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TRNG I HC CN TH
VIN NGHIN CU V PHT TRIN CNG NGH SINH HC
BO CO PHC TRNH
MN THC TP SINH HA
MSHP: CS115
NHM 1.E
CN B HNG DN
i
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
MC LC
Ni Dung
Trang
3.1.
3.2.
IV.
4.1.
4.2.
4.3.
pH ...................................................................................................................... 11
2.2
2.3
2.4
3.2.
Trng HCT
3.3.
Cc bc c bn o pH: .................................................................................... 18
3.4.
4. Bi tp 2: in vo ch trng: .................................................................................. 19
BI 3: CARBOHYDRATE: XC NH M , TRO V HM LNG X TH
20
(CF)
1. Mc tiu:................................................................................................................... 20
2. Khi qut: ................................................................................................................. 20
3. Thit b, dng c, ha cht, mu vt ........................................................................ 21
4. Phng php ............................................................................................................. 21
4.1.
Chun b:............................................................................................................ 21
4.2.
Xc nh m: ................................................................................................. 22
24
1. Mc tiu: .................................................................................................................. 24
2. Khi qut: ................................................................................................................. 24
3. Vt liu v phng php .......................................................................................... 25
3.1.
Vt Liu: ............................................................................................................ 25
3.2.
27
1. Mc tiu:................................................................................................................... 27
iii
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
3.2.
V c ha mu: .................................................................................................. 29
4.2.
Chng ct m: .................................................................................................. 29
5.2.
Kt qu th nghim: ........................................................................................... 30
5.3.
BI 6: CHUN AMONIAC
31
1. Mc tiu:................................................................................................................... 31
2. Gii thiu tng qut .................................................................................................. 31
3. Vt liu v phng php: ......................................................................................... 31
4. Tin hnh th nghim: (ngu nhin 3 ln lp li) ..................................................... 32
5. Kt qu ..................................................................................................................... 33
TNG KT MU THC TP SINH HA
33
1. S liu mu bt bp .................................................................................................. 33
2. Kt lun .................................................................................................................... 34
iv
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
I.
CHUN B HA CHT
mct
100(%)
mdd
mct * M
100(%)
m dd * M '
Trng HCT
m ct
d
Vi:
V: th tch cn ly (ml)
mct : trng lng cht tan (g)
d: t trng cht tan (g/ml)
C % Vdd
100
M MW V 100
C
2
Trng HCT
dung dch hay s mili ng lng gam ca mt cht c trong mt ml dung dch.
ng lng gam (E) ca mt cht l phn phn t gam cht ng vi mt in
tch hot ng. in tch hot ng trong phn ng trao i tnh theo s in tch thc
hin tham gia kt hp vi ion khc, trong phn ng oxi ha kh th tnh theo s in t
cho hay nhn.
II.
Dng c
- Cn in t
-ng ong 10, 25, 50, 100, 1000ml
-Micropipet
-Bnh tam gic
-Cc nha
-C t cc loi
-Mung cn ha cht
III.
3.1.
Chun b ha cht
Tng th
Bi
Ha cht vn
Nng
tch/K.
dng th
lng
nghim
(final)
(final)
Tng lng
ha cht
(final)
(gram/ml)
(gram/ml/t)
3
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Carbonhydrate
Sy 600 C trong
Ht ht m
300g/nhm
3h (tri ht gel
mng 0,5 cm trn
khay inox)
2 bnh (dng
Bnh ht m
cho 6 nhm)
Cc ( m)
3 ci
n khi lng
khng i (4-5h)
Quy trnh thc
hin trong t ht
v eo gng tay,
ta tin hnh nh
sau: Cho 100ml
nc ct vo cc
H2SO4
5%
(v/v)
150 ml
7,65 ml
NaOH
5%
(w/v)
7,5 g
hin trong t ht
v eo gng tay,
4
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
ta tin hnh nh
sau: ong 80ml
H2O cho vo cc
nha loi 250ml,
cn 7,5gr NaOH
ri cho t t vo
cc nha, thm
H2O vo cho
150ml.
