142.phuong Phap Kiem Nghiem Vi Sinh Trong Thuc Pham - 2

AI HOC QUOC GIA THANH PHO HO CH MINH
TRNG AI HOC KHOA HOC T NHIEN

---oOo---

Thac s NGUYEN TIEN DUNG

Tai lieu mon
PHNG PHAP KIEM NGHEM
VI SINH VAT TRONG THC PHAM
(Tai lieu s dung noi bo)

Thanh pho Ho Ch Minh - 2007
http://www.ebook.edu.vn

1
Chng I
CH TIEU VI SINH VAT THNG C KIEM SOAT TRONG NC,
THC PHAM VA MY PHAM

Co rat nhieu vu ngo oc hay cac benh gay ra do thc pham a va ang din ra, mat du
co cac luat ve an toan v sinh thc pham a c ban hanh va ngay cang cht ch va c s
quan tam cua cong ong.
Cho en nay van con co nhng cach hieu va phan biet khong thong nhat ve khai niem
cac benh gay ra do thc pham hay ngo oc thc pham. Song e phan biet hai van e nay thong
thng da vao cac khai niem nay nh sau:
- Ngo oc thc pham la cac bieu hien benh do tieu thu thc pham co cha so lng ln vi
sinh vat, chung nhan len nhanh trong qua trnh che bien hay bao quan. Cac vi sinh vat
co the hien dien mot so lng rat t ban au trong thc pham hay nhiem vao do s tiep
xuc qua qua trnh che bien.
- Cac benh co nguon goc t thc pham do tieu thu nhng thc an cha cac vi sinh vat hay
san pham cua chung, khong phu thuoc vao so lng nhieu hay t do o khong phu thuoc
vao s che bien hay bao quan.
1. Ngo oc thc pham
Ngo oc thc pham dien ra nhieu ngi, co cung mot trieu chng va cung mot thi
iem sau khi tieu thu thc pham. Tuy nhien mc o tac ong en tng ngi se khac nhau bi
v kha nang ap ng vi oc to cua tng ngi khac nhau phu thuoc vao the trang va kha nang
trung hoa oc to cua tng ngi.
Trieu chng cua ngo oc thc pham thng co cac bieu hien nh tieu chay, chong mat,
non ma, au nhc ngi, sot, au au. Cac bieu hien trieu chng phu thuoc vao tng loai vi
sinh vat gay nen. Mc o nguy hiem va trieu chng cua benh co the gay nen do oc to cua
chung tiet vao thc pham hay do chnh te bao cua chung gay nen. e co the gay ngo oc thc
pham, vi sinh phai hien dien vi so lng te bao ln va phu thuoc lieu lng cua tng chung
loai nhiem vao, thc pham phai co cac eu kien ly hoa thch hp cho vi sinh vat o phat trien,
nhiet o va thi gian phai thch hp cho qua trnh tang trng cua chung t khi chung nhiem
vao cho en khi tieu thu e vi sinh vat nhan len en u lieu lng hay san xuat u lng oc
to gay hai.
2. Cac vi sinh vat co the gay ngo oc thc pham
Salmonella
So lng Salmonella u e gay ngo oc la khi chung hien dien ca trieu te bao trong mot
gam thc pham. Cac trieu chng do Salmonella gay ra thng la tieu chay, oi ma, buon non.
Thi gian u benh cho en khi cac trieu chng bieu hien thng sau 12-36 gi ke t khi tieu thu
thc pham b nhiem. Trieu chng thng keo dai t nhat t 2-7 ngay. Khong phai tat ca moi
ngi khi tieu thu thc pham b nhiem Salmonella ieu co bieu hien benh, ngc lai mot so
ngi khong co trieu chng lam sang khi tieu thu phai thc pham nhiem vi sinh vat nay khi o
chung c bai tiet ra ngoai. Cac loai thc pham co nguy c b nhiem Salmonella nh tht gia
cam, san pham tht, trng va cac san pham cua trng, thuy san. Nguon nhiem vi sinh vat vao
cac loai thc pham thng co nguon goc t ng ruot cua ngi va cac loai ong vat, chung
co the c nhiem gian tiep hay trc tiep. Salmonella gay nen benh sot thng han thuoc cac
serotype Salmonella typhi, Salmonella paratyphi A, B, C. cac dong nay thng khong gay benh
cho cac loai ong vat.
Campylobacter
ay la vi sinh vat gay nen benh viem nhiem ng ruot, bang cac phng phap phan
lap a chng minh vi sinh vat nay hien dien khap ni. Campylobacters la mot trong nhng he vi

2
sinh vat cua nhieu loai ong vat va chim. Nhng cac dong co kha nang gay ngo oc thc pham
khong the phat trien khi nhiet o thap hn 30
o
C, ay la vi sinh vat a nhiet bat buot. San pham
sa va tht gia cam la nhng nguon co the gay nen ngo oc do vi sinh vat nay. Nc cung la
mot trong nhng nguon mang benh nay. Campylobacters la vi sinh vat rat nhay vi nhiet o,
chung b tieu diet hoan toan bang phng phap thanh trung Pasteur, chung khong the song sot
trong thc pham co moi trng acid. Chung khong the phat trien trong thc pham bao quan
trong ieu kien hieu kh ma ch phat trien trong cac loai thc pham hut chan khong.
Khi xam nhiem Campylobacter, thi gian u benh thng t 2-11 ngay. Cac trieu chng
do vi sinh vat nay gay nen nh au nhc, tieu chay, sot, au au, kho chu, chuot rut, lanh cong,
me san. Thnh thoang co nhng bieu hien benh giong nh cam cum.
Clostridium perfringens
Quan niem ve s ngo oc thc pham do Clostridium perfringens gay ra a co nhng thay
oi trong nhng nam gan ay. Theo nhng quan niem trc ay cho rang cac dong
C.perfringens khang nhiet, tao bao t va khong lam tan mau mi co the gay ngo o thc pham.
Nhng trong nhng nam gay ay cac dong nhay cam vi nhiet, khong lam tan mau cung c
tm thay trong cac vu ngo oc do vi sinh vat nay gay nen.
V cac bao t cua C. perfringen khang nhiet nen chung thng song sot qua qua trnh
nau chn. Tuy nhien cung phu thuoc vao thi gian tiep xuc vi nhiet. Neu nhng bao t song
sot, khi gap ieu kien thch hp chung se nay mam va nhan len. Khi un nau thc an nhiet o
thap va thi gian ngan co the lam cho cac dong khang nhiet ton tai v the chung se gay tai
nhim sau khi bao quan. Cac nguon thc pham co the gay ngo oc vi cac vi sinh vat nay
thng la tht gia cam, nhat la cac loai gia cam ln ong lanh sau, tht trong cac ham cha. C.
perfringens cung c tm thay trong at, trong phan ngi va trong cac loai thc pham khac.
Cac trieu chng do vi sinh vat nay gay ra thng la au that vung bung, tieu chay. Thi gian u
benh t 12-24 gi. Cac trieu chng lam sang gay nen do oc to cua chung.
Clostridium botulinum.
ay la vi sinh vat phan bo khap ni trong at, trong nc va trong cac gia suc va cac
loai thuy san. Vi sinh vat nay sinh oc to gay benh ngo oc tht cho ngi (botulism). Benh bieu
hien rat nghiem trong ngi. Benh gay ra do oc to c hnh thanh bi C.botulinum nhiem
trong thc pham. Trieu chng lam sang cua benh la oi ma, buon non, sau o co nhng bieu
hien roi loan thanh kinh nh choang vang, roi loan th giac, roi loan cac c co va mieng, au
vung ngc, kho th va te liet, co the dan en t vong. Cac trieu chng tren bieu hien sau 12-
36 gi sau khi tieu thu thuc pham nhiem oc to. Cac trieu chng thng keo dai 2-6 ngay tuy
theo tnh trang nhiem oc va sc khoe cung tng benh nhan.
Cac loai thc pham nh tht, rau qua khong c bao quan ung qui nh hay lay nhiem
t at, phan ong vat hay do che bien khong u nhiet o trc khi dung, cac san pham ong
hop khong ung qui cach cung co nguy c nhiem vi sinh vat nay ray cao. ieu kien thch hp
cho viec hnh thanh oc to cua vi sinh vat nay ieu kien moi trng k kh, pH trung tnh,
khong co cac vi sinh vat khac canh tranh. oc to botuline do C. botulinum tiet ra gom mot so
loai khac nhau nh A, B, C1, C2, D, E, F, G. cac oc to nay la nhng protein co trong lng
phan t ln khoang 1 trieu danton. Nhng nhng dang co tac ong manh en con ngi la A, B,
va E. ay cung la mot trong nhng loai oc to sinh hoc co cng o manh nhat. Trong nhng
nam gay ay, cac vu ngo oc botulism gay ra do C.botulinum dong E thng c phat hien khi
tieu thu ca va cac san pham thuy san. Dong vi sinh vat nay thng xuyen phan lap c t cac
mau bun ay tai cac ca song.
Staphylococcus aureus
Staphylococcus aureus la VSV co kha nang san sinh mot so loai oc to ng ruot ben
nhiet, khong b phan huy khi un 100
o
C trong khoang 30 phut. Khi vi sinh vat nay xam nhiem

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vao trong thc pham, chung tiet oc to vao trong san pham va gay oc. Khi con ngi tieu thu
loai thc pham co cha oc to nay, sau 4-6 gi u benh se boc phat cac trieu chng lam sang
nh tieu chay, non ma, cac trieu chng nay keo dai t 6-8 gi. Cac loai thc pham co cha
ham lng muoi cao thng co nguy c nhiem vi sinh vat nay nh jambon, kem tong hp, nc
soup v cac loai thc pham nay t khi c x ly nhiet o cao hn 40
o
C. Cac loai thuy san
hay thc pham ong hop cung thng hay b nhiem loai vi sinh vat nay. Cac nguon lay nhiem
vao thc pham chu yeu t cac khau che bien trong nha bep. Trong t nhien cac vi sinh vat nay
thng tnh thay tren da, mui, toc hay long cua cac loai ong vat mau nong.
Vibrio spp
Cac loai Vibrio co nguon goc t bien, chung can ion Na
+
e phat trien. Giong Vibrio co
mot so loai co kha nang gay benh cho ngi nh V. cholerae, V. parahaemolyticus, V. vulnificus,
V. hollisae, V. furnsii, V. mimicus, V. fluvialis, V. alginolyticus.
V. cholerae la tac nhan gay nen cac vu dch ta tren toan the gii. Loai vi sinh vat nay
c chia thanh hai kieu huyet thanh chnh o la O1 va non-O1, kieu huyet thanh O1 bao gom
ba kieu huyet thanh phu nh sau: Ogawa; Inaba (hai kieu nay c goi chung la kieu co ien
Classic) va kieu Eltor (kieu Eltor con c goi la kieu O139). Hai kieu huyet thanh Inaba va
Ogawa ngay nay ch con c tm thay tai cac nc thuoc khu vc chau A. Trong khi o cac vu
dch ta tren khap the gii gay ra do kieu Eltor. Khi co cac tran dch do V. cholerae gay ra
thng lan truyen rat nhanh vao trong nc, gay nhiem vao thc pham, neu ieu kien ve sinh
kem, vi khuan se lan truyen qua con ngi va dch benh cang them nghiem trong. Vi sinh vat
nay san sinh oc to cholaratoxin, ay la loai oc to ng ruot co cng o manh, ch can 5g
gay nhiem qua ng mieng co the gay tieu chay cho ngi trng thanh. Mot so oc to khac
cung c vi sinh nay tiet ra nh hemolysine co oc tnh tng t tetrodotoxin (oc to ca noc)
hay oc to tng t shiga-toxin.
Cac loai thc pham co the lan truyen V. cholerae nh nc uong, nc trai cay, rau qua,
sa va cac san pham sa, tham ch bia cung co kha nang nhiem vi sinh vat nay. Cac loai san
pham thuy san ti song, khong qua gia nhiet, gia nhiet nhe hay do s nhiem cheo sau khi gia
nhiet cung c khuyen cac la co nguy c mang V.cholerae kha nghiem trong.
V. parahaemolyticus la loai vi sinh vat ton tai va phat trien trong moi trng co ham
lng muoi cao, chung thng xuyen c phan lap t cac san pham thuy san, trong cac vung
nc am ven b bien. Chung san sinh oc to hemolysine ben nhiet, chat nay chu trach nhiem
cho ac tnh khang nguyen Kanagawa. Nhng trong nhng nam gan ay cac dong
V.parahaemolyticus co phan ng Kanagawa am tnh cung co the gay benh. Trieu chng bieu
hien cua benh co the xuat hien trong khoang 2-96 gi sau khi tieu thu thc pham b nhiem, thi
gian nay phu thuoc vao lieu lng xam nhiem va the trang cua tng benh nhan, loai thc pham
tieu thu va ham lng acid trong da day. Cac bieu hien benh ly khi vi sinh vat nay xam nhiem
va au that vung bung, viem nhiem ng ruot va tieu chay nhe.
Cac loai Vibrio khac khi xam nhiem vao trong thc pham cung co the gay nen cac benh
ng ruot va co bieu hien benh ly tng t nh hai loai tren. D nhien tuy tng loai va lieu
lng ma co nhng bieu hien benh nang nhe khac nhau. Ch rieng loai V. vulnificus khong gay
cac trieu chng benh ng ruot ma chung gay nhiem trung mau cho ngi.
Escherichia coli
E. coli la vi sinh vat hieu kh pho bien trong ng tieu hoa cua ngi va cac loai ong
vat mau nong. Hau het cac dong E. coli ton tai mot cach t nhien va khong gay hai trong ng
tieu hoa, ngc lai chung con ong vai tro quan trong trong viec on nh sinh ly ng tieu
hoa. Tuy nhien co t nhat 4 dong sau ay co the gay benh cho ngi va mot so loai ong vat:
Enterobathogenic E. coli (EPEC)
Enterotocigenic E. coli (ETEC)

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Enteroinvasive E. coli (EIEC)
Enterohaemorrhagic E.coli (EHEC)/ verocytocin E.coli (VTEC) hay Ecoli O157: H7
Ro rang E.coli co the phan lap c de dang khap ni trong moi trng co the b o
nhiem phan hay chat thai. Vi sinh vat nay co the phat trien va ton tai rat lau trong moi trng.
Trong nhng nam gan ay cac nha nhien cu cung chng minh rang E. coli cung co the phan
lap c t nhng vung nc am, khong b o nhiem hu c. Vi s phan bo rong rai nh vay,
E.coli cung de dang phan lap c t cac mau thc pham do nhiem vao t nguyen lieu hay
thong qua nguon nc.
Cac dong E. coli gay benh khi chung xam nhiem vao ngi qua con ng thc pham co
the gay nen cac benh roi loan ng tieu hoa, cac bieu hien lam sang bien ong co the t nhe
en rat nang, co the e doa mang song cua con ngi phu thuoc vao lieu lng, dong gay
nhiem va kha nang ap ng cua tng ngi.
Shigella
Giong Shigella cung la mot thanh vien cua ho vi khuan ng ruot Enterobacteriacae,
chung gom co 4 loai sau: S. dysenteriae, S. sonnei, S. plexneri, S. boydii. ay la giong vi sinh vat
co te bao chu ac hieu, chung ch thch nghi va phat trien trong te bao chu la ngi va cac loai
linh trng. S hien dien cua chung trong moi trng la do s nhiem phan cua ngi va cac loai
mang vi sinh vat nay. Shigella co the ton tai hn 6 thang trong moi trng nc. Cac vu ngo oc
thc pham do Shigella gay ra chu yeu tap trung cac nc kem phat trien, che bien thc pham
trong ieu kien kem ve sinh. Benh cung co the truyen trc tiep t ngi qua ngi.
Shigella chu yeu gay nen cac benh l trc trung. Thi gian u benh sau khi tieu thu thc
pham b nhiem la 1-7 ngay. Cac bieu hien trieu chng benh co the nhe, bieu hien khong ro, ch
thoang qua nh tieu chay nhe, nhng oi khi cung co nhng bieu hien nghiem trong nh i tieu
ra mau, co nhng manh niem mac ruot, mat nc, sot cao va b co rut thanh bung. Cac trieu
chng tren co the keo dai 12-14 ngay hay lau hn. oi vi ngi ln, cac trng hp t vong do
Shigella hiem khi dien ra, nhng benh bieu hien rat nghiem trong oi vi tr em va ngi gia.
Hang nam co khoang na trieu ngi t vong do vi sinh vat gay ra tren khap the gii.
S lay nhiem vi khuan Shigella chu yeu ng mieng. Nc la mot moi trng truyen
benh quan trong, ac biet nhng ni kem ve sinh. Tuy nhien cac loai thc pham cung la
nguyen nhan gay nen cac benh do Shigella. Vi sinh vat nay nhiem vao thc pham qua nguyen
lieu hay qua trnh che bien. i khi nhiem benh do ve sinh ca nhan kem.
Listeria monocytogenes
Trong nhng nam gan ay L. monocytogenes noi len nh mot mot tac nhan gay benh
nguy hiem. oi tng gay benh cua vi sinh vat nay la tre em, phu n mang thai hay nhng
ngi gia. oi vi nhng vi sinh vat gay benh go oc thc pham khac, chung boc phat benh khi
con ngi hap thu u lieu lng, sau thi gian u benh cac trieu chng lam sang bieu hien.
Ngc lai L. monocytogenes hien dien vi mot so lng nho trong thc pham, khi c a vao
c the, chung ton tai va ch c hoi. Khi co ieu kien thuan li, chung nhan len xam nhiem vao
cac mo sau va gay benh. Cac benh do vi sinh vat nay gay nen bat au t ng tieu hoa nh
tieu chay, sot nhe. Sau o chung xam nhiem vao cac ai thc bao gay nen benh nhiem trung
mau, tac ong len he than kinh trung ng, tim mat, va co the xam nhap vao bao thai gay nen
say thai, e non hay nhiem trung thai nhi.
L. monocytogenes thuoc loai vi sinh vat a lanh, chung co the phat trien nhiet o t 2-
44
o
C. Chung thng c phan lap t cac loai thc pham nh phomat sa, tht ca rau qua va
tham ch phan lap c t trong nc mat. Trong tat ca cac cong on che bien thc pham, sa
hay rau qua eu co kha nang xam nhiem vi sinh vat vat nay. at biet trong cong oan bao quan
cac san pham nhiet o thap, vi sinh vat nay co c hoi phat trien thanh so lng ln. Cac san

5
pham thanh trung Pasteur va c bao quan nhiet o thap trong cac tu lanh co nguy c nhiem
vi sinh vat nay rat cao.
Cac virus gay benh trong thc pham
Cac t dch benh gay ra t thc pham do tac nhan virus cho en nay van la van e b
an. Nhng mot so tac gia van tin rang virus trong thc pham la tac nhan gay nen cac benh hiem
ngheo. Nhng tien bo trong cac nhien cu ve virus thc pham van con han che. Cho den nay
cac ac iem sinh ly cua virus ng ruot van con biet rat han che. Cho en nay cac phng
phap nuoi cay e phat hien virus trong thc pham cho en nay van cha the thc hien c.
Nhng vi cac tien bo ve ky thuat sinh hoc phan t nh ky thuat lai phan t, ky thuat PCR co
the phat hien c cac virus co hai cho con ngi trong thc pham.
S lan truyen virus cho ngi qua con ng thc pham c biet t nhng nam 1950.
Cac virus gay benh ng ruot cho ngi chu yeu co nguon goc t cac san pham thuy san. Cho
en nay c biet co khoang hn 100 loai virus ng ruot. Nhng ch mot vai loai trong so o
co kha nang gay benh cho ngi. Theo Kilgen va Cole (1991) cac loai vieus sau ay co the gay
nguy hiem cho ngi.
Hepatitis type A (HAV)
Virus Norwalk
Calicivirus
Astrovirus
Virus NonA va Non B.
Virus ton tai the khong hoat ong khi ben ngoai te bao, chung khong the t nhan len
trong nc hay trong cac san pham thc pham cho du bat ky ieu kien hoa ly nh the nao.
Chung xam nhiem vao thc pham hoan toan do qua trnh che bien, t nc b o nhiem. Cac loai
nhuyen the an loc co kha nang tch luy nhieu virus trong nc. Hang ngay mot con nguyen the
co the loc 1500 lt nc, theo o mot so lng ln virus co the vao c the cua con vat nay va
tch luy tai o. V the mat o virus trong c the nhuyen the cao hn rat nhieu so vi moi trng
nc chung ang sinh song.
Lieu lng gay benh cua virus co the thap hn rat nhieu so vi vi khuan khi con ngi
tieu thu thc pham b nhiem. Lieu lng gay nhiem toi thieu cua mot so loai vurus ng ruot
tng ng vi so lng hien dien trong thc pham ma cac phong th nghien co the phat hien
c bang phng phat nuoi cay.
C the ngi va cac loai ong vat la nguon cha cac virus ng ruot. Virus c tm
thay vi so lng ln trong phan cua nhng ngi b nhiem va ton tai trong nhieu ngay en
nhieu tuan. S nhiem phan vao trong thc pham bang con ng gian tiep hay trc tiep la con
ng xam nhiem virus vao thc pham.
S song sot cua virus trong moi trng hay trong thc pham phu thuoc vao yeu to nh
nhiet o, nong o muoi, cng o bc xa mat tri hay s hien dien cua cac thanh phan hu c
khac. Virus ng ruot cung co kha nang ton tai nhieu thang trong nc bien nhiet o <10
o
C
tham ch co the lau hn. V the ay cung la nhan to ch th o nhiem phan. Tat ca cac virus
ng ruot eu khang vi acid, cac enzym thuy giai, hay muoi mat co trong ng tieu hoa.
Mot so virus co the khang nhiet nh Hepatits type A, mot so khac co the khang vi cac chat tay
ue nh phenolic, ethanol . Ozon va chlorine la nhng tac nhan co the lam bat hoat mot vai
loai virus ng ruot.
e ngan nga cac benh do virus t thc pham, cac loai thc an phai c nau chn,kh
virus trc khi tieu thu, cac loai nhuyen the an loc phai c khai thac tronng nhng vung nc
khong nhiem virus hay c nuoi trong cac vung nc sach trc khi tieu thu.
Coliforms

