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CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERY

NGUYEN NHUT THANH

CHARACTERIZATION OF PATHOGENIC BACTERIA FROM SILVER POMFRET (Pampus argenteus Euphrasen, 1788) CULTURED IN NHATRANG BAY, VIETNAM

GRADUATION THESIS ADVANCED AQUACULTURE

Supervisor’s name Dr. Dang Thi Hoang Oanh Ms. Tran Thi My Duyen

2013

...................................................................................................................................5 2.1 Research objective ...3............................6 2.............................................1 1..................................................................................9 3....................................1 Silver pomfret........................................................................2 Bacterial isolation ..............................4 2......................................... 11 COST ESTIMATION .............. 11 REFERENCE ...................................................................................4.....................................................7 CHAPTER 3 MATERIALS AND METHODS ......................................................................................................................................................3 2.................................................................9 3........................7 2....................................................................................1 Time and sites of study ...............................2 Enrofloxacin ......................................................6 2......3............................................3.............................................................3 Some common bacterial diseases in brackish and marine fishes .................2 CHAPTER 2: LITERATURE REVIEW ....3................3...................4 Florfenicol ................................................................................................................................................................................................................. 10 TIME TABLE ...........................................3 Introduction ....................................................4 Antimicrobial susceptibility testing ........................................................................................................................1 Vibriosis ...........................................................................................................................................................................3 Ciprofloxacin ................................................4.......3 2.........9 3..............................................................................................1 Fish sampling ...................................4...................3 Bacterial identification ..........................................Table content CHAPTER 1 INTRODUCTION ..........................................................3 Streptococcosis .........2 Bacterial hemorrhagic septicaemia ........................3.............................................................................................................4 2............................................................1 Oxytetracycline..............................................2 Research activities ... 14 ..............................................................................4 2............4...........4 Antibiotics in aquaculture ................ 10 3...............3 Methods .................................3 2............................................................2 1..............2 Cause of diseases ............9 3..........................2 Materials ........................3...............................................................1 1.......9 3............6 2................................................................... 12 APPENDIX ...............9 3......................................................................................................

but residues in flesh product are not favorable to consumers. this thesis “Characterization of pathogenic bacteria from silver pomfret (Stromateoides argenteus Euphrasen. 2010). cobia (Rachycentron Canadum). In recent years. especially farmed stripped catfish (Pangasianodon hypophthalmus) and penaeid shrimp (tiger shrimp and white leg shrimp). People prefer using higher dosages of antibiotics when the previous dosages do not have effect. Vietnam has great conditions to develop aquaculture (both fresh and marine aquaculture). Vietnam was ranked the world’s third of aquaculture (FAO 2010). In fact. This is the new specie. they hope to heal illness completely. resistance characteristic could readily and quickly spread out in bacterial populations (Kumarasamy et al. people mainly catch silver pomfret in the wild. Thus. especially this can lead to disease spread out easily on the sea to wild fish. The uncontrolled of using antibiotics can make the presence of antibiotic residues in fish meat and fish products and this also lead to a disease which cause by resistance bacteria and hard to treat. Vietnam” is carried out to provide more information about disease on this species. aquaculture in Vietnam is developing year by year. 1788) cultured in Nhatrang bay. there is a few research about silver pomfret are conducted on over the world. people started to culture silver pomfret in cages with intensive model. Antibiotics are known as the useful treatment for the bacterial disease. In this system. In addition. silver pomfret (Pampus argenteus)… Silver pomfret is one of a new cultured species is Vietnam as it has high nutrition contain and quality of flesh meat. 2008). in the past. Bacterial diseases have been reported as one of the worst problems causing up to 100% mortality of cultured fishes (Bui Quang Te. This can make the fish get stress and diseases. marine aquaculture is contributed an important part for aquaculture with high value species such as sea bass. fish are stocked at high density. but can make bad result if not use at correct ways or dosage. Bacterial resistance often happens and cause treatment failure. Besides.CHAPTER 1 INTRODUCTION 1. usually over feeding.1 Introduction With more than 3200 kilometers of coastline and large area of water surface. grouper (Serranidae). Silver pomfret is easy to find in Northern gulf and central southern of Vietnam.. 1 . or the others cages.

