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TRANSCRIPT
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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 45
170 18
180 7,5
190 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)
Thi gian hp kh trng
(pht)
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1.2 Kh trng m thc vt
Bng3:Ho cht kh trng, nng , thi gian x l v hiu qu thc nghim.
Tc nhn v trng Nng % Thi gian x l( pht) Hiu quCalci hypochlorit 9 10 5 30 Rt tt
Natri hypochlorit 2 5 30 Rt tt
Hydro peroxid 10 12 5 15 Tt
Nc Brom 1-2 2 10 Rt tt
HgCl2 0.1 1 2 10 Trung bnh
Cht khng sinh 4 50 mg/l 30 60 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 cnc 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 trngphng cy 30pht trc khi s dng.
-Cc dng c mang vo phng cy u v trng trc: t o chong, m vi, khutrang 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 ccng cn 960 nhng cc dng c lm vic.
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-Trc khi cy, ngi cy cn ra tay bng x phng v lau k n khu tay bngcn 960.
-Cc dng c bng kim loi nh kp cy, dao m, que cy vng, kim mi nhn cth c kh trng bng cch t di ngn la n cn. Nhng dng c ny trc htphi 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 kt 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 CHMI TRNG:
2.1 Thnh phn mi trng dinh dng
Thnh phn mi trng
Nc ct ng, Acid amin, Vitamin (B1, B6, H, PP)
Cht hu c Auxin, Cytokinin,Gibberellin
Cc cht iu ha sinh trng thc vt a lng N P K Ca Mg S
Cht v c Vi l ng Fe Zn B Co N Mn Cu Al Mo I
Cc hp cht khng bit r thnh phn 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 SKOOG II SKOOG III
NH4NO3 1650 mg/L FeSO4.7H2O 27,8 mg/L KI 0,83 mg/L
KNO3 1900 mg/L MnSO4.4H2O 22,3 mg/L Na2MoO4.2H2O 0,25 mg/L
KH2PO4 170 mg/L H3BO3 6,2 mg/L CuSO4.5H2O 0,025 mg/L
MgSO4.7H2O 370 mg/L ZnSO4.7H2O 8,6 mg/L CoCl2.6H2O 0,025 mg/L
CaCl2.2H2O 440 mg/L
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Na2EDTA 37,3 mg/L
Thnh phn vitamin ca Morel (MorelsVitamin)
Pirydoxine(B6)
Biotin(H)
Meso-inositol
Nicotinic acid(P.P)
Thiamin HCl(B1)
PantotateCalci
1 mg/ L 0,01 mg/L 100 mg/L 1 mg/L 1 mg/L 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 1Lmi trng MS l 100ml/L)
Thnh phn Khi lng
NH4NO3 16500 mg
KNO3 19000 mg
KH2PO4 1700 mg
MgSO4.7H2O 3700 mg
CaCl2.2H2O 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 Khi lngNa2EDTA 3730 mg
FeSO4.7H2O 2780 mg
Cn v ho tan tng cht bng 100mL nc ct trong mi bcher ring.
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t 2 bcher dung dch ln bp v gia nhit cho dung dch m ln 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 Khi lng Th tch dd
KI 8300mg 100ml
Na2MoO4.2H2O 2500mg 100ml
CuSO4.5H2O 250mg 100ml
CoCl2.6H2O 250 mg 100ml
SKOOG III (x100): pha thnh 500ml vi nng s dng khi pha 1lt mi trng
MS l 5ml/l
Thnhphn Khi lng
MnSO4.4H2O 2230 mg
H3BO3 620 mg
ZnSO4.7H2O 860 mg
KI 83 mg/1 ml
Na2MoO4.2H2O 25 mg/1 ml
CuSO4.5H2O 2,5 mg/1 ml
CoCl2.6H 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.
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Thnh phn vitamin
Pha cc stock con
Dung dch Phng php
Pirydoxine (B6) (10mg/ml) Cn 1000mg B6 ha tan vi nc ct cho 100mlBiotin (H) (1mg/ml) Cn 100mg biotin ha tan vi nc ct cho 100 ml
Nicotinic Acid (P.P) (10mg/ml) Cn 1000 mg nicotinic acid ha tan vi nc ct cho
100ml
Thiamin-HCl (B1) (10mg/ml) Cn 1000 mg B1 ho tan vi nc ct cho 100 ml
Pantotate-Ca (10mg/ml) Cn 1000 mg Pantotate-Ca ha tan vi nc ct cho
100ml
Bng thnh phn vitamin Morel (x 100)
Thnh phn Khi lng th tch
Meso-inosito 100 g
Pirydoxine (B6) 10ml
Biotin (H) 1 ml
Nicotinic acid(P.P) 10ml
Thiamin HCl(B1) 10ml
Pantotate Calci 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 uv thm nccho 200ml.
*Thnh phn cc cht iu ha sinh trng thc vt(tnh theo mg)
Dung dch cc cht Phng php thc hin
Dung dch BAP (1mg/ml) Cn 100 mg Benzylaminopurine (BAP) ho tan trong 5ml
NaOH 1N.
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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
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- 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 10mMV d: Cho 1ml dung dch BAP 10mM vo mi trng MS pha c 1lt mi
trng. Nh vy ta c 1lmi 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 lanDendrobium.-Mi trng kho st s bit ha c quan t l Saintpaulia.-Mi trng tng sinh (m so).
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Bi 2. NUI CY NH SINH TRNG LAN DENDROBIUM
1. MC TIU CA BI:
-Thc hin thao tc tch nh chi t chi bnDendrobium.-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 chtv mi trng
Khong a lng Knudson 100ml
Khong vi lng Heller 5mlSkoog II MS : 2ml
Vitamin Morel: 2ml
Glycerin :2ml
Inositol : 5ml
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Vitamin B1: 5ml
ng: 30g
Nc da: 150ml
Khoai ty: 60gThan 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 conDendrobium cao 10cm
- Ra sch chiDendrobium 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 lanDendrobium.-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 BIGip 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 ctv 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 chtv 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 b1lt 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 khcnhau.
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 TRNDENDROBIUM
1. MC TIU
Thc hin thao tc nhn ging in-vitro lan dendrobium bng m soKho 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 trng trong thi giannui 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 tvn, 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 thnhcc 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 cy2. Cc cht kh trng3. Ngun mu cy4. Tp nhim vi sinh t bn trong mu cy5. S nhim t ngi cy6. T cy7. Mi trng8. Dng c
2. PHNG PHP XL 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.
-
7/31/2019 nuoi cay mo lan
23/23
23
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