6
Coliform va Feacal coliform c xem la vi sinh vat ch th, bi v so lng cua chung
hien dien trong mau ch th kha nang co s hien dien cua cac vi sinh vat gay benh khac trong
thc pham. Cac nha nghien cu cho rang so lng Coliform cao trong thc pham th kha nang
hien dien cac vi sinh vat gay benh khac cung rat ln. Tuy vay moi lien he gia so lng vi sinh
vat ch th va vi sinh vat gay benh con ang c tranh cai ve khoa hoc. Cho en nay moi lien
he nay van khong c s thong nhat trong cac hoi ong khoa hoc. Theo nh ngha, nhom
Coliform bao gom ca nhng vi sinh vat hieu kh va k kh tuy nghi, co Gram am, khong sinh
bao t, co hnh que, len men ng lactose va sinh hi trong moi trng nuoi cay long.
Can c vao nhiet o vi sinh vat co the phat trien e chia nhom Coliform thanh hai
nhom. Nhom Coliform co nguon goc t phan cua cac loai ong vat va, nhom nay c goi la
Coliform phan va nhom khong co nguon goc t phan ong vat. Tren thc te, cac phng phap
kiem nghiem ch xac nh Coliform phan la xac nh nhom coliform co nguon ngoc t ruot
ngi va cac ong vat mau nong bao gom cac giong nh Escherichia; Klebsiella va
Enterobater. Mot cau hoi c at ra la co phai tat ca cac thanh vien cua nhom Coliform phan
co y ngha ch th ve sinh nh nhau hay khong? Cho en nay van e nay van con ang c ban
luan, tuy nhien trong cac thanh vien cua nhom nay th E. coli la loai c s quan tam nhieu
nhat cua van e ve sinh an toan thc pham.

Bang 1: Cac loai benh va cac bieu hien lam sang do cac vi sinh vat trong thc pham gay ra
Thi gian u
benh
Vi sinh vat Non
ma
Tieu
chay
au
that
Sot Thi gian
keo dai
Cac loai thc pham
thng gap
30 phut4 gi B.cereus (emertic) +++ (+)
a
(+)
a
- Ngan Gao
8-12 gi B.cereus (diarrhoeal) - ++ ++ - 1-2 ngay Soup va cac san pham sa
4-6 gi S. aureus ++ + ++ - 6-12 gi Tht lanh
San pham sa, salad, kem,
trng, san pham len men ...
8-22 gi C. perfringens - ++ ++ - 12-24 gi San pham tht qua gia nhiet
8-48 gi V. paraheamolyticus - +++ - + 2-3 ngay Hai san
12-48 gi Salmonella (+) ++ - + 48gi-7 ngay Gia cam, th va san pham
trng
24-72 gi C.botulinum - - - -
b
3 ngay o, rau qua ong hop, tht
2-11 ngay Campylobacter - +++ ++ ++ 3 ngay-3 tuan Sa, c uong, tht gia cam,
tht ti, ca nam, sa thanh
trung Pasteur
a
: Co the bieu hien hay khong,
b
: Bieu hien roi loan


7
Chng II
KY THUAT TRUYEN THONG TRONG PHAN TCH VI SINH VAT

1. nh lng vi sinh vat bang phng phap em trc tiep tren knh hien vi
So lng Vi sinh vat trong mau co the c xac nh bang cach em trc tiep tren knh
hien vi. Qui trnh en trc tiep cho phep xac nh c so lng vi sinh vat vi ket qua cao
nhat. Mat du phng phap em trc tiep bang knh hien vi cho phep c lng nhanh chong so
lng vi sinh vat co trong mau. Tuy vay phng phap nay co nhng iem han che nhat nh nh
khong phan biet c so lng te bao song va so lng te bao chet, de nham lan te bao vi sinh
vat vi cac manh v nho cua mau va khong cho phep tm hieu cac ac iem khac cua cua vi
sinh vat c c quan sat.
oi vi cac vi sinh vat co kch thc ln nh nam men, nam moc, tao va protozoa,
phng phap em trc tiep de dang thc hien vi cac loai buong em. Co rat nhieu loai buon
em vi sinh vat khac nhau phu hp vi tng the tch va kch thc cua tng nhom vi sinh vat.
V du co the s dung buong em hong cau Petroff Hauser e em vi khuan. oi vi cac mau
nc va cac mau khac, buong em Holber c s dung rong rai nhat. Buong en Holber la
mot lam knh day 2-3mm co mot vung a em nam gia lame knh vung nay c bao quanh
bi mot ranh. a em thap hn be mat cua lame knh khoang 0,02mm, co hnh tron v the khi
phu len tren bang knh ay vat th o sau cua a em se ong eu nhau. Vung a em co ien
tch 1mm
2
va c chia thanh 400 o vuong nho hn, moi o co dien tch 0.0025mm
2
. the tch cua
moi o la 0,02 x 0,0025 mm
2
, the tch nay tng ng vi 0,00005ml.
Them vai giot formalin vao trong nhng lo cha mau, tron eu. Pha loang mau can em
sao cho trong moi o nho cua buong em co khoang 5 - 10 te bao vi sinh vat. e at c o pha
loang nh vay can phai c lng c so lng vi sinh vat trong mau, ong thi phai th vai
lan trong qua trnh pha loang. Mau phai c pha loang bang dung dch pha loang cha 0,1%
pepton va 0,1% laurylsulphat va 0,01% methyl blue. Tat ca cac dung dch pha loang eu phai
can loc trc khi s dung.
at mot got mau c pha loang vao trong a em tren buong em, ay bang knh ay
vat. Tat ca cac buong em va knh ay vat phai c lau that sach. The tch mau cha trong
buong em la khoang khong gian gia a em va knh ay vat, khong e mau trang ra ben
ngoai ranh cua a. Sau khi at mau, e yen khoang 5 phut e on nh v tr cua cac te bao
trong buong em. em ngau nhien khoang 50-100 o nho. Tnh trung bnh so lng vi khuan
trong tat ca cac o a em. Sau o nhan vi 20 000 va vi o pha loang mau trc khi em e
co c so lng te bao trong 1ml. Lap lai hai lan hay nhieu hn e lay gia tr so em trung
bnh. oi vi cac vi sinh vat tu thanh tng cum, khong the phan biet tng te bao th moi cum
c xem la mot vi khuan.
Vi nhng kinh nghiem cua moi ca nhan ve o pha loang, thao tac ky thuat thanh thao
co the nhan c cac so em chnh xac. Tuy vat ket qua co chnh xac hay khong con phu thuoc
vao s sach se cua buong em, knh ay vat nham am bao khong co vi khuan khac khong phai
t mau hien dien trong buong em va trong knh ay vat.

Ky thuat em Breed
Ky thuat nay rat hu ch trong viec xac nh tong so vi sinh vat bang phng phap em trc
tiep. Lame knh Breed co mot vung co dien tch 1cm
2
c anh dau tren lame. Dung bm
tiem, hay que cay vong cho 0,01ml mau can em vao trong vung nay, say kho bang ngon la
sau o nhuom vi methyl blue. Khi em, cho dau nhung len dien tch vung can em. Thong

8
thng vung nay co ng knh 0,16 mm. Dien tch vung em la r
2
hay 3,14 x 0,08 x 0,08
=0,02mm
2
. Vi dien tch nay chiem 1/5000 trong tong dien tch at mau la 100mm
2
hay the tch
trong vung em la 1/5000 x 0,01ml. Nh vay neu co 1 vi sinh vat trong vung em th tng
ng vi 5000/0,01ml hay 500 000 te bao/ml. So em cua cac vi sinh vat trong cac vung khac
nhau cua vung qui c c tnh bang cong thc nh sau:

2
4
10 4
d
N
C

=

d: ng knh vua vung em
N: so lng te bao trong 1 vung
C: so te bao trong 1ml

Khi s dung knh hien vi huynh quang vi cac chat nhuom khac nh acridin cam
(AODC), 4,6-dianidino-2-phenyl-indol (DAPI), fluorescein isothiocyanate (FITC) cung c s
dung rong rai e em so lng vi khuan. Cac nghien cu cua K.G. Porter va Y.S. Feig cho thay
rang khi s dung DAPI e em so lng vi khuan trong nc cho ket qua tot hn khi s dung
chat nhuom AODC khi trong mau nc co nhieu chat dinh dng va nhieu phieu sinh vat khac.
Ve o ben tnh phat huynh quang cua DAPI cung cao hn AODC. Hai tac gia tren cung chng
minh rang khi nhuom vi DAFI co the bao quan trong toi en 24 tuan ma cng o phat quan
khong thay oi. Trong khi o AODC cng o va so lng phat quang b giam khi bao quan
trong vong 1 tuan cung ieu kien.
Mot ky thuat khac da tren nguyen tac nhuom mau ac hieu DNA c goi la Hoechst
33258 cung c s dung e nhuom va em so lng vi sinh vat tren knh hien vi huynh quang.
Phng phap nay nh sau: Bisbenzumide ket hp vi vung DNA giau adenine va thyamine sau
khi gan phc hp nay se phat huyng quang. Hoechst 33258 c s dung nhieu trong viec nh
lng vi sinh vat tren be mat. at biet tren be mat co gan cac acridine orange. Mot s thuan li
khac khi s dung phng phap nhuom mau bisbenzimide cho phep em so lng vi sinh vat
trong cac dung dch chat tay ra, trong muoi dung cho cac phong th nhiem hay trong cac che
pham sinh hoc khac ma khong co s hien dien cua DNA. Phng phap em so lng vi sinh
vat bang phng phap phat huynh quang c ng dung rong rai trong viec phan tch cac mau
moi trng nh nc at .
S dung dau nhung phat quang thap la mot phat minh quan trong trong viec han che s
phat huynh quang cua cac mang loc. Mang carbonate Nuclepore la mot mang cellulose cao cap
dung trong viec em trc tiep vi khuan bi v chung co kch thc lo ong nhat va be mat mang
phang, chung co the gi lai tat ca cac vi sinh vat tren be mat cua chung. Mang Nuclepore co
the nhuom vi thuoc nhuom Irgalan en hay cac loai thuoc nhuom phu hp khac e tao nen
mot nen mau en tng phan vi mau phat quang cua vi sinh vat v the viec em c thc
hien de dang. Khi s dung thuoc nhuom la acridine cam vi khuan va cac vi sinh vat khac phat
quang mau xanh hay mau cam. Mau phat quang xanh hay cam la phu thuoc vao tnh trang sinh
ly cua tng vi sinh vat. Nhng kha nang phan biet te bao ang song hay te bao a chet bi mau
phat quang oi khi a en s nham lan. S dung ADPI nhuom DNA cua te bao vi khuan chung
a en s phat quang mau xanh dng c cho la toi u hn so vi viec s dung acridin cam
e nhuom cac vi khuan co kch thc nho.
So em nhan c t phng phap em trc tiep tren knh hien vi phat huynh quang
luon cho ket qua cao tng ng hai lan so vi ket qua nhan c bang ky thuat nuoi cay

9
khac. Nhng vi khuan co kch thc nho, co hnh dang bat thng khac cung eu co the nhn
thay bang phng phap phat huynh quang. Mot so dang vi sinh vat khong the nuoi cay trong cac
moi trng tong hp trong phong th nghiem. Hien tng khong the nuoi cay cua cac vi sinh vat
cho en nay van cha ro la do chung thoai hoa, khong the nhan len trong cac moi trng nhan
tao hay do khong ap ng c cac ieu kien sinh ly can thiet cho s phat trien cua chung.
Trong mot so trng hp, s tng quan cao gia phng phap em trc tiep bang phng phap
phat huynh quang va phng phap em khuan. Tuy nhien trong mot so trng hp khac s
tng quang nay rat kem. S khac biet gia phng phap em trc tiep va phng phap em
khuan lac phan anh s chon loc cua moi trng va ieu kien nuoi cay, mot phan te bao b chet
hay b thng ton va so loai cu the trong mau.

Bang 2: Ket qua tong so vi khuan nhan c t phng phap em trc tiep tren knh hien
vi va phng phap em khuan lac 20
o
C va 4
o
C.
em khuan lac Mau em trc tiep
4
o
C 20
o
C
Nc bien
Nc a
Nc t nhien
Nc t vnh
Alaska
Alaska
9.9 x10
4

8.2 x10
5

1.8 x10
5

5.2 x10
5

3.0 x10
5

1.4 x10
5

6.6 x10
1

9.6 x10
3

6.1 x10
2

5.0 x10
4

1.0 x10
2

1.3 x10
2

4.5 x10
1

7.3 x10
3

1.1 x10
1

2.7 x10
4

5.2 x10
2

2.4 x10
2

Gia tr cua so em trc tiep bang phng phap phat huynh quang tren knh hien vi tng
ng vi vi so lng vi sinh vat that s co the co vi tat ca cac loai khac nhau. ieu o cho
phep c oan c c so lng that s trong nc bien, trong nc t nhien, trong trong mot
loai mau nao o, mat du cac loai nay co s khac biet ln ve so lng cua tng chung loai, khac
biet ve ac iem sinh ly dien ra trong cung ieu kien cua mau. So em trc tiep thng phan
anh ung vi sinh khoi, v the thng c dung e c lng sinh khoi vi sinh vat co trong
mau. Tuy nhien e co c so em chnh xac bang knh hien vi phat huyng quang can phai em
nhieu lan va nhieu v tr khac nhau tren mau.
Them vao o neu em nhieu te bao ang phan chia co the cho phep c oan toc o
phat trien cua vi sinh vat trong mau. Tan suat cua te bao ang phan chia c nhn thay co moi
lien he vi ch so o khac cua toc o phat trien vi sinh vat nh toc o tong hp RNA c o
bi s phong thch cua adenine c anh dau. Tang xuat cua te bao ang phan chia c tnh
bang thi gian gia luc bat au s that cua te bao en khi te bao phan chia la mot hang so. Con
so nay khong phai la bat bien. That kho khan e nhan ra s phan chia te bao khi s dung knh
hien vi quang hoc ma khong s dung knh hien vi phat huynh quang.

2. Cac ky thuat nh lng vi sinh vat trong mau bang phng phap nuoi cay
Co hai cach e xac nh so lng vi khuan bang phng phap nuoi cay: phng phap
em khuan lac va phng phap c oan so lng vi khuan (MPN Most probable number).
Tat ca cac phng phap nh lng vi khuan trong mau bang phng phap nuoi cay eu yeu cau
cac te bao phai c tach ri nhau, qua qua trnh nuoi cay cac te bao nay phat trien thanh cac
dong rieng biet. Tat cac qui trnh nh lng bang phng phap nuoi cay nham chon loc mot
hay nhieu nhom vi khuan nhat nh nao o, mc o chon loc phu thuoc va tng qui trnh qui
trnh cu the. e xac nh tong so vi sinh vat trong mau phai chon qui trnh giam toi thieu kha

10
nang chon loc. Nhng du mc o chon loc mc toi thieu cung ch nh lng c so lng vi
sinh vat co the nuoi cay. Con mot so lng ln vi sinh vat khac khong the phat trien c trong
qua trnh nuoi cay th khong the nh lng c bang phng phap nay. Co cac phng phap
nh lng nuoi cay nh sau nh sau:

1.1 Phng phap em khuan lac
Phng phap em khuan lac tren a c s dung rong rai e nh lng vi sinh vat,
ac biet la vi khuan, nhng phap nay co nhng khuyet iem va a co nhng cach nhn nhan
khac nhau ve mat khoa hoc. Khuyet iem cua phng phap nay nam trong s lam dung e bieu
at sai cac ket qua. Tat ca moi ngi eu mac sai lam khi nhan ra rang phng phap nay khong
the dung e thu c ket qua tong so vi sinh vat.
Phng phap em khuan lac s dung nhieu loai moi trng va cac ieu kien nuoi cay kha c
nhau. Agar la chat thng c dung e lam ac cac moi trng nuoi cay v hau her cac vi sinh
vat eu mat cac gen tong hp enzym phan giai agar. Cac mau a c pha loang e trai len be
mat moi trng agar (phng phap trai) hay khuyet tan vao trong noi trng trc khi lam ac
(phng phap o a). Mot van e can phai can nhac la cac vi sinh vat can xac nh co the ton
tai va phat trien trong moi trng agar hay khong. Mot so vi sinh vat co the b chet khi trai tren
be mat moi trng trong qui trnh cay trai be mat. Mot so loai vi khuan khac khong the song
trong ieu kien nhiet o duy tr trang thai long cua agar trong qui trnh o a. Bi v agar ch la
tac nhan lam ran moi trng nuoi cay trong qui trnh nh lng vi sinh vat v the trong mot so
trng hp agar co the c thay the bang cac tac nhan lam ran khac. Trong mot so trng hp
cac vi sinh vat co cac nhu cau dinh dng ac biet khac, cac chat nh silicagel co the c thay
the e lam ran moi trng. Nh ng v chuan b moi trng silicagel kho khan hn chuan b moi
trng agar nen ch dung moi trng silicagel khi khong the thay the c moi trng agar.
Phng phap ong cuon c dung e nh lng cac vi sinh vat k kh bat buoc. ay la
mot phng phap cai bien cua phng phap o a. Cac a hay cac ong sau khi cay vi khuan
c u trong ieu kien nhiet o ac biet trong mot thi gian nhat nh e cho sinh vat phat trien
thanh cac khuan lac co the nhn thay bang mat trng, so lng khuan lac xuat hien tren a se
c em. So lng khuan lac se c gan cho so te bao n le trong mau ban au. e so
lng khuan lac phan anh c so lng te bao vi khuan, mau phai c phan tan eu vao
trong moi trng hay tren be mat moi trtng ran. Nhng a co qua nhieu khuan lac xuat hien
th khong the cho so lng em chnh xac bi v chung co the chong lap len nhau. Nhng a co
qua t khuan lac cung phai bo i v theo nguyen tac cua xac suat.
Nhng van e chnh yeu can xem xet trong qui trnh em khuan lac la thanh phan moi
trng, ieu kien va thi gian nuoi u. Moi trng e nh lng cac vi sinh vat d dng khong
the co nh nit phai cha cac nguon nit, carbon, phosphate de s dung va cac cation, anion
can thiet nh sat, mangie, calci, natri, clo va sulphate. Thanh phan cua cac ham lng khong
nhat nh, chung at thu cho tng loai moi trng va tng loai mau nhat nh e co the nhan
c so lng vi sinh vat muc tieu cao nhat. Nh vay moi trng nuoi cay phai co cac thanh
phan dinh dng phu hp vi tng yeu cau cua loai vi sinh vat, bao gom ca cac nhan to can
thiet khac cho s phat trien cua mot he vi sinh vat nhat nh. Muc ch chung cua moi trng la
ham lng dinh dng phai cao hn cac thanh phan co trong cac mau ang phan tch. Ham
lng dinh dng trong cac loai moi trng em tong so vi sinh vat phai u cao, va oc tnh cua
moi trng oi vi sinh vat mc thap nhat.
e nang cao hieu qua cua qui trnh nh lng vi sinh vat bang phng phap em khuan
lac, cac ieu kien can phai ieu chnh sao cho ch co cac thanh vien vi sinh vat can phat hien

