ging hoa hng bng cch tng sinh chi. Thc hin nhn ging in-vitro cy thn g

2. DNG C, HA CHT V MI TRNG NUI CY.

2.1 Dng c: Bnh tam gic Becher ng nghim + mi trng Kp inox a petri v trng Dao cy, n cn Pipet thy tinh Bnh nh mc, ng ong Qu bp cao su Giy cy v trng

2.2. Ha cht v mi trng - Mi trng MS b sung 30g/l ng, 100mg/l myo-inositol, vitamin MS, 8g/l agar, v 1M BA - Cn 960 v 700 - Nc ct v trng - Dung dch chlorine 10% - X phng bt
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- Than hot tnh - Tween 20

3. CC BC THC HIN:
- Cch chn v thu nhn mu: thu ly chi pht trin mnh. Nhng chi c th ly t vn, vn m hoc cy trong chu . - Loi b l - Kh trng mu: kh trng 10-15 pht trong chlorine 10% (v/v) vi vi git tween 20 (2 git/ 100ml dung dch). Ra sch vi nc ct v trng 3 ln. - Ct thn thnh cc on cha 1 chi ngn hay 1-3 t thn. - Cy mu vo mi trng - vi ngy (16-24 gi chiu sng) - Khi chi mc ln t cc mm cao khong 1cm, tch ly chng v cy ln mi trng mi. Loi b phn ngn ca cc ch i s lm tng s to nhnh. Theo di s thay i v cht lng chi (tnh n nh) khi s ln cy chuyn tng ln. (C th kho st thm s nh hng ca nng BA ln s ti sinh chi: 0, 1, 4 v 10 M) - Nhng chi cao khong 2cm c em ra ngoi mi trng trng.

4. BO CO THC HNH
- Thao tc thc hnh th nghim - Quan st v ghi nhn kt qu

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BI 7: KHO ST TP NHIM
1. MC TIU
Ghi nhn cc kt qu th nghim. Xc nh nguyn nhn gy nhim mu . Cc nguyn nhn c th gy nhim: 1. Mu cy 2. Cc cht kh trng 3. Ngun mu cy 4. Tp nhim vi sinh t bn trong mu cy 5. S nhim t ngi cy 6. T cy 7. Mi trng 8. Dng c

2. PHNG PHP X L V HN CH TP NHIM

nh gi nh hng ca s tp nhim ln mc thnh cng ca cng ngh nui cy m t bo. Xem xt li thao tc thc hin k thut nui cy m.

3. BO CO THC HNH
Quan st v ghi nhn tp nhim mu. S lng cy con t yu cu khng nhim mi nhm.

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BI 8: VIT BO CO TNG HP
1. MC TIU
nh gi tng hp cc kt qu thc hnh

2. YU CU
- 5 bi th nghim - Quan st v ghi nhn y cc kt qu

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