TRNG I HC CN TH

KHOA THY SN

B MN SINH HC V BNH THY SN

DNG V M HNH

XC NH NNG C CH TI THIU (MIC)

CA THUC NATSOL (KHNG SINH THC VT)

LN VI KHUN

Trung tm Hc EdwardsiellaliuHCnictaluriTh@VTiAeromonasliuhchydrophilatpvnghin cu

LUN VN TT NGHIP I HC

NGNH NUI TRNG THU SN

CHUYN NGNH BNH HC THY SN

2008

TRNG I HC CN TH
KHOA THY SN

B MN SINH HC V BNH THY SN

DNG V M HNH

XC NH NNG C CH TI THIU (MIC)

CA THUC NATSOL (KHNG SINH THC VT)

LN VI KHUN

Edwardsiella ictaluri V Aeromonas hydrophila

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

CN B HNG DN

T THANH DUNG

NGUYN TH NH NGC

LUN VN TT NGHIP I HC

NGNH NUI TRNG THU SN

CHUYN NGNH BNH HC THY SN

2008

MC LC

MC LC

DANH SCH HNH 3

DANH SCH BNG

LI CM T 5

TM TT

CHNG 1: GII THIU 7

CHNG 2: TNG QUAN TI LIU

2.1 Bnh do vi khun E. ictaluri gy ra

2.1.1 Mt s c im ca vi khun E. ictaluri

2.1.2 Phn b ca vi khun E. ictaluri

2.2 Bnh do vi khun Aeromonas hydrophila

10

2.2.1 Mt s c im v vi khun Aeromonas hydrophila

10

2.2.2 Hnh dng khun lc 10

2.2.3 Hnh dng t bo

10

2.2.4 Phn b ca Aeromonas hydrophila 11

2.2.5 Bnh xut huyt do vi khun A. hydrophila 11
2.3 Cy tho dc

12

2.3.1 S lc v cy tho dc

12

2.3.2 Mt vi cy thuc tho dc v cng dng phng tr bnh thy sn

12

2.3.3 Mt s kt qu khoa hc s dng cy tho dc phng v tr bnh thy sn13

2.4 Natsol

14

2.5 Chloramphenicol 14
Trung tm Hc2.6MtliusktHqu xcCnnh Thnng@c chTitiliuthiu (MIC)hc trtpnthuvc khngnghinsinh cu

tho dc

14

CHNG 3: PHNG PHP NGHIN CU 16

3.1 Thi gian v a im nghin cu

16

3.1.1 Thi gian nghin cu 16

3.1.2 a im nghin cu 16
3.2 Vt liu nghin cu

16

3.2.1 Ho cht v mi ttrng

3.2.2 Thuc khng sinh

16

3.2.3 Dng c th nghim

16

3.2.4 Vi khun th nghim

17

3.3 Phng php nghin cu

16

17

3.3.1 Chun b nguyn liu nghin cu

3.3.1.1 Chun b vi khun

17

17

3.3.1.2 Chun b dung dch thuc khng sinh

18

3.3.2 Thc hin nghin cu 20

3.3.2.1 Nghin cu thm d 20
3.3.2.1 Phng php lp khng sinh

21

3.3.2.2 Phng php xc nh nng c ch ti thiu (MIC) trn mi trng thch

(Lila Ruangpan, 2004):

22

3.3.2.3 Phng php xc nh nng c ch ti thiu (MIC) bng phng php

pha long (Geert Huys, 2002):

23

CHNG 4: KT QU THO LUN

25

4.1 Kt qu nghin cu thm d khong nng c ch ca thuc khng sinh thc

vt natsol ln vi khun gy bnh trn c tra

4.2 Xc nh nng MIC 26

25

4.2 Kt qu kim tra khng sinh ca thuc natsol ln vi khun E. ictaluri v A.

hydrophila

27

4.3 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun E.

ictaluri v A. hydrophila bng phng php pha long

28

4.3.1 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun A.

hydrophila

28

4.3.2 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun E.

ictaluri 30
4.2.3 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun E.
ictaluri v A. hydrophila bng phng php thch.

32

CHNG 5: KT LUN V XUT 35

5.1 Kt lun 35
5.2 xut

35

TI LIU THAM KHO

36

Ph lc A: Pha dung dch nhum gram vi khun

41

Ph lc B: Phng php nhum Gram vi khun

42

Ph lc C: Pha mi trng

43

Ph lc D: Pha nc mui sinh l v pha mi trng NB 44

Ph lc E: Pha thuc khng sinh chloramphenicol v natsol

45

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

DANH SCH HNH

Hnh 2.1: Kt qu nhum gram vi khun E. ictaluri vt knh 40X (gram m

hnh que)

10

Hnh 2.2: Kt qu nhum gram vi khun A. hydrophila vt knh 40X (gram

m hnh que)

11

Hnh 4.3: Kt qu MIC trn mi trng thch ca vi khun A. hydrophila (a

i chng (tri), a mi trng cha thuc nng 375ppm (phi))

34

Hnh 4.4: Kt qu MIC trn mi trng thch ca vi khun A. hydrophila (a

i chng (tri), a mi trng cha thuc nng 750ppm (phi))

34

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

DANH SCH BNG

Bng 3.1: Pha long nng thuc natsol cho hai chng vi khun theo phng

php pha long hai ln

18

Bng 3.2: Pha long nng thuc chloramphenicol cho hai chng vi khun

theo phng php pha long hai ln

19

Bng 3.3: Nui vi khun cc hm lng thuc khc nhau (cho 1 chng vi

khun)..

19

Bng 3.4: Nui vi khun cc hm lng thuc khc nhau (cho 1 chng vi

khun)..

20

Bng 4.1: Kt qu nghin cu thm d nng c ch ti thiu ca thuc

Natsol trn vi khun E. ictaluri.

26

Bng 4.2: Nng c ch ti thiu ca thuc chloramphenicol trn E. coli

27

Bng 4.3: Kt qu khng sinh kim tra nhy ca thuc natsol bng a

khng sinh t ch

28

Bng 4.4: Nng c ch ti thiu (MIC) ca thuc khng sinh natsol trn vi

khun A. hydrophila chng CAF2 ..

29

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

Bng 4.5: Nng c ch ti thiu (MIC) ca natsol trn vi khun A.

hydrophila chng CAF133

29

Bng 4.6: c kt qu pha long ng MIC s 6 v 7 pha long 105 trn vi

khun A. hydrophila 30

Bng 4.7: Nng c ch ti thiu (MIC) ca natsol trn vi khun E. ictaluri

chng vi khun E.3B3

31

Bng 4.8: Nng c ch ti thiu (MIC) ca natsol trn vi khun E. ictaluri

chng vi khun E.223

31

Bng 4.9: c kt qu pha long ng MIC s 8 v 9 pha long 104 trn vi

khun E. ictaluri

32

Bng 4.10: Kt qu MIC trn mi trng thch trn vi khun A. hydrophila vi

cc a mi trng 5, 6, 7 ng vi cc nng 1500; 750; 375ppm 32

Bng 4.11: Kt qu MIC trn mi trng thch trn vi khun E. ictaluri vi cc

a mi trng 6, 7, 8 ng vi cc nng 750; 375; 187,5ppm

33

LI CM T

Ti c bit bit n c T Thanh Dung v Nguyn Th Nh Ngc n cn hng dn v gip

ti thc hin nghin cu v hon thnh ti tt p.

Chn thnh cm n c c vn hc tp c Nguyn Th Thu Hng nhit tnh dy d v

dn dt lp v ti thi gian qua.

Ti xin cm n tt c thy c ca Khoa thu sn ni chung v thy c ca B mn Sinh hc

v bnh thu sn ni chung truyn dy cho ti nhng kin thc qu gi trong sut 4 nm
hc va qua v to iu kin cho ti thc hin ti ny.

Cm n bn b trong lp cng nh bn b khc lp tn tnh trao i vi ti nhng kin

thc khim khuyt v gip ti trong hc tp cng nh trong qu trnh thc hin ti.

Ti gi li cm n n gia nh, ngi thn nui dy ti v to iu kin cho ti n hc.

Ti c c thnh qu nh ngy hm nay th khng th k ht cng n tri bin ca cha
m dnh cho ti.

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

TM TT

Th nghim thm d nng c ch ti thiu ca thuc khng sinh thc vt natsol ln vi

khun E. ictaluri v xc nh c khong nng c ch ca natsol ln vi khun E.
ictaluri l 50-100ppm.

Thc hin nghin cu xc nh nng c ch ti thiu ca thuc khng sinh thc vt

natsol bng ba phng php:

Xc nh nng c ch ti thiu (MIC) bng phng php lp khng sinh , nhng qua th
nghim trn thuc natsol khng cho kt lun g.

Xc nh nng c ch ti thiu (MIC) bng phng php pha long, xc nh c nng c

ch ti thiu (MIC) ca natsol ln vi khun E. ictaluri l 93,75ppm v ln vi khun A. hydrophila l
375ppm.