11
theo nh ngha c em. Cac moi trng em a c chon loc hay phan biet vi cac thanh
vien vi sinh vat khac. Qui trnh em a chon loc c thiet ke cho thch hp vi s phat trien
cua vi sinh vat mong muon. S phat trien cua cac nhom vi sinh vat khac c loai bo bi cac
thanh phan cua moi trng hay ieu kien nuoi u. Moi trng phan biet la moi trng khong loai
bo cac nhom vi sinh vat khong mong muon ma ngc lai cho phep chung phat trien cung tren
moi trng o nhng co the biet phan biet cac nhom vi sinh vat mong muon vi cac nhom khac
bang cac ac iem nhan dang.
Moi trng cung co the c thiet lap e chon loc cac loai nam moc. Mat du ky thuat
em khuan lac khong phai la phng phap c chon cho viec xac nh so lng nam moc
trong cac mau thc pham va trong moi trng. Bi v ky thuat nay ch thch hp cho viec nh
lng cac loai vi sinh vat khong co si hay bao t. D nhien phng phap em khuan lac cung
thch hp cho viec nh lng cac loai nam men. V so lng cua vi khuan luon nhieu hn so
lng cua nam moc nen e ngan can s phat trien cua vi khuan trong cac khuan lac cua nam
moc, cac tac nhan c che vi khuan c them vao trong cac moi trng nuoi cay. Cac thuoc
nhuom bengal rose va cac khang sinh nh streptomycine, neomycine thng c s dung la
nhng tac nhan c che vi khuan. Mot bien phap n gian e c che s phat trien cua vi khuan
la ha thap pH cua moi trng nuoi cay xuong khoang 4,5 - 5,5. Hau het cac nam moc eu
khong b tac ong khi phat trien trong khoang pH nay nhng khi o vi khuan b c che.
Moi trng chon c xac lap cong thc e c che s phat trien cua nhom vi sinh vat va
cho phep mot nhom vi sinh vat khac phat trien. V du moi trng Saubouraud Dextrose Agar
c dung e nh lng nam moc da tren nguyen tac pH thap va nguon carbohydrate. Bo
sung vao moi trng cac chat khang sinh khac nh peniciline hay methyl bule la nhng tac nhan
c che vi khuan gram am. Cac vi sinh vat khang khang sinh cung co the c nh lng tren
moi trng bang cach them vao o mot lieu lng khang sinh.
Moi trng phan biet c thiet lap da tren nhieu cach. Cac thuoc th c them vao
trong moi trng e cho phep nhan ra cac s khac biet cua nhng vi sinh vat mong muon. Co
the them thuoc th vao moi trng sau khi nuoi cay e nhan ra cac loai vi sinh vat ac trng
can tm. Cha khoa nang cao kha nang phan biet cua moi trng la qui trnh nuoi cay e cho
phep moi trng phan biet mot cach tot nhat vi vi sinh vat can nh lng va cac vi sinh vat
khac co trong mau.
Moi trng Eosin methyl blue (EMB) va MacConkey agar la nhng moi trng c s
dung rong rai trong viec phan tch vi sinh vat trong nc. Nhng moi trng nay bao gom tnh
chon loc va tnh phan biet. Chung chon loc cho s phat trien cua cac vi khuan gram am bi
trong o co them vao cac tac nhan c che cac vi khuan gram dng. Moi trng nay co the
phan biet cac vi khuan co the s dung lactose bi s hnh thanh cac khuan lac co the phan biet
bang mau sac. Tren moi trng EMB, vi khuan thuoc nhom coliforms la nhom gram am tao nen
nhng khuan lac co mau tm anh kim. nh lng so lng coliform bang cach em so lng
khuan lac co cac ac iem nay. Cach nay thng xuyen c s dung trong viec phan biet cac
vi sinh vat ch th trong nc va trong thc pham.
Vi ky thuat en khuan lac, cac mau can phan tch phai c pha loang thanh chuoi pha
loang lien tiep va c xac nh mot vai nong o pha loang nhat nh e cay vao cac moi
trng a chon phu hp vi cac oi tng can xac nh. Moi khuan lac c hnh thanh c
gan cho mot n v hnh thanh khuan lac (cfu-colony forming units). Ky thuat thng qua cac
bc nh sau:
Dung dch pha loang: mot so dung dch dung e pha loang co the lam chet te bao nh:
nc muoi, nc cat. Cac dung dch pha loang khong c dung ngay khi lay ra t tu lanh co

12
the gay soc va lam cho vi sinh vat khong the phat trien c. Dung dch pepton water la dung
ch pha loang c s dung nhieu nhat va c xem la dung dch pha loang cho tat ca cac loai
mau. Neu trong mau con co mot t cac chat kh trung th phai them vao dung dch pha loang tac
nhan giam thieu kha nang kh trung cua chung, v du: neu trong mau con d lng cua cac hp
chat amonium (NH
4
-
) th co the them vao o Tween 80 hay lecithin. Ngc lai neu trong mau
co cha cac hp chat chlorin hay iodin th can them vao o cac muoi nh sodium thiosulphate.
Chuan b cac chuoi pha loang mau
Bm chnh xac 9ml dung dch pha loang vao trong ong nghiem co nap ay. Neu s dung
cac ong nghiem co nap khong ong chac c th phai kh trung ong nghiem, sau o bm dung
dch pha loang vao, cac thao tac nay phai c thc hien mot cach vo trung. Phan phoi dung
dch pha loang vao cac ong sau khi kh trung con co y ngha am bao the tch dung dch pha
loang khong b mat do qua trnh kh trung. Cac thao tac pha loang tuy thuoc vao tng loai mau.
Cu the nh sau:
- Neu pha loang cac mau la chat long, trc khi pha loang mau phai c lac ky, cam
au pippette sau vao trong mau khoang 1 hut ra 1ml, cho vao trong ong cha dung dch pha
loang au tien, lac ky ong nghiem cha mau, bo pippette va s dung, dung mot pipptte mi
thc hien lai thao tac nh tren vi cac o pha loang tiep theo. C tiep tuc nh vay cho en khi
at c o pha loang mong muon. Moi o pha loang ch c phep s dung 1 pippette. Ham
lng mau trong 1ml dung dch cac o pha loang cua day nh sau:

Ong so 1 2 3 4
T le pha loang 1/10 1/100 1/1000 1/10000
The tch mau
ban au (ml)
0,1 0,01 0,001 0,0001
o pha loang 10
-1
10
-2
10
-3
10
-4

- oi vi cac mau ran hay ban long, cac thao tac c tien hanh nh sau: can 10 g mau
vao trong cac bao dap mau hay cac may xay vo trung, them 90ml dung dch pha loang va ong
nhat mau, phai am bao sao cho vi khuan phan tan eu vao trong dung dch. Sau khi mau va
dung dch pha loang c ong nhat ta c dung dch pha loang 1/10. Cac o pha loang sau
c tien hanh tng t nh phan pha loang mau long. Trong 1ml cac dung dch pha loang cha
ham lng mau nh sau:

Ong so 1 2 3 4
T le pha loang 1/10 1/100 1/1000 1/10000
Khoi lng mau
ban au (g)
0,1 0,01 0,001 0,0001
o pha loang 10
-1
10
-2
10
-3
10
-4

Cac ong nghiem cha cac o pha loang khac nhau phai c anh dau e tranh nham lan.

Cay mau bang phng phap o a
Cac moi trng agar c un chay trong cac chai hay trong cac ong nghiem co the tch
phu hp. c lam mat trong cac be ieu nhiet 45
o
C.
Hut 1ml moi dung dch pha loang cho vao trong a petri vo trung. Moi o pha loang
thng c cay vao hai a petri hay nhieu hn. Cung s dung cac pippette sach cho moi o
pha loang. Ch chon cay vao a petri nhng o pha loang co mat o vi sinh vat phu hp, thng

13
trong khoang 25-300 te bao/ml. o vao moi a a cay 10-15ml moi trng a c un chay
va lam nguoi, lac a theo chieu va ngc chieu kim ong ho khoang 5-6 lan, ac a len mat
phang ngang va i cho en khi moi trng trong a ong ac. Lat ngc a va u trong ieu
kien nhiet o va thi gian xac nh.
Cay mau tren be mat
ay la phng phap thay the cho phng phap o a e phan tch cac ch tieu vi sinh trong
thc pham, nhat la cac ch tieu vi sinh vat nhay cam vi nhiet o nh Pseudomonas, cac vi sinh
vat nay se b suy yeu khi tiep xuc vi nhiet o cua moi trng agar trang thai long. Phng
phap nay cung c dung e phan tch cac ch tieu ma cac dong nhan c phai cay chuyen
cac bc tiep theo.
Cay 0,1ml hay the tch phu hp len be mat bang pipettes hay bang que cay len a moi
trng a c lam kho va trai eu bang que cay trang hay que cay vong e trai mau tren khap
be mat moi trng. U cac a a cay ieu kien nhiet o va thi gian xac nh tuy theo tng
ch tieu phan tch, em cac khuan lac ac trng hay tat ca cac khuan lac xuat hien tren a.
Khi em cac khuan lac, trong mot so trng hp xuat hien cac khuan lac moc lan, thong
thng cac khuan lac khac cung c nhn thay ben trong cac vet khuan lac loang. Khi em ta
ch coi khuan lac loang la mot n v hnh thanh khuan lac va en cac khuan lac khac trong
khuan lac loang. Cac khuan lac loang nay la do cac vi khuan tap trung thanh tng tap oan
trong qua trnh phat trien.
em khuan lac trong trng hp cay trang hay o a
em tat ca cac khuan lac n le tren cac a cay a chon, thong thng chon cac a co
so lng khuan lac trong khoang 30 - 300 khuan lac. Thng dung cac but co the viet len thuy
tinh e anh dau cac khuan lac a em pha sau a petri. Co the dung cac thiet b ho tr trong
qua trnh em so lng khuan lac nh may em hay knh lup. Tnh toan so n v hnh thanh
khuan lac theo cac cong thc thong ke da tren the tch mau cay, so lng khuan lac tren cac
a, o pha loang a cay va so lng a cua moi o pha loang. Thong thng so n v hnh
thanh khuan lac trong mot gram mau hay 1ml c tnh toan t nhat t hai nong o pha loang
lien tiep cua mau. Cac tng trnh ket qua c bieu dien di dang so n v hnh thanh khuan
lac/g (ml) (cfu/g). Khong bieu dien ket qua di dang so vi khuan/g(ml).
Phng phap em khuan lac tren ong
Thay v dung a petri, co the dung ong nghiem co ng knh ln hay cac chai nho co
nap e thay the. Cay mau vao, cho moi trng vao, lac theo chieu ngang cho en khi moi
trng trong chai hay ong ong ac.
Phan phoi 2-4 ml moi trng co ham lng agar cao hn cac moi trng dung cho o a
khoang 0,5-1% vao ong nghiem co nap vi the tch 25ml, moi trng a c lam nguoi en
45
o
C, cay vao ong nghiem 0,1ml mau a c pha loang. Lac cac ong theo chieu ngang trong
nc lanh cho en khi agar ong ac tao thanh mot mang film ong nhat tren thanh ong
nghiem. V the ky thuat nay can co nhng ky nang kheo leo. Mot phng phap khac co the
c dung la s dung mot mau nc a, at len tren mot manh vai va lam mot ng ranh
bang vi ong nghiem bang cac xoay tron ong nghiem at ngang tren mieng bang. Cac ong
nghiem a cay mau va moi trng c xoay tren manh cua mieng bang. Vi phng phap nay,
cac ngi thc hien co the tiet nhien c nhieu thi gian va sc lao ong.
Cac ong sau khi cay mau c lat ngc e tranh s ngng tu hi nc gay nen vet
loang sau khi u. em cac khuan lac xuat hien trong lp mang fiml agar xung quanh ong, cac
khuan lac nang sau ben trong co the c quan sat bang knh lup.

14
Phng phap dung ky thuat ong xoay c s dung nhieu trong viec phan tch cac mau
sa va cac san pham cua sa, cac loai mau thc pham cong nghiep. Cho en nay a co nhng
thiet b thay the cho viec xoay ong, am bao cac mang film tren thanh ong ong nhat va tiet
kiem c thi gian cung nh cong lao ong.
Phng phap cay theo ng xoan oc
Phng phap nay da tren s ho tr cua mot thiet b xoay. Thiet b nay co the hoat ong
hoan toan t ong va cung co the hoat ong khong t ong. Thiet b nay se phan phoi mot
lng mau xac nh len be mat a ang xoay tron. iem tiep xuc cua mau va a moi trng
bat au t trung tam a va di chuyen dan ra ben ngoai theo ng xoan oc. Sau khi lam kho be
mat moi trng, a c u trong ieu kien xac nh, vi khuan se phat trien theo ng xoan oc.
Cang xa tam cua a, mat o vi sinh vat se giam dan va se xuat hien cac khuan lac rieng biet.
Can c vao toc o xoay cua thiet b, the tch mau phan phoi va kch thc cua a, mat
o khuan lac xuat hien vong xoan cuoi cung se tnh c mat o vi sinh vat trong mau. Ket
qua tong so n v hnh thanh khuan lac c xac nh bang phng phap nay c cho la tng
ng vi phng phap o a hay cay trai tren be mat. Cac he thong t ong c thiet ke da
tren nguyen tac cay xoay c thiet ke vi s ho tr cua cac may em khuan lac ban tia laser
a c ban ngoai th trng rat thuan tien cho viec phan tch tong so vi sinh vat va cho ket qua
chnh xac trong thi gian nuoi cay ngan.
Phng phap em khuan lac tren mang loc
Phng phap nay thng c ap dung cho cac mau long nh nc, sa va c anh
gia la cho ket qua chnh xac hn so vi phng phap c oan MPN. Cac mau chat long c
loc qua mang loc co kch thc lo loc nho hn kch thc cua vi sinh vat. Vi sinh vat c gia
lai tren mang loc. Mang c at tren moi trng agar hay tren giay tham c ngam sun moi
trng nuoi cay long a chon va u trong ieu kien xac nh. Sau khi u, em cac khuan lac se
xuat hien tren be mat mang loc.
Thiet bi loc co the bang thuy tinh, bang kim loai hay bang nha, bao gom pheu loc gia
mang loc, bo thiet b ho tr hut chan khong va bnh cha. Mang loc c lam bang cellulose
ester co lo loc mam trong khoang 0,1-1 m, cac loai mang loc dung cho viec phan tch tong so
vi sinh vat thng co kch thc lo loc la 0,47m, ng knh mang loc co nhieu kch c khac
nhau phu hp vi ng knh cua pheu loc, be day cua mang khoang 120m. Khi loc tat ca cac
vi khuan eu c gi lai tren mang. Khi ac mang loc sao cho be tren cua mang hng len
pha tren, vi khuan khong tiep xuc trc tiep vi moi trng nhng, cac thanh phan cua moi
trng de dang tham qua mang va nuoi vi khuan phat trien thanh khuan lac. Mot so loai mang
loc co gan cac li k nc ch cho phep khuan lac phat trien trong moi o cua li va khong cho
lan sang cac o khac nham tao ieu kien thuan tien khi em.

1.2 Phng phap c oan so lng vi sinh bang ky thuat MPN (Most Probable Number):
Phng phap MPN la phng phap co the thay the phng phap em khuan lac e xac nh
mat o vi sinh vat trong mau, phng phap nay c da tren nguyen tac xac suat thong ke s
phan phan bo vi sinh vat trong cac o pha loang khac nhau cua mau. Moi o pha loang c
nuoi cay lap lai nhieu lan trong cac moi trng long a c chon, thong thng phai cay lap
lai t 3-10 lan tai moi nong o pha loang. Cac o pha loang c tien hanh sao cho trong cac
lan lap lai co mot so lan cho dau hieu dng tnh va mot so lan cho dau hieu am tnh. So lan lap
lai cho dau hieu dng tnh va am tnh c ghi nhan e oi chieu vi bang thong ke se c
gia tr c oan so lng vi sinh vat trong mau. Qui trnh MPN cho gia tr so lng vi sinh vat
trong mau co y ngha thong ke theo xac suat phan bo vi sinh vat trong mau khi s dung mot so

15
lan lap lai, v the khoang tin cay la rat ln. Phng phap MPN e nh lng vi sinh vat cho en
nay a co nhieu cai tien cho phep tien hanh qui trnh de dang va ton t sc lao ong hn va cho
gia tr chnh xac cao hn.
Chon nong o pha loang sao cho trong cac lan lap lai co mot so lan cho ket qua dng
tnh va mot so lan cho ket qua am tnh , s la chon nay rat quan trong. Trong nhieu trng hp
qui trnh MPN c thiet lap sao cho gia tang so lng lan lap cho ket qua dng tnh bang tn
hieu phat trien cua vi sinh vat. Trong mot so trng hp khac, qui trnh c thiet lap co nhieu
th nghiem khang nh sau khi cac lan lap lai cac o pha loang cho tn hieu phat trien cua vi
sinh vat nh phat hien protein hay mot emzym nao o. Cac th nghiem sau nay cho ket qua
dng tnh th cac lan lap lai ban au mi c khang nh la dng tnh. Cung giong nh qui
trnh em khuan lac, qui trnh MPN cung c s dung cac moi trng chon loc, khong chon loc
hay moi trng phan biet.
Phng phap MPN cung c dung e nh lng virus ng ruot. Trong phng phap
nay, mot chuoi pha loang cua mau can kiem tra c cho vao trong cac ong nghiem cha te bao
chu thch hp c nuoi cay. Sau khi u, cac ong nghiem c kiem tra hieu ng cytopathic
(CPE), o la cac bieu hien lam chet te bao b xam nhiem. So lng virus trong mau cung nhan
c t bang MPN bang s tham chieu so lng cac ong nuoi cay cho hieu ng CPE dng tnh.
So lng cua virus cung co the c nh lng bang ch so TCID 50 (Tissue culture infectious
dose 50%). Nong o pha loang thap nhat co s hien dien cua virus o la nong o co t so CPE la
50% trong cac ong nghiem.
Cung giong nh qui trnh em a, trong phng phap MPN, moi trng va nong o nuoi
cay cung c ieu chnh e chon loc cho mot nhom vi sinh vat hay phan biet cac nhom vi sinh
vat nay vi cac nhom vi sinh vat khac vi cac ac iem mong muon, ro rang rang s ket hp
gia ieu kien nuoi cay va moi trng cung co the nh lng nhng nhom vi sinh vat ac biet
nao o theo nh ngha cu the. Moi qui trnh phai c chon loc mot cach cu the va can than e
co the thu c ket qua mot cach chnh xac.
Theo quan iem cua C.H. Collins va Patricia M. Lyne thc chat con so MPN bieu dien
so lng vi sinh vat trong mau la so lng trung bnh sau cac lan lap lai. Neu so lng vi sinh
vat trong mau ln th s khac biet cua cac mau gia cac lan lap lai la nho, ket qua rieng le cua
tat ca cac lan lap gan vi ket qua trung bnh. Neu so lng vi sinh vat trong mau nho, s khac
biet nay se ln. Neu trong mot mau chat long cha 100 vi sinh vat/100 ml, th trong 10 ml mau
se cha trung bnh 10 te bao. D nhien co mot so phan mau nhieu hn 10 te bao, tham ch co
nhng phan mau co the cha 20 te bao trong 10ml mau va mot so phan mau cha t hn. Neu
tat ca cac phan mau tren c nuoi cay trong moi trng va ieu kien thch hp, cac vi sinh vat
trong mau se phat trien va cho tn hieu dng tnh.
Tng t nh vay, 1ml se cha trung bnh 1 te bao vi sinh vat, nh vay co nhng lan lay
se co 2 hay 3 te bao va co nhng lan lay se khong co te bao nao. Neu cac lan hut nay c
nuoi cay trong moi trng va ieu kien thch hp se thu c nhng ong nghiem cho ket qua
dng tnh va nhng ong cho ket qua am tnh.
Neu hut 0,1ml th sau 10 lan hut mi co kha nang nhan c 1 lan hut co 1 te bao, nh
vay hau het cac lan nuoi cay khi lay 0,1ml th eu cho ket qua am tnh.
Co the tnh toan con so chac chan so lng vi sinh vat trong trong 100ml mau bang s
ket hp cac ket qua nhan c t cac chuoi cay nh tren. Bang gia tr MPN khi s dung he
thong cay vi cac day 5 ong 10ml, 5 ong 1ml va 5 ong 0.1ml a c tnh san va dung cho phan
tch cac mau nc hay cac mau a pha loang. Cho en nay co rat nhieu bang gia tr MPN c

16
s dung vi o chnh xac va cac khoang tin cay khac nhau va c s dung cho cac muc ch
khac nhau.