Antibiotics susceptibility testing and determination of concentration of those antibiotics to isolated bacterial strains. Classification to species level of bacteria that was isolated from silver pomfret.2 Research objective The aim of this research is to investigate the bacterial pathogen which are isolated from silver pomfret (Pampus argenteus) in Nhatrang bay.3 Research activities This thesis will focus on the following contents: 1. minimal inhibitory 2 . 2. then find down the most effective antibiotic to treat the disease.1. 1.

high flesh meat quality. Inanimate etiological agents are factors without life of their own and can originate within a host (endogenous) or outside of a host (exogenous). Both fish species and strain of fish are important because all are not equally susceptible to a specific disease organism. 1999). which include viruses. with viscosity about 0. protozoa. electrical shock. 2. which they live in. bacteria. and dietary deficiencies. inanimate agents include trauma. temperature shock.1 Silver pomfret Silver pomfret (Pampuss argenteus) is a valuable food fish with a wide geographical distribution from East China Sea to Southest Asia. In the past. Silver pomfret is found in coastal water from 5 to 100 meters depth. it has high economic value base on its nutrition value. Endogenous. Etiological agents can be classified as either inanimate or animate. because they exposure directly with the water.6m/s. heredity. 3 . it has maximum size 80 centimeters but usually see at 30 centimeters (FAO). Feed quality. and fish species. Cages are placed in clean area.2 Cause of diseases Aquatic organisms are sensitive with the diseases. These etiological agents may serve as sublethal stressors that predispose fish to infectious disease. helminthes and copepods (Plumb.CHAPTER 2: LITERATURE REVIEW 2. but in recent years. it is stared being culture in cages.20. Exogenous. inanimate factors are those associated with genetic and/or metabolic disorders of the host. This is new species cultured in Vietnam. the India Ocean. gender. it was only caught on the sea. There is not many researches done about this species have been reported in Vietnam. and intrinsic factors include age. Disease can be caused by an etiological (specific cause) or a nonetiological (contributing cause) agent. Nonetiological causes of disease are characterized as extrinsic (from outside the body) or intrinsic (within the body). fungi. chemical toxicity. Arabian Gulf and the North Sea (Davis and Wheeler 1985). Animate etiologies are living communicable infectious agents. Extrinsic factors are usually associated with environmental conditions or dietary problems.

3. In some cases. 2010). reddisease. They are usually chronic. also referred to as motile aeromonad infection. 2007). and water temperature extremes can be classified as either etiological or nonetiological extrinsic factors and can contribute to infectious disease (Plumb. 2010). 1999). Vibrio salmonicida. 1999 cited by Toranzo. as with other bacterial septicaemias. turbot. and Vibrio harveyi (FAO. is among the most prevalent fish diseases caused by bacteria of genus Vibrio (Woo and Bruno. ponds…) and are considered as the primary pathogen or the opportunistic pathogen. 2. boil-disease. rainbow trout. the species causing the most economically serious diseases in marine culture are Listonella Vibrio) anguillarum. exophthalmos.anguillarumis the most common fish-pathogenic vibrio (Noga. and swollen abdomen (Noga. sea bass. canals. 2011). Most bacterial agents causing diseases to fish are Gram-negative. or ulcer-disease (Austin and Austin.1 Vibriosis Vibriosis. Vibriosis has been reported on many fish species. 2004). sea bream.Within Vibrionaceae.. They are ubiquitous in the environment (sea. and organic pollution (Noga.This type of disease is commonly considered stress mediated with the predisposing factors of handling. 2. 2006). acute or subacute diseases.Vibriosis. lakes. 1998). moving from fresh to salt water (Plumb and Hanson. Vibrio ordalii. but where outcreaks occur. bacterial diseases can cause 100% of mortality (Bui Quang Te. Infected fish may havered areas on body. infectious dropsy.water quality factors.3 Some common bacterial diseases in brackish and marine fishes Bacteria are one of the most important-causative agents causing adverse diseases to fish. red sore. and eel (Actis et al. V. including salmon. high temperature. corneal ulcers. 2010). also known as salt-water furunculosis.2 Bacterial hemorrhagic septicaemia Bacterial hemorrhagic septicaemia.3. cod. 2006). crowding. some of them are Gram-positive. Vibrio vulnificus biotype 2 (Toranzo et al. 2005). striped bass. can be controlled by maintaining good water quality. rivers. rubella. treatment with an oral antibiotic is the only option (Woo and Brono. depression. and others (Plumb and 4 . red pest. skin ulcers.. 1998) 2.