Xc nh nng c ch ti thiu (MIC) bng phng php thch, xc nh c nng c

ch ti thiu (MIC) ca natsol ln vi khun E. ictaluri l 187,5ppm v ln vi khun A. hydrophila
l 750ppm.

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

CHNG 1

GII THIU

C tra (Pangasianodon hypophthalmus) l loi c da trn nc ngt c nui ph bin ng

Bng Sng Cu Long (BSCL). T nhng nm 1998-2000 thnh cng trong nghin cu Quy trnh
k thut sinh sn nhn to v ng nui c tra thng phm ca Khoa Thu sn - i hc Cn
Th, y mnh phong tro nui c tra thng phm pht trin vng BSCL, nng sut v sn
lng nui khng ngng tng. Theo Dng Nht Long (2006) th sn lng c tra nm 1994 t
30.000 tn, nm 2001 t 150.000 tn, nm 2002 t 200.000 tn, nm 2003 t 220.000 tn, nm
2004 t trn 300.000 tn, nm 2005 vt qua 500.000 tn. Song bn cnh mt thun li cn
tn ti nhng vn kh khn, l tnh hnh bnh trn c tra nui. Trong nhiu nm qua ngi
nui c tra b thit hi kh ln do dch bnh xy ra, nh bnh m , trng da, ph u ph mt,
bnh do k sinh trng ngoi k sinh(Ferguson & ctv, 2001). Theo Lng Trn Thc oan (2006)
bnh trng gan (cn gi l bnh m gan) do vi khun Edwardsiella ictaluri (E. ictaluri) gy ra l bnh
ph bin, xut hin hu nh quanh nm c tra, gy thit hi nghim trng trong nui c

Trung tmtraHcthm liucanh. MHtkhc,CnbnhThm@ (cnTigliuilbhcnhxuttphuytv) donghinvikhuncu

Aeromonas hydrophila (A. hydrophila) gy ra trn nhiu loi c nc ngt trong c c tra
nui (T Thanh Dung, 2005).

T Thanh Dung (1996) cho rng bnh vi khun l mt vn quan trng trong nui trng thu sn,
vic iu tr ch yu bng ho cht hoc s dng thuc khng sinh penicillin, streptomycin,
chloramphenicol, oxytetracylin, tetracylin.... Tuy nhin s dng ho cht phng v tr bnh vi khun
trong thi gian di, c bit l khng sinh li gp nhiu kh khn, nh gi thnh cc loi thuc
khng sinh cao, li gy ra hin tng khng thuc ca cc tc nhn gy bnh, lm gim hiu qu
phng v tr bnh v sau. Thm vo l vn nhim mi trng nc, tch t d lng khng
sinh trong sn phm thu sn nh hng n cht lng sn phm xut khu, nht l khng tt
cho sc kho ngi tiu dng. Theo Direkbusarakom (1995), mt gii php phng tr khc l s
dng cy tho dc iu tr bnh vi khun trong thu sn, tuy hiu qu khng nhanh chng nhng
khc phc c nhng kh khn gp khi s dng ha cht v thuc khng sinh. V vy, Thi
Lan, Trung Quc, n k c Vit Nam, mt vi cy tho dc c sn trong t nhin c dng
lm dc phm cho ngi v gia sc, cng c th nghim lm thuc tr bnh trong ngnh thu
sn (Sivarajan, 1994). Cht chit cy u (Clinacanthus nutans) c ch bnh Yellowhead
Baculovirus trn tm s (Penaeus monodon)

(Direkbusarakom & ctv, 1995). Theo Kamei (1988) cho rng chit sut tho dc t cy i (Psidium
guajava) phng tr c bnh vi rt nh IHNV, IPNV, OMV trn c. T Thanh Dung (1996)
nghin cu thnh cng nh hng ca cy tho dc ln vi khun A. hydrophila gy bnh xut
huyt trn c tr lai (Clarias macrocephalus x C.gariepinus). Hin nay vn cha c nhiu ti liu
khoa hc cp n cch phng tr bnh c tra bng tho dc. V vy, lm phong ph thm
cho phng php iu tr cng nh lm a dng danh mc thuc phng tr bnh cho c l l do
chnh ti Xc nh nng c ch

ti thiu (MIC) ca thuc Natsol (khng sinh thc vt) ln vi khun E. ictaluri v A.
hydrophila c thc hin.

Mc tiu nghin cu

Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun A. hydrophila v E.

ictaluri.

Ni dung nghin cu

Kim tra nng c ch ti thiu (MIC) bng phng php pha long v mi trng thch
trn vi khun A. hydrophila v E. ictaluri.

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

CHNG 2

TNG QUAN TI LIU

2.1 Bnh do vi khun E. ictaluri gy ra

2.1.1 Mt s c im ca vi khun E. ictaluri

Theo Hawke & ctv (1981) E. ictaluri l loi thuc Enterobacteriaceace, gram m, hnh que ngn,
kch thc 0,75x1,5-2,5m, di ng yu 25-300C, khng di ng khi nhit cao hn. Catalase
dng tnh, cytochrom oxidase m tnh v ln men glucose. Khng sinh H2S v indole m tnh. Vi
khun E. ictaluri pht trin trn mi trng TSA (trypton soya agar) chm 36-48 gi ti 18-280C.

Ging vi khun Edwardsiella cn pht trin tt trn mi trng BHI (brain heart infusion) v mi
trng TSA, vi khun c dng khun lc nh mu xanh c nhn en trn mi trng EIA (E. ictaluri
agar) (T Thanh Dung, 2005).

Trung tm Hc liu v nghin cu

Hnh 2.1: Kt qu nhum gram vi khun E. ictaluri vt knh 40X

(gram m hnh que)

2.1.2 Phn b ca vi khun E. ictaluri

E. ictaluri c th truyn bnh qua mi trng nc, t v cc ng vt khc.

Vi khun E. ictaluri l mm bnh nguyn thu trong ngnh nui c nheo M. Bnh do vi khun
E. ictaluri c chun on vng nui c da trn ti
Mississippi, Arkanas, Alabana, Louisiana, Georgia v Florida. Theo Hawke & ctv (1998), bnh cng
thng xuyn xy ra Virginia, Texas, Idaho, Kentucky,

California, Arizona v Maryland. Vit Nam, vng BSCL bnh m gan xut hin u tin vo ma
l nm 1998 cc tnh nui c tra thm canh pht trin mnh nh An Giang, ng Thp v Cn
Th. Sau , bnh ly lan sang cc

vng ln cn. c bit, nhng nm gn y bnh ny cng xut hin mt s tnh ven bin mi
pht trin nui c tra nh Tr Vinh, Bn Tre v Sc Trng

(T Thanh Dung & ctv, 2004). Bnh m gan ch yu xut hin trn c tra ( tt c cc giai
on pht trin), thnh thong xut hin trn c basa. T l hao ht cao c tra ging,
nhng gy thit hi ln nht v kinh t giai on c tra tht c 300-500g (T Thanh Dung
& ctv, 2004).

2.2 Bnh do vi khun Aeromonas hydrophila

2.2.1 Mt s c im v vi khun Aeromonas hydrophila

A. hydrophila l vi khun gram m hnh que, c tm thy trong mi trng nc, gy

bnh trn ng vt c xng sng mu lnh v ng vt hu nh, tn ti t do trong nc
(Ho & ctv, 1990). A. hydrophila l mm bnh ch yu ca ng vt thy sn vng nc
ngt, ng vt trn cn v k c con ngi (Bi Quang T, 2006).

Trung tm Hc liu v nghin cu

Hnh 2.2: Kt qu nhum gram vi khun A. hydrophila vt knh 40X

(gram m hnh que)

2.2.2 Hnh dng khun lc

A. hydrophila c th sng n c trong hu ht cc mi trng. Khun lc ca vi khun A.

hydrophila l nhng cm mu vng, hnh trn, nh mt trng, ni

(T Thanh Dung, 2005).

2.2.3 Hnh dng t bo

A. hydrophila c nhng c trng sau: gram m, que thng, kch thc khong

0,5x1,4-4,0m, c roi cc c th, k kh, ln men cacbonat hnh thnh acid hoc kh gas,
sn phm ca 2,3-butandiol, oxydase dng tnh, kh nitrat. (Shotts & Rimler, 1973).

10

2.2.4 Phn b ca Aeromonas hydrophila

Thng thng, bnh rt d pht sinh do kt qu tng tc gia vt ch, mm bnh v mi

trng. Mc d khi iu kin mi trng khng thch hp l nguyn nhn chnh ca tc
nhn gy bnh nhim trng, th tc nhn ngu nhin v tc nhn c hi ch c th l
nguyn nhn mm bnh khi c hai yu t tnh trng sinh l ca vt ch v iu kin mi
trng ca h thng nui khng thch hp (Subasingha, 1995).