Phng phap MPN c dung chu yeu e phan tch Coliforms va cac vi sinh vat khac
khi chung phat trien trong moi trng nuoi cay long cho cac tn hieu de dang nhan dang nh
sinh hi, lam uc moi trng chon loc, thay oi pH moi trng V du nam men va nam moc
trong nc trai cay hay rau qua, vi sinh vat k kh hay cac bao t Clostridia.
1.3 Phng phap lai khuan lac
Lai khuan lac la phng phap ket hp gia phng phap lai phan t va phng phap
nuoi cay truyen thong trong viec phan tch cac ch tieu vi sinh vat trong cac mau. Sau thi gian
nuoi cay tren be mat moi trng thach, cac khuan lac vi khuan c chuyen len mang lai, cac
khuan lac nay c ly giai trong moi trng kiem hay x ly bang emzym, sau o tien hanh lai
phan t. Phng phap nay phu thuoc vao kha nang phat trien cua vi sinh vat muc tieu tren moi
trng, chung khong phu thuoc s phat trien cac cac quan the vi sinh vat khac. S phat trien
cua vi sinh vat muc tieu trong moi trng lam gia tang so lng ban sao cua cac gen muc tieu,
v the chung se gia tang kha nang phat hien cua cac mau do.
Nguyen tac cua phng phap nay c phat trien au tien bi M. Grunstein va S.D.
Hogness (1975) nham muc ch sang loc tren a co mat o cao cho cac dong nuoi cay thuan.
Trong qui trnh nuoi cay khuan lac vi khuan phat trien tren moi trng ran c chuyen len
mot gia phu hp nh mang nitrocellulose, ly giai e tach DNA va bien tnh chung sau o co
nh chung tren mang. Mang loc co nh DNA c u vi mau do. Mau do c tach ra t mot
oan thong tin di truyen. Va c anh dau bang cac cac ong v phong xa nh
32
P hay cac
chat phat quang khac nh biotin. Hien tng lai c din ra neu cac trnh t base cua cac te bao
c ly giai tng ong vi cac trnh t tren mau do e hnh thanh cac oan lai mach oi. Cac
oan lai nay c phat hien bang bang cac film phong xa t ghi khi cac mau do c anh dau
bang cac ong v phong xa, hay cac film nhay sang khi cac mau do c anh dau bang biotin.
Bang phng phap nay, cac ac iem di truyen ac trng c phat hien mot cach chuyen biet.
Neu chon mau do ac trng cho mot nhom vi sinh vat, phng phap nay co the phat hien va
nh lng nhom vi sinh vat o trong mau.
Mau do c lai vi cac vi sinh vat co nguon goc t mau, hay cac dong thuan c phan
lap va nuoi cay th cap. Bang phng phap nay co the cai thien c cac bat tien trong qua
trnh phan tch bang phng phap nuoi cay thuan tuy nh:
- Tranh c nhng sai lech trong qua trnh em khuan lac tren moi trng nuoi cay
chon loc.
- Chac chan phat hien c cac dong mang kieu gen muc tieu can phat hien du cac
gen o khong hay bieu hien rat kem trong mot so ieu kien nuoi cay.
- Co the phan tch c cac vi sinh vat b tress hay khong the nuoi cay c tren cac
moi trng chon loc bang cach thay the bang moi trng khong chon loc hay moi
trng dinh dng toi a.
- Giam c thi gian phan tch bang cach giam thi gian nuoi cay, qua trnh nh
lng gia nh va thi gian khang nh kieu gen hay kieu hnh.
Phng phap lai khuan lac cung c s dung e khang nh cac dong vi sinh vat nghi
ng a c lam thuan.
ng dung chu yeu cua phng phap lai khuan lac la phat hien, nh lng va phan lap
cac vi sinh vat co kieu hnh va kieu gen ac trng. Va c s dung ac biet trong viec kiem
soat s hien dien va hoat ong cua cac dong vi sinh vat trong moi trng. Nghien cu s phan

17
bo cac vi sinh vat khang khang sinh, khang kim loai nang trong nc trong cac mau moi trng
va trong thc pham.

18
Chng 3
CAC PHAN NG SINH HOA TRONG XAC NH VI DINH VAT

e xac nh vi sinh vat da tren nhng ac iem ve kieu hnh th nhng vi sinh can phat
hien trong mau phai co cac ac iem e nhan ra chung va co cac bieu hien khac biet ve kieu
hnh trong suot thi gian nuoi cay In vitro (Atlas 1991). Trong mot so trng hp, quan sat mot
ac iem rieng biet co the u e nhan ra mot so vi sinh vat muc tieu. Trong nhng trng hp
khac, can thiet phai xac nh lan lc nhieu ac iem khac biet nhau e phan biet cac vi sinh
vat muc tieu vi cac vi sinh vat khac. Viec xac nh ro rang mot vi sinh vat tren c s kieu hnh
la cong viec kho khan bi v ngay ca nhng vi sinh vat co moi quan he ho hang xa cung co the
co kieu hnh tng t mot so ac iem.
Phng phap co ien e xac nh vi sinh vat la cay chung len moi trng ran (qui trnh
cay a) hay trong moi trng canh (qui trnh tang sinh) cha tat ca cac chat dinh dng can
thiet cho s phat trien cua vi sinh vat muc tieu, u moi trng a nuoi cay trong ieu kien thch
hp cho s phat trien cua vi sinh vat ong thi chung bieu hien kieu hnh co the nhan ra.
Qua trnh cay len a nham tach biet cac te bao cac vi sinh vat e cho chung ri nhau
nhng v tr oc lap tren moi trng ran, khi te bao vi sinh vat nhng v tr rieng re sinh san,
chung hnh thanh nhng khuan lac rieng biet bao gom nhng te bao co nguon goc t mot te bao
khi au. Qui trnh cay a bao gom hai giai oan: 1. Phan lap cac vi sinh vat t tap hp cac vi
sinh vat trong quan the t nhien sao cho kieu hnh cua moi vi sinh vat c xac nh tren c s
cua phong phap nuoi cay thuan khiet cua vi sinh vay hoc, 2. Chung phat trien va the hien
nhng dau hieu (kieu hnh) e co the nhan ra thong qua s sinh san cua te bao. ac iem kieu
hnh cua hang trieu te bao c cho la giong nhau trong mot khuan lac co the c quan sat de
dang hn nhieu so vi cac vi sinh vat rieng le.
Phng phap em a co hieu qua trong viec xac nh mot vi sinh vat cu the khi nhng
vi sinh vat muc tieu o hien dien mot so ln trong tap hp vi sinh vat, s hien dien vi so lng
thap co the khong c phat hien trong phng phap nay. Trong trng hp mot loai vi sinh vat
ch chiem mot so lng nho trong tap hp o, th co the dung moi trng nuoi cay long trong
ieu kien thch hp at hieu cho s phat trien cua vi sinh vat o e tang sinh chung.
Vi s ieu chnh cac thanh phan hoa hoc trong moi trng va ieu kien nuoi cay, qui
trnh cay a va tang sinh c ap dung e phan biet hay chon loc e phat hien cac vi sinh vat
muc tieu. Kha nang phat trien cua mot vi sinh vat tren mot moi trng cu the da vao kha nang
s dung cac thanh phan dinh dng hu c hay vo c ac hieu trong moi trng o. Moi trng
nuoi cay co tnh chon loc la do cac thanh phan cau thanh nen chung (nh la ngn carbon cu
the, nong o muoi va cac phan khac). ieu kien nuoi cay cung co the gay chon loc bi s ieu
chnh ieu kien sinh ly tang trng (nh nhiet o, nong o oxygen...). Bo sung them cac hp
chat gay c che vao moi trng e han che s phat trien cua mot so ln cac vi sinh vat va kch
thch s phat trien cac loai khac mong muon.
V ac iem kieu hnh cua vi sinh vat phu thuoc vao ac iem trao oi chat ac hieu vi
thanh phan dinh dng co mat trong moi trng, thuoc nhuom, tac nhan oxy hoa va nhieu thanh
phan khac co mat trong moi trng e phan anh s trao oi chat cac chat dinh dng ac hieu
bi cac quan the khac nhau va t o a en s bieu hien khac nhau gia cac vi sinh vat muc
tieu.
Phng phap phat hien vi sinh vat bang cac dung moi trng nuoi cay thng theo mot
qui trnh co he thong nham vao s tien li cua nhng ac iem ac hieu cua vi sinh vat nh la
kha nang s dung dinh dng, tnh khang vi cac loai khang sinh, t o vi sinh vat muc tieu co

19
u the phat trien hn so vi cac loai khac. Tom lai, ac iem co the nhan thay (nhu la sac to),
kha nang s dung mot c chat ac hieu hay tnh khang lai cac khang sinh hay kim loai nang
c dung e phan biet cac vi sinh vat muc tieu t tap hp cac vi sinh vat.
Trong kiem nghiem vi sinh vat, cac th nghiem sau day thng xuyen c s dung:
1. Th nghiem Bile esculin
Nguyen tac: Nham xac nh kha nang cua mot vi sinh vat co the thuy giai glucoside
esculin thanh escuiletin va glucose khi co s hien dien cua muoi mat.
C s sinh hoa: Esculin la mot glucoside, ay chnh la dan xuat acetal cua mot
monosaccharide. Khi mot goc khong phai la mot hydratcarbon lien ket vi mot ng tao
thanh mot hp chat goi la mot glycoside, phan khong phai ng c goi la aglucon. Aglucon
lien ket vi ng qua nguyen to oxy (lien ket glucoside).

HO CH
2
OH
CH
2
OH H OH
H H
H HO
OH H
H
OH H H OH
OH H
H OH

Trong phan ng nay esculin b thuy giai tai lien ket vi goc acetal b thuy giai bi acid
tao thanh esculetin va giai phong phan t glucose t do.
Esculetin c phong thch phan ng vi muoi sat tao thanh phc hp mau en hay
mau nau en. Trong th nghiem nay muoi nitrat sat c s dung trong moi nh la c chat nhan
dang esculetin c hnh thanh. Sau khi cay moi trng c u qua em, cac vi sinh vat cho
phan ng dng tnh la nhng vi sinh vat phan huy c esculin, chuyen moi trng thanh mau
en hay mau nam en.
Cac chung vi sinh vat oi chng cho cac th nghiem nay nh sau: oi chng dng: S.
marcessens; oi chng am: E. tarda.
2. Th nghiem kha nang len men cac nguon Carbonhydrate
Nguyen tac:
Nham th nghiem kha nang len men mot nguon carbohydrate xac nh nao o trong moi
trng nuoi cay e tao acid va co the tao hi.
C che sinh hoa:
Carbonhydrate c chia thanh cac nhom nh sau 1. monosaccharide, polyhydroxylic
aldehyde hay ketone, 2. polysaccharide hay oligosaccharide, 3. polyhydric alcohol va cac
cyclitol, cac san pham kh cua monosacchride.
Mot so alcohol c goi la ng nh adonitol, mannitol, sorbitol, tat ca cac san pham
nay la ket qua cua qua trnh kh cac monosaccharide. Cac polysachcaride, trisaccharide va
disaccharide la nhng hp chat phc tap, vi sinh vat khong the hap thu mot cach de dang cho
qua trnh chuyen hoa. e chung co the s dung c cac carbonhydrate nay, vi sinh vat phai
tng hp va tiet ra ngoai cac emzym ngoai nao nham phan huy cac cac phan t nay thanh cac
phan t n gian hn, sau o tham qua thanh te bao e chuen hoa ben trong.
+
Esculine (C
15
H
16
O
9
)
6,-Glucosido-7-hydroxycoumarin
HO
HO
Esculetin (aglycone)
6,7-dihydroxycoumarin
-D-glucose

20
Len men la mot qua trnh trao oi chat oxy hoa kh ma chat nhan ien t cuoi cung
khong phai la oxy ma la c chat hu c khac. S len men cua cac c chat hu c nh
carbohydrate thu uc hai san pham la chat kh va chat oxy hoa. Cac san pham cuoi cung nhan
c t s len men cac c chat hydrate carbon nay phu dthuoc vao cac nhan to nh: (1) dong vi
sinh vat len men; (2) c chat t nhien dung e len men; (3) thi gian, nhiet o va pH cua moi
trng. Cac san pham cuoi cung cua qua trnh len men cac nguon hydrate carbon va cac alcohol
thng la cac dang kh (H
2
, CO
2
), cac acid hu c, cac alcohol va mot vai keton
oi vi nguon carbon la glucose, mot so vi sinh vat co the len men nguon c chat nay
trong ieu kien k kh, ngc lai, trong ieu kien hieu kh chung chuyen hoa theo con ng
oxy hoa. Mot so dong vi sinh vat khac co the chuyen hoa c chat nay bang ca hai cach. S khac
biet ve cac con ng chuyen hoa cac nguon c chat hu c phu thuoc vao nhom vi sinh vat,
giong hay loai, v the can c vao cac con ng khac biet nay cung co the phan biet c cac
loai vi nhau.
Cac th nghiem kha nang chuyen hoa cac nguon hydrate carbon c tien hanh tren cac
moi trng long hay ran. Trong moi trng cha mot hay mot vai nguon hydratecarbon can tie n
hanh th nghiem va mot chat ch th, thong thng s dung chat ch th pH e phat hien cac san
pham acid c tao ra trong qua trnh chuyen hoa. e phat hien cac chat kh c tao ra, mo t
ong nghien co kch thc nho lat ngc c cho vao trong cac moi trng long, cac ong nay se
gi cac san pham kh tao ra trong qua trnh nuoi cay. Trong cac moi trng ran, phat hien cac
san pham kh tao ra bang cach cay sau vao trong phan thach ng. Cac san pham kh tao ra se
pha v phan thach nay. Thong thng phat hien s tao thanh cac san pham kh c tien hanh
trong cac phan ng len men cac nguon mono hay disaccharide va c thc hien trong moi
trng nuoi cay long. e gay k kh trong qua trnh nuoi cay th nghiem cac chat kh thng
c them vao trong moi trng nuoi cay. Mot lng nho muoi sat cung co the gay nen moi
trng k kh.
Cac th nghiem kha nang s dung cac nguon Carbonhydrate c tien hanh tren moi
trng ran phai them vao cac chat ch th pH, khi cac vi sinh vat phat trien tren be mat hay, cac
san pham acid c tao thanh lam thay oi mau cac chat ch th xung quanh khuan lac.


21

Cac san pham tao thanh trong qua trnh chuyen hoa cac carbonhydrate)

3. Th nghiem catalase
HCOOH
Acid formic
H
2
+ CO
2
Carbonhydrate
Disaccharide, trisaccharide, polysaccharide
HOOCH
2
CH
2
COOH
Acid succinic
CH
3
CH
2
COOH + CO
2

Acid propyonic
CH
3
COCHOHCH
3
+ CO
2

Acetylmethylcarbinol
CH
3
CHOHCHOHCH
3

2,3-Butylence glycol
CH
3
COCH
3

CO2 + acetone
CH
3
CHOHCH
3

Isopropyl alcohol
C
6
H
12
O
6

Glucose
CH3COCOOH
Acid pyruvic
CH
3
CHOHCOOH
Acid lactic
CH
3
COOH
Acid acetic
+
CH
3
CHO + CO
2

Acetaldehyde
CH
3
CH
2
OH
Ethyl alchol
CH
3
COOH
Acid acetic
CH
3
COCH
2
COOH
Acetoacetic acid
CH
3
CHOHCH
2
COOH
-hydroxybutyric acid
CH
3
CH
2
CH
2
COOH
Butyric acid
CH
3
CH
2
CH
2
COOH
Butyl alcohol

22
Nguyen tac: th nghiem nham phat hien s hien dien cua enzym catalase vi sinh vat.
C s sinh hoa: Enzym catalase hien dien trong hau het cac vi sinh vat hieu kh va k
kh co he thong cytochrom. Thong thng nhng vi sinh vat khong co he thong cytochrom th
cung khong co enzym catelase, v the chung khong co kha nang phan huy hydropeoxide. Nhng
vi sinh vat ky kh bat buoc nh Clostridium co he thong peroxydase thay cho enzym catalase.
Tuy the enzym catalase hoat ontg khong chuyen biet va chung co the can thiep vao hoat ong
cua enzym peroxydase. Ca hai enzym catalase va oxydase c chia vao nhom enzym
hydroperoxydase, ay la hai nhopm enzym thng c tm thay trong thc vat (peroxydase) va
trong ong vat (catalase). Catalase la mot homoprotein bao gom bon cau t protein va mot nhan
Fe
3+
.
Nhng theo Burrow va Moulder co nhng bang chng cho rang mot so vi khuan co
nhng emzym catalase khong cha nhan Fe
3+
, cac nghien cu cua Dacre va Sharpe cho thay
hoat tnh catalase co hai dang manh va yeu trong lactobacillus, cac nghien cu cua Whittenbury
cho thay cao hai dang catalase trong vi khuan lactic: nhom catalase phan huy hydroperoxyde,
nhom nay khong nhay vi ieu kien moi trng acid; va nhom gia catalase, nhom nay bi mat
hoat tnh trong moi trng acide. Mot so nghien cu khac cung chng minh rang co hai kieu
catalase.
Catalase xuc tac phan ng phan huy hp chat hydroperoxide, trong phan ng nay mot
phan t hydroperoxide ong vao tro la mot chat cho ien t va mot phan t ydroperoxide khac
ong vai tro la mot chat nhan ien t. C chat b kh bi nhan hydrogen cung cap t phan t
cho ien t, qua trnh dien ra giai phong oxy phan t. C che phan ng nh sau:
Catalase
H
2
O
2
+ H H
2
O + O
2

H

C che phan ng phan huy hydropeoxyde bi catalase
Phng phap tien hanh th nghiem
Cach 1: Cho hon hp nuoi cay vi sinh vat vao 0,5ml dung dch Tween 80, tat ca cho vao
trong ong nghiem co nap ay. Them vao 0,5ml dung dch hydroperoxyde 20% vao, lac va quan
sat. Neu co hien tng sui bot kh la co s hien dien cua catalase.
Cach 2: Nho hon hp co t le 1:1 hydroperoxyde va Tween 80 len cac khuan lac vi sinh vat
phat trien tren moi trng agar. Quan sat sau 5 phut, neu co hien tng sui bot kh th khuan lac
vi khuan co catalase.
Cac chung vi sinh vat oi chng: oi chng dng S. epidermidis, oi chng am: E. feacalis

4. Th nghiem citrate
Nguyen tac: Nham xac nh kha nang vi sinh vat s dung nguon citrat nh la nguon
carbon duy nhat.
C s sinh hoa: Nang lng cung cap cho vi sinh vat khi trong moi trng khong co
nguon nguyen lieu s dung cho qua trnh len men hay tao cac san pham lactic bang cach s
dung citrate nh la nguon carbon duy nhat. Trong thng, s trao oi chat citrate bao gom cac
bc ket hp acetyl-CoA e tao thanh oxalacacetate e cho chung i vao chu trnh Kreb. Con
ng ong hoa citrate cac vi khuan thng rat nhanh qua chu trnh tricarboxylic acid hay con
ng len men citrate. vi khuan s tach citrate bao gom mot he thong enzym khong co s

C chat Chat cho
C chat b
kh
Chat cho b
oxy hoa

23
tham gia cua enzym acetyl CoA, enzym nay c c goi la citratase (citrate axaloacetat
lyase) hay citate demolase. Enzym nay oi hoi mot cation co hoat tr 2 cho hoat ong cua chung
, thong thng cac cation nay la magne ( Mg
2+
) hay mangan (Mn
2+
).
Bc au tien vi sinh vat tach citrate thanh oxaloacetate va acetate. ay la hai san
pham trung gian trong qua trnh ong hoa citrate, con san pham nhan c t qua trnh nay phu
thuoc vao pH cua moi trng. Neu pH kiem th trong moi trng se nhan c nhieu acetate va
formate, neu pH acid th san pham tao ra la lactate va CO
2
. pH tren 7,0 khong co san pham
lactate trong moi trng va san pham nhan c nh sau:
Citrate CO
2
+ Formic acid + 2 Acetic acide
pH acid acetyl methycarbinol (acetoin) va latate la san pham chnh cua qua trnh s
dung citate.
Moi trng s dung cho qua trnh len men citrate con cha muoi vo c amonium. Mot
vi sinh vat co kha nang s dung citrate la nguon carbon duy nhat cung co the s dung
ammonium nh la nguon nit duy nhat, amonium b phan huy e tao thanh amonia, chat nay se
lam kiem moi trng nuoi cay.
Deffner va Franke thanh lap cong thc cho moi trng citrate cho cac vi sinh vat ng
ruot, san pham thu c sau qua trnh nuoi cay nh sau:
4 Citrate - 7 acetate + 5 CO
2
+ formate + Succinate
S s dung cac acid hu c dang muoi cua chung nh la nguon carbon tao nen cac
carbonate hay bicarbonate kiem tnh.
e s dung cho viec th nghiem citrate, co the s dung moi trng Simmon citrate agar
hay moi trng long Koser. Nen cay mot lng vi sinh vat va phai, neu cay vi mat o qua
day co the gay nen hien tng dng tnh gia. Sau khi cay, u nhiet o 30-37
o
C, quan sat s
phat trien cua vi sinh vat va s chuyen sang km cua moi trng.
Cac chung vi sinh vat oi chng: oi chng dng: P. rettgeri; oi chng am: S.
epidermidis.

