B GIO DC & O TO

TRNG I HC K THUT CNG NGH TP.HCM

KHOA MI TRNG & CNG NGH SINH HC




BI GING

THC HNH
PHN TCH NH GI CHT LNG THC PHM

Bin son: KS. PHM MINH NHT

ThS. BI C CH THIN

- 2009 -

GII THIU
Trong x hi ngy nay, i sng ca con ngi ngy cng c nng cao v
nhu cu v n ung cng phi c ci thin. Tuy nhin, cuc sng ca con ngi
cng c nng cao th thi gian dnh cho vic n ung cng gim xung. Chnh v
th, nhng ba n nhanh, nhng qun cm va h l nhng la chn thch hp i vi
nhng con ngi bn rn. Do , vic nh gi v qun l ngun thc phm l iu
ht sc cn thit c th hn ch ti a kh nng ng c thc phm.
y chnh l tin ra i mn hc Phn tch nh gi cht lng thc
phm, gp phn gip cho sinh vin c ci nhn khi qut v nhng nguyn nhn gy
ng c thc phm ng thi bit c cc phng php phn tch, nh gi cht
lng ca ngun thc phm. V ni dung thc hnh ca mn hc ny gip cho sinh
vin c th t tay mnh c th phn tch v nh gi mc v sinh an ton ca thc
phm i vi mt s cc ch tiu vi sinh vt v mt s cc ch tiu ha l thc phm.
Trong ni dung ca mn thc hnh ny, sinh vin s c lm quen vi quy
trnh phn tch cc ch tiu vi sinh nh quy trnh nh lng tng vi sinh vt hiu kh,
coliform tng s, Staphylococcus aureus, quy trnh nh tnh E. Coli, Salmonella. Cc
quy trnh phn tch ny da trn tiu chun ngnh v tiu chun Vit Nam v cc quy
trnh ny cng c p dng rng ri kh nhiu phng th nghim vi sinh ti cc c
quan, cng ty. Thng thng, cc quy nh v vi sinh vt hin din trong mu thc
phm thng thng cho php c s hin din ca TPC, coliform tng s v S. aureus
vi s lng nht nh do phi tin hnh nh lng cn i vi hai ch tiu E. Coli
v Salmonella tuyt i khng c php hin din trong thc phm nn ch cn phn
tch nh tnh.
Ti hy vng sau mn hc thc hnh ny, sinh vin s tch ly c t nhiu kin
thc thc t v phn tch nh gi cht lng thc phm v hy vong n s tr thnh
mt phn trong hnh trang cc bn bc vo i.

MC LC
PHN 1: KIM NGHIM VI SINH VT GY BNH TRONG THC PHM
Bi s 1: nh lng tng s vi sinh vt hiu kh (TPC) trong thc phm
Bi s 2: nh lng tng s Coliform trong thc phm
Bi s 3: nh tnh E. Coli trong thc phm
Bi s 4: nh lng Staphylococcus aureus trong thc phm
Bi s 5: nh tnh Salmonella trong thc phm
PHN II: KIM NGHIM HA L THC PHM
Bi s 6: Phng php pht hin nhanh mu c v khng c
Bi s 7: Xc nh hm lng cht bo trong sa theo phng php Adam Rose
Gottlieb
Bi s 8: Xc nh hm lng protein trong sa theo phng php kt ta
Bi s 9: Xc nh m nguyn liu
Bi s 10: Phng php pht hin nhanh hn the

PHN I

KIM NGHIM VI SINH VT GY BNH

TRONG THC PHM

CC QUY TC AN TON TRONG PHNG KIM NGHIM VI SINH

Thao tc an ton l yu cu cc k quan trng trong kim nghim vi sinh vt. Khi
lm vic vi vi sinh vt, chng ta thng thao tc vi s lng rt ln v m c t
bo vi sinh vt ( mc 109 t bo/ml). Nhiu chng vi sinh vt l tc nhn gy bnh
nn cn lun lun cn thn vi tt c cc chng ang thao tc.Mt khc, nhn vin
kim nghim cng phi s dng nhiu loi ha cht, trong c cc acid hoc nhng
ha cht c c tnh. Do vy, cn tun th mt s quy tc an ton m bo an ton
cho bn thn v cho nhng ngi khc trong phng th nghim nh sau:
-

Nm vng nguyn tc, phng php lm vic vi vi sinh vt.

Khng n ung, ht thuc trong phng kim nghim. Mang khu trang khi thao

tc vi vi sinh vt.
-

Mc o blouse trong thi gian lm vic.

Trc khi bt u lm cn st trng mt bn bng giy lau tm cn 700 hoc

dung dch cht dit khun khc (lysol 5%, amphyl 10%, chlorox 10%), kh. Thc
hin tng t cho hai tay. Ch cha t n cn hoc n Bunsen khi tay cha kh
cn. Lp li vic st trng ny sau khi hon thnh cng vic.
-

Cn ghi ch tn chng, ngy thng th nghim ln tt c cc hp petri, ng

nghim mi trng, bnh nui cy.

-

Khi l tay lm , nhim vi sinh vt ra ni lm vic, dng khn giy tm cht

dit khun lau k, sau thc hin kh trng li bn lm vic.

-

Cn thn khi thao tc vi n cn hoc n Bunsen. Tt ngn la khi cha c

nhu cu s dng hoc ngay sau khi thc hin xong mi thao tc. Lu trnh a tay,
tc qua ngn la. Cn c cch bo v tc thch hp trng hp tc di.
-

S dng qu bp cao su khi thao tc ng ht nh lng (pipette), khng ht

bng ming.
-

Khi lm v dng c thy tinh, cn thn mang gng tay thu gom tt c mnh v

vo mt ti rc ring.
-

Tch ring cht thi rn v cht thi lng.

Tt c cht thi rn, mi trng cha hoc nhim vi sinh vt cn c hp kh

trng trc khi thi b vo cc bi rc. Cc dng c, bnh cha nhim vi sinh vt cn
c ngm vo dung dch cht dit khun (nc javel) trc khi ra v ti s dng.
5

Cn gi hoc rng bng bng keo khi t chng cc hp petri ln nhau.