Sy giy lc
600C n khi
lng khng i.
Giy lc (15x
3 t
15cm2)/nhm
Sau em giy
vo bnh ht m
v em cn bng
cn phn tch.
Lipid
Chun b 4 chai
Diethy ether
(500ml/chai).
Diethy ether
Giy lc
600C n khi
(15x15cm2/
lng khng i.
nhm)
Sau em giy
vo bnh ht m
5
Trng HCT
v em cn bng
cn phn tch.
Ct 6 si ch c
Dy ch
1 cun
chiu di khong
30cm.
Nit tng
Se: 0,1g
Se:K2SO4:CuSO4
1:100:10
11,1g
K2SO4: 10g
Ha cht c
thy chun b sn.
CuSO4: 1g
Quy trnh thc
hin trong t ht
v eo gng tay,
ta tin hnh nh
sau: Rt H2SO4
H2SO4 chun
0,1N/ ng
0,5 N
1 lt
50 ml
0,1N c sn vo
bnh nh mc
loi 1 lt c
50ml nc ct,
sau thm nc
ct vo cho n
vch c bnh.
6
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Cn 80g NaOH,
cho t t vo cc
nha loi 250ml
c sn 100ml
NaOH
40%
(w/v)
200 ml
80 g
nc ct, khuy
cho tan, khi tan
hon ton cho
nc ct vo n
vch 200ml,
khuy u.
Do khi lng
Thuc th acid
boric
Methyl Red v
10 lt
Bromoresol green
qu nh nn ta
pha hn hp vi t
Acid bric
100 g
2,5 g
l ca 5 lt thuc
th dng chung
cho 6 nhm.
250 ml
(1) Ha tan 33mg
Methyl Red vi
Methyl Red
66g
1,65 mg
50ml ethanol vo
cc thy tinh
100ml bao giy
Bromoresol
green
bc.
99 g
2,475 mg
(2) Ha tan
49.5mg
7
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Bromoresol green
vi 50ml ethanol
vo cc thy tinh
100ml bao giy
bc.
Cho (1) v (2) vo
cc 250ml bao
giy bc, khuy
u.
Mi nhm ly
Ethanol
200 ml
5 ml
5ml hn hp trn
vo bnh cc
250ml bao giy
bc, sau cho
2,5gr acid boric
vo, khuy u.
Cho thm nc
ct n 250ml.
Cho vo bnh ti
mu dn nhn,
y kn.
6
NH3 v Formol
8
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
1000 ml
200 ml
5) vo cc 250ml.
Cho nc ct vo
n vch 200ml.
Cho 100ml NaOH
1000 ml
200 ml
5) vo cc 250ml.
Cho nc ct vo
n vch 200ml.
Formol 37%
3.2.
Kt qu v tho lun.
Tn nhm.
Tho lun: S dng cc cng thc tnh ng khi lng, th tch cc ha cht,
bo co vi cn b ging dy v trnh by vi c nhm. Trong qu trnh chun b
c sai s do cc yu t cn, o, ong, m.
IV. ................................................................................................................ Cch
s dng micropipette:
4.1.
Trng HCT
thn.
4.2.
Cch cm micropipette:
a.
Ci
mc:
cm
Pipet
pipet lu.
c.
d.
Cch ly mu:
Th pittng ra t t.
BI 2. pH V DUNG DCH M
10
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Mc tiu
1.