5. Th nghiem coagulase.
Nguyen tac: Th nghiem kha nang cua mot so vi sinh vat lam ong tu huyet tng bi
hoat ong cua emzym coagulase.
C s sinh hoa: Coagulase la emzym c tao ra bi S. aureus, la mot enzym lien quan en kha
nang ben nhiet, co the ben en nhiet o 60
o
c trong gian 30 phut. Enzym nay la mot protein t
nhien, chung c tiet ra ben ngoai te bao bi cac vi sinh vat S. aureus, chung cung de dang bat
hoat bi cac protease.
Coagulase la enzym ong vai tro ong tu huyet tng, chung ket hp cac cau t trong
huyet tng thanh tng khoi hay thanh cuc.
Coagulase vi khuan
Huyet tng khoi Fibrin
C che thong thng bieu dien qua trnh ong tu huyet tng nh sau:

Prothrombin

Fibrinogen Fibrin
Thrombokinase enzym (throboplastin)
Ca
++

Thrombin

24

Mo hnh c che ong tu huyet tng

Oginsky va Umbreit cho rang co nhng bang chng chng minh rang coagulase co mot
c che ngng ket huyet tng khac, chung la nhng tac nhan hoat hoa cac thanh phan trong
huyet tng e chuyen fibrinogen thanh khoi fibrin. Smith va cac ong s cho rang coagulase la
mot c chat giong priothrombin, chat nay phan ng vi cac huyet tng tao thanh phc chat
giong thrombin va chat nay ket hp cac fibrinogen thanh khoi fibrin.
Theo Burrow va Moulder, s ong tu huyet tng dien ra qua hai bc nh sau:
Bc I: Phan ng gia enzym do vi khuan tiet ra (proenzym) vi cac nhan to hoat ong
hien dien trong huyet tng e tao thanh coagulase.
Bc II: Coagulase hoat ong ngng ket cac thanh to fibrinogen thanh fibrin.
Cung theo Burrow va Moulder nhan to do vi khuan tiet ra la procoagulase ket hp vi
nhan to trong huyet tng la mot dang globulin, nhng chat nay khong c chng minh co phai
la prothrombin hay khong. Tacgia Dithrie chng minh rang enzym coagulase do Burrow va
Moulder thanh lap chnh la procoagulase, chung hien dien dien hai dang: dang coagulase gii
han ben trong te bao va dang t do. Trong cac th nghien cogulase c tien hanh tren lam
knh ch co cac coagulase gii han tham gia vao viec chuyen fibrinogen thanh fibrin con trong
th nghien coagulse tren ong ca hai dang gii han va t do tham gia vao viec ngng ket nay
Con theo cac nghien cu cua Soulier, Tager va Zajden ket luan rang, coagulase va
prothrombin khong co hoat tnh enzym, nh s tham gia cua chung tao nen cac phc hp ben
vi cac hoat ong ly giai ac hieu goi la taphylothrombin, staphylocogulase khong co hoat tnh
ly giai, chung phan ng mot cach chuyen biet vi cac prothrobin va hoat hoa hp chat nay e
a en s ket hp cac figrinogen thanh cac khoi fibrin. S o hoat ong nh sau:

S o c che tac o cua Staphylocoagulase len s ngng ket huyet tng

Cach tien hanh: e phat hien coagulase cua vi khuan co hai cach tien hanh nh sau:
Cach I: Tien hanh th nghiem tren Lam knh
Lay 1 hay 2 khuan lac cua vi khuan huyen phu vao trong got nc tren lam knh, quan
sat trong khoan 10 giay, neu khong co hien tng ngng ket, dung que cay a huyet tng tho
hay ngi a c co nh bang EDTA hoa vao trong huyen phu vi khuan. Quan sat trong sau
10 giay. Hien tng ngng ket xay ra co the quan sat c bang mat thng. Tien hanh kiem
chng song song vi cac dong vi sinh vat co coagulase dng tnh a biet.
Cach II: Tien hanh th nghiem tren ong, ay la phng phap nham khang nh cac mau
a th nghiem am tnh tren lame.
Cho 0,2ml huyet tng vao 0,8ml moi trng Nutrient Broth a cay vi khuan khong co
glucose at trong be ieu nhiet 37
o
C, quan sat sau 3 gi, neu khong co hien tng ngng ket
dien ra, co the e nhiet o phong qua em va quan sat tr lai.
Tren th trng hien nay co cac loai huyet tng c co nh trong cac gia the khac
nhau nh EDTA, citrate hay trong oxalate. Neu s dung huyet tng citrate co the gay ngng
Staphylocoagulase
Prothrombin
Staphylothrombin
Fibrinogen Fibrin

25
ket bi cac dong vi sinh vat s dung citrate hay fecal Streptococci. V the e kiem tra
Staphylocoagulase ch nen s dung loai huyet tng co nh trong EDTA hay trong oxalate.
Cac th nghiem tren lam ch phat hien cac coagulase gii han, chung se hoat ong trc
tiep len cac fibrinogen. Th nghiem tren cac ong nghiem nham phat hien ca cogulase t do va
coagulase gii han, chung hoat ong lam ngng ket cac thanh phan trong huyet tng. Hien nay
co nhieu kit a c thng mai hoa nham phat hien S. aureus nh nha ngng ket (latex kit)
nh slidex (biomerieux); Staphylex (Oxoid); Staphynuclease (API)
6. Th nghiem decarboxylase va dehydrolase cua cac amino acid
Nguyen tac: Th nghiem dehydrolase va decarboxylase nham xac nh kha nang cua
mot vi sinh co the tong hp cac emzym kh nhom carboxyl hay tach hydrogen t cac acid amin
nh lysine, ornithin, hay arginine, qua o chung lam kiem moi trng nuoi cay.
C s simh hoa: kh nhom carboxyl cua mot acid amin la qua trnh vi sinh vat tac ong
len nhon carboxyl (-COOH) cua mot acid amin e giai phong ra cac amin, hay di amin va CO
2
.
R-CH-NH
2
-COOH RCH
2
-NH
2
+ CO
2

Amino acid amin
Enzym decarboxylase co nhat nhieu loai khac nhau, trong o mot loai se tac ong len
mot c chat khac nhau. Trong cac phong th nghiem nghien cu vi sinh vat gay benh ba nhom
enzym decarboxylase quan trong thng hay s dung la lisine, ornithine, arginine. ay la nhng
enzym cam ng, chung ch c tong hp khi trong moi trng nuoi cay co pH acid va co c
chat ac hieu. Cac san pham cua chung se lam cho moi trng chuyen sang kiem. Qua trnh
nay dien ra tren cac aminoacid co nhieu hn mot goc NH
2
ngoai nhom NH
2
C
.
trong ieu
kien moi trng k kh, va can co mot coenzym, thong thng coenzym nay la pyridoxal
phosphate.
Khi enzym lisine decarboxylase tac ong len amino acid L-lisin va kh nhom carboxyl,
chung tao thanh mot i amin la cadaverine va CO
2
, qua trnh dien ra theo phan ng nh sau:
NH
2
NH
2

CH
2
CH
2

(CH
2
)
3
Lisine decarboxylase (CH
2
)
2
+ CO
2

CH
2
CH
2

NH NH
2

COOH

Phan ng kh amin bi lysine decarboxylase

NH
2
NH
2

CH
2
CH
2

(CH
2
)
2
(CH
2
)
2
+
CH NH
3
CH
2

COOH NH
2

L-lisine Cadaverine (diamin)
CO
2
Phan ng kh amin bi Ornithine
decarboxylase
Ornithine
decarboxylase
-CO
2
L-Ornithine
Putrescine (diamin)

26

Acid amin L-ornithine b kh nhom carboxyl bi enzym onithine decarboxylase se thu
c mot diamin la putrescine va CO
2
. Ca hai chat putrescine va cadaverine eu ben trong ieu
kien k kh. V the khi nuoi cay vi sinh e th nghiem, phai nuoi trong ieu kien k kh, tren be
mat moi trng nuoi cay phai c phu mot mi parafin hay dau khoang e ngan can s khuech
tan cua oxy. Sau qua trnh nuoi cay, pH cua moi trng se thay oi ve pha kiem, pH cua moi
trng co the c kiem soat do o co the nhan biet phan ng trong moi trng nuoi cay bi
cac chat ch th pH. Cac chat ch th pH thng c s dung trong th nghiem nay la
Bromcresol purple hay cresol red.
Rieng oi vi L-arginine co the c trao oi bi hai con ng trong qua trnh nuoi cay,
thong qua hai enzym: arginine dehydrolase va Arginine decarboxylase. Hai con ng nay co
the dien ra ong thi trong qua trnh nuoi cay hay co the dien ra rieng le:
+ Phan ng arginine decarboxylase: qua trnh trao oi arginine nh enzym arginine
dehydrolase c tien hanh theo s o sau:

NH
CNH
2
CH
2
NH
2

NH (CH
2
)
2
+ CO
2

(CH
2
)
2
CH
2
NH
2

CH NH
2

COOH

Hoat ong cua toan he thong nh sau

Decarboxylase
L-Arginine Putrescine
L-Arginine L-Agmatine + CO2
Arginine dehydrolase
Agmatinase (agmatine
ureohydrolase)
Agmatinase (agmatine
ureohydrolase)
Putrescine + Urea
Urease
2 NH
3
+ CO
2
NH
3

+
Monocarbaminyl-
putrescine
Putrescine
+
CO
2

+
2 NH
3


27

Theo he thong nay, san pham sau qua trnh trao oi chat nhan c agmatine va mot
lng ln putrescine, nhng chat nay khong phai la san pham trao oi chat cuoi cung ma chung
tham gia vao mot loat cac phan ng khac, cuoi cung se thu c cac san pham la CO
2
va NH
3
.
+ Phan ng arginine dehydrolase: qua trnh trao oi chat theo hng kh hydro cua
arginine dien ra theo s o nh sau:

He thong hoat ong cua vi khuan co he enzym arginine dehydrolase

Bc au tien cua qua trnh la tach hydro cua goc NH
2
t arginine bi enzym arginine
dehydrolase e tao thanh citrulline va NH
3
va mot phosphate vo c. Bc tiep theo citrulline
di tac dung cua enzym citrulline ureidase co s hien dien cua H
3
PO
4
e tao thanh ornithine
va carbarmyl phosphate, chat nay se c thuy giai e thu nhan ATP. Nh vay ket thuc qua
trnh se thu nhan c ATP cho cac hoat ong khac cua vi sinh vat.
San pham cuoi cung cua qua trnh phan huy arginine cung nhng san pham NH3 va
CO2, ay la nhng chat lam kiem moi trng nuoi cay. Co the nhan biet phan ng nay qua cac
chat ch th pH hien dien trong moi trng.
Phng phap th nghiem cua Falkow c s dung oi vi cac vi sinh vat co hnh que,
gram am, nhng phng phap cua Moeller cho ket qua tot hn oi vi cac vi sinh vat thuoc ho
Enterobacteriaceace. Moi trng Falkow c s dung ch cho th nghiem Lisine
Decarboxylase, trong khi o moi trng Moeller c s dung cho tat ca cac th nghiem oi vi
Lysine, Arginine va Ornithine.
Vi sinh vat c nuoi cay trong moi trng Falkow, u 37
o
C trong 24 gi, chat ch th
trong moi trng la bromocresol purple. Neu phan ng dng tnh, moi trng gi nguyen mau
tm ban au, canh khuan uc, neu phan ng am tnh, moi trng chuyen t mau tm sang vang.
Neu s dung moi trng Moeller cho cac th nghiem nay, phai nuoi cay vi sinh vat trong
ieu kien k kh vi dau parafin hay dau khoang tren be mat, u 37
o
c trong thi gian 24-28
Arginase
L-Arginine Ornithine + Urea
Urease
2 NH
3
+ CO
2

Arginine dehydrolase
L-citruline + NH
3
Citrulline ureidase
2 NH
3
+ CO
2
+ L-ornithine
Ornithine
decarboxylase
Putrescine + CO
2

28
gi. Trong moi trng co hai chat ch th pH: brommothymol blue va cresol red. Neu sau khi
nuoi cay moi trng chuyen thanh vang la tn hieu am tnh, ngc lai moi trng gi nguyen
mau ban au va uc canh nuoi cay la tn hieu dng tnh.
Cac vi sinh vat oi chng:
Lisin: dng tnh: S.marcescens,am tnh: P.rettgeri
Arginine: dng tnh: E.cloacae, am tnh: E.aerogenes
Ornithine: dng tnh: S.marcescens, am tnh: P.rettgeri

7. Th nghien DNAse va thermonuclease
Nguyen tac
- th nghiem DNAse nham phat hien kha nang tong hp enzym deoxyribonuclease
(Dnase) e phan huy DNA.
- Th nghiem thermonuclease nham th nghiem tnh ben nhiet cua Dnase t
Staphylococcus aureus khi vi sinh vat c un nong.
C s sinh hoa
Th nghiem DNAse: Enzym nuclease la enzym phan huy acid nucleic c chia thanh
hai nhom nh sau: Nhom 1. Endonuclease la nhng enzym phan huy cac noi phosphodiester
ben trong mach DNA e tao ra cac au 3-hydroxyl va 5-phosphoryl hay 5-hydroxyl va 3-
phosphoprryl. Nhom 2. Exonuclease ch phan huy cac nucleotide cac au tan cung cua chuoi
DNA . Mot so nuclease co the phan huy ca hai mach tren si DNA, mot so khac ch co the phan
huy mot mach n. DNA co cac ac tnh vat ly va hoa hoc khac vi nucleotide va
oligonucleotide. S khac biet nay c dung e phat hien s ly giai DNA bi cac enzym
Dnase.
Dnase la mot enzym ngoai bao, chung ch phan huy cac acid nucleic. Hau het tat ca cac
Dnase vi khuan eu can mot cation hoa tr hai e cho enzym nay hoat ong. Cation thong
thng hien dien trong cac pepton c s dung trong moi trng nuoi cay th nghiem. Hoat
ong cua enzym xay ra trong khoang pH 5,5-8,5 nhng thch hp nhat tai pH 7,2. Co the phan
biet nhieu loai DNAse bang cac khang the hay bang cac tac nhan c che.
Khi phan giai, khoan so noi phosphodiester hay noi hydrogen b phan huy va thu c
mot hon hp cac oligonucleotide trong moi trng nuoi cay. Trong hon hp nay gom co cac
oan DNA co tan cung la 3-hydroxyl va 3-phosphoryl; 5hydroxyl va 5-phosphoryl. Khi DNA
con lai cac mach ngan, nang nhiet o hay pH cao qua bnh thmg se lam bien tnh cac oan
nay. Khi co hien tng bien tnh, hon hp DNA se giam o nht va gia tang mc o hap thu UV
bc song 260nm. Hieu ng thay oi nay la do co nong o deoxyribonucleotide gia tang trong
dung dch so vi c chat DNA ban au.
DNA hien dien trong cac dch chiet vi sinh vat. Nhng DNAse ngoai bao ch hien dien
mot so loai nh S. aureus hay Streptococcus nhom A
- Thermonuclease cua S. aureus: cung nh tat ca cac vi sinh vat khac S. aureus eu co
the tong hp DNAse ngoai bao, nh co iem khac biet so vi tat ca cac loai vi sinh vat khac,
DNAse cua S. aureus ben nhiet hn va can ligand Ca
2+
cho s hoat ong cua chung, chung phan
huy DNA manh pH kiem 8,6-9,0. S hien dien cua DNAse ben nhiet S. aureus co lien quan
en s co mat coagulase loai vi sinh vat nay. DNAse cua S. aureus co the bao toan hoat tnh
khi nang nhiet o en 100
o
C trong 15phut.

29
Phng thap th nghiem: trai mot tham vi khuan day len moi trng Dnase agar, u qua
em. Cho acid gap tren be mat moi trng sau khi u. Quan sat hien tng tua cua cac muoi co
trong moi trng. Neu xung quanh khuan lac hay tren a co nhng quang trong suot do khong
co tua cac cac muoi trong moi trng la phan ng dng tnh.

8. Th nghiem kha nang sinh H
2
S
Nguyen tac: Xac nh kha nang sinh H
2
S t cac hp chat hu c va vo c cha lu huynh
trong qua trnh phat trien cua vi khuan tao nen cac vet mau en tren moi trng nuoi cay,
C s sinh hoa: Khi phan giai protein thu c cac aminoacid, cac vi sinh vat d dng
co the s dung cac aminoacid e trao oi trong qua trnh sinh trng va phat trien va phong
thch kh H
2
S t nhng amonoacid cha lu huynh. Cac nguon pepton, cystein, cystine, sulphite,
thiosulphate la nhng nguon cha lu huynh, nhng cac loai vi sinh vat khac nhau ch co the s
dung mot so hp chat cha lu huynh nhat nh e tao H
2
S. Cac enzym cua vi sinh vat tham gia
vao qua trnh nay la desulphohydrase.
Mot vi sinh vat tao H
2
S khi nuoi cay trong moi trng cha cac hp chat lu huynh ma
chung co kha nang ong hoa se kh cac hp chat nay e tao nen kh H
2
S.

CH
2
SH Cysteine desulfurase CH
3

CH NH
2
+ H
2
O C = O + H
2
S + NH
3

COOH COOH

Trong moi trng co cha cac ion kim loai nh ion sat la dau hieu e nhan biet H
2
S. Cac
hp chat co the nhan biet H
2
S nh sat, FeSO
4
, ferric amonium sulphate, sodium thiosulphate,
bismuth sulphite. Nguyen tac e nhan biet H
2
S bang cac hp chat tren nh sau:
H
2
S + Fe
2+
= FeS + 2 H
+

Cac hp chat sulphur kim loai eu co dau hieu mau en.
Trong cac moi trng khac nh Kligler Iron agar chat ch th e nhan biet H
2
S la cac
muoi ferric ammonium citrate va mot chat vo c sodium thiosulphate. Ca hai chat ch th nay
eu phai hien dien ngay t au trong moi trng. Ket qua nhan biet s tao thanh H
2
S qua hai
bc nh sau:
Bc I: Vi sinh vat phat trien trong moi trng se kh hp chat thiosulphate e tao thanh
sulpite va sulphate. Cac nguon carbohydrate co trong moi trng se tao thanh moi trng acid
trong phan sau cua moi trng KIA thong qua qua trnh ho hap k kh. Moi trng acid trong
phan sau cua ong nghiem se ho tr cho phan ng kh tren. Qua o lu huynh ong vai tro la
chat nhan dien t cho qua trnh oxy hoa-kh cac hp chat hu c. Thiosulphate thay the
sulphate nh la chat nhat ien t va la nguon cung cap sulphur cho vi sinh vat.

4H
+
+ 4e + 3S
2
O
3
2-
2SO
3
2-
+ 2 H
2
S

H
2
S tao thanh la mot chat kh khong mau. Mot chat ch th th hai can thiet e nhan
dang chat kh nay.
Bc II: Chat kh khong mau H
2
S phan ng vi cac muoi kim loai nh ferric ammonium
citrate e hnh thanh cac hp chat ket tua co mau en FeS. Ferric Ammonium citrate c s
Thiosulphate
Reductase
Sulphite
Hydrogen sulphur Thiosulphate
Cysteine Acid pyruvic

30
dung nhieu hn citrae sat bi vi chung tan nhieu hn trong moi trng. FeSO
4
cung co the c
thay the cho nguon sat trong moi trng. Ngoai ra kh H
2
S cung co the c phat hien bi cac
kim loai khac nh ch, bismuth, e hnh thanh nen cac hp chat sulphur kim loai co mau en.