Khng m hp petri v dng mi ngi trnh nhim vi sinh vt vo ng h

hp.
-

Khi t que cy c dnh sinh khi vi sinh vt, cn t vng hoc u que cy

vo chn ngn la trnh s vng nhim vi sinh vt vo khng kh.

-

St trng v ra tay sch s trc khi ri phng th nghim.

Bi s 1: XC NH TNG S VI SINH VT HIU KH (TPC)

----------------------------------1.1. nh ngha
Vi sinh vt hiu kh l nhng vi sinh vt tng trng v hnh thnh khun lc trong
iu kin c s hin din ca oxy phn t. Tng s vi khun hiu kh hin din trong
mu ch th mc v sinh ca thc phm.
1.2. Nguyn tc
Tng s vi sinh vt hiu kh c m bng cch a v trong iu kin hiu
kh 300C/72h + 6h hoc 370C/48h + 6h
1.3. Mi trng s dng
-

Mi trng pha long mu: Saline Pepton Water (SPW) hoc Buffer Pepton
Water (BPW)

Mi trng nui cy: Plate Count Agar (PCA)

1.4. Dng c
-

a petri

ng nghim

T m 300C

1.5. Quy trnh phn tch

25g mu + 225ml SPW  ng nht trong 30 giy 
pha long 10-1

Pha long trong nc mui sinh l

 pha long 10-2, 10-3, 10-4

T mi pha long cy 1ml trn 2 a petri v trng. Sau

mi trng PCA c lm ngui n 450C

Lc u v ch a thch ngui  p ngc  em

nhit 300C trong 72h

c kt qu cc kt qu t 25 250 khun lc

Tnh ton kt qu:

N
A =-----------------------------------n1V1f1 + + niVifi
1.6. Thuyt minh quy trnh
1.6.1. Chun b mu
Cn chnh xc 10 g (hoc 25 g) mu cho vo bao PE v trng, sau thm vo 90
ml (hoc 225 ml) dung dch pha long mu. Tin hnh ng nht mu bng my dp
mu (stomacher). Thi gian dp mu ty thuc vo tng loi mu nhng khng qu
2,5 pht. Tt c cc thao tc trn phi thc hin trong iu kin v trng. Khi , ta s
c c dung dch pha long l 10-1.
Dich pha long s c pha long theo dy thp phn bng cch dng micropipette
(pipetman) v trng chuyn 1ml vo ng nghim cha 9ml dung dch pha long 
ng nht, ta s c c dch pha long 10-2. Tip tc thc hin tng t c c
cc pha long cn thit.
1.6.2. a
Chn 2 hay 3 pha long lin tip d kin cha t 30 300 t bo vi sinh vt.
Dng micropipette vi cc u tip v trng chuyn 1ml dch pha long vo gia a
petri v trng. Tng ng vi mi pha long cy t 2 3 a. Sau khi cy, vo
mi a 10 15ml mi trng PCA nu chy v lm ngui n 45 500C. Trn u
mu vo mi trng bng cch xoay trn a petri xui v ngc chiu kim ng h t
3 5 ln. t a ln mt phng ngang cho thch ng c
1.6.3.
Cc a c lt ngc li v nui 300C trong 72 gi
1.6.4. c kt qu

Hnh 1: Tng s vi sinh vt hiu kh

m tt c cc khun lc xut hin trn cc a sau khi . Chn cc a c s m
t 30 300 t bo vi sinh vt tnh. Mt tng vi sinh vt hiu kh trong 1g mu
c tnh nh sau:
N
A (CFU/g)

=
n1Vf1 + + niVfi

Trong :

A: s t bo vi khun (khun lc) trong 1g mu

N: Tng s khun lc m c trn cc a chn
n: s lng a cy ti pha long th i
V: th tch dch mu (ml) cy vo mi trng
f: pha long tng ng

Bi s 2: XC NH TNG S COLIFORMS
-------------------------2.1. nh ngha Coliforms
Coliforms l nhng trc khun gram m, khng sinh bo t, hiu kh hoc k kh
ty nghi, c kh nng ln men lactose sinh acid v sinh hi 370C trong 24 48 gi.
Nhm coliforms hin din khp ni trong t nhin, trong rut ngi v ng vt.
Coliforms c xem l nhm vi sinh vt ch th: s lng hin din ca chng trong
thc phm, nc hay cc loi mu mi trng c dng ch th kh nng hin din
ca cc vi sinh vt gy bnh khc.
2.2. Nguyn tc
Mu c ng nht c cy mt lng nht nh ln mi trng thch chn
lc thch hp cha lactose. Sau khi 370C + 10C trong 24 48 gi, m s lng
khun lc Coliforms in hnh. Xc nh li bng cc phn ng c trng. Mi trng
chn lc Coliforms l mi trng cha lactose, y l ngun carbon duy nht, ng
thi mi trng cn cha mui mt nh mt tc nhn chn lc v cc tc nhn ch th
nh neutral red, crystal violet. Khng nh cc dng khun lc c trng bng mi
trng canh chn lc nh canh BGBL.
2.3. Mi trng s dng
-

Mi trng Tryptone Soya Agar (TSA)

Violet Red Bile Agar (VRB)

Brilliant Green Bile Lactose broth (BGBL)

2.4. Dng c
-

a petri

ng nghim

ng Durnham

Chai thy tinh 250 ml

T m 370C

2.5. Quy trnh phn tch

25g mu + 225ml SPW  ng nht trong 30 giy  pha long 10-1

10

T mi pha long cy 1ml trn 2 a petri v trng. Sau mi

trng TSA c lm ngui n 450C v ch trong 30

mi trng VRB ln mi trng TSA

nhit 370C trong 24h

Chn v m cc khun lc c mu n sm, c qung ta mui

mt, ng knh > 0.5 mm

mi a chn 3 5 khun lc c trng cy sang mi trng BGBL

370C + 0,5 trong 24 gi

T l xc nhn R:
S khun lc sinh hi trong BGBL
R = --------------------------------------------------S khun lc cy

Tng s Coliforms (cfu/g)