Bnh xut huyt do A. hydrophila c th ly truyn qua nc, t c bnh sang c khe, c tp
lm thc n nhn to, v kt hp ng thc vt k sinh bn ngoi v bn trong c th
(Newman, 1993). Cc yu t gy sc nh mt nui cao, thc n tha, p sut nc cao,
nng oxy ha tan thp, ngho dinh dng l nguyn nhn c d mc bnh (Shotts & ctv,
1972).

Vi khun A. hydrophila tn ti trong mi trng nui, c bit trong nc c hm lng hu c

cao (Snieszko & Axelrod, 1971). Vi khun c th tn ti bn ngoi c th c khe (Trust & Sparrow,
1974). Nhit nh hng n c ca vi khun. Ging A. hydrophila sng n c trong nc
ly truyn vi hm lng vi khun thp trong c bnh v mi cu tm nc ngt. Mt vi chng vi
khun ca A. hydrophila c c t ngoi l i vi c, khng c c khi vi
khun yu i.

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

A. hydrophila c th xm nhp qua nhng tn thng do k ch trung gian. Nhiu trng
hp bnh bc pht c lin quan n yu t gy sc, chng hn hm lng oxy ho tan thp
kt hp vi kh ammonia v kh cacbonic cao, lm tng p sut nc v nng nitrat. C
cng tr nn nhy cm vi A. hydrophila (Pai & ctv, 1995).

2.2.5 Bnh xut huyt do vi khun A. hydrophila

Triu chng ca bnh ny ging triu chng ca cc bnh xut huyt do vi khun khc, ch
khc bn im ph bin: nhy vi c thp, hnh dng ph v hnh dng vt lot. Biu
hin bnh xut huyt c chp (Cyprinus carpio) l nhng m trn thn, c tr lai th
b ph (Rogers & Burke, 1981).

A. hydrophila gy bnh trn nhiu loi c: c lc (Ophicephalus striatus) (Bloch), c tr (Clarias

batrachus), c dic (Glossogobius giurus) (Hamilton), c hi m en (Oncorhynchus mykiss)
(Walbaum) (Lio-Po & ctv, 1992).

Bnh ph xy ra c tr lai c nh. Tonguthai & ctv (1993) tm thy nguyn nhn ca bnh ny l
A. hydrophila, Vibrio sp, v Pseudomonas sp vi cc triu

11

chng lm sng: xut huyt, ph, sng ty vy ngc, tc nhn quan trng nht l vi khun A.
hydrophila, gy thit hi nhiu cho ngi nui thy sn.

2.3 Cy tho dc

2.3.1 S lc v cy tho dc

Tho dc l nhng cy c hng thm chi, l, hoa, ht v r c dng lm hng liu, gia v
hoc lm thuc theo tng mc ch (Eisenbrand & ctv, 1992). Cy tho dc l mt cy mm,
mng nc, hu ht pht trin t ht, khng pht trin t thn g, x cng dai. Mt vi tho dc
nh hng liu lm nc gii kht c th tm thy trong t nhin nhng hu ht tho dc c
trng v rt sch. Chng c sy kh v d tr lm hng liu cho nm. Tho dc lm thuc
cha bnh c cy hoang d ln cy c trng u tt v

c sy kh hoc ch bin k.

T xa n nay, trong thy sn s dng thuc t thc vt (Sivarajan & ctv, 1994). Cch y
hng ngn nm khai ha, t Ai Cp, Trung Quc, n , Rp, Ba T, cc ti liu ghi chp cc
bng chng v s hiu bit tinh vi v l lun truyn thng vi vic trng nhng cy tho dc v
cy gia v.

c bit l trong ch bin lm thc n, v chng giu dinh dng v c tnh st

Trung tmkhuHcnttliucho HsckheCn.HngThtrm@cyTith oliudchcv giatpv vcnghindnglmcu

thuc v m phm, dng trong git ra v tm gi, hun khi trong nh v theo mc ch tn
ngng (Eisenbrand & ctv, 1992).

Theo Tt Li (1991) c trn 600 loi cy thuc Vit Nam, H (1993) bo co kt qu

ban u trn cy thuc l phng v iu tr c bnh trn c v tm Vit Nam. Hu ht
chit sut tho dc cho kt qu chng li vi khun mt vi bnh c v tm nhng
phng thc chit sut, c c cho iu tr cha hon thin.

2.3.2 Mt vi cy thuc tho dc v cng dng phng tr bnh thy sn

+ Cy inh hng (Eugenia caryophyllus)

Cy inh hng c s dng lm hng liu kch thch nh l v cay ca t, lm gia v nm v

lm du xng hi. Cy inh hng dng lm thuc cha bnh. Du inh hng dng trong cng
nghip hng thm v lm x phng, cng nh lm thuc gim au, c tr au rng (Trease &
Evans, 1973).

+ Cy xuyn tm lin (Andrographus panicullata)

Cy xuyn tm lin c tc dng thanh nhit, gii c, tiu thng, c ch vi khun, tng
cng hin tng thc bo ca t bo bch cu, dng tr bnh vim rut cho c trm c
(Bi Quang T, 2006)

12

+ Cy nh ni (Eclipta alba)

C nh ni c tc dng cm mu, khng gy tng huyt p, khng lm gin mch ngi, iu

tr vim gan (Dixit & Achar, 1979). Eclipta alba c tc dng trong iu tr tn thng gan v vt
bng (T Thanh Dung, 1996).

Kt qu th tc dng ca cao tch chit tho dc cao nh ni c tc dng vi

3 vi khun V. harveyi, V. alginolyticus v A. hydrophila (Bi Quang T, 2006).

+ Cy ch (Phyllathus debilis)

Cy ch c tc dng khng sinh, cha inh ru, au mt, mn nht ngi. th nghim
tc dng khng sinh vi vi khun A. hydrophila, E. tarda gy bnh hoi t c tr, vng khng
khun 11-20mm (Bi Quang T, 2006).

+ Cy ti (Allium sativum)

Kt qu th cao tch chit tho dc t ti c tc dng vi 6 loi vi khun:

Vibrio parahaemolyticus, V. harveyi, V. alginolyticus, A. hydrophila, E. tarda,

Hafnia alvei) gy bnh c nc ngt, l mn. Ti tch chit thnh cao du phi ch thnh thuc
cha bnh tm c, c tc dng phng tr bnh xut huyt, hoi t ni tng (bnh m trng) do vi
khun cho c tra nui. Kt qu ch phm phi ch t hot cht tch chit ca ti v si t c tc
dng phng tr bnh n
mn v kitin do vi khun Vibrio spp cho tm nui (Bi Quang T, 2006).

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

2.3.3 Mt s kt qu khoa hc s dng cy tho dc phng v tr bnh

thy sn

T Thanh Dung (1996) kim tra tnh nhy ca 8 loi chit sut tho dc trn 6 chng vi khun
A. hydrophila gy bnh trn c tr lai (Clarias batrachus x C.gariepinus). Kt qu ch c 2 loi tho
dc nhy: Phyllanthus debitis (PD) v Eugenia caryophyllus (EC). ng thi xc nh nng
c ch ti thiu

(MIC) ca cao bt PD l 10.000g/l, ca chit du EC l 1.000l/l. Cn c vo kt qu MIC v nng

gy cht LC50, th nghim c tin hnh trn b nui nh gi kh nng ngn nga bnh
nhim trng huyt do Aeromonas trn c tr bng cch ngm vo thc n hoc phng php tm.
i vi EC, d dng ngm thuc vo thc n vi hm lng di 10ml/kg thc n, v vy hm
lng EC c c trong thc n l 1ml/kg thc n (khong gi tr nng nh nht MIC). Cn PD,
khong gi tr MIC trong thc n l 10g/kg thc n v khong MIC trong phng php tm gim 10
ln ch dng 1.000g/ml.

Theo Goujun Yin v ctv (2005) kim tra nh hng ca 2 loi tho dc

Astragalus radix v Scutellaria radix ln kh nng min dch khng c hiu c r phi
(Oreochromis niloticus). C Astragalus radix v Scutellaria radix u c kh nng iu chnh h
thng min dch bm sinh ca c r phi. Thc n

13

cha 0,1% v 0,5% Astragalus radix trong 1 tun u lm tng hot ng ca lisozyme v kch
thch thc bo sau 3 tun, tuy nhin hot ng h hp ca t bo thc bo khng tng. Khi cho
c r phi n Scutellaria radix vi liu lng cao th hot ng h hp v thc bo b c ch,
lisozym khng hot ng. Kt qu th nghim sai khc khng ng k gia 2 loi thc n vi
nhng nng khc nhau ca c 2 loi cy tho dc. Liu tt nht ca Astragalus radix l
0,1% v 0,5% trong 3 tun. Vic cn lm l xc nh liu kch thch v thi gian cho n tt nht
ca Scutellaria radix.

2.4 Natsol

Natsol l cht chit sut t cy t nhin, l sn phm ca cng ty BIOFLAVONOIDS. Natsol

l dung dch dng tinh du, trong, hi vng nh mt ong.