Mt s khi nim
2.1.
pH
H2O OH- + H+
[OH-] x [H+] = 1x10-14
pH= -log10[H+]
2.2
S phn ly ca cht in ly yu
H+ +A-
Dung dch m
Trng HCT
3,6
3,8
4,0
4,2
4,4
4,6
4,8
5,0
5,2
5,4
5,6
46,3
44,0
41,0
36,8
30,5
25,5
20,0
14,8
10,5
8,8
4,8
3,7
6,0
9,0
13,2
19,5
24,5
30,0
35,2
39,5
41,2
45,2
Dung dch m phosphate (Sorensen, Biochem, z 21, 131, 1909, 22, 352, 1909) Stock
Solutions:
A: 0,2M NaH2PO4 (27,8g, in 1000ml)
B: 0,2M Na2HPO4 (53,6g Na2HPO4.7H2O or 71,7g Na2HPO4.12H2O in
1000ml)
x ml A + y ml B diluted to a total of 200ml.
pH
5,7
5,8
5,9
6,0
6,1
6,2
6,3
6,4
6,5
6,6
6,7
6,8
93,5 92,0 90,0 87,7 85,0 81,5 77,5 73,5 68,5 62,5 56,5 51,0
6,5
8,0
7,0
10,0 12,3 15,0 18,5 22,5 26,5 31,5 37,5 43,5 49,0
pH
6,9
7,1
7,2
7,3
7,4
7,5
7,6
7,7
7,8
7,9
8,0
45,0 39,0 33,0 28,0 23,0 19,0 16,0 13,0 10,0 8,5
7,0
5,3
55,0 61,0 67,0 72,0 77,0 81,0 84,0 87,0 90,5 91,5 93,0 94,7
12
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
pH
5,2
5,4
5,6
5,8
6,0
6,2
6,4
6,6
6,8
7,2
10,5
15,3
20,8
26,9
33,0
38,0
41,6
44,4
Thit b: Cn in t, my khuy t, pH k
Dng c: Cc thy tinh cc loi, mung cn ha cht,
Ha cht: HAc, NaAc, Na2HPO4, NaH2PO4, sodium carbonate, NaHCO3.
3.2.
Dng ng ong, ong 20,5ml dung dch CH3COOH 0,2M (va pha trn)
v dng 1 ng ong khc ong 4,5ml dung dch CH3COONH4 0,2M (va
pha trn), ri cho c hai dung dch ny vo cc 50ml, thm H2O vo cho
n vch 50ml, khuy u ta c dung dch cn pha.
13
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Dng ng ong ong 7,4ml dung dch CH3COOH 0,2M (pha trn) v
dng 1 ng ong khc ong 17,6ml dung dch CH3COONH4 0,2M (va
pha trn), ri cho c hai dung dch ny vo cc 50ml, thm H2O vo cho
n vch 50ml, khuy u ta c dung dch cn pha.
3.2.2. Chun b dung dch m A.Acetate pH 5,6:
-
14
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Dng ng ong 9,75ml dung dch NaH2PO4 0,2M (pha trn) v dng 1 ng
ong khc ong 15,25ml dung dch Na2HPO4.12H2O 0,2M (va pha trn),
ri cho c hai dung dch ny vo cc 50ml, thm H2O vo cho n vch
50ml, khuy u ta c dung dch cn pha.
Dng ng ong 4ml dung dch NaH2PO4 0,2M (pha trn) v dng 1 ng
ong khc ong 21ml dung dch Na2HPO4.12H2O 0,2M (va pha trn), ri
cho c hai dung dch ny vo cc 50ml, thm H2O vo cho n vch 50ml,
khuy u ta c dung dch cn pha.
3.2.5. Chun b dung dch m Maleate pH 6,0
- Cn 0,16 g NaOH v 0,464 g maleic acid ri cho vo cc c cha sn 10 ml
H2O ct, khuy u, sau thm nc ct cho 20ml, khi ta c dung
dch acid Na-maleate 0,2M.
- Dung dch cn dng th 2 l NaOH 0,2M cn 0,08g NaOH ri cho vo cc
c cha sn 5ml H2O ct, khuy u, sau thm nc ct cho 10ml, khi
ta c dung dch NaOH 0,2M.