N
A = --------- x R
nVf

11

2.6. Thuyt minh quy trnh

2.6.1. Chun b mu
Qu trnh chun b mu tng t nh phn nh lng tng s vi sinh vt hiu kh.
Nhng qu trnh pha long mu sao cho trong 1ml dung dch pha long mu cha
khong <100 khun lc
2.6.2. Cy mu
Cy chuyn 1ml dch pha long mu chn vo a petri, mi nng cy t nht
vo 2 a v chn 2 nng pha long lin tip cy sao cho sau khi mi a xut
hin t 10 100 khun lc.
Cho vo mi a cy mu 5ml mi trng TSA c un chy v lm ngui
n 450C, trn u dch mu vi mi trng bng cch xoay trn a petri xui v
ngc chiu kim ng h. n nh nhit phng trong 1 2 gi phc hi
kh nng ca Coliforms. thm 10 15ml mi trng VRB. Ch mi trng ng
c, lt ngc a v 37 + 10C trong 24 gi.
2.6.3. c kt qu
Chn cc a c s m t 10 100 khun lc Coliforms tnh. Khun lc
Coliforms c mu n m v ng knh >0,5mm, xung quanh khun lc c
vng ta mui mt

Hnh 2: Khun lc Coliforms trn mi trng VRB

2.6.4. Khng nh
Quy trnh khng nh thc hin nh sau: chn 5 khun lc nghi ng trn a
m c vi cc hnh dng khc nhau cy chuyn sang mi trng canh BGBL v
370C trong 24 gi. Phn ng dng tnh khi vi sinh vt sinh kh trong ng Durnham.
T l xc nhn l t s gia s khun lc cho kt qu dng tnh vi s khn lc khng
nh

12

Hnh 3: Kt qu khng nh Coliforms trn mi trng BGBL

2.6.5. Tnh ton kt qu
Da vo s khun lc m c v t l xc nh, tnh mt ca Coliforms theo
cng thc:
N
A (CFU/g hay CFU/ml)

xR
n1vf1 + + nivfi

Trong :

N: tng s khun lc m c
ni: s a c s khun lc c chn ti mi pha long
v: th tch cy vo mi a
fi: pha long c s khun lc c chn ti cc a m
R: t l xc nhn

Kt qu Coliforms c lm trn chn chc v c biu din dng s m c c

s thp phn.

13

Bi s 3: NH TNH E.COLI
---------------3.1. nh ngha
Coliforms l nhng trc khun gram m, khng sinh bo t, hiu kh hoc k kh
ty nghi, c kh nng ln men lactose sinh acid v sinh hi 370C trong 24 48 gi.
Coliforms chu nhit l coliforms c kh nng ln men lactose, sinh acid v sinh
hi nhit 440C trong 24 48 gi.
Coliforms phn (feacal Coliform) l coliform nhit c th nghim indol trong mi
trng tryptom dng tnh.
E.Coli l coliforms phn c nghim php IMViC ln lt l + + - -.
3.2. Nguyn tc
Phng php ny dng nh tnh v kt lun pht hin hay khng pht hin
E.Coli trong mt khi lng mu xc nh.
Cy mu vo mi trng tng sinh (BGBL), kim tra trn mi trng phn lp
(EMB) v th nghim bng cc phn ng sinh ha ph hp (nghim php IMViC)
3.3. Mi trng s dng
-

Mi trng TSA

Canh BGBL

Mi trng EMB

Canh Trypton

Canh MR - VP

Thch Simmon Citrate

Thuc th Kovacs

Thuc th Methyl Red

Thuc th -naphtol 5%

KOH 40%

3.4. Dng c
-

a petri

ng nghim - ng Durnham

B iu nhit 440C

T m 370C
14

25 g mu
3.5. Quy trnh phn
tch + 225 ml SPW, ng nht mu trong 30 giy
 pha long 10-1

Ly 1 ml mu pha long 10-1 cho vo 10 ml mi trng BGBL

440C + 0,5 trong 24 gi

Chn cc ng sinh hi cy chuyn sang mi trng EMB.

Mu khng sinh hi c coi l m tnh E. Coli

370C + 0,5 trong 24 gi

Khun lc c trng ca E. Coli trn EMB: trn li, ng knh

<0,5mm, c nh kim tm

Cy chuyn sang TSA  370C + 0,5 trong 24 gi

Cy vo mi trng 1 trypton, 2 MR-VP, 1 Simmons Citrate

370C + 0,5 trong 24 gi

S dng cc loi thuc th test th nghim IMViC

Kt lun
15

3.6. Thuyt minh quy trnh

3.6.1. Chun b mu
Chun b mu tng t nh bi 1
3.6.2. Tng sinh
Cy 1ml dch mu pha long nng 10-1 vo ng nghim cha 10ml canh
BGBL, 440C trong 24 gi.
3.6.3. Phn lp
Sau 24 gi, chn cc ng nghim cho phn ng dng tnh (mi trng c v c
sinh hi) v cy chuyn sang mi trng phn lp EMB. 370C trong 24 gi
Nhn dng khun lc E.Coli: khun lc trn, mu tm, c b u, ng knh 0,5
mm, c nh kim tm.

Hnh 4: Khun lc c trng ca E.Coli trn mi trng EMB

3.6.4. Khng nh
Nhng khun lc nghi ng c thc hin qua cc bc th nghim sinh ha (thc
hin nghim php IMViC).
Chn t nht 2 khun lc nghi ng t mi trng phn lp cy chuyn sang mi
trng rn khng chn lc (mi trng TSA), 370C trong 24 gi.
Cc th nghim sinh ha c dng khng nh E.Coli nh sau:

3
2
Hnh 5: Cc th nghim sinh ha

(1): Th nghim Indole

(2): Th nghim Methyl red
16

(3): Th nghim Voges Proskauer

(4): Th nghim Citrate
STT

Th nghim sinh ha

Kt qu

Indol

Methyl Red

Voges Proskauer

Kh nng s dng citrate

3.6.5. Bo co kt qu
Pht hin hay khng pht hin E.Coli trong 10 g (25 g) mu

17

Bi s 4: NH LNG STAPHYLOCOCCUS AUREUS

---------------------4.1. Nguyn tc
Cy trang mt lng mu xc nh trn b mt mi trng thch chn lc. Sau khi
v m nhng khun lc c cc c im c trng v khng c trng ca S.
aureus. Xc nhn cc khun lc m bng phn ng coagulase v cc c trng
khc. Kt qu c xc nh bng s khun lc m, th tch cy, nng pha
long v h s xc nhn.
4.2. Mi trng s dng
-

Mi trng BP

Mi trng TSA

Huyt tng th

4.3. Quy trnh thc hin

25g mu + 225ml SPW  ng nht trong 30 giy 
pha long 10-1

Ly 1 ml mu pha long 10-1 cho vo a mi trng

BP c b sung egg yolk  cy trang

nhit 370C trong 48h

KL c trng ca S.aureus trn BP: trn, li, c tm en v

c qung sng bao quanh

Chn 5 KL c trng cy chuyn trn mi trng TSA

Cy chuyn vo ng nghim cha 0,2 ml huyt tng th

18

 370C v theo di sau 2, 4, 6, 8 gi. Nu khng c biu hin ngng

kt s tip tc n 24 gi. Xc nh t l ngng kt R

Tng s S. aureus (cfu/g)

N
S = --------------nVf
4.4.

