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PHAN LAP VA DAC TINH
H 6 AVI
K H U A NLACTIC
DOI KHANG v o l Vmho SPP. GAY BENH TlT AO NUOI TOM Of IHUA THIEN HUE
NGO THI TUdNG CHAU, NGO HOANG SONG UYEN Trudng Dgi hpc Khoa hpc, Dgi hpc Hui Trii dai tii cira sdng 6 Lau din cira biln Tu Hien, hf dim phaTam Giang - Ciu Hai vdi difn tich 22.000ha, Idn nhit Ddng Nam A, da tao cho Thira Thien Hue mgt tilm nang to Idn vl nghi
•nudi tdm. Tuy nhien, frong nhirng nam gin day, nghe pudi tdm d Thira Thien Hue dang phii dii mat vdi dich bfnh tdm mi chu ylu la bfnh do vi khuan, die bigt Ii Vibrio spp., gay nen. Tmdc tinh hinh dd, mgt sd bifn phap d i dugc de nghi v i i p dyng nham kiem soat cic benh do Vibrio spp. giy ra tren tdm nhu bd sung khing sinh, hda chat va sir dyng che pham sinh hgc. Tuy nhien cic bien phip niy khdng nhirng khdng giii quyet dugc van de cdt yeu ma cdn bdc Ig nhieu han chi. Viec sir dyng hoa chit, khing sinh khdng diing quy cich, khdng diing lilu lugng di gay nen tic hai Idn nhu la tao cac ddng vi khuan khing thudc, lam suy thoii mdi frudng, anh hudng khdng tdt len sire khde con ngudi. Tuong ty, vifc sir dung cic che pham sinh hgc chii yeu la nhip ngoai, chua dugc cdng bd ve xuit xii ngudn gdc, cd the Ii nguy co lan tmyen cic vi sinh vat (VSV) dgc hai, VSV bien ddi gen lim mat an toin sinh hgc. Vi vay, vifc nghien ciru tao ra cac chi phim VSV ban dia phii hgp vdi dilu kien khi hau dia phuong, an toan trong khi sii dung vi gii thanh re nhim phuc vy cho myc dich ban che djch bfnh trong nudi tdm that sy Ii mgt vin de cap thiet. Trong dd die bift quan tam den Ii nhdm vi khuan lactic vdi kha nang ddi khing cao vdi Vibrio spp. gay bfnh tren tdm.
L PHLTOIVG PHAP NGHIEN CUtJ
Ddi tugng nghien ciiu la cac chiing vi khuan lactic cd kha ning doi khang vdi Vibrio spp.
gay bfnh phan lip tir mau tdm vi bun ao nudi tdm va d Thira Thien Hue. Phan lap Vibrio spp.
gay bfnh: tdm bfnh dugc tich rieng mgt, nghien nit, can lg, nghien trong cdi chiy siir va hda trong Iml nude cit vd trimg. Nhd dich nay vao cac hgp petri chira mdi tmdng TCBS (Thiosulphate Citrate Bile salt Sucrose agar) va dimg que gat din deu tren mit thach. Nudi ciy d 37°C trong 24 gid. Cic khuan lac rieng re dugc cay truyen sang mdi trudng thach nghieng de giir gidng. So bg djnh danh cic loii Vibrio spp. dugc phan lap. Phan lap cac chung vi khuan lactic: dimg giu chuyen dyng thu bim sft ldp mat diy, mau tdng hgp dugc thu d 5 vj tri ngau nhien rdi frgn deu. Phan lap vi khuan lactic theo phuong phip Koch tren mdi trudng MRS (DeMan, Rogosa, Sharpe Agar) d 30°C trong 24 gid. Cac khuin lac rieng re dugc tilp tuc thuan khiet tren mdi tmdng thach dTa, sau do ciy truyen sang mdi trudng thach nghieng de giir gidng.
Xic nhan vi khuan lactic thdng qua die tinh sinh trudng tot v i chilm im thi fren mdi frudng MRS, Gram (+), catalase (-) v i oxydase (-). Xic dinh khi ning doi khing vdi Vibrio spp. cua vi khuan lactic bing phuong phip dia thach hai Idp: ciy vi khuin lactic len tren cic dTa petri chiia mdi tmdng MRS vi nudi d 30°C trong 24 gid. Dd mdi trudng TCBS d 50 - 55°C len tren dTa petri da cd sy hien dien cua khuan lac vi khuan lactic d l tao ldp mdi trudng thur hai. Khi be mat mdi trudng TCBS da khd, nhd lOO^I dich huyIn phii cua Vibrio spp. va dan diu tren be mit thach, nudi d 32 - 37°C trong 24 gid. Quan sit sy hinh thinh vdng khang khuin. Dinh gia kha ning phan giai chit hiiu co cua vi khuin lactic bing phuang phap khich tan tren thach.