9. Th nghiem kha nang sinh indol
Nguyen tac: Phat hien kha nang cua vi khuan co the tao indol trong moi trng canh
trypton
C s sinmh hoa:Tryptophan la mot aminoacid co the b oxy hoa bi mot so vi sinh vat
nhat nh tao nen cac hp chat indol hay cac dan xuat cua chung nh: indol, skatol (methyl
indol) va indolacetic. Mot so enzym noi bao c goi chung la trytophanase tham gia trong qua
trnh tao thanh san pham indol trong hoat ong thuy giai tryptophane.
Cac hp chat trung gian trong qua trnh thuy giai tryptophan la acid indolepyruvic, t o
indol c hnh thanh trong qua trnh kh amin, hnh thanh skatol la qua trnh kh carboxyl cua
acid indol acetic. Qua trnh thuy giai tryptophan co the c mo ta qua qua trnh nh sau:
NH
3

CH
3
C COOH

N
H

CH
3
C COOH

N N
H H

CH
2
CHO

N
H

CH
2
COOH
N
H

CH
3

N
H

Enzym tryptophanase xuc tac phan ng kh amin bi s tan cong vao phan t tryptophan
v tr mach nhanh va tach nhan thm ra khoi phan t tryptophan hnh thanh indol.
CH
3
CH - COOH
NH
2
+ CH
3
C COOH + NH
3

N N
H H
H
L-Tryptophane
Indolepyruvic
Indoleacetaldehyde
Indole
Indoleacetic acid
Skatole
Tryptophanase

31
S tach amin t tryptophane la mot dang phan ng kh. Qua qua trnh nay nhom amin
c tach ra va chuyen thanh NH
3
, qua trnh kh giai phong mot phan nang lng c s dung
cho hoat ong tang trng cua vi sinh vat. ong thi qua trnh nay con tao thanh acid pyruvic,
c chat tham gia vao chu trnh Krebs e tiep tuc thu nang lng.
Phat hien indol va cac dan xuat cua chung trong moi trng nuoi cay bang thuoc th
Kovacs hay Ehrlichs. C chat chnh tham gia phat hien indol trong moi trng nuoi cay la p-
dimethylaminobenzaldehyde (DMAB). Chay nay phan ng vi indol tao thanh mot phc hp
mau o. Phan ng nay la do nhan pyrrol cua indol phan ng vi nhom aldehyde cua DMAB qua
hai giai oan e tao thanh nhan quinone co mau o, phan ng nh sau:

Phng phap th: Vi sinh vat th nghiem c nuoi trong moi trng canh trypton khoan
24 - 48 gi. Nho vai giot ether vao canh khuan nham keo indol len be mat mot trng, them vai
giot thuoc th Kovacs hay Erhlich. Quan sat sau vai phut, phan ng dng tnh neu tren be mat
moi trng co vong mau o xuat hien. Ngc lai neu khong co s xuat hien cua mau o hong
c coi la phan ng am tnh.
Thuoc th
- Thuoc th Erhlich: Hoa tan 4g p-dimethylaminobenzaldehyde trong hon hp gom 80ml
acid HCL am ac va 380 ethanol tuyet oi. Dung dch sau khi pha co mau vang nau.
- Thuoc th Kovacs hoa tan 5g p-dimethylaminobenzaldehyde trong hon hp gom 75 ml
amyl alcohol va 25 ml H
2
SO
4
am ac.

10. Th nghiem tren moi trng Kligler Iron Agar hay tren moi trng Triple Sugar Iron agar
Nguyen tac: Th nghien nay nham phat hien kha nang s dung cac nguon
carbonhydrate co mat trong moi trng, kha nang sinh H
2
S va sinh kh trong moi trng.
C s sinh hoa: Moi trng Kligler Iron agar (KIA) va Triple Iron agar (TSI) la cac moi
trng ran dung trong th nghiem sinh hoa. Moi trng c phan phoi vao trong cac ong
nghiem, gom hai phan: phan ng sau khoang 2,5cm va phan nghieng ben tren. Th nghiem
nay nham hai muc ch: phat hien kha nang len men cac nguon Carbonhydrate co trong mo i
trng va phat hien kha nang sinh H
2
S.
Moi trng KIA cha hai nguon carbohydrate: 1% lactose va 0,1%. glucose, moi trng
TSI tng t nh moi trng KIA nh co them 1%. Sucrose. C che sinh hoa cua hai moi
trng nay tng t nhau.
Tren moi trng KIA, vi sinh vat co the s dung ca hai nguon carbohydrate tren hay ch
s dung glucose. Qua trnh len men nay co the tao kh trong moi trng hay khong tuy thuoc
vao tng loai vi sinh vat. S s dung cac nguon carbonhydrate khac nhau hai ieu kien hieu
kh tren phan nghieng va k kh trong phan ng. Tren phan nghieng, glucose c trao oi
theo con ng oxy hoa trong chu trnh Krebs e tao san pham la H
2
O va CO
2
ong thi thu
c nang lng. Lactose la ng oi, e vi sinh vat co the s dung chung phai phan giai
thanh galactose va glucose, cac ng n nay se i vao trong te bao va tham gia vao qua trnh
chuyen hoa noi bao.
Co ba trng hp se xay ra khi nuoi cay vi sinh vat tren moi trng KIA: ch len men
glucose, len men ca hai nguon carbonhydrate trong moi trng, khong len men ca hai nguon
tren.

32
Neu vi sinh vat ch len men glucose sau 18-24 gi nuoi cay, tren phan nghieng cua moi
trng co pH kiem va phan sau cua ong co pH acid. Hien tng nay c quan sat khi co
chat ch th pH trong moi trng nuoi cay, thong thng tren moi trng nay thng s dung
chat ch th pH la phenol red. Tren phan nghieng, sau 24 gi nuoi cay, mot lng nho glucose
trong moi trng c vi sinh vat s dung het hoan toan. Sau o vi sinh vat s dung pepton
trong moi trng, qua trnh trao oi pepton lam giai phong NH
3
, san pham nay se lam kiem
phan nghieng cua ong moi trng. phan sau moi trng co pH acid bi s len men k kh
glucose, cac san pham thu c la nhng acid hu c, chnh cac acid nay lam cho pH giam. Neu
trong moi trng co chat ch th pH la phenol red th tren phan nghien se co mau o va phan sau
se co mau vang.
Neu vi sinh vat co the s dung ca hai nguon carbohydrate: ca hai phan sau va phan
nghien eu co tnh acid sau 18-24 gi nuoi cay v lng ng lactose u e vi sinh vat s dung
trong thi gian nay, cac san pham tao thanh la cac acid hu c. Neu keo dai thi gian nuoi cay
en sau 24 gi, tren phan nghien co the thu c mau o do moi trng chuyen sang kiem.
Trong trng hp ca hai nguon carbon tren khong c vi sinh vat s dung, nguon
pepton trong moi trtng tr thanh nguon cung cap nang lng va vat lieu cho qua trnh tang
trng va phat trien cua vi sinh vat. Nhng thng pepton ch c chuyen hoa trong ieu kien
hieu kh do o hien tng lam kiem moi trng ch dien ra tren phan nghien va moi trng
chuyen thanh mau o. Trong kh o phan sau moi trng se khong co hien tng chuyen mau.
Trong cac trng hp tren neu qua trnh chuyen hoa cua vi sinh vat co the tao thanh cac
san pham kh, chung se tch tu trong moi trng va co the lam v thach hay tao thanh cac bot
kh ben trong moi trng.
Trong th nghiem nay co the phat hien kha nang sinh H
2
S t viec chuyen hoa cac thanh
phan trong moi trng. Sodium thiosulphate trong moi trng c s dung nh nguon cung cap
lu huynh. San pham chuyen hoa cac hp chat lu huynh la H
2
S, ay la chat kh khong mau,
e nhan ra chung, mot chat ch th khac la Fe
2+
c cung cap vao di dang ferric ammonium
citrate. H
2
S phan ng vi Fe
2+
se tao thanh FeS ket tua mau en trong moi trng. Nh vay vi
th nghiem nay co the nhan biet c kha nang s dung cac nguon carbon khac nhau, kha nang
tao kh va tao H
2
S cua vi sinh vat can th nghiem.
Trong trng hp s dung moi trng TSI, cac hien tng cung xay ra tng t. Tuy
nhien trong trng hp ca hai phan nghien va phan sau eu co hien tng acid la co the co mot
trong hai hay ca hai nguon lactose va sucrose c vi sinh vat s dung. Trong trng hp nay
can phai co cac th nghiem tren cac nguon lactose hay sucrose mot cach rieng le.
Phng phap th nghiem
Moi trng KIA hay TSI c chuan b trong cac ong nghiem vi phan nghieng cach
nap ong nghiem khoang 2,5cm va phan sau co chieu cao khoang 2,5cm. Dung que cay nhon
a vi sinh vat vao phan sau cua ong nhng khong ung ay ong, sau o cay ria tren phan
nghieng. U cac ong a cay 37
o
C trong 18-24 gi. Ghi nhan cac bieu hien phan sau, phan
nghieng, s tch tu kh va tao thanh H
2
S.

11. Th nghiem malonate
Nguyen tac: Th nghiem nham xac nh kha nang cua vi sinh vat s dung malonate nh
la nguon carbon duy nhat trong moi trng.
C s hoa hoc: Malonate la mot tac nhan c che enzym c phat hien lan au tien khi
nhan thay acid malonic c che s chuyen hoa succinic thanh fumaric bi s tan cong vao tam
hoat ong cua enzym succinic dehydrogenase. Acid malonic c che qua trnh chuyen hoa

33
succinic bang phng thc c che canh tranh do cau truc phan t cua malonic va succinic tng
t nhau.
COOH COOH
CH
2
(CH
2
)
2

COOH COOH

Khi b malonate c che con ng thu hoi nang lng trong chu trnh Krebs, vi sinh vat
chuyen sang mot so con ng chuyen hoa khac e thu hoi nang lng nh qua trnh chuyen t
chu trnh Krebs sang chu trnh Glyoxylic acid. Tuy vay e am bao cho s phat phat trien bnh
thng khi trong moi trng co malonate khi vi sinh vat o s dung c c chat nay nh la mot
nguon cung cap nang lng hay nguon carbon. Neu vi sinh vat khong s dung malonate th c
chat nay se tr thanh mot chat diet khuan. Leifson cho rang vi sinh vat s dung malonate nh la
mot c che len men e thu nang nang lng. Khi vi sinh vat s dung malonate e chuyen hoa
nh la nguon carbon th ong thi chung cung s dung ammonium sulphate nh la nguon cung
cap nit. Ket qua cuoi cung la lam moi trng nuoi cay chuyen thanh kiem do co s tao thanh
NaOH.
Phng phap tien hanh: Cay vi sinh vat vao trong moi trng malonate broth co cha
chat th mau pH la bromothylmol blue nuoi u qua em nhiet o 37
o
C. Phan ng dng tnh khi
moi trng co dau hieu chuyen sang mau xanh dng. Phan ng am tnh khi vi sinh vat khong
lam thay oi mau moi trng.

12. Th nghiem lam tan gelatin
Nguyen tac: Th nghiem kha nang cua vi sinh vat co the tong hp cac enzym ngoai bao
lam thuy giai gelatin.
C s sinh hoa: Gelatin la mot dang protein co kch thc phan t ln, vi sinh vat muon
s dung cac protein nay nh mot nguon cung cap nang lng hay vat lieu xay tong hp cac
thanh phan khac cua te bao, chung phai co kha nang san xuat cac loai enzym ngoai bao lam
phan giai cac phan t gelatin thanh cac thanh phan nho hn hay thanh cac monomer. Cac
enzym nay thuoc nhom gelatinase c tiet ra ben ngoai te bao.
Qua trnh thuy phan gelatin qua hai giai oan nh sau:

gelatinase
Protein + H
2
O polipeptide

gelatinase
Polipeptide + H2O aminoacidu1
Phng phap thc hien: e nhan biet kha nang lam tan gelatin cua vi sinh vat, c chat
nay c them vao trong moi trng nuoi cay, khi cha cay vi sinh vat, moi trng cha gelatin
se dang ong ac. Sau khi nuoi cay, vi sinh vat co kha nang tiet gelatinase se lam moi trng
tan chay thanh dang long. Phng phap tien hanh nh sau: cay am sau vi sinh vat vao trong
moi trng dinh dng cha gelatin, u ong sau khi cay nhiet o phong cung vi mot ong
nghiem cha moi trng nhng khong cay vi sinh vat vao trong khoang 14 ngay. Ca hai ong
sau khi u c cho vao trong tu lanh qua em. Phan ng dng tnh khi ong oi chng trang
Acid malonic Acid succinic

34
thai ong ac va ong co vi sinh vat b tan chay. Phan ng am tnh khi ca hai ong trang thai
ong ac.
Co the cay vi sinh vat len moi trng cha gelatine tren a e tach c cac khuan lac
rieng biet sau khi u 3 ngay nhiet o phong. Nho len tren be mat a 5-10ml dung dch acid
trichloacetic, cac vi sinh vat cho khuan lac co quan trong ro tren moi trng la cac vi sinh vat
lam tan chay gelatin.

13. Th nghiem methyl red (MR)
Nguyen tac: Th nghiem methyl red nham xac nh kha nang cua vi sinh vat san xuat va
duy tr cac san pham acid ben trong moi trng t trong qua trnh len men glucose
C s sinh hoa: Th nghiem methyl red tren c s s dung chat ch th pH la methyl red
e nhan biet lng ion H
+
co trong moi trng sau khi vi sinh vat len men glucose. Ham lng
ion H
+
co trong moi trng thu thuoc vao t le CO
2
va H
2
, ham lng cac chat nay lai tuy thuoc
vao con ng chuyen hoa cua tng loai vi sinh vat.
oi vi cac vi sinh vat ng ruot, thi gian nuoi cay e th nghiem phan ng MR
thng c xac nh trong khoang 18-24 gi. Tuy nhien cac vi sinh vat nay eu tao acid trong
moi trng ngay t khi chung bat eu phat trien. Khi keo dai thi gian nuoi cay cac vi sinh vat
co phan ng MR dng tnh se tch luy acid trong moi trng ngay cang nhieu hn, o pH trong
moi trng ngay cang giam.
oi vi cac vi sinh vat cho phan ng MR am tnh, ngay ban au mot lng acid c
hnh thanh trong moi trng, nhng keo dai thi gian muoi cay vi sinh vat nay tiep tuc chuyen
hoa cac san pham acid bang cac phan ng nh decarboxyl tao nen cac san pham trung tnh nh
acetoin (acetyl methylcarbinol) va ket qua la lam cho pH trong moi trng chuyen dan ve pha
trung tnh.
Trong mot so trng hp khac vi sinh vat cung san sinh acid trong moi trng nhng cac
acid nay cho ham lng ion H
+
thap nh acid lactic, acid acetic, acid formic bi v cac acid
acid nay co xu hng chuyen ve trung tnh do hien tng tao thanh cac carbonate va tao thanh
cac san pham nh CO
2
va cac hp chat amonium trong moi trng cac san pham nay lam tang
pH trong moi trng.
Th nghiem MR phu thuoc rat ln vao thi gian nuoi cay e xac nh s khac nhau trong
cac con ng chuyen hoa glucose. Th nghiem nay thng c tien hanh trong thi gian
khoang 2-5 ngay 37
o
C.
Phng phap tien hanh: Nuoi cay vi sinh vat trong moi trng glucose phosphate (MR-VP
broth) u trong khoang 2-5 ngay. Them vao vai giot thuoc th methyl red c pha theo t le 0,1g
trong 300ml ml con va cho nc vao e at the tch 500ml. Phan ng dng tnh khi moi trng
chguyen sang mau o. Phan ng am tnh khi moi trng gi nguyen mau vang.

14. Th nghiem tnh di ong trong moi trng thach mem
Nguyen tac: Nham xac nh vi sinh vat co di ong hay di ong. Vi sinh vat di ong
thng la cac vi sinh vat co tien mao. Tien mao thng c tm thay tai cac vi sinh vat hnh
que. Tuy nhien co mot so vi sinh vat hnh cau cung co kha nang di ong. Vi sinh vat di ong co
the co 1 hay nhieu tien mao co the phan bo tai cac vi tr khac nhau tren te bao vi sinh vat. Mot
so vi sinh vat co the chuyen t trang thai di ong sang trang thai khong di ong nhng a so
khong co kha nang chuyen t trang thai khong di ong sang trang thai di ong. Thong thng
cac vi sinh vat t di ong thanh khong di ong la do mat tien mao.
Th nghiem tnh di ong

35
Cay am sau vi sinh vat vao trong moi trng thach mem cha 0,5 % agar. U qua em
nhiet o thch hp vi tnh di ong cua tng vi sinh vat. Quan sat hien tng sau nuoi cay. Neu
vi sinh vat di ong se lam uc moi trng, vi sinh phat trien lan ra khoi vet cay. Ngc lai vi
sinh vat khong di ong se phat trien sinh khoi xung quanh ng cay, moi trng khong v uc
bi sinh khoi vi khuan.
15. Th nghiem kh nitrate
Nguyen tac: Nham th nghiem kha nang vi sinh vat kh nitrat thanh nitrite hay thanh
nit t do. Tat ca cac qua trnh dien ra trong te bao vi sinh vat di s xuc tac cua enzym
nitratase.
C s sinh hoa: S kh nitrate thanh nitrite hay nito t do thng dien ra trong ieu kien
k kh. Oxy t nitrate c coi nh la nhan to nhan dien t va proton cuoi cung. Hau het cac vi
sinh vat hieu kh kh nitrate la nhng vi sinh vat hieu kh tuy nghi, chung ch thc hien qua trnh
kh nitrate khi song trong ieu kien k kh, khong co s hien dien cua oxy phan t. Trong chuoi
chuyen ien t, nitrate ong vai tro nh mot chat oxy hoa.
S kh nitrate thanh nitrite: qua trnh nay dien ra nh phan ng sau ay:
NO
3
-
+ 2e
-
+ 2H
+
NO
2
-
+ H
2
O
Nitrate Nitrite
S kh cac san pham nitrate thanh oxy phan t theo phan ng nh sau:

NO
3-
+ 10e
-
+ 12H
+
N
2
+ 6H
2
O
Nitrate Nit
San phan cuoi cung cua s kh nay co the la cac chat nh sau: nitrite (NO
2
-
); ammonia
(NH
3
); nit phan t (N
2
), mot hydroxylamine hay nitric oxyde (NO). S hnh thanh cac san
pham cuoi cung nay phu thuoc vao tng loai vi sinh vat. Thong thng cac san pham cuoi cung
la kh N
2
do s kh tiep tuc cac san pham nitrite tao thanh. S hnh thanh san pham cuoi cung
con phu thuoc vao ieu kien moi trng.
Phng phap thc hien: Co the phat hien s co mat cua nitratase trong moi trng bang
cac cach nh sau:
Cach 1: Cay vi sinh vat vao trong moi trng nitrate broth va u qua em.
Cach 2: Khuech tan day ac vi sinh vat th nghiem vao trong dung dch sodium nitrate
0,01mol/l trong dung dch em phosphate pH 7,0, u 37
o
C trong 24 gi.
Cach 3: Nho vai giot dung dch sodium nitrat 1% vao moi trng canh khuan day a
c nuoi cay trc o. U nhiet o 37
o
C trong 4 gi.
Acid hoa cac dung dch th nghiem tren bang vai giot HCl 1N, them vao moi th
nghiem 0,5ml dung dch sulphanilamide 0,2% va 0,5ml N-napthylethylenediamine
hydrochloride, tat ca cac thuoc th nay phai c bao quan trong tu lanh. Neu co xuat hien mau
hong la dau hieu cua nitratase dng tnh. Neu khong co mau hong xuat hien co the co mot
trong hai trng hp nh sau:
Khong co nitatase trong moi trng, nitrate van con dien dien. Them vao mot lng rat
nho bui kem vao cac th nghiem e chuyen nitrate thanh nitite, khi o mau hong se xuat hien.
Nitrate khong con trong moi trng: them bui kem vao moi trng van khong xuat hien
mau hong.

16. Th nghiem urea
Nguyen tac: Xac nh vi sinh vat co kha nang phan huy urea thanh ammonia va CO
2

di s xuc tac cua enzym urease do vi sinh vat tiet ra.

36
C s sinh hoa: Urea la mot hp chat carbamide rat de b thuy giai. S thuy giai cua
urea bi s xuc tac cua urease se giai phong hai phan t ammonia. Trong dung dch cac thanh
phan sau phan ng se chuyen hoa thuan nghch, san pham thu c sau phan ng se c bieu
dien trong can bang sau:
H
2
N
C =O + 2 H
2
O CO
2
+ H
2
O + 2 NH
3
(NH
4
)
2
CO
3

H
2
N

Enzym urease la mot enzym quan trong trong te bao vi sinh vat, trong mot so vi sinh vat
ay la enzym cau truc, nhng trong mot so loai khac ay la enzym cam ng. Khi co c chat
urea hien dien trong moi trng, enzym nay c tong hp va phong thch ra ben ngoai te bao,
xuc tac phan ng thuy giai urea. Cac san pham sau phan ng lam cho moi trng hoa kiem va
lam chuyen mau chat ch th pH.
Phng phap tien hanh: Cay vi sinh vat vao moi trng canh urea co cha chat ch th la
phenol red, u nhiet o 37
o
C trong khoang 12-18 gi. Phan ng dng tnh khi moi trng
chuyen sang mau o hong, phan ng am tnh khi moi trng khong chuyen mau.