- ong 12,5ml dung dch acid Na-maleate 0,2M bng ng ng cho vo cc
50ml, dng ng ong khc ong 6,725ml dung dch NaOH 0,2M cng cho
vo cc trn, thm H2O ct vo cho n vch 50ml th ta c dung dch cn
pha.
15
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Tng th
BI
Ha cht vn
dng th ngim
Nng
tch/K.
lng
(final)
(final)
(gram/ml)
Tng lng
ha cht
(final)
(gram/ml/t)
ng ong) sn 20ml
0,2M
50ml
0,5525 ml
NaAc
(CH3COONH4)
50ml
0,7708g
0,2M
100ml
3,1202g
NaH2PO4 cho vo
cc ong (bng
16
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
ng ong) sn 80 ml
H2O ct, khuy u,
sau thm nc ct
vo cho 100ml th
ta c dung dch
NaH2PO4 0.2M.
Cn 7,1628g
Na2HPO4.12H2O cho
vo cc ong sn
80ml H2O (bng ng
Na2HPO4
0,2M
100ml
7,1628g
0,2M
20ml
0,16g
khuy u, sau
thm nc ct cho
20ml, khi ta c
dung dch NaOH
0,2M.
Cn ln lt 0,16 g
Maleic acid
200ml
4,64g
NaOH v 0,464 g
maleic acid ri cho
17
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
vo cc c cha sn
10 ml H2O ct, khuy
u, sau thm
nc ct cho
20ml, khi ta c
dung dch acid Namaleate 0,2M
Tin hnh o pH bng pH k bn:
Cc bc c bn o pH:
3.3.
Kt qu:
Chun b dung dch m Maleate pH = 6,0, kt qu pH ca dung dch m
sau khi pha o c l 5,947. So vi pH yu cu l chnh lch 0,053 =>
cha t yu cu, sai s cn qu ln (sai s ti a cho php l 0,02).
Nhn xt:
Nguyn nhn sai s:
-
Trng HCT
4. Bi tp 2: in vo ch trng:
1/. 7.
2/. 5.
3/. pH= 8.
4/. Hydrophilic.
5/. Solvent.
6/. pH value
7/. Equilibrium constant (Kw)
8/. A- ; HA
9/. Hydrophopic.
10/. logarithmic
11/. exchange reaction
12/. Titration.
13/. hydren, ion, hydrophohic, interactions, vanderwaals force.
14/. base solution.
15/. Buffer.
16/. Donor.
17/. equilibrium.
18/. Acceptor.
19/.pH=pKa.
19
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
20/. Phosphate.
21/. Bicarbonate.
22/. Acidic solution.
23/. Nng ca acid acetic v mui acetat trong dung dch bng nhau v bng
1,738105
24/. pH l ch s o hot ng ca cc ion H+ trong dung dch v v vy n th
hin acid hay base ca mt dung dch, thang o pH l 14, v l thuyt pH=7 l
trung tnh, pH<7 l c tnh acid v pH>7 l c tnh base, pH c tnh bng cng
thc pH = log10[H+].
pKa l pH m ti c 50% phn t acid phn ly thnh H+ v base tip hp, v
50% phn t cn li khng phn ly. Tnh acid ca nhm phn ly cng cao th gi tr
pKa cng thp, => pKa = -log10[Ka].
S khc bit gia pH v pKa: pH biu th mnh hay yu v tnh acid hay base
ca mt dung dch; cn pKa th mc phn ly ca acid v da vo pKa ta c th
xc nh c acid no mnh - acid no l yu, cn da vo pH th kh phn bit
hn.
25/. acid acetic l acid yu hn acid formic. Acid yu c khuynh hng cho proton
d dng hn acid mnh.
BI 3: CARBOHYDRATE:
XC NH M , TRO V HM LNG X TH (CF)
1. Mc tiu:
Kho st hm lng x th trong bt bp.
2. Khi qut:
- Phng php nh lng hm lng cellulose da vo kh nng ha tan nhng hp
cht khng phi cellulose (hemicelluloses, lignin, pectin) s b ha tan bng dung dch
acid sulphuric (H2SO4) v sodium hydroxide (NaOH).