Natsol c tc dng ngn chn cc bnh khc nhau ca vi khun v nm. Natsol c m
rng s dng n 3 nm trc v thnh cng trong bc u iu tr bnh thi chy,
thi mang, ct ph b. Ngoi ra Natsol cn dng iu tr bnh vi rt trn tm c. Thnh
phn gm flavonon sinh hc v khng c cha kim loi, ha cht hay c t.

2.5 Chloramphenicol

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

Khng sinh l cc cht hu c c cu to ha hc phc tp, c ngun gc sinh hc hay do
con ngi tng hp nn. C tc ng mt cch ring bit trn mt giai on chnh yu ca
s bin dng ca cc vi khun, nm v vi rt (L Th Kim Lin, 2007). Ngoi ra, theo Bi
Th Tho (2003) cho rng khng sinh l nhng cht c tc ng chng li s sng ca vi
khun, ngn nga vi khun nhn ln bng tc ng mc phn t hoc tc ng vo
mt hay nhiu giai on chuyn ha cn thit ca i sng vi khun hoc tc ng vo s
cn bng l ha.

Chloramphenicol l thuc khng sinh c hot tnh khng khun cao, nhanh, v mnh, c bit ph
khng khun c m rng vi c vi khun Gram m v Gram dng (Edwardsiella (gram m)
v Aeromonas (gram m), Pseudomonas

(gram m), Streptococcus (gram dng)) (L Th Kim Lin v Nguyn Quc

Thnh, 2006).

2.6 Mt s kt qu xc nh nng c ch ti thiu (MIC) trn thuc

khng sinh tho dc

Mullika & ctv (2005) tin hnh th nghim xc nh nng c ch ti thiu ca 19 loi tho
dc (andrographic paniculata, azadirachta indica, barleria lupulina, carthamus tinctorius,
centella asiatica, clinacanthus nutans,

14

cymbopogon citrates, eupatorium odoratum, garcinia mangostana, hibiscus

sabdariffa, houttuynia cordata, lawsonia inermis, lycopersicon esculentum, murdennia
lorifomis, psidium guajava, senna alata, senna occidentalis, senna siamea, tagetes
erecta) ln hai loi vi khun Probionibacterium acnes v

Staphylococcus epidermidis bng phng php thch. Kt qu cho thy 13 loi

thuc tho dc c nh hng n s pht trin ca hai loi vi khun trn mi
trng thch. Trong , Garcinia mangostana l thuc c tc dng mnh nht,
vi gi tr MIC nh nht (39ppm). iu c bit l gi tr MIC ca tho dc ny
trn c hai loi vi khun th bng nhau. Kumar & ctv (2007) cng tin hnh th
nghim xc nh nng c ch ti thiu ca tho dc ln hai loi vi khun
Probionibacterium acnes v Staphylococcus epidermidis, vi 12 loi thuc tho
dc hemidesmus indicus, eclipta alba, coscinium fenestratum, curcubito pepo,
tephrosia purpurea, mentha piperita, pongamia pinnata, symplocos racemosa,
euphorbia hirta, tinospora cordyfolia, thespesia populnea, jasminum officinale.
Qua phng php thch c 7 loi thuc cho kt qu c ch s pht trin ca hai
loi vi khun ny trn mi trng thch. V qua phng php pha long xc nh
c nng c ch ca Coscinium fenestratum trn hai loi vi khun ny bng
nhau v nh nht. Gi tr MIC trn hai loi vi khun l 49ppm.

Trung
tmSupayangHcliu&ctvH(2005)CnkimThtratc@dngTicaliuethylhcacetatetpvvnbutanolnghinchitcu sut t v qu Punica granatum ln E. coli O157:H7, E. coli O26:H11, E. coli

O111:NM v E. coli O22 bng phng php thch. Xc nh c gi tr MIC ca c

hai chit sut ny ln E. coli O157:H7 l nh nht (50ppm). Gi tr MIC ca ethyl
acetate chit sut t Centaurea ln vi khun Staphylococcus aureus l 62,5ppm, thp
hn so vi gi tr MIC ca chloramphenicol (125ppm), ngha l ethyl acetate c tc
dng mnh hn chloramphenicol ln vi khun

Staphylococcus aureus (Kiymet & ctv, 2005).

Chit sut methanol v acetone t nhiu cy nm c tc dng trn vi khun

baccilus cereus, Staphylococcus aureus, listeria monocitogenes, Escherichia coli,
salmonella infantis. Qua phng php pha long xc nh c khong gi tr
MIC ca cc chit sut ny t 165-2640mg/ml. B. cereus nhy vi hu ht cc
chit sut t cinnamomum cassia, azadirachta indica, ruta graveoln, rumex
nervosus vi khong MIC t 165-660mg/ml. Chit sut t

Cinnamomum cassia ch c hiu qu trn vi khun E.coli, S. infantis gi tr

MIC cao nht (2640mg/ml) (Mohan & ctv, 2004).

15

CHNG 3

PHNG PHP NGHIN CU

3.1 Thi gian v a im nghin cu

Thi gian nghin cu: Bt u t thng 04/2008 n thng 05/2008

a im nghin cu: Phng th nghim b mn sinh hc v bnh thu

sn, khoa Thu sn, trng i hc Cn Th.

3.2 Vt liu nghin cu

3.2.1 Ho cht v mi ttrng

Mi trng nui cy vi khun: nutrient agar (NA, Merck), nutrient broth

(NB, Merck), muller hinton agar (MHA, Merck).

Cc loi ho cht nhum Gram: dd I (ammonium oxalate, crystal violet),

dd II (iodine), dd III (cn tuyt i, ethanol (Merck)), dd IV (safranin).

NaCl.

Du xem knh hin vi.

Glycerol.

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

Nc ct.

3.2.2 Thuc khng sinh

Tho dc: natsol. Chloramphenicol.

3.2.3 Dng c th nghim

Que cy, pel, khay nha, bnh xt cn, giy v sinh, bt lng, knh hin vi, lame, n cn,
ht qut.

Cc thu tinh, l thu tinh, que tri thu tinh, a petri, ng ong, ng nghim.

Pipet 1-5ml, 100-1000l, 10-100l, 5-40l.

u col 5ml, 1ml, 100l; ng falcon 50ml thanh trng.

Cn in t, ni thanh trng, my li tm, my so mu quang ph, cuvette 3ml, my lc, my

trn mu, my m khun lc.

Giy lc lm a khng sinh.

T m, t lnh, t sy, t cy, ni chng ct (water-bath). My nh, s ghi chp theo di

hng ngy.

16

3.2.4 Vi khun th nghim

Vi khun E. coli LMG8223 t ngun i hc Ghent, vng quc B.

Vi khun E. ictaluri v A. hydrophila c phn lp trn c tra, ngun cung cp t B mn Sinh

hc v Bnh Thy sn, Khoa Thy sn, i hc Cn Th. Tt c vi khun c tr -80 0C
trong mi trng BHI lng + glycerol.

Hai chng vi khun E. ictaluri: E.223 v E.3B3

Hai chng vi khun A. hydrophila: CAF2 v CAF133

3.3 Phng php nghin cu

3.3.1 Chun b nguyn liu nghin cu

3.3.1.1 Chun b vi khun

Nui cy v phc hi vi khun

Vi khun tr c phc hi bng cch cho vi khun vo ng nghim cha 10ml dung dch NB lng,
trong t m 28-300C hoc lc u trn my lc (200 vng/pht) 18-24 gi, ri cy sang a mi
trng kim tra tnh thun (da vo cc ch tiu v hnh thi: hnh dng, mu sc khun lc, kt qu
nhum gram).
Hai loi vi khun E. ictaluri v A. hydrophila c cy phc hi v tch rng

Trung tm Hc liu H Cn Th 0@ Ti liu hc tp v nghin cu

trn mi trng thch NA, 28 C trong 24 gi i vi A. hydrophila v 48 gi i vi E. ictaluri. Vi

khun thun c nui tng sinh trong mi trng

NB lng, trong t m 28 C hoc lc u trn my lc t 18-24 gi (Rahman & Kawai,

2000).

Ly tm vi khun

Cho vi khun vo ng falcon 50 ml, ly tm vi tc 4000 vng/ pht, ly tm trong 15 pht,

s dng nc mui sinh l (0,85% NaCl) tit trng ra vi khun. Lp li thao tc 2 3
ln. Sau ln ly tm cui cng cho mi trng NB vo v trn u mu.

Xc nh mt vi khun

Ly tm xong, cho mi trng NB vo nh tan phn vi khun lng, ri tin hnh so mu bng my
so mu quang ph. Mt vi khun c o bng my so mu quang ph bc sng 590nm, vi
OD=0,10,02 tng ng vi mt vi khun A. hydrophila l 1x108cfu/ml v OD=0,010,002
tng ng vimt vi khun E. ictaluri l 1x107cfu/ml (Lng Trn Thc oan, 2006).

Sau mi chng vi khun c cy trn mi trng NA kim tra thao tc k thut chun b
dung dch vi khun trc khi thc hin nghin cu.