Thuyt minh quy trnh

4.4.1. Chun b mu
Chun b mu nh tng t bi 1
4.4.2. Cy mu
Dng micropipette chuyn 0,1ml dch mu pha long vo mi trng BP. Dng
que cy tam gic trang u trn b mt cho n khi kh. Thc hin lp lai 3 a BP
mi nng pha long. 370C trong 48 gi i vi mi trng BP.
4.4.3. c kt qu
Sau 48 gi, trn mi trng Baird Parker khun lc S. aureus c ng knh
khong 0,5 1 mm, li, en bng, c vng sng rng khong 1 2 mm bao quanh.
nh du trn mt y ca a c cc khun lc c cc c im trn v tip tc n
48 gi. Sau 48 gi, cc khun lc S. aureus c ng knh khong 1 1,5 mm, vng
sng rng khong 2 4 mm. C mt s dng S. aureus khng to cc khun lc c cc
c im trn. Cn m v nh du c 2 dng khun lc.

Hnh 6: Khun lc c trng ca S.aureus trn mi trng BP

4.4.4. Khng nh
19

Trn mi trng BP: Chn 5 khun lc c trng v 5 khun lc khng c trng

t mi trng BP cy vo mi trng TSA. 370C trong 24 gi. Cy sinh khi vi
sinh vt vo ng nghim cha mi trng huyt tng th. 370C. Theo di phn
ng ng t huyt tng trong 2, 4, 6, 8, 24 gi. Tnh t l khng nh trn cc khun
lc c trng v khng c trng.
4.4.5. Tnh ton kt qu
S lng S. aureus trong mu c tnh nh sau:
10
S S. aureus (CFU/g)

(Nt x Ht + Na x Ha)
F1 + F2

Trong : F: pha long

Nt: tng s khun lc c trng
Na: tng s khun lc khng c trng
S khun lc c trng cho phn ng HT dng tnh
Ht =
S khun lc c trng
S khun lc khng c trng cho phn ng dng tnh
Ha =
S khun lc khng c trung

20

Bi s 5: NH TNH SALMONELLA
--------------5.1. Nguyn tc
Pht hin c hay khng c Salmonella trong khi lng mu xc nh. Quy trnh
pht hin Salmonella trong thc phm c thc hin qua 4 bc:
-

Tin tng sinh

Tng sinh chn lc

Phn lp

Khng nh

5.2. Mi trng s dng

-

Mi trng canh RV

Mi trng XLD

Mi trng TSA

Mi trng TSI

Mi trng Mannitol Phenol Red both

Mi trng Urea broth

Mi trng LDC

5.3. Quy trnh thc hin

25 g mu + 225 ml BPW, ng nht mu trong 30 giy
 pha long 10-1

em nhit 370C trong 24h

Ly 0,1 ml canh trng cho vo 10 ml mi trng RV

420C + 0,5 trong 24 gi

21

Cy chuyn trn mi trng XLD v nhit 370C, 24h

KL c trng ca Salmonella trn XLD: trn, li, trong sut, c tm

en i khi tm en qu ln bao trm ca khun lc, mi trng quanh
chuyn sang mu

Cy chuyn sang mi trng TSA

370C trong 24 gi

Cy chuyn vo mi trng th nghim sinh ha: mi trng TSI,

Mannitol phenol red broth, Urea broth, LDC both

370C trong 24 gi

Biu hin c trng ca Salmonella: Trn TSI: /vng/ H2S (+)/ gas
(+); Mannitol (+); Urea (-), LDC (+)

Kt lun: Pht hin hay khng pht hin Salmonella trong 25 g mu

5.4. Thuyt minh quy trnh
5.4.1. Tin tng sinh

22

Tin tng sinh cc loi mu thng thng: cn 25g mu cho vo ti v trng. Thm
225ml dung dch pepton m v ng nht mu. Thi gian ng nht mu c th l 15
hoc 30 giy ty theo tng loi mu. 370C trong 24 gi.
5.4.2. Tng sinh chn lc
Trn mi trng canh sau khi 24 gi trc khi cy chuyn 0,1ml sang mi
trng tng sinh chn lc RV. Sau , mu nhit 420C trong thi gian 24 gi
5.4.3. Phn lp
Dng que cy vng ly mt vng sinh khi vi sinh vt t mi trng tng sinh
chn lc RV cy ria ln mi trng chn lc cho Salmonella nh: XLD, BPLS, BSA,
HE Cng chn lc, mc c trng v hnh thi biu hin ca khun lc
Salmonella trn tng mi trng khc nhau. nhn dng v chn lc c tt c cc
dng Salmonella trn tng mi trng khc nhau nh sau:
-

Mi trng XLD: khun lc c mu hng trong sut, c hay khng c tm en.

Mt s dng c th c tm en rt ln bao trm c khun lc. Mi trng XLD chuyn

sang mu hng.

Hnh 7: Khun lc Salmonella trn mi trng XLD

-

Mi trng BPLS: cc khun lc Salmonella c mu hng nht, trong sut,

xung quanh khun lc mi trng chuyn thnh .