20
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
4.
Ha cht: H2SO4 1,25%, NaOH 1,25%, nc lc, giy lc, cc tro ha.
Mu: bt bp.
Phng php
4.1.
Chun b:
21
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
trc mt ngy).
-
Sy cc 70oC n khi lng khng i (4-5 gi) hoc 105oC trong 2 gi.
4.2.
-
Xc nh m:
Cn 1 gram mu bt bp (M1) cho vo cc ( xc nh trng lng khng
i) ri sy 1050C n trng lng khng i (2-3 gi) xc nh trng
lng khng i ca mu (M2)
nng.
-un si mu trong 30 pht.
Trng HCT
8. Ph lc s liu
Bng gi tr 3 ln lp li m trong 1 gram bt bp:
Khi
Cc lng mu
(g)
Khi lng cc
(g)
Khi lng
cc + cht x
(g)
Khi lng
m (g)
% m
1,0090
23,7132
24,6532
0,94
6,84%
1,0070
24,8621
25,8001
0,938
6,90%
1,0095
26,6304
27,5674
0,9371
7,90%
23
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Cc + Tro
(g)
(g)
1,0042
22,50
1,0097
1,0086
Cc
Mu
Tro (g)
% Tro
22,6722
0.1722
17.15%
19,83
19,9973
0,1673
16.57%
22,44
22,6213
0,1813
17.98%
X th (g)
% x
1,8431
0,0356
3,56%
1,8275
1,8845
0,057
5,69%
1,8679
1,9382
0,0703
7,03%
Cc
Mu
Giy lc (g)
1,0011
1,8075
1,0025
1,0002
th (g)
BI 4: LIPID
1. Mc tiu:
-
2. Khi qut:
-
Lipid thuc nhm hp cht hu c khng ng nht, khng tan trong nc, nhng
d tan trong dung mi hu c khng phn cc nh: diethyl ether, chloroform,
benzene trong alcol cht bo tan c mc .
Trong phn t lipid c t nht mt chc ester ca acid bo cao phn t vi alcol.
Khi thy phn lipid trong mi trng kim mnh nh NaOH (hay KOH) trong alcol
un nng cho x phng v glycerin.
24
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Trong c th sinh vt, lipid c phn gii nh enzyme thy phn lipase to
thnh acid bo tng ng v alcol
Lipid: Lipid trong ht bp ton phn t 4-5%, phn ln tp trung mm. Trong
cht bo ca ng c 50% l axit linoleic, 31% l axit oleic, 13% l axit panmitic v
3% l Stearic. - Gluxit: Gluxit trong ng khong 69% ch yu l tinh bt. ht ng
non c thm mt s ng n v ng kp.
3. Vt liu v phng php
3.1. Vt Liu:
-
Thit b, dng c: bp un, h thng soxhlet dng trch ly cht bo, giy
lc, ch cun.
Ha cht: diethylether
Mu vt: bt bp.
Trng HCT
26
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
5. Kt qu v tho lun
Hm lng lipid: mu 2 l 11,9%; mu 3 l 16,41%. Hm lng lipid gia 2 mu
chnh lch ng k. Trung bnh t 3 mu lp li l 13,99 tng i cao.
Nguyn nhn c th do sai trong qu trnh thc hin th nghim. Thi gian cng di
th hm lng lipid cn li trong b cng t. Mu tht thot trong qu trnh thc nghim.
So snh vi nhm 1.a ( mu u nnh):
Hm lng lipid: 23,07% ln hn nhiu so vi lng lipid ca mu bt bp
Khi lng
Giy lc
mu (g)
(g)
1,0006
1,7681
2,6319
0,1368
13,67%
1,0032
1,8048
2,6886
0,1194
11,90%
1,0071
1,7871
2,6289
0,1653
16,41%
Mu
mu (g)
sy
Khi lng
lipid
% lipid
Mc tiu:
Xc nh hm lng Protein trong mu bt bp.