17

3.3.1.2 Chun b dung dch thuc khng sinh

Natsol: c cung cp t cng ty BIOFLAVONOIDS ca n . Chun b dung dch gc

(stock) Natsol nh sau: ha tan 5ml Natsol trong 100ml nc ct c nng 50000l/l, sau
pha long theo phng php pha long hai ln (double dilution method) c cc nng
24000; 12000; 6000; 3000; 1500; 750; 375;187,5; 93,75; 46,875 v 26,4375l/l (Bng 3.1).
Trong , cc nng 24000ppm v 1500ppm c pha t nng 50000ppm.

Bng 3.1: Pha long nng thuc natsol cho hai chng vi khun theo phng php pha
long hai ln

ng
Nng cn pha
Th tch
Th tch

thuc khng sinh natsol

nc ct

nghim
(ppm)

(ml)
(ml)

1
24000
6 (50000ppm, dung dch gc 1)
6

2
12000
6 (24000ppm)
6

3
6000
6 (12000ppm)
6

4
3000
6 (6000ppm)
6

Trung tm

cu

5
1500
6 (3000ppm, dung dch gc 2)
6

Hc liu H Cn Th @ Ti liu hc tp v
nghin

6
750
6 (1500ppm)
6

7
375
6 (750ppm)
6

8
187,5
6 (375ppm)
6

9
93,75
6 (187,5ppm)
6

10
46,875
6 (93,75ppm)
6

11
23,4375
6 (46,875ppm)
6

Ch :

ng nghim c nng thuc 24000 v 1500ppm s c pha long t dung dch gc 50000ppm
vi natsol v chloramphenicol th cc nng 512 v 128ppm pha long t dung dch gc
1.024ppm (Bng 3.3).

Cn lc u dung dch thuc trc khi pha long cc nng tip theo. Nng thuc s gim i
phn na khi cho dung dch vi khun vo. Ghi tn thuc v nng thuc trc khi bt u th
nghim.

Phi lc u dung dch thuc trc khi pha long nng tip theo. iu cn ch l mi
nng pha long, hm lng thuc gim i phn na sau khi cho dung dch vi khun vo
(Bng 3.2 v Bng 3.4).

18

Bng 3.2: Nui vi khun cc hm lng thuc natsol khc nhau (cho 1 chng vi khun)

ng
Nng tht thuc natsol
Th tch
Th tch

thuc natsol
vi khun

MIC
(ppm)

(ml)
(ml)

1
12000
2 (24000ppm, ng 1)
2

2
6000
2 (12000ppm, ng 2)
2

3
3000
2 (6000ppm, ng 3)
2

4
1500
2 (3000ppm, ng 4)
2

5
750
2 (1500ppm, ng 5)
2

6
375

2 (750ppm, ng 6)
2

7
187,5
2 (375ppm, ng 7)
2

8
93,75
2 (187,5ppm, ng 8)
2

9
46,875
2 (93,75ppm, ng 9)
2

10
23,4375
2 (46,875ppm, ng 10)
2

11
11,72875
2 (23,4375ppm, ng 11)
2

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

Bng 3.3: Pha long nng thuc chloramphenicol cho hai chng vi khun

theo phng php pha long hai ln

Th tch
Th tch

ng
Nng cn pha
thuc khng sinh

nc mui sinh l

nghim
(ppm)
chloramphenicol

(ml)
(ml)

1
1024

2
512
5 (1024ppm,
5

dung dch gc 1)

3
256
5 (512ppm)
5

4
128
5 (256ppm,
5

dung dch gc 2)

5
64
5 (128ppm)
5

32
5 (64ppm)
5

7
16
5 (32ppm)
5

8
8
5 (16ppm)
5

9
4
5 (8ppm)
5

19

Bng 3.4: Nui vi khun cc hm lng thuc chloramphenicol khc nhau (cho 1 chng
vi khun)

ng
Th tch tht thuc khng sinh
Th tch
Th tch

thuc khng sinh

vi khun

MIC
chloramphenicol (ppm)

(ml)
(ml)

1
512
2 (1024ppm, ng 1)
2

2
256
2 (512ppm, ng 2)
2

3
128
2 (256ppm, ng 3)
2

4
64
2 (128ppm, ng 4)
2

5
32
2 (64ppm, ng 5)
2

6
16

2 (32ppm, ng 6)
2

7
8
2 (16ppm, ng 7)
2

8
4
2 (8ppm, ng 8)
2

9
2
2 (4ppm, ng 9)
2

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

Chloramphenicol (Oxiod).

Chun b dung dch gc chloramphenicol: Pha 2 ng nghim thuc gc, mi ng 50 ml (l

thuc khng sinh) (ng th nht c hm lng thuc l 1024 ppm v ng th 2 c hm lng
thuc l 256 ppm). Thuc phi c pha bng dung mi ph hp (theo hng dn ca nh sn
xut).

Pha thuc vi pha long 2 ln t 4-1024 ppm trong ng nghim 50ml (Bng 3.2). Hm lng
thuc 512 ppm v 256 ppm c pha t ng thuc gc th nht (1024 ppm) bng cch thm
nc mui sinh l. Hm lng thuc 128, 64, 32, 16, 8 v 4 ppm c pha t ng gc th 2
(256 ppm).

3.3.2 Thc hin nghin cu

3.3.2.1 Nghin cu thm d

Tin hnh nghin cu thm d gm cc bc nh sau:

Chun b dung dch gc natsol: th nghim thm d nng thuc natsol trn vi khun E.
ictaluri vi chng E.223 c tin hnh t nng gc

(50000ppm) pha long cc nng : 10000; 8000; 6000; 5000; 4000; 3000; 2000; 1000;
500; 400; 300; 200; 100; 50; 45; 40; 35; 30; 25; 20; 15 v 10ppm. Pha nng 50000ppm
theo hng dn ca nh sn xut.

20

Chun b dung dch vi khun (nh 3.3.1.1)

Tin hnh th nghim theo phng php xc nh MIC bng phng php pha long

Cho 2 ml vi khun vo mi ng nghim cha 2 ml thuc khng sinh vi cc nng 1010000ppm.Tt c cc ng nghim v a cy kim tra vi iu kin nhit 28-30 oC. Sau 24
gi c kt qu cc ng MIC: so snh c gia cc ng MIC. Kim tra khong ng MIC c
c kh phn bit bng cch kim tra s pht trin ca vi khun trn a petri mi trng
thch. Tu theo tng c khc nhau m ta c th pha long theo nng gim dn sao cho
c th d dng m cc khun lc trn a thch. Chn nng no c mt vi khun thp
nht (gim 80% so vi mt vi khun gc) l nng c ch ti thiu (MIC).. V sau 24 gi
cha thi gian cho vi khun E. ictaluri pht trin trn a mi trng. Do , ghi nhn kt qu
cc ng MIC sau 24 gi ri li trong t m, 48 gi c kt qu cc ng MIC li cng vi
a mi trng vi khun E. ictaluri.

3.3.2.1 Phng php lp khng sinh

Kim tra nhy ca natsol ln vi khun E. ictaluri v A. hydrophila bng a khng sinh
natsol, gm cc bc sau:

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

Chun b dung dch gc natsol (nh 3.3.1.2 )

Chun b a khng sinh tho dc:

Chun b a khng sinh: s dng giy lc v to thnh nhng khoanh giy trn nh (s
dng dng c bm giy) c ng knh tng ng vi a khng sinh thng mi
0

(6mm). Dng giy nhm gi nhng a giy v mang i tit trng khong 2 gi 180 C.

Chun b dung dch thuc khng sinh: dng chai tit trng pha thuc khng sinh vi nng tng
t a khng sinh thng mi. Ly khong 50 a giy tit trng cho vo 10ml dung dch thuc
khng sinh, ngm khong 24 gi (hoc ngm qua m). Sau 24 gi ngm a, dng pel tit trng
nht tng a thuc khng sinh chuyn qua a petri tit trng a kh iu kin nhit
phng, sau cho vo chai l tit trng gi 4 - 6oC v c th s dng lu di.

Chun b dung dch vi khun (nh 3.3.1.1)

Lp khng sinh

Dng pipet tit trng ht ln lt 0.2ml dung dch vi khun cho ln mi trng thch MHA. Dng
que tri thy tinh tit trng tri u n va kh. Sau yn khong 1 pht ri dng pel tit trng
ly a thuc khng sinh t vo a

21

petri sao cho khong cch gia 2 tm ca a thuc khng sinh khong 24mm v khong
cch gia tm a khng sinh vi mp a petri 10-15mm. Mi a mi trng t ti a 6
a khng sinh.

Tt c cc nng thuc c kim tra lp li 2-3 ln v 28 C. c kt qu sau 48 gi

i vi vi khun E. ictaluri, sau 24 gi i vi vi khun A. hydrophila v o ng knh vng
v trng. Vng c ch l ng knh vng v trng 24-48 gi sai khc gn nht cc ln
lp li.