23

Hnh 8: Khun lc Salmonella trn mi trng BPLS

Tt c cc a mi trng sau khi cy c nhit 370C trong 22 26 gi.
Sau khi , chn nhng khun lc nghi ng Salmonella khng nh bng cc th
nghim sinh ha v th nghim cc c tnh khng nguyn.
5.4.4. Khng nh
Khun lc nghi ng l Salmonella phi c kim tra sinh ha v khng huyt
thanh. T mi mi trng phn lp, cy chuyn t nht 5 khun lc nghi ng sang mi
trng khng chn lc (mi trng TSA), 370C trong 24 gi, cc khun lc xut
hin trn mi trng ny c s dng th nghim sinh ha v khng huyt thanh.
a. Khng nh bng th nghim sinh ha
Cc th nghim sinh ha chnh c s dng cho Salmonella l ln men glucose,
lactose, saccharose, H2S, urea, indol, VP, ODC, LDC, mannitol. Cc chng
Salmonella cho kt qu th nghim nh sau:
-

Th nghim trn mi trng KIA hoc TSI: Salmonella ch ln men c

glucose trong cc mi trng ni trn v th phn nghing c mu , phn su c mu

vng. a s cc dng Salmonella u c kh nng sinh H2S nn c xut hin mu en
trn mi trng. C th c hin tng sinh hi qua lm v thch mi trng ny hoc
mi trng b y ln trn ro ra mt khong trng bn di ng nghim.

Hnh 9: Kt qu trn mi trng TSI

-

Th nghim LDC (+): sau khi nui cy, mi trng chuyn thnh kim, mu

mi trng c chuyn sang mu ban u

24

Hnh 10: Th nghim LDC (+)

-

Th nghim urea (-): Salmonella khng phn gii urea nn khng lm thay i

pH mi trng; sau khi nui cy mi trng vn gi nguyn mu vng cam.

Hnh 11: Th nghim Urea (-)

-

Ln men mannitol (+): mi trng sau khi nui cy b acid ha chuyn sang

mu vng.

Hnh 12: Th nghim Mannitol (+)

-

Th nghim Indol, VP (-)

b. Khng nh bng th nghim khng huyt thanh

Th nghim khng huyt thanh phi c thc hin song song vi mu trng (dung
dch nc mui sinh l) nhm loi tr kh nng ngng kt gi. Dng khng huyt
thanh Salmonella polyvalent O v Salmonella polyvalent H. Phn ng dng tnh khi
25

vi sinh vt th nghim ngng kt vi khng huyt thanh nhng khng c hin tng
ngng kt vi nc mui sinh l.
5.4.5. Bo co kt qu
Vi quy trnh kim nghim ny, cho php kt lun c hoc khng c Salmonella
c pht hin trong 25 g mu tuy nhin khng cho php phn bit cc dng
Salmonella hin din trong mu. khng nh c dng Salmonella no nhim vo
trong thc phm th phi thc hin th nghim ngng kt vi cc loi khng huyt
thanh n gi khc nhau, ng thi c th gi chng n cc PTN chuyn nh danh vi
sinh vt.

26

PHN II

KIM NGHIM HA L THC PHM

27

Bi s 6: PHNG PHP PHT HIN NHANH PHM MU C V

KHNG C
6.1. Nguyn l
-

Phm mu kim tnh (chromobase) dn xut t than c tnh cht c hi,

khng c php s dng trong thc phm. Phm mu axit dn xut t than c
php s dng.
-

Phm mu dn xut than c tnh kim tan c trong nc hay trong cn.

Dung dch phm mu ny nu cho tc dng vi mt cht kim mnh (NH3), s lm

gii phng cht mu ca kim phm.
-

C hai loi phm mu kim tnh:

+ Chromobase: sn phm chit c mu s ha tan c trong ether v nhum mu

ether
+ Leucobase: sn phm chit khng mu, ha tan c trong ether v khng
nhum mu ether.
-

Dung dch ether c cha cht mu kim tnh nu cho tc dng vi acid long

(acid acetic), cht mu ban u li chuyn sang dung dch acid v nhum mu dung
dch acid (c laucobase v chromobase).
6.2. Ho cht dng c
a- Ho cht
- Acid acetic 5%
- Ether ethylic
- Dung dch hn hp: cn 750 + NH4OH c (1:1) hoc
nc ct + NH4OH c (1:1)
b- Dng c
-

ng nghim c np: 3 ci

a thy tinh hoc a inox c u bt

6.3. Tin hnh

-

nh s th t 3 ng nghim: 1, 2, 3

Cho vo ng 1: 5ml dung dch hn hp cn 750 + NH4OH c (1:1)

ng th 2: 5ml ether ethylic

ng th 3: 5ml acid acetic 5%

28

Cn 3g mu, ct hoc nghin nh mu cho vo ng 1. y nt, lc k yn.

Gn nc ng 1 vo ng 2. Lc nh, y nt, yn phn lp.

Gn lp ether trn (ng 2) sang ng 3. Lc u, yn v quan st

1.4. Kt qu
-

Dung dch acid acetic (di) c mu: Phm mu kim khng c php dng

Dung dch acid acetic (di) khng mu: phm mu acid c php dng

1.5. Yu cu sinh vin thc hin:

-

Thc hin th nghim vi hai mu thc phm (tht ngui hoc xi gc v ko

hoc bnh c mu thc phm)

-

Sinh vin t mang mu n PTN phn tch

29

BI S 7: XC NH HM LNG CHT BO TRONG SA THEO

PHNG PHP ADAM ROSE GOTTLIEB
7.1. L THUYT
Trong mi trng ammoniac v cn, lipid c chit xut di tc ng ca ether v
ether du ha.
-

Ammoniac c nhim v ha tan protein, lm thay i sc cng b mt ca cht

bo, ct lin kt gia lipid v protein, gip lipid ha tan d dng.

Cn c kh nng ht nc khin cho lipid d ha tan trong ether hn; gip cho vic
ct lin kt gia lipid v protein.

Ether ngoi kh nng ha tan lipid cn c kh nng ha tan mt s tp cht khc,

do phi s dng thm ether du ha.

Ether du ha mang tnh y nc cao nn c kh nng y cc tp cht ha tan

trong nc b ln trong ether thng. Tuy nhin, ether du ha khng c kh nng
ha tan lipid c cha trong nc, do vy phi s dng kt hp ether v ether du
ha.