17. Th nghiem Voges Proskauer (VP)
Nguyen tac: Phat hien kha nang vi sinh vat tao thanh mot so san pham trung tnh
acetylmethylcarbimol (acetoin) trong qua trnh len men glucose.
C s sinh hoa: Th nghiem VP nham muc ch phat hien acetoin, mot san pham trung
tnh trong qua trnh trao oi glucose trong te bao vi sinh vat. Qua trnh phan huy glucose trong
te bao vi sinh vat tao ra san pham trung gian la acid pyruvic, t ay co the chia vi sinh vat thanh
hai nhom theo hai cong ng chuyen hoa pyruvic khac nhau nh sau: Nhom trao oi khong tao
thanh 2,3-butanediol, v du nh E.coli c goi la nhom VP am tnh (VP-); nhom th hai qua
trnh trao oi nay tao thanh san pham trung gian la 2,3-butanediol, v du nh Klebsiella, c
goi la nhom VP dng (VP+). Tuy nhien th nghiem VP nham muc ch phat hien acetoin
(acetylmethylcarbimol, AMC) mot tien chat cua 2,3-butanediol.
Phan t acetoin c tao thanh do qua trnh kh nhom carboxyl cua acid pyruvic, phan
ng nh sau:
2 Pyruvate acetoin + 2CO
2

V acetoin la mot san pham trung gian trong bc tao thanh 2,3-butanediol nen trong
moi trng nuoi cay tch luy mot lng rat t tien chat nay. e acetoin tch luy nhieu, de dang
cho viec phat hien, vi sinh vat phai nuoi trong ieu kien hieu kh. Qua trnh chuyen hoa gia
acetoin va 2,3-butanediol la mot qua trnh thuan nghch, co the bieu dien qua trnh nay nh sau:
Oxyhoa
2,3-butanediol acetoin
2,3-butanediol dehydrogenase

Kh
Acetoin 2,3-butanediol
diacetyl (acetoin) reductase
Urea
Amonia
Amonia
carbonat

37
e phat hien acetion trong moi trng nuoi cay 1-napthol c s dung nh mot thuoc
th phat hien trong moi trng kiem manh c tao ra do KOH 40% hay NaOH 40% c cho
vao moi trng th nghiem.
C che phan ng nhan biet acetoin c bieu dien nh sau:

CH
3

C = O
C
6
H
12
O
6
CHOH
CH
3

OH CH
3
CH
3

+ C = O C = O + H
2
O
CHOH C = O
CH
3
CH
3

CH3
C = O
Glucose Acetoin
Acethylmethylcarbinol, AMC
KOH 40%
O
2
- Napthol Acetoin Diacetyl
NH
2

C = NH
NH - R
+
Diacetyl Guanidine trong pepton

38
C = O
CH3

Trong cac thanh phan tham gia phan ng e tao phc chat co mau o hong co hp chat
guanidine, v du arginine NH:C(NH
2
)-NH-(CH
2
)
3
-CH(NH
2
)-COOH, chat nay hien dien trong
pepton c s dung trong moi trng nuoi cay.
Phng phap tien hanh: Cay vi sinh vat vao trong moi trng canh glucose phosphate
(MR-VP broth), u nhiet o 37
o
C trong khoang 2-5 ngay. Them vao canh khuan sau nuoi cay
3ml dung dch 1-napthol 5% trong con, bo sung them 3ml dung dch KOH hay NaOH 40%, lac
manh. Quan sat phan ng trong khoang 5 phut. Phan ng VP dng tnh khi moi trng chuyen
sang mau o hay hong sang, phan ng VP am tnh khi khong co s chuyen mau nay.

18. Th nghiem len men oxy hoa ( Th nghiem Hugh&Leifson)
Nguyen tac: Xac nh vi sinh vat trao oi carbonhydrate bang qua trnh oxyhoa hay qua
trnh len men.
C s sinh hoa: Vi sinh vat s dung carbonhydrate trong qua trnh trao oi chat thong
qua hai qua trnh: oxy hoa va len men. Mot so vi sinh vat ch co the s dung carbonhydrate
trong ieu kien hieu kh, trong khi o mot so vi sinh vat khac co the trao oi c chat nay trong
ca hai ieu kien hieu kh va k kh. ay c goi la vi sinh vat k kh tuy nghi.
Len men la mot qua trnh k kh, vi khuan len men la nhng vi khuan k kh tuy nghi.
Qua trnh len men glucose se chia phan t nay thanh hai ng triose (ng 3C). San phan
cuoi cung cua con ng len men phu thuoc vao tng loai vi sinh vat va phu thuoc vao ieu
kien moi trng. Tuy vay iem cot yeu cua con ng len men la phan t glucose b phosphoryl
hoa bc au tien e thanh glucose-6-phoshate. Qua trnh len men can mot chat nhan ien t
cuoi cung la mot hp chat hu c. Glucose tham giam trong qua trnh len men thng c trao
oi theo con ng Embden-Meyerhof, cung co trng hp trao oi theo con ng Entner-
Doudoroff va con ng theo hng trao oi pentose.

+++
Phc hp mau o hong
1 mol glucose Glucose 6 - phosphate
Cach Embden - Meyerhof
Cach Embden - Meyerhof
Fructose 6 - Phosphate
Fructose 1, 6 di phosphate
2 mol Glyceraldehyde 3 - Phosphat
2 mol acid pyruvic
6-phosphogluconic acid
Cach Entner-Doudoroff
2-keto-3-deoxy-6-phosphogluconic acid
Glyceraldehyde 3 - Phosphat
Cach Pentose Shunt
Ribulose 5 - Phosphate
+ CO
2

Phosphoryl hoa

39

S o cac con ng len men

Oxy hoa la qua trnh ch dien ra trong ieu kien co oxy. Trong qua trnh nay phan t
glucose khong b chia thanh hai triose nh tren, thay vao o chung oxy hoa nhom chc aldehyde
(-CHO) e tao thanh acid gluconic. Khac vi qua trnh len men, qua trnh oxy hoa khong b
phosphoryl hoa giai oan au cua qua trnh phan giai glucose. ong thi trong qua trnh oxy
hoa chat nhan ien t cuoi cung khong phai la phan t hu c ma co the la cac chat vo c nh
oxy phan t nitrat hay sulphate. Qua trnh len men thng tao moi trng acid cao hn trong
qua trnh oxy hoa.

Con ng oxyhoa glucose

Phng phap thc hien: Cay am sau vi sinh vat th nghiem vao hai ong nghiem moi
trng Hugh &Leifson co cha 2-5 g agar/ lt. Mot trong hai ong c phu len be mat 1ml dau
khoang hai parafine long vo trung e ngan can s tiep xuc vi oxy khong kh. U ca hai ong
trong cung ieu kien khoang 24-48 gi.
Phan ng len men khi ca hai ong eu bi acid hoa eu tren mat va phan sau cua moi
trng.
Phan ng oxy hoa khi ong k kh b acid hoa, ong hieu kh khong b acid hoa tren mat
ma ch co acid phan ay ong.

19. Th nghiem oxydase
Nguyen tac: th nghiem nay nham phat hien he enzym oxydase cua vi sinh vat.
C s sinh hoa: Th nghiem oxydase nham muc ch phat hien enzym oxydase, mot san
pham noi bao cua vi sinh vat. Phan ng oxydase xay ra do s co mat cua he thong cytochrom
oxydase oxyhoa cac cytochrom dang kh bi oxy phan t.
Tat ca cac vi sinh vat nhan nang lng t cac nguon vat vat chat bang c che ho hap,
ay la qua trnh oxy hoa. He thong ho hap la mot chuoi cac enzym va cac chat chu trach nhiem
cho s van chuyen ien t t cac c chat en oxy phan t. Oxy la chat nhan proton hydro cuoi
cung trong chuoi ho hap e tao thanh nc hay hydro peroxyde, san pham cuoi cung c hnh
thanh phu thuoc vao tng loai vi sinh vat va he thong enzym cua chung.
Acid pyruvic
Acid pyruvic
Glucose
Gluconic acid
2-keto - Gluconic acid
2-keto-6-phospho-Gluconic acid
2-pyruvic acid

40
He thong cytochrom thng hien dien ch trong cac loai vi sinh vat hieu kh hay k kh
tuy nghi. Cac vi sinh vat k kh bat buoc khong co he thong nay. Co nhieu loai he thong
cytochrom oxyadse khac nhau tuy thuoc vao tng loai vi sinh vat, mot so loai ch co mot
oxydase trong khi mot so loai khac co hai hay ba oxydase. Cytochrom oxydase la he enzym
cuoi cung trong toan bo he thong cytochrom, ngoai ra con co cac cytochrom khac nh b, c
1
,c.
Phat hien he thong cytochrom oxydase nh thuoc th p-phenylenediamin, ay la mot
dan xuat t hp chat phenol amin. Cytochrom oxydase khong phan ng trc tiep vi thuoc th
nay ma phai qua trung gian oxyhoa cytochrom c. Qua trnh nay nh sau:
Cytochrom oxydase
2 cytochrom c kh + 2H
+
+ O
2
2 Cytochrom c oxy hoa + H
2
O
oxy hoa
2 Cytochrom c oxy hoa + thuoc th hp chat co mau xanh

Hp chat co mau xanh nay la mot indolphenol blue, ay la mot loai thuoc nhuom co
nhan quinon.
Cac tien hanh: Nuoi cay vi sinh vat tren moi trng nutrien agar qua em, lay mot t sinh
khoi vi sinh vat bang que cay vong c lam bang nha hay go at len giay loc may trang,
anh dau v tr at sinh khoi vi sinh vat. Nho len v tr ang dau mot giot thuoc th N,N,N,N-
tetramethyl-p-phenylenediamine (TMPD), quan sat sau vai phut. Phan ng oxydase dng tnh
khi sinh khoi vi sinh vat chuyen thanh nay xanh dng am. Phan ng am tnh khi khong co
hien tng chuyen mau.

41

http://www.ebook.edu.vn 39
Chng 4
PHNG PHAP KHONG TRUYEN THONG

Phat hien vi sinh vat bang phng phap nuoi cay c xay dng va phat trien trong thi
gian dai, c nhieu nc cong nhan va chung a tr thanh nhng phng phap tieu chuan. Tuy
vay nhc iem ln nhat cua phng phap nuoi cay la ton nhieu thi gian, cho ket qua cham,
mat nhieu cong sc, cong kenh va ngay cang to ra khong ap ng c cac yeu cau phan tch
phuc vu nhu cau thc te hien nay. e khac phuc nhng nhc iem tren cua phng phap nuoi
cay, nhieu phng phap nhanh va t ong a c hnh thanh va phat trien da tren nhieu
nguyen tac sinh hoc va vi sinh vat hoc khac nhau. Cac phng phap mi nay nham muc ch rut
ngan thi gian phan tch, giam thieu s phc tap trong thao tac, de dang thc hien va co o
nhay va o chnh xac cao.
Cac phng phap nhanh va t ong hoa trong phan tch vi sinh vat thc pham eu da
tren nguyen tac cua vi sinh hoc, sinh hoa hoc, hoa ly, mien dch hoc, sinh hoc phan t hoc e
phat hien va nh lng vi sinh vat muc tieu hay san pham oc hai cua chung trong thc pham,
moi trng. Tat ca cac yeu cau ky thuat lien quan en viec thu va chuan b mau, phan tch
eu c nghien cu cai tien theo hng t ong hoa, n gian hoa cho ket qua nhanh va chnh
xac.
1. Phng phap phat quang sinh hoc ATP
Phan t Adenosine triphosphate (ATP) co mat trong tat ca cac te bao song, nen s phat
hien ATP la dau hieu e nhan biet vat chat song ang ton tai. ATP co the c phat hien va
nh lng thong qua cng o anh sang phat ra do s ket hp vi enzyme Luciferase. K thuat
nay co the phat hien c 1pg ATP, tng ng vi khoang 1000 te bao vi khuan (10
-15
g ATP/ te
bao). Qua trnh phan tch ch dien ra trong vai phut va v the phng phap nay c xem la
nhanh hn va thuan li hn so vi phng phap em khuan lac.
Viec dung phng phap o ham lng ATP e xac nh ro so vi sinh vat ang hien dien
a c biet en vao nam 1960. Tuy nhien, phng phap nay oi hoi nhieu s cai tien trong
viec thiet ke thiet b o cng o anh sang phat ra va nhng hoa chat e on nh s phat sang.
Ngay nay phng phap nay c ng dung trong 3 lnh vc: giam sat ve sinh, kiem tra nhng
loai chat long nh nc ra lam sach he thong, anh gia chat lng vi sinh cua thc pham. e
anh gia chat lng vi sinh cua thc pham th lng ATP cua vi sinh vat phai c o va ieu
nay oi hoi nhng quy trnh tach chiet ATP ra khoi te bao vi sinh vat.
Phan ng phat sang sinh hoc trai qua hai giai oan :

E + LH
2
+ ATP E LH
2
AMP + PP
i
(1)

E LH
2
AMP +O
2
Oxyluciferin + AMP + CO
2
+ hv (2)
Phan ng phat sang sinh hoc om om co the viet lai nh sau:

E + LH
2
+ ATP +O
2
Oxyluciferin + AMP + CO
2
+ hv +PP
i
E : luciferase
LH
2
:luciferin
Anh sang phat ra co bc song 562nm, co mau vang.
Mg
2+
Mg
2+
Mau c lay bang cach quet nhng vi sinh vat tren be mat dung cu va thiet b, sau o
o lng ATP thong qua mot loat qua trnh ly trch. Nhng thiet b c che tao au tien oi hoi
ngi thc hien quet mau tren mot v tr (thng la 10cm
2
), sau o nhung hoan toan que quet
mau vao trong dung dch cha nhng tac nhan lam phong thch ATP, tron vi dung dch
luciferase luciferin va at vao trong mot cuvette e oc tr so anh sang.

S o cac bc nh lng nhanh vi sinh vat hien dien tren be mat bang
phan ng phat sang

2. PHNG PHAP ELISA (Enzyme-Linked ImmunoSorbent Assay)
Nhng tin b cua khoa hoc trong nhng nam gan ay a thuc ay s phat trien cua cac
ky thuat chan oan bang huyet thanh. Cac phng phap nay to ra rat hieu qua trong viec chan
oan nhanh va chnh xac cac vi sinh vat gay benh. Ngay nay phng phap nay con c phat
trien e xac nh cac chat oc hai trong moi trng nh oc to, d lng khang sinh Nguyen
tac cua phng phap ELISA da tren phan ng ket hp gia khang nguyen vi mot khang the
ac hieu. Phan ng mien dch xay ra c phat hien bang cach s dung nhng khang the a
c anh dau (bang chat nhuom phat huynh quang, ong v phong xa, hay enzyme).
Phng phap ELISA la phng phap hap phu mien dch lien ket enzyme (enzyme-linked
immunosorbent assay). Nguyen tac ky thuat ELISA la s dung khang the n dong (khang the
s cay) phu ben ngoai nhng gieng (well) nho nham muc ch thu gi nhng khang nguyen
muc tieu. Nhng khang nguyen thu gi c phat hien bang cach s dung khang the th hai co
gan vi enzyme phat tnh hieu (thng la horseradish peroxidase hay alkaline phosphatase).
Khi cho vao hon hp phan ng mot c chat ac hieu cua enzyme, phan ng xay ra va tao ra cac
san pham lam oi mau phan ng. Nh vay, chung ta co the phat hien c s hien dien cua
khang nguyen .

khang Iha Ihu
khang nguyan
khang nguyan
khang Iha mang
antyma danh dau
8andWIrh
6o rhaI
FhaI quang

Nguyen tac phan ng ELISA - S: c chat, E: ensyme, P: phat quang

Qui trnh thc hien phan tch bang ELISA co cac chnh nh sau: ac mau vao trong cac
gieng co cha khang the s cap, cho khang the th cap co lien ket vi enzym tao mau vao e
tao sandwich, ra e loao bo cac khang the mang enzym khong tham gia phan ng, cho c chat
tao mau vi emzym lien ket qua hom hp, o cng o mau tao thanh e xac nh lng khang
nguyen trong mau.
Hien nay ELISA a c s dung rong rai e phan tch Salmonella, E. coli gay benh,
Listeria, oc to Staphylococus, thuoc tr sau, d lng kham sinh oi vi vi sinh vat, phng
phap ELISA co the s dung phat hien va nh lng vi sinh trong thc pham sau vai gi tang
sinh nham tang o nhay cua phng phap.

3. PHNG PHAP MAU DO (Gene probes)
Vao nhng nam 1980, co nhieu nghien cu ng dung ky thuat sinh hoc vao lnh vc thc
pham, ac biet la trong chan oan vi sinh. Phng phap s dung mau do (probe) trong viec phat
hien vi sinh vat trong thc pham thong qua s phat hien mot trnh t DNA ac trng. Cac mau
do thng s dung RNA ribosome (rRNA) lam muc tieu, do trong c the so lng ban sao
rRNA ln, u e lam tang o nhay cua phng phap phan tch. Nguyen tac cua phng phap
mau do la lai phan t, o la bat cap gia hai trnh t DNA tng ong. Mot trong hai trnh t
(thng la trnh t ch, tc la trnh t DNA cua te bao vi sinh vat) c co nh tren mot gia
the ran hoac tren te bao hay mo. S lai phan t xay ra khi cac trnh t bo sung gap nhau do
chuyen ong nhiet va khi nhiet o moi trng thap hn T
m
t nhat vai o. S lai phan t con co
the xay ra gia DNA va RNA. Qua trnh lai phan t chu anh hng rat nhieu bi cac yeu to:
nong o DNA trong moi trng, nhiet o va thi gian phan ng, kch thc cac trnh t lai va
lc ion cua moi trng.
Mot v du ve he thong phat hien bang mau do la thong Gentrak (Framingham, USA).
Phng phap phan tch nay s mau do e phat hien Listeria trong mau b sa va mau moi
trng. Mau do la nhng oan oligomer DNA anh dau bang hoa chat phat quang. Quy trnh
phan tch co the c chia thanh 6 bc: 1. Pha v te bao thu nhan rRNA, 2. mau do DNA thu
gi co uoi deoxyadenylic nucleotide (dA) va mau do phat hien (reporter probe) cha
fluorescein isothiocyanate (F) au 5 va 3 cua phan t, 3. Que th c bao boc bi
polydeoxythymidine (dT) c at vao vi muc ch gan nhng mau do vi vi que th, 4. que
th c at vao ong cha enzyme lien ket vi mau do phat hien, 5. Sau khi ra loai phan
enzyme tha, que th c at vao ong cha c chat tao mau, 6. Sau khi u e hien mau, mau
c phat hien bc song 450 nm.