3.3.2.2 Phng php xc nh nng c ch ti thiu (MIC) trn mi

trng thch (Lila Ruangpan, 2004):

Xc nh nng MIC ca natsol ln vi khun E. ictaluri v A. hydrophila bng phng php

thch theo Lila Ruangpan (2004), nhng c iu chnh do iu kin phng th nghim khng c
dng c hon chnh th nghim nh phng php. Phng php th nghim gm cc
bc sau:

Bc 1: Chun b dung dch khng sinh thc vt natsol (nh 3.3.1.2)

Bc 2: Chun b hai loi vi khun E. ictaluri v A. hydrophila. Vi khun cng c phc hi, nui
cy v nui tng sinh, ly tm v iu chnh mt bng my so mu quang ph (nh 3.3.1.1). Sau
pha long dung dch vi khun va
iu chnh theo phng php pha long 10 ln c mt khong 105-106

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

cfu/ml v cy kim tra thao tc chun b dung dch vi khun.

Bc 3: Chun b mi trng c thuc khng sinh

Mi trng MHA c tit trng 1210C trong 15 pht, c gi m 40-450C trong ni chng ct
(water bath). Cho thuc khng sinh vo mi trng MHA vo a theo t l 1:1, lc tht nh v u
( thuc ho tan u trong mi trng ng thi khng to bt). ra mi trng a petri
c tit trng k. Tt c thao tc chun b c thc hin trong iu kin v trng.

Bc 4: c kt qu: Xc nh nng c ch ti thiu (MIC) ca thuc khng sinh tho

dc natsol ln vi khun E. ictaluri v A. hydrophila

Mi a c chia lm hai phn ng vi hai chng vi khun ca mt loi vi khun. Nh 5 l vi

khun vo phn a ng vi chng vi khun . Dng que cy tri mng git vi khun v lt a
li, 28-300C trong 24 gi i vi vi khun A. hydrophila v 48 gi vi E. ictaluri. Kt qu
c tnh l s khun lc pht trin ca vi khun sng st. bt k nng no vi khun sng
st khng qu 20% th c xc nh l nng c ch ti thiu (MIC).

22

3.3.2.3 Phng php xc nh nng c ch ti thiu (MIC) bng phng

php pha long (Geert Huys, 2002):

Xc nh nng c ch ti thiu ca natsol bng mi trng NB lng ln vi khun E.

ictaluri v A. hydrophila, c thay i cc bc:

Bc 1: Chun b mi trng ha cht, ha cht

Mi trng NB, NA tit trng.

Nc mui sinh l tit trng, nc ct tit trng. ng nghim tit trng, u col tit trng.

Bc 2: Chun b dung dch thuc natsol v dung dch thuc khng sinh chloramphenicol
(nh 3.3.1.2)

Bc 3: Chun b dung dch vi khun

Chun b dung dch vi khun (nh 3.3.1.1)

Thao tc thc hin: Cho 2 ml vi khun iu chnh mt vo mi ng nghim c hm

lng thuc t 4ppm - 1024ppm v 23,4375ppm - 24000ppm.

Lu : Khng nn cho vi khun vo thuc lu hn 1 gi sau khi o mt vi khun. Lc u dung

dch vi khun trc khi cho vo ng nghim c
thuc. Tt c cc ng nghim c 28oC - 30oC trong 24 gi (hoc lu

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

hn n khi no thy vi khun trong ng i chng dng pht trin).

Mi chng vi khun sau khi iu chnh mt phi c cy ln mi trng NA kim tra

s thun chng v phi c trong cng iu kin vi cc ng MIC.

Mi th nghim cn c hai i chng:

i chng m (2ml NB + 2ml nc mui sinh l (0,85% NaCl))

i chng dng (2ml vi khun + 2ml nc mui sinh l (0,85%

NaCl))

Tt c cc ng nghim c 28 C, trong 18 - 24 gi.

Bc 4: c kt qu

Kim tra s thun chng ca chng vi khun xt nghim trn a TSA. Nu pht hin c s
tp nhim th khng c kt qu MIC.

S pht trin ca vi khun c xc nh bng cch so snh c ca mi ng MIC vi

ng i chng m.

Lot ng nghim ca chng vi khun no pht trin khng lin tc th loi b, khng ly kt
qu, thc hin li th nghim (v d: vi khun pht trin ng 5 & 7 nhng khng pht trin
ng 6).

23

Gi tr MIC c xc nh l hm lng thuc trong ng nghim c nng thuc nh nht khng

c vi khun pht trin. Trng hp vi khun pht trin tt c nng th gi tr MIC c xc nh
ng nghim c hm lng thuc m s pht trin ca vi khun gim khong 80% so vi

cc ng trong lot ng kim tra.

* Ch : Khi c kt qu ta thy mt s ng MIC c c gn bng nhau, kh phn bit bng mt

thng th ta tin hnh kim tra s pht trin ca vi khun trn a petri mi trng thch. Tu theo
tng c khc nhau m ta c th pha long theo nng gim dn sao cho c th d dng m
cc khun lc trn a thch. Dng pipet vi u col tit trng ht 0,5 ml dung dch vi khun cn xc
nh mt cho vo 4,5 ml nc mui sinh l (0,85% NaCl) (ng nghim 10-1), trn u mu. Tip
tc dng pipet ht 0,5 ml dung dch t ng nghim 1 cho vo 4,5 ml nc mui sinh l ng nghim 2
(ng 10-2), thc hin pha long nh vy cho n nng thch hp. Sau khi pha long xong nh

20l dung dch vi khun ln a NA bng pipet, mi nng lp li t nht 3 ln. a trong t
m ti iu kin nhit 280C - 300C, c kt qu sau 24 gi i vi A. hydrophila hoc 48 gi
i vi E. ictaluri. m s khun lc vi khun pht trin trn mi trng thch v p dng cng
thc sau:

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

Mt vi khun (cfu/ml) = trung b nh s khun lc * 50* pha lo ng

Chn nng no c mt vi khun thp nht (gim 80% so vi mt vi khun gc) l

nng c ch ti thiu (MIC).

24

CHNG 4

KT QU THO LUN

4.1 Kt qu nghin cu thm d khong nng c ch ca thuc khng sinh thc

vt natsol ln vi khun gy bnh trn c tra

Do thuc khng sinh natsol cha c nghin cu s dng trong iu tr bnh do vi khun E. ictaluri
v A. hydrophila gy bnh trn c tra nn cha bit nng thuc c th s dng trong iu tr
bnh cho c, cng nh cha bit khong nng c ch ca thuc trn cc vi khun ny. Theo T
Thanh Dung (1996), nng c ch ti thiu (MIC) ca thuc khng sinh c ngun gc t Eugenia
caryophyllus (EC) trn vi khun A. hydrophila gy bnh trn c tr l 1000ppm. Gi tr MIC ca
thuc chit sut t c roi nga (Verbe seriphioides) ln vi khun Bacillus subtilis l 31,2ppm (Luis
Scardapane & ctv, 2006). Nng thuc khng sinh tho dc s dng trong iu tr bnh c
tng i cao hn so vi s dng thuc khng sinh. V vy, th nghim thm d nng thuc
natsol trn vi khun E. ictaluri vi chng E.223 c chn tin hnh
cc nng cao: 10000; 8000; 6000; 5000; 4000; 3000; 2000; 1000; 500; 400;

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

300; 200; 100; 50; 45; 40; 35; 30; 25; 20; 15 v 10ppm.

Th nghim thm d thc hin theo phng php xc nh nng c ch ti thiu bng mi
trng lng. Kt qu so snh c ca cc ng MIC cho thy thuc nhy vi vi khun cc nng
cao (200-10000ppm). Vi khun cn pht trin cc nng thp (10-40ppm). c cc ng
MIC cc nng

45; 50; 100ppm c khc bit r vi cc ng c nng cao v thp. Kim tra s pht trin ca
vi khun trn a mi trng t cc ng MIC ny thy vi khun khng pht trin nng
100ppm nhng pht trin rt t nng 45 v 50ppm. Do khong nng 45-100ppm
c chn l khong nng c ch ca thuc natsol trn vi khun E. ictaluri (Bng 4.1). Do
cc nng thm d tng i gn nhau, t nng cao nht cho n nng nh nht, v
vy kt qu th nghim thm d ny c th s dng lm c s cho kt qu th nghim xc nh
nng c ch (MIC) ca thuc natsol trn vi khun E. ictaluri cng nh trn vi khun A.
hydrophila gy bnh trn c tra.

25

Bng 4.1: Kt qu nghin cu thm d nng c ch ti thiu ca thuc khng sinh tho
dc natsol trn vi khun E. ictaluri.

S ln lp li
1
2
S ln lp li
1
2

Nng (ppm)

Nng (ppm)

10000
200

8000
100
---

6000
50
----

5000
45
-----

4000
40

+
+

3000
35
+
+

2000
30
+
+

1000
25
++
++

500
20

++
++

400
15
+++
+++

300
10
+++
+++

Ch thch:

Du (-) tng trng cho trong ca ng MIC, khng c vi khun pht trin.

