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 LC1

DANH SCH HNH3

DANH SCH BNG4

LI CM T5

TM TT6

CHNG 1: GII THIU7

CHNG 2: TNG QUAN TI LIU92.1 Bnh do vi khun E. ictaluri gy ra92.1.1 Mt s c im ca vi khun E. ictaluri92.1.2 Phn b ca vi khun E. ictaluri92.2 Bnh do vi khun Aeromonas hydrophila102.2.1 Mt s c im v vi khun Aeromonas hydrophila102.2.2 Hnh dng khun lc102.2.3 Hnh dng t bo102.2.4 Phn b ca Aeromonas hydrophila112.2.5 Bnh xut huyt do vi khun A. hydrophila112.3 Cy tho dc122.3.1 S lc v cy tho dc122.3.2 Mt vi cy thuc tho dc v cng dng phng tr bnh thy sn122.3.3 Mt s kt qu khoa hc s dng cy tho dc phng v tr bnh thy sn13

2.4 Natsol142.5 Chloramphenicol14Trung tm Hc2.6MtliusktHqu xcCnnh Thnng@c chTitiliuthiu (MIC)hc trtpnthuvc khngnghinsinh cutho dc14

CHNG 3: PHNG PHP NGHIN CU16

3.1 Thi gian v a im nghin cu163.1.1 Thi gian nghin cu163.1.2 a im nghin cu163.2 Vt liu nghin cu163.2.1 Ho cht v mi ttrng163.2.2 Thuc khng sinh163.2.3 Dng c th nghim163.2.4 Vi khun th nghim173.3 Phng php nghin cu173.3.1 Chun b nguyn liu nghin cu173.3.1.1 Chun b vi khun173.3.1.2 Chun b dung dch thuc khng sinh183.3.2 Thc hin nghin cu203.3.2.1 Nghin cu thm d203.3.2.1 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):23CHNG 4: KT QU THO LUN25

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 tra254.2 Xc nh nng MIC26

1

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

hydrophila274.3 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun E.ictaluri v A. hydrophila bng phng php pha long284.3.1 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun A.hydrophila284.3.2 Xc nh nng c ch ti thiu (MIC) ca thuc Natsol ln vi khun E.ictaluri304.2.3 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 XUT35

5.1 Kt lun355.2 xut35

TI LIU THAM KHO36

Ph lc A: Pha dung dch nhum gram vi khun41

Ph lc B: Phng php nhum Gram vi khun42

Ph lc C: Pha mi trng43

Ph lc D: Pha nc mui sinh l v pha mi trng NB44

Ph lc E: Pha thuc khng sinh chloramphenicol v natsol45

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

2

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

3

DANH SCH BNG

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

php pha long hai ln18

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

theo phng php pha long hai ln19

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. coli27

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

khng sinh t ch28

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

khun A. hydrophila chng CAF2 ..29Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cuBng 4.5: Nng c ch ti thiu (MIC) ca natsol trn vi khun A.

hydrophila chng CAF13329

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

khun A. hydrophila30

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

chng vi khun E.3B331

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

chng vi khun E.22331

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

khun E. ictaluri32

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; 375ppm32

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; 187,5ppm33

4

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

5

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

6

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)

7

(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

8

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 liuv 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 tiMississippi, 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

9

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 liuv 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 vikhun 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 nmn 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 ChloramphenicolTrung 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@dngTicaliuethylhcacetatetpvvn-butanolnghinchitcu 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

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

3.1.2 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 cuNc 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.

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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 -800C 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 rngTrung 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 280C 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.

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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

ngNng cn phaTh tchTh tch

thuc khng sinh natsolnc ct

nghim(ppm)

(ml)(ml)

1240006 (50000ppm, dung dch gc 1)6

2120006 (24000ppm)6

360006 (12000ppm)6

430006 (6000ppm)6

Trung tmcu

515006 (3000ppm, dung dch gc 2)6

Hc liu H Cn Th @ Ti liu hc tp vnghin

67506 (1500ppm)6

73756 (750ppm)6

8187,56 (375ppm)6

993,756 (187,5ppm)6

1046,8756 (93,75ppm)6

1123,43756 (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)

ngNng tht thuc natsolTh tchTh tch

thuc natsolvi khun

MIC(ppm)

(ml)(ml)

1120002 (24000ppm, ng 1)2

260002 (12000ppm, ng 2)2

330002 (6000ppm, ng 3)2

415002 (3000ppm, ng 4)2

57502 (1500ppm, ng 5)2

63752 (750ppm, ng 6)2

7187,52 (375ppm, ng 7)2

893,752 (187,5ppm, ng 8)2

946,8752 (93,75ppm, ng 9)2

1023,43752 (46,875ppm, ng 10)2

1111,728752 (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 tchTh tch

ngNng cn phathuc khng sinh

nc mui sinh l

nghim(ppm)chloramphenicol

(ml)(ml)

11024

25125 (1024ppm,5

dung dch gc 1)

32565 (512ppm)5

41285 (256ppm,5

dung dch gc 2)

5645 (128ppm)5

6325 (64ppm)5

7165 (32ppm)5

885 (16ppm)5

945 (8ppm)5

19

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

ngTh tch tht thuc khng sinhTh tchTh tch

thuc khng sinhvi khun

MICchloramphenicol (ppm)

(ml)(ml)

15122 (1024ppm, ng 1)2

22562 (512ppm, ng 2)2

31282 (256ppm, ng 3)2

4642 (128ppm, ng 4)2

5322 (64ppm, ng 5)2

6162 (32ppm, ng 6)2

782 (16ppm, ng 7)2

842 (8ppm, ng 8)2

922 (4ppm, ng 9)2

Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cuChloramphenicol (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 10-10000ppm.Tt c cc ng nghim v a cy kim tra vi iu kin nhit 28-30oC. 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 cuChun 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 (6mm). Dng giy nhm gi nhng a giy v mang i tit trng khong 2 gi 1800C.

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 280C. 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 vaiu chnh theo phng php pha long 10 ln c mt khong 105-106Trung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cucfu/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 luTrung tm Hc liu H Cn Th @ Ti liu hc tp v nghin cuhn 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 280C, 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 cuMt 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 cu300; 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.

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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 li12S ln lp li12

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

4.2 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