H. KET QUA NGHIEN CLTU
1. Phan lap va so bg djnh danh Vibrio spp.
Tiln hinh phan lap Vibrio spp. tir miu tdm bfnh, kit qui thu dugc 2 loai Vibrio sp. (ki hifu Ii VI vi V2). Qua quan sat hmh thii, budc diu xac dinh VI thugc loai Vibrio harvey va V2 thugc loii Vibrio alginolyticus (bang 1).
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Phan lap va so bg djnh danh Vibrio spp. tir tdm bfnh phim d TT Hue Bdng I
Dac diem hinh thai Hinh dang khuan lac Mau sac khuan lac Be mat khuan lac Ria khuan lac
Kich thudc khuan lac (mm) Hinh dang te bao
Nhupm Gram
VI Tron to, khong deu
Xanh Tron bong
Nhan 2,9-3,2 Hinh que, cong nhe
-
V2 Tron nho, deu
Vang Tron bong
Nhan 1,2- 1,4 Hinh que, cong nhe
- 2. Phan lap cac chung vi khuan lactic
Tiln hinh phan lap cac chung vi khuin lactic tir bim ao nudi tdm d Thira Thien Hul, kit qui thu dugc 31 chiing. Trong dd, 6 chiing dugc phan lap tir miu biin diy cua ao nudi tdm d Phu Lgc, Thira Thien-Hue (ki hieu tir Bl den B6), 12 chiing tir mau biin diy ciia ao nudi tdm d Phu My, Phu Vang, Thira Thien-Hue (ki hifu tir B7 den B18), 13 chiing tir bim diy ciia ao nudi tdm d Ling Cd, Thira Thien-Hue (ki hifu tir B19 den B31) (bang 2). Cic chimg vi khuin nay deu cd khi ning sinh tmdng tot tren mdi tmcmg MRS, Gram (+), boat tinh catalase (-) va oxydase (-) dip img tieu chuin phin loai vi khuin lactic theo TCVN (0013:2006).
Bdng 2 So lugng cac chijng vi khuan lactic dugc phan lap tir ao nudi tdm d Thira Thien-Hue STT
1 2 3
Dia diem thu mau Phii Lpc, Thira Thien-Hue Phu My, Phil Vang, Thira Thien-Hue
Lang Co, Thira Thien-Hue
So chung 6 12 13
Ky hifu chiing B1-B6
B7-B18 B19-B31 3. Xac djnh hoat tinh doi khang cua vi khuan lactic vai Vibrio sp.
jpn-'' ' ^-
•. ^.-i.iirtBBftWUlto*
Hinh 1: Kha nang doi Hinh 2: Kha nang doi khang voi VI ciia chung BIO (trai) khing vdi VI cua chung B9 vi chung B13 (phai)
Tiln hanh xic dinh boat tinh doi khang cua vi khuin lactic vdi Vibrio sp. bing phuang phip dia thach 2 ldp, kit qua tuyln chgn dugc 3 chimg B9, BIO vi B13 cd khi ning doi khing vdi Vibrio sp. (bang 3 vi hinh 1, 2). Trong dd, chimg B9 thi hifn kha nang ddi khing Ii cao nhit.
Bdng 3 Kich thudc vdng khang khuan cua cac chiing vi khuan lactic dugc tuyen chgn
Chung B9 BIO B13
Kich thuffc vong khang khuan (mm) 13,89± 1,50
10,23 ±0,97 13,56 + 0,75
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4. Mgt so dac dilm ciia cac chung vi khuin lactic dugc tuyen chgn + Dgc diim vi hinh thdi cda vi khudn lactic
Dac dilm ve hinh thai va sinh hoa cua cac chiing vi khuan lactic
Bdng 4
Dac diem hinh thai Hinh dang khuan lac Mau sac khuan lac Ria khuan lac Be mat
Hinh dang te bao Kich thudc khuan lac
Chiing B9 Trdn to, khong deu
Trang due Ggn sdng Xii xi Hinh que 3,2-4,7mm
Chung BIO Tron, deu Tring frong
Nhan Tran, lang Hinh oval 2,5-3,4mm
Chung B13 Tron, deu Trang sira
Nhan Tran, bdng
Hinh que l,9-2,4mm + Dgc diim sinh hoa ciia chiing 39: Tien hanh xic dinh mgt sd die diem sinh hda ciia chimg B9 Ii chimg cd kha nang ddi khang cao nhat vdi Vibrio spp. frong sd cac chiing dugc myln chgn. Kit qua dugc trinh biy d bing 5. Dya tren mgt sd die diem nghien ciiu, budc diu chiing tdi xac dinh chimg B9 thudc giong Lactobacillus.