Du (-): ng MIC c trong rt trong, rt r rng

Du (--): ng MIC c trong khng r rng

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

Du (---): ng MIC c c kh phn bit bng mt thng

Du (+) tng trng cho c ca ng MIC, c vi khun tn ti v pht trin trong ng

MIC.

Du (+): ng MIC c c kh phn bit bng mt thng

Du (++): ng MIC c c hi c

Du (+++): ng MIC c c rt c, rt r rang

Xc nh nng MIC

Kt qu th nghim cho gi tr MIC ca thuc khng sinh Chloramphenicol trn vi khun

E.coli l 4l/ml. Theo Lila Ruangpan ctv (2004), thuc khng sinh Chloramphenicol c gi tr
MIC <=8l/ml trn hu ht cc tc nhn gy bnh cho ng vt mu lnh.

26

Bng 4.2: Nng c ch ti thiu ca thuc chloramphenicol trn E. coli

ng MIC
Nng tht

Th nghim lp li

(ppm)

1
512
-

2
256
-

3
128
-

4
64
-

5
32
-

6
16
-

7
8
-

--

8
4
--

--

9
2
++

++

ng i chng (-)
-

ng i chng (+)
+

4.2 Kt qu kim tra khng sinh ca thuc natsol ln vi khun E.

ictaluri v A. hydrophila.

Tin hnh th nghim kim tra kh nng dit khun ca thuc khng sinh tho

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

dc natsol trn vi khun E. ictaluri v A. hydrophila vi nhiu nng thuc khc nhau (50000;
24000; 12000; 6000; 3000; 1500; 750; 375; 187,5; 93,75; 46,875; 23,4375 v 11,71875ppm). Kt
qu cho thy thuc c hiu qu cc nng cao (50000; 24000; 12000; 6000; 3000; 1500ppm)
(Bng 4.2). Mt khc, theo NCCLS (2003), c a giy khng sinh cha 10g thuc nh mong
mun th dung dch thuc cn pha phi c nng l 400-500ppm, tc l nng thuc cn chun
b phi gp 40-50 ln so vi lng thuc c c trn

a khng sinh. Nh vy cc a giy khng sinh t ch gi c hm lng thuc khng sinh

natsol l 1250; 600; 300; 150; 75ppm. Tuy vy m kt qu lm khng sinh ca thuc khng sinh
tho dc natsol cng khng cho c kt lun g, do ng knh vng v trng o c ( 8mm)
nh hn 10mm.

iu ny chng t giy lm a khng sinh khng gi c lng thuc nh mong mun.

27

Bng 4.3: Kt qu khng sinh kim tra nhy ca thuc Natsol bng a

khng sinh t ch

Thuc

ng knh vng v trng trung bnh (mm)

khng

Nng thuc (ppm)

sinh

Vi khun
1.500
3.000
6.000
12.000
24.000
50.000

E. ictaluri
0
1,50.1
2,10.1
2,20.1
4,70.1

7,90.1

A. hydrophila
0
1,20,1
1,40.1
1,50.1
2,00.1
6,30.1

4.3 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun

E. ictaluri v A. hydrophila bng phng php pha long

4.3.1 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun A.

hydrophila

Qua kt qu th nghim so snh c gia cc ng MIC v tnh gi tr trung

bnh gia hai chng vi khun A. hydrophila, xc nh c gi tr MIC ca natsol ln vi khun A.

hydrophila l 375ppm. Trong , kt qu trn chng
Trung tmCAF2Hcr liurthn,Ht lCnvikhunThsng@stTich cliun5%hctingtpMICvsnghin6(nngcu

375ppm) ln th nghim th nht. Ln lp li th hai nng ny th khng cn vi khun

(Bng 4.3 v Bng 4.5). Nhng i vi chng vi khun CAF133 th 2 ln lp li u cn vi
khun vi t l vi khun sng st tnh trung bnh hai ln lp li l 11,65% (Bng 4.4 v Bng
4.5). T l vi khun sng st cng nng trn hai chng vi khun CAF2 v CAF13 khc
nhau khng ng k nhng kt qu ny cng cho thy c gi tr MIC ca thuc natsol ln hai
chng l khc nhau. Gi tr nng c ch ti thiu (MIC) trn cc chng vi khun khc nhau

ca cng loi vi khun cng khc nhau (T Thanh Dung & ctv, 1996). S khc nhau ny do
nhiu nguyn nhn. ng MIC s 7 ca hai chng vi khun, t l vi khun sng st rt ln
(245-315%), chng t nng thuc ng MIC s 7 khng c ch c vi khun m ngc
li vi khun vn c th pht trin v tng mt s.

Gi tr MIC trn vi khun A. hydrophila gy bnh trn c tr ca thuc tho dc Phyllanthus debitis
(PD) (dng bt cao) l 10000g/ml v Eugenia carryophyllus (EC) (dng tinh du) l 1000ppm (T
Thanh Dung & ctv, 1996). Theo Pithai & ctv (2005), chit sut t tri chui xanh (Musca saientum
Linn)

c ch vi khun A. hydrophila nng thp nht l 37,5mg/ml, cn chit sut t i hoa hng
(Hibiscus sabdariffa Linn) c ch vi khun A. hydrophila

28

nng thp nht l 4,2mg/ml. iu ny cho thy nng c ch ti thiu (MIC) ca thuc
natsol nhy vi vi khun A. hydrophila tng i cao.

Bng 4.4: Nng c ch ti thiu (MIC) ca thuc khng sinh Natsol trn vi khun A.
hydrophila chng CAF2

ng MIC

Nng tht

Th nghim lp li

(ppm)

12000

6000

3000

1500

750

375

---

--

187,5

93,75

46,875

++

++

10

23,4375

+++

+++

ng i
chng (-)

ng i chng (+)

Bng 4.5: Nng

c ch ti thiu
(MIC) ca Natsol trn
vi khun A.

hydrophila chng CAF133

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

ng MIC
Nng tht
Th nghim lp li

(ppm)
1
2

12000
-

6000
-

3000
-

1500
-

750
-

375
+
---

187,5
+
+

93,75
+
+

46,875
++
+

10

23,4375
+++
+++

ng i chng (-)
-

ng i chng (+)
+
+

29

Bng 4.6: c kt qu pha long ng MIC s 6 v 7 pha long 10 trn vi khun A.

hydrophila

Chng vi khun
CAF133
CAF2

ng MIC
ng MIC
ng MIC
ng MIC

s 6
s 7
s 6
s 7

S ln lp li

1
4
61
0
47

2
2
67
3
43

3
1
61
0
57
Trung bnh

2,33
63
1
49
Mt vi khun sng st
11,65x106

315x106
5x106
245x106

(cfu/ml)

(%) vi khun sng st

11,65
315
5
245

4.3.2 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi

khun E. ictaluri

Qua bng 4.6, nng c ch ti thiu (MIC) ca thuc khng sinh tho dc natsol ln chng vi
khun E.3B3 c xc nh l 93,75 ng MIC s 8, kt qu th hin r qua hai ln lp li u
khng cn vi khun sng st (Bng 4.8).

chng E.223, cng xc nh c nng c ch ti thiu (MIC) ca thuc

khng sinh natsol ln chng vi khun ny l 93,75ppm (Bng 4.8), ng MIC

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

s 8 khng cn vi khun sng st. Mt ln thc hin th nghim ng MIC s 9 (nng

46,875ppm) cng khng cn vi khun nhng ln th nghim lp li c ti 343,35% vi khun
sng st (Bng 4.7). T l vi khun sng st tnh gi tr trung bnh gia hai ln lp li ln
hn 20%, do nng ny khng c xc nh lm nng MIC.

So snh kt qu trn hai loi vi khun thy c nng c ch ti thiu (MIC) ca thuc
khng sinh natsol ln vi khun A. hydrophila cao hn ln vi khun E. ictaluri. Chng t vi khun
A. hydrophila c kh nng chng li tc dng ca thuc natsol mnh hn vi khun E. ictaluri
hay ni cch khc thuc natsol nhy trn vi khun E. ictaluri hn trn vi khun A. hydrophila.