7.2. DNG C V THIT B

-

1 bnh lng c np

1 ng ong 100 ml

1 becher 250 ml

1 pipet 10 ml

ng nh git

Cn phn tch

Bnh ht m

T sy

T ht

7.3. HA CHT
-

Dung dch cn ammoniac

Cn 900: 208,5 ml
Ammoniac: 7,5 ml
Nc ct va : 250 ml

Ether thng
30

Ether du ha

DD phenolptalein 1 %

7.4 THC HNH

7.4.1. Chun b mu
-

Cho vo bnh lng:

10 ml thc phm nu l thc phm lng. Trong trng hp l thc phm
c: cn 1,4 g mu sau thm 10 ml nc ha tan mu ri cho vo bnh
lng.
Dung dch cn ammoniac: 10ml

Ether:

11 ml

DD phenolptalein:

1 git

Lc u lc nh, sau lc mnh dn v cui cng lc mnh (ch nh k phi

dc ngc, m kha bnh lng cho thot hi) trong 5 pht.

yn 30 pht, trong bnh lng s chia thnh 2 lp:

Lp di l ammoniac ha tan protein, nc v cc thnh phn khc ca
thc phm.
Lp trn l ether ha tan trong cht bo, nc v mt s cht khc

Tch b lp di (Phn ny c chuyn sang bi sau), ly lp trn.

7.4.2. Phng php xc nh

-

Cho thm vo 10 ml ether du ha, lc mnh trong 5 pht, yn 5 pht.

Cc cht khng phi l cht bo s lng xung di. Cc cht bo cng ether bn
trn.

Chuyn ether cha cht bo vo becher sy kh n khi lng khng i v

cn trc. Trng li bnh lng 2 ln bng ether, mi ln 5 ml. Dn ht ether vo
becher.

bc hi nhit thng, sau cho vo t sy 100 1050C trong 30 pht.

Ly ra ngui trong bnh ht m v tin hnh cn.

7.4.3. Tnh ton kt qu

Hm lng cht bo c trong 100 g hoc 100 ml thc phm l:
X=

(m1 m2 ) x100
m

31

Trong :
m: khi lng thc phm cn ban u (g), hoc th tch ca thc phm o
ban u
m1: trng lng ca becher cha lipid (g)
m2: trng lng ca becher

32

BI S 8: XC NH HM LNG PROTEIN TRONG SA THEO

PHNG PHP KT TA
8.1. L THUYT
-

Cc phng php xc nh protein

Tnh cht ca protein trong sa

8.2. DNG C V THIT B

-

My ly tm

4 ng ly tm chu nhit

1 ni cch thy

a thy tinh

ng nh git

Cn phn tch

Bnh ht m

T sy

8.3. HA CHT
-

Acid trichlor acetic 50 %

Acid trichlor acetic 1%

8.4. THC HNH

8.4.1. Chun b mu
-

Tng t nh bi xc nh cht bo theo phng php Adam Rose Gottlieb.

Ly phn chit lng xung di y bnh lng.

8.4.2. Phng php xc nh

-

Cho phn lng vo ng ly tm sy kh, cn sn.

t ng ly tm ln ni cch thy cho bay ht hi ether, cn v ammoniac (khong

30 35 pht).

Cho tng git acid trichlor acetic 50 % vo ng ly tm cho n khi kt ta. Ch ,

nh tng git theo thnh ng v quan st ch tip xc gia 2 dung dch.

Dng a thy tinh khuy u.

t ln ni cch thy un si khong 30 pht protein kt ta ht.

Quay ly tm, protein s lng xung di y ng. B kt ta pha trn.

Ra ta nh trn thm 2 ln na, cui cng cht kit nc.

33

Cho ta v ng ly tm vo t sy nhit 100 1050C n khi lng khng i

(khong 4 h).

Ly ra cho vo bnh ht m, ngui v cn.

8.4.3. Tnh ton kt qu

Hm lng cht bo c trong 100 g hoc 100 ml thc phm l:
X=

(m1 m2 ) x100
m

Trong :
m: khi lng thc phm cn ban u (g), hoc th tch ca thc phm o
ban u
m1: trng lng ca ng ly tm cha m (g)
m2: trng lng ca ng ly tm

34

Bi s 9: XC NH M NGUYN LIU
9.1. Khi nim m nguyn liu
Nguyn liu m, c th coi nh hn hp c hc gm cht kh tuyt i v nc
t do:
m = mo + w (1)
Trong : m - Khi lng chung ca nguyn liu m
mo - Khi lng cht kh tuyt i (khng c m)
w - Khi lng nc cha trong nguyn liu.
+ m tng i () ca nguyn liu m: L t s gia khi lng nc trn
khi lng chung (m) ca nguyn liu m, tnh bng phn trm:
=

w
w
100 o o =
100 o o
m
mo + w

(2)

+ m tuyt i (o) ca nguyn liu m: l t s gia nc (w) v khi

lng cht kh tuyt i (mo) ca nguyn liu m, %:
o =

w
100 o o
mo

Mun quan st qu trnh sy bng ng cong ly mt cch r rng (to thnh

cc im un cc giai on sy) ngi ta thng s dng m tuyt i, cn m
tng i biu th trng thi m ca nguyn liu.
V vy trong sn xut, xc nh thnh phn m hay m ca nguyn liu
ngi ta thng xc nh v tnh ton m tng i () bng % theo biu thc (2)
trn.
9.2. Xc nh m tng i
9.2.1. Phng php sy khi lng khng i
9.2.1.1. i vi cc loi nguyn liu c m khng qu 18%: qu, c, ht,
vt liu ri v bt (vt liu rn)
Dng c th nghim
-

Cc thy tinh hay hp kim loi ng mu

My nghin hoc ci nghin, a thy tinh

T sy, bnh ht m

Cn phn tch

35

Cch tin hnh

 X l nguyn liu
-

i vi qu, c ti c th tho hoc bm nh, nghin nh i vi qu, c

i vi cc loi ht, cc loi bnh quy, vt liu dn c kch thc ln

kh

em nghin nh
-

i vi vt liu ri nh ng ct, bt ngt, cc loi bt kch thc nh

khng cn x l
-

X l nguyn liu thc hin nhanh, x l xong cho vo hp kn

Dng cn phn tch cn chnh xc 5 g mu c x l cho vo cc sch, kh

bit trc khi lng
Dng a thy tinh san u lng mu trong hp ng mu m bc hi
nhanh v u
a mu vo t sy nhit 1050C n khi khi lng khng i. Sau khi
sy cho mu vo bnh ht m lm ngui v cn. Khi kt qu gia 2 ln cn cui
cng c sai s + 0,5% coi nh khi lng khng i.
Tnh kt qu: Nu gi
-