2.
-
Khi qut:
Protein c cu to t nhiu nguyn t nh C, H, O, N, S, P trong s cc
nguyn t ny, nit chim t l tng i cao (khong 15-17%) v tng i n
nh trong thnh phn ca protein. V vy v nguyn tc, protein trong thc phm
27
Trng HCT
3.
Mu vt: Bt bp.
3.2.
Phng php:
28
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Sau chun dung dch ny bng dung dch chun H2SO4 0,1N, NH3 c gii
phng v xc nh c lng Nit theo cc phn ng sau:
4.1.
-
Cn 1gram bt bp cho vo ng
nghim ng mu (ng Kjeldahl)
ca my phn tch m.
bt
xc
tc
(Se:K2SO4:CuSO4=1:100:10) vo
ng Kjeldahl.
-
Lp ng vo h thng l v c
ha mu, m my v tin hnh v
c ha mu.
4.2.
-
Chng ct m:
Dng 15ml thuc th Boric cho vo bnh tam gic 250ml; t vo v tr thu
kh NH3 bay ra ( u thu mu ngp trong dung dch acid Boric); chuyn mu
( v c ha) vo bnh cha mu ca h thng.
29
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Mu rn:
b.
Mu lng:
Kt qu th nghim:
Bng gi tr Nit tng trong 3 ln lp li 1 gam mu bt bp:
Hm lng nit
Mu
1,0006
4,9
0,69%
1,0019
7,8
1,09%
1,0008
2,5
0,35%
tng s (%)
30
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
Gii thch:
Trong nc vn c ngun m.
Hp th cha ht sn phm.
Do chun sai.
Tho lun v kt lun: Rt kinh nghim v phng php tin hnh th nghim cho
ng quy cch hn ch sai s, hm lng m trong cc loi lng thc hay trong
cng mt loi c trng cc iu kin khc nhau v trong cc giai on sinh trng
khc nhau th c hm lng khc nhau, do kh c th cho mt kt qu th nghim
chnh xc.
BI 6: CHUN AMONIAC
1. Mc tiu:
Xc nh hm lng amoniac trong mu nc
mm c cm Hng i.
2. Gii thiu tng qut
Vt liu: mu nc mm c cm Hng i.
Hnh 6.1: Nc
mm Hng i
Vin NC&PT Cng ngh Sinh hc
Trng HCT
32
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
dch tr li. Chng ct trong khong 15 pht t khi thuc th bt u i mu. Sau
hng khong 50ml dung dch c mu xanh nc bin.
Sau khi chng ct xong cc mu ta tin hnh mu th khng (thay lng
5. Kt qu
Bng gi tr hm lng Nit amoniac:
Mu
2,7
1,82
3,2
2,17
4,9
3,36
m amoniac: =
0,7 xV V0 0,0007xV Vo
x1000 2,45( g / l )
m
M
33
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
STT
Thnh phn
7,21
Cht x
5,52
Tro
17,23
Lipid
13,99
Protein
4,44
2. Kt lun
-
Tng phn trm hm lng cc cht trn khng t 100% v mt s cht khc
khng c tin hnh o c v tht thot trong qu trnh thc hin th nghim.
34
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
PH LC BNG
Ni dung
Trang
BI 2. pH V DUNG DCH M
10
12
13
16
20
23
24
24
BI 4: LIPID
24
27
27
30
BI 6: CHUN AMONIAC
31
33
33
PH LC HNH
Ni dung
Trang
1
10
v
Chuyn ngnh Vi Sinh Vt K38
Trng HCT
BI 2. pH V DUNG DCH M
10
S phn ly ca cht in ly yu
11
20
Hnh 3.1: Bt bp
21
22
BI 4: LIPID
24
26
27
27
29
29
BI 6: CHUN AMONIAC
31
31
33
PH LC NGUN
-