30

Bng 4.7: Nng c ch ti thiu (MIC) ca Natsol trn vi khun E. ictaluri chng vi
khun E.3B3

ng MIC
Nng tht
Th nghim lp li

(ppm)
1
2

12000
-

6000
-

3000
-

1500
-

750
-

375
-

187,5
-

93,75
-

46,875
-----

10

23,4375
+
+

11

11,71875
++
++

ng i chng (-)
-

ng i chng (+)
+
+

Bng 4.8: Nng c ch ti thiu (MIC) ca Natsol trn vi khun E. ictaluri chng vi
khun E.223

ng MIC
Nng tht
Th nghim lp li

Trung tm Hc liu H
Cn Th @
Ti liu hc tp v nghin
cu

(ppm)
1
2

1
12000
-

2
6000
-

3
3000
-

4
1500
-

5
750
-

6
375
-

7
187,5
-

8
93,75
-

9
46,875
----

10

23,4375
+
+

11
11,71875
++
++

ng i chng (-)
-

ng i chng (+)
+
+

31

Bng 4.9: c kt qu pha long ng MIC s 8 v 9 pha long 10 trn vi khun E.

ictaluri

Chng vi khun
E.223
E.3B3

ng MIC
ng MIC
ng MIC
ng MIC

s 8
s 9
s 8
s 9

S ln lp

1
0
70
0
63

2
0
63
0
57

li

3
0
73
0
57

Trung bnh

0
68,67
0
59

Mt vi khun sng
0
343,35x10

0
295x10

st (cfu/ml)

(%)vi khun sng st

0
343,35

0
295

4.2.3 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun E. ictaluri

v A. hydrophila bng phng php thch.

Kt qu th nghim bng 4.9 v bng 4.10 xc nh c nng MIC ca thuc natsol ln vi

khun A. hydrophila trn mi trng thch l 750ppm. nng ny th khng c vi khun pht
trin trn mt thch (Hnh 4.1B). Nng MIC ca thuc natsol ln vi khun A. hydrophila trn mi
trng thch cao hn gi tr MIC ca thuc trn vi khun ny trong mi trng lng (375ppm).
Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

T i nng 375ppm, vi khun cn pht trin rt nhiu trn a mi trng thch (Hnh
4.1A). Gi tr MIC ca natsol trn vi khun E. ictaluri trn mi trng thch (375ppm) cng
cao hn trong mi trng lng (187,5ppm). S chnh lch kt qu MIC ca thuc trn cng
mt loi vi khun hai phng php l do phng php thch khng dng c thc
hin th nghim. Theo Lila & Eleonor (2004), xc nh nng c ch ti thiu (MIC) ca
thuc khng sinh cn s dng b dng c xc nh MIC a knh (MIC multiple inoculating
apparatus set).

Bng 4.10: Kt qu MIC trn mi trng thch trn vi khun A. hydrophila vi cc a mi

trng 5, 6, 7 ng vi cc nng 1.500; 750; 375ppm

Chng vi khun
S ln lp li
Trung bnh

A. hydrophila

1
2
3

CAF2

a mi trng s 5
0
0
0
0

a mi trng s 6
56
48
75
59,67

a mi trng s 7
175
154
189
172,67
CAF133

a mi trng s 5
0
0
0
0

a mi trng s 6
104
97
71
90,67

a mi trng s 7
215
102
133
150

32

Bng 4.11: Kt qu MIC trn mi trng thch trn vi khun E. ictaluri vi cc a mi

trng 6, 7, 8 ng vi cc nng 750; 375; 187,5ppm

Chng vi khun

Lp li

Trung bnh

E. ictaluri

1
2
3

E.223

a mi trng s 6
0

0
0
0

a mi trng s 7
76
58
105
79,67

a mi trng s 8
155
149
178
160,67

E.3B3

a mi trng s 6
0
0
0
0

a mi trng s 7
51
47
41
46,33

a mi trng s 8
210
132
153
165

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

33

Trung tm

cu

Hnh 4.1: Kt qu MIC trn mi trng thch ca vi khun A. hydrophila (a i chng (tri), a
mi trng cha thuc nng 750ppm (phi))

34

CHNG 5

KT LUN V XUT

5.1 Kt lun

Th nghim t c mt s kt qu sau:

Th nghim thm d xc nh c khong nng c ch ca natsol ln vi khun E.

ictaluri l 50-100ppm.

Xc nh c nng c ch ti thiu (MIC) ca thuc khng sinh thc vt Natsol trn vi

khun E. ictaluri l 93,75ppm bng phng php pha long v 187,5ppm bng phng
php thch.

Xc nh c nng c ch ti thiu (MIC) ca thuc khng sinh thc vt Natsol trn vi

khun A. hydrophila l 375ppm bng phng php pha long v 750ppm bng phng
php thch.

Phng php lp khng sinh ca thuc khng sinh natsol khng hiu qu trn vi khun
E. ictaluri v A. hydrophila.

5.2 xut

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

Trang b thm dng c, thit b h tr cho phng php xc nh nng c ch ti thiu
(MIC) bng mi trng thch.

35

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Pai, R., Karunasagar, I., Shetty, H. P. C and Karunasagar, I., 1995. The Effect
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Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

40

Ph lc A: Pha dung dch nhum gram vi khun

1. Dung dch I (ammonium oxalate, crystal violet)

Crystal violet (colour index No.42555)

2g
Ethanol (95%)
20ml
Ammonium oxalate
0,8g
Nc ct
80ml

Ho tan crystal violet vo ethanol

Ho tan ammonium oxalate vo nc ct

Trn hai dung dch ny li, sau 24 gi lc qua giy lc loi b

phn cn

Dung dch II (iodine)

Iodine
1g
Potassium iodine
2g

Nc ct
300ml

Trung tm Hc -liuHoHtanCnpotassiumThiodine@ trongTi 20mlliu nchccttp v nghin cu

Cho tip iodine vo, gi qua m

Sau cho thm phn nc cn li vo dung dch

Dung dch III (dung dch ra mu)

Hn hp ethanol/acetone theo t l 95%:5%

4. Dung dch IV (Safranin)

Safranin (colour index No.50240)

0,25g
Ethanol (95%)
10ml
Nc ct
90ml

Ho tan safranin trong ethanol v nc ct.

41

Ph lc B: Phng php nhum Gram vi khun

Nh mt git nc ct ln lame, dng que cy nht mt t vi khun tri u ln git nc

ct. kh nhit phng sau h lt lame trn ngn la n cn c nh vi
khun trn lame.

Cc bc nhum Gram:

Nh dung dch crystal violet (dung dch I) ln lame. 1 pht Ra bng nc ct cho ht mu tm
trn lame (khong 2 giy). Nh dung dch Iodine (dung dch II). khong 1 pht.

Lt nghing lame cho ht dung dch Iodine trn lame.

Dung dch cn: aceton (dung dich III) ty mu bng cch lt nghing lame nh t t
dung dch III cho n khi git nc cui cng tren lame khng cn mu tm. Ra v vy cho
ro nc.

Nh dung dch safranin (dung dch IV) ln lame. khong 2 pht. Ra v vy cho ro
nc. kh nhit phng .Quan st vt knh 100X vi du soi knh.

Trung tmcHckt qu:liu H Cn Th @ Ti liu hc tp v nghin cu

Vi khun Gram m (G ) c mu hng .

Vi khun Gram dng (G ) c mu tm xanh.

42

Ph lc C: Pha mi trng

NA (nutrient agar)
20g
Nc ct
1000ml
TSA (triptic soy agar)
40g
Nc ct
1000ml
MHA (muller hinton agar)
38g
Nc ct
1000ml

Ho tan mi trng cn s dng vo 1000ml nc ct, sau tit trng nhit

1210C trong 15 pht. nhit h xung khong 50 0C, tin hnh ra a
Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cu

petri vi dy kho ng 4mm.

43

Ph lc D: Pha nc mui sinh l v pha mi trng NB

1. Pha nc mui sinh l (0.85% - NaCl)

NaCl
0.8g
Nc ct
100ml
Ho tan 0.8g NaCl vo 100ml nc ct.

2. Chun b mi trng NB lng

NB
8g
Nc ct
500ml

- Ho tan NB trong nc ct, vn cht np, ri tit trng 121 C trong 15 pht.

Trung tm Hc liu

Vn

kn

Cn

np,gi

Th

nhi

Ti

phng

liu hc tp v nghin cu

44

Ph lc E: Pha thuc khng sinh chloramphenicol v natsol

Cn 0.1024g ca thuc khng sinh (chloramphenicol) cho vo chai tit trng, sau cho
vo100ml dung dch ethanol. Lc u cho thuc tan hon ton, nu thuc khng sinh khng tan
th cn s dng my nh sng m, khi nng ca thuc khng sinh l 1024ppm (nng
gc).

ong 5ml thuc khng sinh natsol cho vo 100ml nc ct tit trng, lc u cho thuc
tan hon ton, lc ny nng thuc khng sinh l 50000ppm theo hng dn ca nh sn
xut.

Cc nng thuc khng sinh chloramphenicol v natsol c pha long p dng theo cng thc
C1V1 = C2V2. Chng hn chun b 10ml dung dch thuc khng sinh chloramphenicol c nng
256ppm (dung dch gc th hai) t

Trung tm

nng

Hc

c1024ppm

liu

p dng cng thc: C1V1 = C2V2.

Vy: V1 = C2V2/C1

HCn Th @ Ti liu hc tp v nghin cu

256 x 10/1024

2,5ml thuc khng sinh gc

Do , t 10ml thuc khng sinh nng 256ppm cn 2,5ml thuc khng sinh (nng
gc) v 7,5ml nc mui sinh l.

Cc nng khc p dng phng php pha long hai ln nng gim i phn na t
cc dung dch gc.

45