S o quy trnh phat hien Listeria vi mau do phat quang

4. PHNG PHAP PCR (Polymerase Chain Reaction)

Tat ca cac DNA polymerase eu can nhng moi chuyen biet e tong hp mot mach DNA
mi t mach khuon. Mach khuon thng la nhng nhng trnh t DNA cua gen quy ac trng
cho loai vi sinh vat muc tieu hoac cac gen quy nh viec tong hp mot loai oc to chuyen biet.
Moi la nhng oan DNA ngan, co kha nang bat cap bo sung vi mot au cua mach khuon va
nh hoat ong cua DNA polymerase oan moi nay c noi dai e hnh thanh mach mi. Khi
cung cap hai moi chuyen biet bat cap bo sung vi hai au cua mot trnh t DNA, vi ieu kien
DNA polymerase hoat ong trong phan ng PCR, mot oan DNA nam gia hai moi se c tao
nen. Nh vay, e khuech ai mot trnh t DNA xac nh, ta phai co nhng thong tin toi thieu ve
trnh t o u e tao cac moi bo sung chuyen biet. Cac moi nay gom mot moi xuoi (sens
primer) va mot moi ngc (antisens primer) so vi chieu phien ma cua gen.
Phan ng PCR la mot chuoi nhieu chu ky noi tiep nhau. Moi chu ky gom ba bc: bien
tnh (denaturation), lai (anealation), tong hp (elongation). Phan tch san pham khuyech ai
bang s ien di tren gel agarose. ac trng cua phan ng khuech ai oi vi tng vi sinh vat
muc tieu thong qua kch thc cua san pham khuech ai.

hk mur IIau Mau do
Ihu gIu
Mau do
phaI hIan
ua Ihu
6o rhaI phaI
quang

S o phan ng PCR

S phn ng PCR
u iem cua phng phap PCR:
- Thi gian cho ket qua nhanh
- Co the nhan dien nhng vi sinh vat kho nuoi cay. Viec nuoi cay tang sinh la n
gian hn va co khi khong can thiet.
- Hoa chat can cho phan ng PCR san co hn va de ton tr hn so vi trng hp
huyet thanh hoc. Khong can dung cu va moi trng chan oan phc tap, co the thc
hien hien trng.
- t ton kem ve mat nhan s. Co the c t ong hoa e lam giam chi ph phat hien
cac vi sinh vat gay benh trong thc pham.
Mac du vay phng phap PCR van con mot so khuyet iem can khac phuc:
- S c che hoat tnh cua Taq DNA polymerase bi thanh phan cua mau vat. Mau thc
pham thng co nhng thanh phan phc tap. Viec chiet tach va tinh che DNA t
thc pham hay moi trng trc khi thc hien phng phap PCR cho phep loai bo
nhng hp chat c che. Tuy nhien, mot vai quy trnh phat hien vi sinh vat gay benh
thc pham bang PCR khong can tach chiet, tinh che DNA.
- Mat o vi sinh vat gay benh hien dien trong mau thc pham thng thap, nen trong
a so trng hp can co bc nuoi cay lam giau e co c mat o u e phat hien
bang PCR.
- Phng phap nay khong phan biet c te bao song vi te bao chet. Do vay co the
dan en trng hp dng tnh gia do DNA t te bao chet. Ngc lai phng phap
nay cho phep phat hien bao t, dang tiem sinh, hay te bao a chet cua cac vi sinh
vat gay benh hoac gay ngo oc.

5. MOT SO PHNG PHAP TH NHANH KHAC
5.1. Ky thuat phan tach va tang mat o

Trong qua trnh ong nhat mau, mau thng c pha loang bac 10 tao mot the tch ln
nguyen lieu ban au (250ml). Nhng co the ch dung vai mililite cho nhng bc tiep theo trong
quy trnh phat hien. Nh vay chung ta co the dung bc tang mat o cac vi sinh vat muc tieu
trong mau e rut ngan thi gian va tang hieu qua phat hien.
Mot ky thuat phan tach c s dung rong rai la mien dch phan tach (Immunomagnetic
separation - IMS). Vi phng phap nay, giai oan phan lap co the c rut ngan bang cach
thay the moi trng tang sinh chon loc bang quy trnh tng t khong can nuoi cay. IMS s
dung nhng hat sieu thuan t c bao boc ben ngoai bi nhng khang the cua vi sinh vat muc
tieu. Cac hat nay co chc nang phan tach chon loc vi sinh vat muc tieu nay ra khoi mot hon hp
quan the. Cac vi sinh vat muc tieu nay co the c phat hien bang cac quy trnh vi sinh truyen
thong. Tnh sieu thuan t cua cac hat ch c the hien khi chu s tac ong cua mot lc t tnh
ben ngoai tac ong.
Dynabeads
(Dynal A/S, Oslo, Norway) la mot san pham da tren ky thuat IMS. Quy
trnh nay s dung nhng hat polystyren sieu thuat t co ng knh 2,8m (Dynabeads M-
280) va 4.5m (Dynabeads M-450). Ca hai loai M-280 va M-450 eu co the c bao ben
ngoai bi lp khang the do ngi dung la chon. Mot v du ve chat hap thu t tnh khac co the
c la chon la BioMag (Metachem Diagnostics Ltd, Northampton). Cac hat t tnh oxide sat
(ng knh 0.5 - 1.5m) c bao phu ben ngoai bi cac nhom amino-, carboxy- hoac thiol-.
Ngoai ra mot so he thong khac con co kha nang tao t tnh cho cac te bao vi sinh vat bang cach
cho chung hap thu trc tiep nhng hat sieu hien vi oxide sat mang t tnh len be mat te bao
(Safark va cong s, 1995).
5.2. Ky thuat mang loc phat huynh quang trc tiep (Direct Epifluorscent Technique
DEFT) va mang loc li k nc (Hydrophobic Grid Membrane)
C s cua viec s dung phng phap mang loc la thu nhan te bao t mot lng ln the
tch c loc. Sau o co the kiem tra bang knh hien vi hoac bang cach em khuan lac. Phng
phap nay thch hp oi vi mau co mat o te bao thap.
Mang loc co the c lam bang nitrocellulose, cellulose acetate ester, nylon, polyvinyl
chloride va polyester (Sharpe, 1994). Kch thc lo s dung la 0,45m (hoac 0,22m) ng
knh 13mm en 150mm. Tao lc ay qua loc bang hut chan khong hoac lc ep.Mang loc c
s dung nh mot bien the cua ky thuat truyen thong vi nhieu muc ch: tang mat o vi sinh vat
muc tieu trong mot the tch ln nham tan dung gii han phat hien; loai bo tac nhan c che s
tang trng.
o nhay cua ky thuat phat huynh quang trc tiep (DEFT) phu thuoc vao mat o te bao
trc khi nhuom c loc bi mang loc. Co the phan biet te bao song va te bao chet bang cach
nhuom nhan vi fluorochrome acridine orange. Mau sac phat quang trong te bao thay oi trong
suot cac qua trnh tang trng. Thuoc nhuom phat mau o vi RNA va mau xanh vi DNA.
Thong thng th te bao song cho mau o da cam trong khi cac te bao chet cho mau cho mau
xanh luc. Nam 1991, phng phap ISO-GRID
ng dung tren oi tng Salmonella a dc

AOAC cong nhan ap dung cho moi loai thc pham (Method 991.12).

Hnh 8: He thong ISO-GRID

5.3. Ky thuat mang petri (Petrifilm)
Moi trng dinh dng dang ong kho c co nh vao cac mang mong goi la Petrifilm.
Khi s dung, lp mang bao ve ben tren c nang len va nho vao mot 1ml dch mau roi ay
lai. Mot a petri nha c at len mang bao ve e tao mot khuon tron. Moi trng dinh
dng se ho tr cho s phat trien cua vi sinh vat trong thi gian u. Co the em trc tiep so
khuan lac tren Petrifilm.
Petrifilm a c dung e kiem tra tong so vi sinh vat hieu kh, so coliform, coliform
phan, nam moc, nam men. u iem cua ky thuat Petrifilm la de thao tac, tiet kiem khong gian
u va bao quan. Thi han s dung lau do dung moi trng ong kho va khong can xu ly nhiet
nh phng phap thong thng. Co the dung nc cat vo trung e lam t lai moi trng ong
kho. Sau khi moi trng ong lai, co the dung trc tiep Petrifilm e em tap khuan be mat.

k
8
at mau len mang Petrifilm
Dan eu mau tren mang
U Petrifilm, em trc tiep hoac phan lap

Cach s dung he thong Petrifilm cua 3M Microbiology

5.4. Ky thuat Redigel

Ky thuat nay s dung cac chat dinh dng va pectin gel cha trong mot ong nghiem. Co
the s dung ong nghiem nay bat c luc nao ma khong can phai un chay thach. Trc tien nho
1ml mau vao ong nghiem, tron eu. Sau o o tat ca vao mot a petri ac biet a c trang
san mot lp calci. Khi chat long tiep xuc vi calci, gel Ca-pectate se hnh thanh va phc chat
nay se trng len nh moi trng thach thong thng. Sau khi u che o thch hp co the em
khuan lac giong nh phng phap em a thong thng.

Hnh 10: Thao tac s dung he thong Redigen cua 3M

5.3. Ky thuat tr khang vi sinh vat (conductance / impedance)

Vi sinh tr khang dung e phat hien trc tiep vi sinh vat thong qua tnh ion trong san
pham cua qua trnh trao oi chat hoac trc tiep t s giai phong CO
2
(carbon dioxide). Nhng
mo ta chi tiet ve ky thuat nay c cong bo trong bao cao cua Kell & Davey (1990) va Silley &
Forsythe (1996).
Ngi ta s dung moi trng nuoi cay chon loc lam dung dch ien phan. S trao oi chat
cua vi sinh vat tao ra nhng san pham mang tnh ion trong moi trng nuoi cay (acid hu c va
ion amonium) va v vay lam tang tnh dan ien cua moi trng. S thay oi ve ien dan c
ghi nhan bi cac thiet b o phan anh s hien dien cua vi sinh vat trong moi trng nuoi cay.
Phng phap nay khong the ap dung cho nhng moi trng co lc ion cao, nh moi trng chon
loc Listeria long, v trong moi trng nay, ngay t ban au, gia tr ien dan a nam ngoai gii
han o cua thiet b.
Ky thuat giam sat trc tiep o dan phc tap hn do lien quan en cau KOH (potassium
hydroxide). KOH c co nh trong moi trng (agar) va tao thanh cau noi dan ien gia hai
au ien cc. Cau noi nay va mau phan tch c ngan cach vi nhau bang mot khoang khong
gian nhat nh. Khi qua trnh phat trien, vi sinh vat sinh ra CO
2
va kh nay lam phan ra cau noi
KOH. Ket qua cua hien tng nay la lam giam tnh dan ien va s thay oi nay co the quan sat
c bang thiet b giam sat. Thi gian tnh dan thay oi c goi la thi gian phat hien.
Thong thng th cac thiet b giam sat bang tr khang eu co nhng chng trnh t ong
xac nh s hien dien cua vi sinh vat khi o dan vt qua mot gia tr quy c. Gii han phat
hien cua phng phap nay la mot te bao song. Bi theo ly thuyet, t mot te bao nay se sinh ra
nhieu te bao khac va c phat hien do lam thay oi o dan. Hien nay a co nhieu thiet b
giam sat s thay oi ien dan tren th trng nh Bactometer 123 (Bactomatic Ltd.), Malthus
2000 (Malthus Instruments Ltd) va ky thuat RABIT (Rapid Automated Bacterial Impedance
Technique, Don Whitley Scientific Ltd). Nhng phng phap tr khang vi sinh vat hau nh ng
dung rat sm trong cong nghiep thc pham va cong nghiep sa.
Phng phap nay ap dung trong nhieu trng hp tng quan vi phng phap em khuan
lac ( nhieu loai san pham); giam ganh nang ve thi gian phat hien. Tuy nhien no yeu can ieu
kien moi trng chuan, on nh; cac thiet b va moi trng ac biet.
5.4. Ky thuat nh lng bang o vi lng calorie (Microcalorimetry)

Phng phap nay s dung nhng thiet b rat nhay e o nhiet lng rat nho sinh ra trong
qua trnh trao oi chat cua vi sinh vat. Qua o co the nh lng ca the trong mau bang cach o
lng nhiet tao ra hoac xac nh thi gian lng nhiet sinh ra en ngng o. Phng phap nay
con dung e nh danh vi sinh vat bang cach o nhiet lng tao ra cua vi sinh vat o tren nhng
nen c chat khac nhau.
5.5. Ky thuat nh lng vi sinh vat bang o mc phong xa (Radiometry)

Ngi ta s dung c chat co carbon 14 anh dau ong v phong xa (C
14
labelled) trong moi
trng nuoi cay vi sinh vat. Trong qua trnh trao oi chat, vi sinh vat giai phong ra CO
2
co cha
C
14
. Ngi ta co the nh lng vi sinh vat da vao lng C
14
giai phong hoac da thi gian can
thiet e lng C
14
at en ngng phat hien. He thong phat hien ong v phong xa nay rat nhay
nhng do tnh oc hai nen khong c a dung trong cong nghiep thc pham.
PHU LUC

Bang 1. Mot so bo kit sinh hoa va he thong t ong nhan dang vi khuan gay benh trong thc pham
c ban tren th trng

He thong phan
tch
ac iem
phan tch
Nha san xuat oi tng vi sinh vat
API
b
Sinh hoa bioMerieux Enterobacteriaceae, Listeria,
Staphylococcus, Campylobacter,
khong len len, k kh
Cobas IDA Sinh hoa Hoffmann LaRoche Enterobacteriaceae
Micro-ID
b
Sinh hoa REMEL Enterobacteriaceae, Listeria
EnterotubeII Sinh hoa Roche Enterobacteriaceae
Spectrum 10 Sinh hoa Austin Biological Enterobacteriaceae
RapID Sinh hoa Innovative Diag. Enterobacteriaceae
BBL Crystal Sinh hoa Becton Dickinson Enterobacteriaceae,
Vibrionaceae, khong len men,
k kh
Minitek Sinh hoa Becton Dickinson Enterobacteriaceae
Microbact Sinh hoa Microgen Enterobacteriaceae, Gram am,
khong len men, Listeria
Vitek
b
Sinh hoa
a
bioMerieux Enterobacteriaceae, Gram am,
Gram dng
Microlog Oxy hoa C
a
Biolog Enterobacteriaceae, Gram am,
Gram dng
MIS
b
Acid beo
a
Microbial-ID Enterobacteriaceae, Listeria,
Bacillus, Staphylococcus,
Campylobacter

Sinh hoa
a
MicroScan Enterobacteriaceae, Listeria,
Campylobacter
Replianalyzer Sinh hoa
a
Oxoid Enterobacteriaceae, Listeria,
Campylobacter
Riboprinter Acid nucleic
a
Qualicon Salmonella, Staphylococcus,
Listeria, Escherichia coli
Cobas Micro-ID Sinh hoa
a
Becton Dickinson Enterobacteriaceae, Gram am,
khong len men
Malthus
b
o dan
a
Malthus Salmonella, Listeria,
Campylobacter, E. coli,
Pseudomonas, coliforms
Bactometer Tr khang
a
bioMerieux Salmonella
* Feng, P., App.I., FDA Bacteriological Analytical Manual, 8A ed.
a
He thong t ong
b
He thong c AOAC chnh thc chap nhan.


Bang 2. Mot so bo kit thng mai da tren ky thuat phan tch nucleic acid dung trong phat hien vi
khuan gay benh trong thc pham

oi tng vi sinh vat Ten thng mai Phng phap
phan tch
Nha san xuat
Clostridium botulinum Probelia PCR BioControl
AccuProbe probe GEN-PROBE Campylobacter
GENE-TRAK probe GENE-TRAK
Escherichia coli GENE-TRAK probe GENE-TRAK
BAX PCR
a
Qualicon E. coli O157:H7
Probelia PCR BioControl
GENE-TRAK
c
probe GENE-TRAK
AccuProbe probe GEN-PROBE
BAX PCR Qualicon
Listeria
GENE-TRAK
c
probe GENE-TRAK
BAX PCR Qualicon
BIND
b
phage BioControl
Salmonella
AccuProbe probe GEN-PROBE Staphylococcus aureus
GENE-TRAK probe GENE-TRAK
Yersinia enterocolitica GENE-TRAK probe GENE-TRAK
* Nguon trch dan: Feng, P., App.I, FDA Bacteriological Analytical Manual, 8A ed.
a
Polymerase chain reaction
b
Bacterial Ice Nucleation Diagnostics
c
He thong c AOAC chnh thc chap nhan


Bang 3. Mot so bo kit thng mai da tren ky thuat phan tch khang the dung trong phat hien tac
nhan gay benh va oc to trong thc pham

Vi sinh vat/ oc to Ten thng mai Kieu phan tch
a
Nha san xuat
TECRA ELISA TECRA Bacillus cereus
diarrhoeal toxin BCET RPLA Unipath
Campyslide LA Becton Dickinson
Meritec-campy LA Meridian
MicroScreen LA Mercia
VIDAS ELFA
b
bioMerieux
EiaFOSS ELISA
b
Foss
Campylobacter
TECRA ELISA TECRA
Clostridium
botulinum toxin
ELCA ELISA Elcatech
C. perfringens
enterotoxin
PET RPLA Unipath
Escherichia coli
RIM LA REMEL
E. coli O157 LA Unipath
Prolex LA PRO-LAB
Ecolex O157 LA Orion Diagnostica
Wellcolex O157 LA Murex
E. coli O157 LA TechLab
O157&H7 sera Difco
PetrifilmHEC Ab-blot 3M
EZ COLI Tube-EIA Difco
Dynabeads Ab-beads Dynal
EHEC-TEK ELISA Organon-Teknika
Assurance
e
ELISA BioControl
HECO157 ELISA 3M Canada
TECRA ELISA TECRA
E. coli O157 ELISA LMD Lab
Premier O157 ELISA Meridian
E. coli O157:H7 ELISA Binax
E. coli Rapitest ELISA Microgen
Transia card ELISA Transia
E. coli O157 EIA/capture TECRA
VIP
e
Ab-ppt BioControl
Reveal Ab-ppt Neogen
Quix Rapid O157 Ab-ppt Universal
HealthWatch
ImmunoCardSTAT Ab-ppt Meridian
VIDAS ELFA
b
bioMerieux
EHEC
**c
O157:H7
EiaFOSS ELISA
b
Foss
VEROTEST ELISA MicroCarb
Premier EHEC ELISA Meridian
Shiga toxin (Stx)
Verotox-F RPLA Denka Seiken
ETEC
c

Labile toxin (LT) VET-RPLA RPLA Oxoid
Stabile toxin (ST) E. coli ST ELISA Oxoid
Microscreen LA Microgen
Listeria Latex LA Microgen
Listeria-TEK
e
ELISA Organon Teknika
TECRA
e
ELISA TECRA
Assurance
e
ELISA BioControl
Transia Listeria ELISA Transia
Pathalert ELISA Merck
Listertest Ab-beads VICAM
VIP
e
Ab-ppt BioControl
Clearview Ab-ppt Unipath
RAPIDTEST Ab-ppt Unipath
VIDAS
e
ELFA
b
bioMerieux
EiaFOSS ELISA
b
Foss
Listeria
UNIQUE Capture-EIA TECRA
Bactigen LA Wampole Labs
Spectate LA Rhone-Poulenc
Microscreen LA Mercia
Wellcolex LA Laboratoire
Wellcome
Serobact LA REMEL
RAPIDTEST LA Unipath
Screen Ab-beads VICAM
CHECKPOINT Ab-blot KPL
1-2 Test
e
diffusion BioControl
SalmonellaTEK
e
ELISA Organon Teknika
TECRA
e
ELISA TECRA
EQUATE ELISA Binax
BacTrace ELISA KPL
LOCATE ELISA Rhone-Poulenc
Assurance
e
ELISA BioControl
Salmonella ELISA GEM Biomedical
Transia ELISA Transia
Bioline ELISA Bioline
VIDAS
e
ELFA
b
bioMerieux
OPUS ELISA
b
TECRA
PATH-STIK Ab-ppt LUMAC
Reveal Ab-ppt Neogen
Clearview Ab-ppt Unipath
Salmonella
UNIQUE
e
Capture-EIA TECRA
Bactigen LA Wampole Labs Shigella
Wellcolex Laboratoire
Wellcome
Staphyloslide LA Becton Dickinson
AureusTest
e
LA Trisum
Staphylococcus
aureus
Staph Latex LA Difco
S. aureus VIA ELISA TECRA
SET-EIA ELISA Toxin Technology
SET-RPLA RPLA Unipath
TECRA
e
ELISA TECRA
Transia SE ELISA Transia
RIDASCREEN ELISA R-Biopharm
VIDAS ELFA
b
bioMerieux
enterotoxin
OPUS ELISA
b
TECRA
choleraSMART Ab-ppt New Horizon
bengalSMART Ab-ppt New Horizon
choleraScreen Agglutination New Horizon
Vibrio cholera
bengalScreen Agglutination New Horizon
enterotoxin VET-RPLA
d
RPLA Unipath
* Nguon trch dan: Feng, P., App.I, FDA Bacteriological Analytical Manual, 8A ed.
a
Ch viet tat: ELISA, enzyme linked immunosorbent assay; ELFA, enzyme linked
fluorescent assay; RPLA, reverse passive latex agglutination; LA, latex agglutination; Ab-
ppt, immunoprecipitation.
b
ELISA t ong
c
EHEC - Enterohemorrhagic E. coli; ETEC - enterotoxigenic E. coli
d
Cung phat hien oc to ng ruot LT cua E. coli (E. coli LT enterotoxin)
e
He thong c AOAC chnh thc chap nhan
** Chu y: Mot so san pham tren ch ac hieu phat hien chung O157 nhng khong hoan
toan thuoc serotype H7 (nhng chung O157 khong phai H7 thng khong tao oc to,
Shiga toxin, nen thng khong gay benh cho ngi). Mot so khang the O157 co the phan
ng cheo vi Citrobater, E. hermanii va nhng vi sinh vat ng ruot khac.

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AI HOC QUOC GIA THANH PHO HO CH MINH

TRNG AI HOC KHOA HOC T NHIEN

ng dung tren oi tng Salmonella a dc

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