G1: Khi lng hp v nguyn liu trc khi sy (g)

G0: Khi lng hp khng (g)

G2: Khi lng hp v mu sau khi sy (g)

G: Khi lng mu cn xc nh (g)

G = G1 G0

m tng i hay m tng i c xc nh theo biu thc sau:

=

G1 G 2
x100
G

9.2.1.2. i vi cc loi nguyn liu c m ln hn 18 %: bt nho, bt

st, ng non, du thc vt
i vi nguyn liu ny nn trn u vi mu ban u trn 0,5kg, sau ly mu
th nghim trn 20g.
Dng cc hp ng mu c th tch ln hn hp dng ng vt liu rn, a
thy tinh ngn dng trn mu, c th yn mu trong hp sau khi sy. Trc khi

36

cho mu vao hp, hp v a thy tinh cng c ra sch, sy kh, lm ngu v cn

bit trc khi lng.
Sy nhit 1050C cho n khi m mu t 18%, nghin nh, cn 5g, sy
1300C trong 40 pht ri lm tip tc nh phn 4.1. Cng thc tnh:
W = 100 G.g (%)
Trong :

G: Khi lng 20g nguyn liu sau khi sy s b nhit

1050C n m di 18% (khong 30 pht)
g: Khi lng 5 g nguyn liu (ly t G) sau khi sy 1050C n
khi lng khng i

9.2.1.3. Xc inh m nguyn liu dung dch c

- Nguyn tc: Cho nc bc hi ht, ta thu c lng m trong dung dch
- Dng c:


Cc ng mu

a thy tinh, mung

Ni un cch thy

T sy

Bnh ht m

Cn phn tch

- Tin hnh: Dng dng c thch hp ly 10 gam mu cho vo cc cng a thy

tinh bit trc khi lng. t cc c mu v a thy tinh vo ni nc cch thy
c cn nc trong cc. Tip theo ly cc ra cng a thy tinh cho vo t sy
1050C n khi lng khng i. Ch trong qu trnh c cn ni un cch thy
cng nh trong qu trnh sy dng a thy tinh khuy rt ngn thi gian.
- Tnh kt qu:
=

Trong :

G1 G 2
x100
G

G1: Khi lng mu + hp + a thy tinh trc khi sy

G2: Khi lng mu cn li + hp + a thy tinh sau khi sy kh
n khi lng khng i.
G: Khi lng mu (ban u)

9.2.1.4. Xc nh m nguyn liu dng dung dch ha tan

37

i vi loi dung dch ny thng hm lng m ln hn rt nhiu so vi lng

cht kh trong dung dch. V vy, ngi ta thng xc nh hm lng cht kh suy ra
hm lng m ca dung dch.
Cc phng php xc nh kh hay xc nh thnh phn cc cht ha tan hin
nay thng dng cc phng php sau:
-

Phng php sy kh

Phng php t trng

Phng php quang hc

Phng php ha hc

9.2.2. Xc nh m bng phng php o dn in

- Nguyn tc
+ Cht kh tuyt i ca mi loi nguyn liu c mt gi tr dn in nht
nh.
+ dn in tng theo t l thun vi m ca nguyn vt liu.
+ Da trn cc nguyn tc trn ngi ta c th o dn in ca nguyn vt
liu ri tnh ra m ca vt liu.
- Dng c:
+ My nghin hoc ci nghin
+ My xc nh m "Freuchtron"
+ My xc nh m Gralner II PM - 300
- Cch tin hnh
+ i vi loi my Freuchtron c th xc nh m ca nhiu vt liu khc
nhau: cc loi ng cc, ca cao, ch, c ph, thuc l si, bng, len, si v.v. s dng
loi my ny, nguyn vt liu phi c nghin nh, kch thc bt nghin ln nht
bng 1mm. Cn bng, len, si phi c ct ngn t 2 - 3mm. Cho nguyn vt liu
vo y o (khong 2 - 3gam), bt cng tc, my o lm vic s ch trn thang o
dn in ca mu bng "ohm", t gi tr ny tra theo bng lp sn, ta s tnh c
m ca nguyn vt liu theo %. Loi my ny c chnh xc cao, sai s 0,5%.
+ i vi loi my Grainer II PM - 300 thng dng cho cc loi ng
cc: la m, thc, go v.v. Nguyn vt liu dng cho loi my ny c th nguyn
ht, nhng tt nht l nghin nh. Cho nguyn vt liu cn o m vo y o, bt
38

cng tc, my o lm vic s cho ngay gi tr m ca nguyn vt liu tnh bng %,

v khi chuyn t "ohm" ra %, ngi ta nhn h s chuyn (h s ny ph thuc vo
cht kh tuyt i ca mi loi vt liu). Do my ny ch o vi mt s nguyn vt
liu c thnh phn ha hc gn ging nhau, c bit l hm lng tinh bt.
* u im: ca loi my ny dng pin, gn, nh, tin li cho vic s dng trn cc
cnh ng trng ng cc, kho tng bo qun, sn phi, trm sy... xc nh m
ca nguyn vt liu kp thi phc v cho cng ngh thu hoch.
* Nhc im: Ch xc nh m c i vi mt s ng cc c tnh cht gn ging
nhau. chnh xc thp, sai s 2 - 3%.
9.2.3. Phng php kt hp
- Nguyn tc: i vi mt s nguyn vt liu nh rau, qu thng c m ban
u rt cao t 60 - 90%. Nu ch dng phng php nhit (sy n khi lng khng
i) th thi gian sy di, nn ngi ta c th s dng phng php nhit kt hp
phng php o in tr bng cch: giai on u dng phng php nhit sy n
m cn li trong vt liu < 30%, sau tip tc dng phng php o in tr, tt
nht nn dng loi my o Freuchtron v n cho chnh xc kh cao.
- Cch tin hnh: xem cch tin hnh ca phng php nhit v phng php
o in tr rt ra qu trnh tin hnh thch hp.