Bdng 5 Mgt so dac diem sinh hda ciia chung B9
STT 1 2 3 4 5 6 7 8 9
Phan irng sinh hoa Glucose (Kha ning len men dudng) Lactose (Kha nang len men dudng)
Clank Clubs: Dd methyl (Type len men acid formic) Phan ling PV (Type len men butylen glycol) Urea (Phan giii urea)
Peptone (Kha ning sinh idol) Citrate (Kha nang sir dyng citrate) Thach mem (Khi ning di dgng) Oxydase (Khi nang sir dyng oxidase)
Ket qua +
- - - - - + + + + ThO: hogt tinh phan gidi chat hiru ca ciia vi khuan lactic
Hmh 3: Kha nang phan giai tinh bft (trai) vi gelatine (phai) ciia chiing BIO
Hinh 4: Kha nang phan giai CMC ciia chung B9
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Tiln hanh thii hoat tinh phan giai chat huu co ciia vi khuin lactic theo phuang phap khulch tin tren thach, ket qui dugc trinh bay d cic hinh 3 va 4.
HL KET LUAN
Da phan lap dugc 31 chung vi khuin lactic tir cac miu biin ao nudi tdm d Thira Thien Hul, trong dd da tuyen chgn dugc 3 chung B9, BIO vi B13 cd hoat tinh doi khang cao vdi 2 loai Vibrio sp. gay benh (ki hieu la VI vi V2) dugc phan lap tir mau tdm bfnh d Thira Thien Hul.
Budc diu xic dinh chimg vi khuan B9 cd khi nang ddi khing cao nhit thugc gidng Lactobacillus, mau VI thudc loii Vibrio harveyi va miu V2 thudc loai Vibrio alginolyticus.
Ben canh dd, di dugc xic dinh khi ning phan giii chit him co ciia mgt sd chiing vi khuin lactic dugc tuyen chgn. Qua dd, the hien tiem ning urng dyng ciia chimg trong sin xuit chi phim sinh hgc han che dich bfnh tren tdm.
TAI L i f u THAM KHAO
1. Tran Linh Thudc, 2007: Phuang phap phan tich vi sinh vat trong nude, thyc phim va mT pham. NXB. Giio due.
2. Trudng Dai hgc Y Dugc Hue, Bg mdn Vi sinh, 2007: Tii lieu thyc tip Vi sinh vat Y hgc.
3. Felipe do Nascimento Vleira, Fabiola Santiago Pedrotti, Celso Carlos Buglione Neto, Jose Luiz Pedreira Mourino, Elpidio Beltrame, Mauricio Later<;a Martins, Cristina Ramirez and Luis Alejandro Vinatea Arana, 2007: Brazilian Joumal of Oceanography 55(4): 251-255.
4. You J. L., L. X. Cao, G. F. Liu, S. N. Zhou, H. M. Tan, Y. C. Lin, 2005: Worid Journal of Microbiology & Biotechnology 21: 679- 682.
ISOLATION AND C H A R A C T E R I Z A T I O N O F L A C T I C ACID BACTERIA ANTAGONISTIC T O P A T H O G E N I C VIBRIO SPP. F R O M SEDIMENTS O F
S H R I M P P O N D S IN T H U A THIEN HUE P R O V I N C E
NGO THI TUONG CHAU, NGO HOANG SONG UYEN SUMMARY
Thirty one (31) strains of lactic acid bacteria were isolated from sediments of shrimp ponds in Thua Thien Hue province. Subsequently, three strains (B9, BIO and B13) screened to be antagonistic to pathogenic Vibrio spp. (VI and V2) were isolated from diseased shrimps of shrimp ponds in Thua Thien Hue. The preliminary results revealed that B9 strafri belongs to Lactobacillus, and VI and V2 belong to different species. Vibrio harveyi and Vibrio alginolyticus, respectively. The ability to degrade organic substances of some lactic acid bacteria was also confirmed. These lactic acid bacteria could have potential application in biocontrol of shrimp diseases in Thua Thien Hue.
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