39

Bi s 10: PHNG PHP PHT HIN NHANH HN THE

10.1. Nguyn l
Hn the c phn ng kim vi phenoltalein cho dung dch mu hng. Nu cho
dung dch ny tc dng vi glycerin trung tnh, dung dch s chuyn thnh acid, s mt
mu hng (khng mu) do to thnh acid glycero boric.
10.2. Ha cht v dng c
a- Ha cht
-

Ch th mu phenoltalein 1% trong cn

Glycerin trung tnh

b- Dng c
-

Cc 100 ml

Giy lc

ng nghim

Phu lc

a thy tinh

Ko

10.3. Tin hnh

-

Cn chnh xc 1g thc phm cho vo bnh kjedahl c di, cho vo 10ml H2SO4

m c v 5g cht xc tc. Cho vo thm khong 50ml nc ct

-

t bnh Kjeldahl ln bp in trong t ht, nghing bnh v un t t cho n

khi bc hi v hnh thnh khi trng SO2, khi bt tan un si cho n khi dung dch
mu trong sut hoc c mu xanh l ca CuSO4. ngui.
-

nh mc dung dch c v c ha thnh 250 ml vi nc ct. Cho c dung

dch trn vo bnh cu ca my ct m, trng bnh bng nc ct v cho vo bnh cu.

-

Nh vo vi git thuc th tashiro, trung ha bng NaOH 50% cho n khi

dung dch chuyn sang mu xanh l cy th dng (khong 20ml).

-

Lp bnh cu vo my ct m.

Chun b bnh hng: cho vo bnh tam gic 50 ml acid boric v 5 git thuc th

Tashiro
-

Bt my ct m, khong 15 pht. Khi dung dch trong bnh hng chuyn sang

mu xanh un tip 15 pht th dng.

40

Dng H2SO4 0,1N chun (mu xanh l cy chuyn tr li mu tm nht).

Ghi li s ml H2SO4 0,1N dng.

10.4. Kt qu
-

Nu c mu hng xut hin, nh tip 2-3 git glycerin trung tnh, mu hng s

mt i thnh khng mu: kt lun sn phm c hn the.

-

Nu khng c mu hng xut hin: kt lun sn phm khng c hn the.

10.5. Sinh vin t chn b mu th:

-

Thc hin th nghim vi hai mu thc phm khc nhau.

Sinh vin t mang mu n PTN phn tch.

41

TI LIU THAM KHO

1. Trn Linh Thc, 2002. Phng php phn tch vi sinh vt trong nc, thc phm
v m phm. Nh xut bn Gio Dc, 230 trang
2. B Mn Cng ngh Thc phm, 2004. Gio trnh thc hnh Kim nghim thc
phm 1, 2. Trng i Hc Cng Nghip Tp.HCM.
3. Quy trnh kim nghim Salmonella tht. Tiu chun Vit Nam, 1998.

42

PH LC: THNH PHN MT S MI TRNG S DNG TRONG

PHN TCH CC CH TIU VI SINH
4. Mi trng Violet Red Bile Agar (VRB)
-

Cao nm men

3g

Peptone hoc gelysate

7g

NaCl

5g

Mui mt

1,5 g

Lactose

10 g

Neutral red

0,03 g

Crystal violet

0,002 g

Agar

15 g

Nc ct

1000 ml

5. Mi trng Brilliant Green Bile Lactose broth (BGBL)

-

Pepton

10 g

Lactose

10 g

Mt b

20 g

Brilliant green

0,0133 g

Nc ct

1000 ml

6. Mi trng Baird Parker

-

Trypton

10 g

Cao tht

5g

Cao nm men

1g

Sodium pyruvate

10 g

Glycine

12 g

Lithium chloride.6H2O

5g

Agar

20 g

7. Mi trng Trypticase Soya Agar (TSA)

-

Trypticase peptone

15 g

Phytone Peptone

5g

NaCl

5g

Agar

15 g
43

Nc ct

1000 ml

8. Mi trng Eosine Methylene Blue Agar (EMB)

9. Mi trng MR VP
-

Buffered Pepton Water Powder

7g

Glucose

5g

K2HPO4

5g

Nc ct

1000 ml

10. Mi trng Simmons Citrate Agar (SCA)

-

Sodium citrate

2g

NaCl

5g

K2HPO4

1g

NH4H2PO4

1g

MgSO4

0,2 g

Bromothymol Blue

0,08 g

Agar

15 g

Nc ct

1000 ml

11. Mi trng Xylose Lysine Deoxycholate Agar (XLD)

-

Cao nm men

3g

L Lysine

5g

Xylose

3,75 g

Lactose

7,5 g

Sucrose

7,5 g

Sodium deoxycholate

2,5 g

Ferric ammonium citrate

0,8 g

Sodium thiosulfate

6,8 g

NaCl

5g

Agar

15 g

Phenol red

0,08 g

Nc ct

1000 ml

12. Mi trng Triple Sugar Iron Agar (TSI)

-

Cao tht

3g
44

Cao nm men

3g

Peptone

15 g

Proteose peptone

5g

Glucose

1g

Lactose

10 g

Sucrose

10 g

FeSO4

0,2 g

NaCl

5g

Na2S2O3

0,3 g

Phenol red

0,024 g

Agar

12 g

Nc ct

1000 ml

13. Mi trng Urea broth

-

Urea

20 g

Cao nm men

0,1 g

Na2HPO4

9,5 g

K2HPO4

9,1 g

Phenol red

0,01 g

Nc ct

1000 ml

14. Mi trng Lysine DeCarboxylase (LDC)

-

Dextrose

1g

KH2PO4

0,5 g

Nc ct

100 ml

L Lysine HCl

0,5 g

15. Mi trng Trypton

-

L tryptophan

1g

NaCl

1g

K2HPO4

3,13 g

KH2PO4

0,27 g

Nc ct

200 ml

16. Mi trng Rappaport Vassiliadis (RV)

45

Mi trng c bn
-

Tryptone

5g

NaCl

8g

KH2PO4

1,6 g

Nc ct

1000 ml

Dung dch MgCl2

-

MgCl2.6H2O

400 g

Nc ct

100 ml

Dung dch Malachite green oxalate

-

Malachite green oxalate

0,4 g

Nc ct

1000 ml

Mi trng hon chnh

-

Mi trng c bn

1000 ml

Dung dch MgCl2

100 ml

Dung dch Malachite green oxalate 10 ml

Tng th tch

1110 ml

46