Although S. sulphonamides. 1996. pyogenes.(FAO.iniae can affect various freshwater and coastal fish species (Austin and Austin. Creeper and Buller. 2007). erythromycin. 2012). 2007). 2. 2007). appearing in chains (Roberts. but were very sensitive to enrofloxacin (Brag and Todd. fish pathogen S.Hanson. 2007). streptomycin. chloramphenicol. enrofloxacin (Stoffregenet al. They occur as Gram-negative. novobiocin. 2010). cited by Austin and Austin.0 x 1. including ampicillin. 2007).iniae can cause disease in human hemorrhaging (Austin and Austin. 2007). hydrophila. equi. irregular reddened skin ulcerations. 1988. exophthalmia. motile.Diverse host species have been reported with streptococcus infection. This species is a pathogenic species mainly to fresh water fish.. Even though. Disease syndromes may include lethargy. 2012). 1994) and sea bass (Bromage et at.. oxytetracycline (Aoki. and pale gills (Austin and Austin. 2006). cited by Roberts. agalactiaeare the two that mostfrequently cause serious disease in tilapia. parauberis. S. Besides. pale gill. nitrofurentoin. tilapia. dysqalactiae. reddened fluid and organs in infected fish (Noga. By far the most significant fish pathogen is A. More importantly. straight rods (0.A. S.. 2011).3-1. hydrophila is widely distributed in the aquatic environment (Roberts. cited by Austin and Austin. and occasionally to marine fish (Austin and Austin. This disease can cause darkening. tetracycline. S. agalactiae. This bacterial agent can be treated with fluoroquinolone compound. iniaeand S. cited by Austin and Austin. 2006). anorexia.3. dysgalactiae. S. 1988. has been associated with several members of genus Aeromonas such as A.Fish infected by this species often get damaged brain. S. and S. 2012). facultative anaerobic cocci. including rainbow trout. 2010). hydrophila (Noga. hybrid triped bass (Eldar et al. 1988.S. surface and internal. Streptococcus iniae are small. including S. and Pseudomonas sp. A.. this bacterial species is more commonly isolated from fresh-water fish such as rainbow trout and tilapias than from marine fish such as flounders and sardines (Kusada and Salati. sobria. reddened abdominal fluid.03. 1999. 1999. many species of Streptococcosis.3 Streptococcosis Streptococcosis is sometimes called “popeye” because exophthalmos (exophthalmia) is very common. De Paoplaet al. This bacteria species showed resistant to many types of antibiotics. Gram-positive. A. iniae. laboratory studies also showed the efficacy of oxytetracycline and 5 . caviae. 2012). 2007).5µm) (Roberts. have been reported from fish.

other antibiotics such as chlortetracycline. oxolinic acid.4. It has a basic dihydroquinoline (4-quinolone ring) chemical core with an ethyl group at the 4th position. 2007). Tetracyclines are produced by Streptomyces spp. ciprofloxacin.4. Isidori et al.. 2006. sarafloxacin..4 Antibiotics in aquaculture Antibiotics are known as the best treatment when fish get bacterial diseases. 2004. such as vibriosis and furunculosis (Capone et al. Oxytetracycline is a bacteriostatic antibiotic that exerts its antimicrobial effect against protein synthesis. 2003. Enrofloxacin is a derivative of nalidixic acid.. This prevents the addition of amino acids to the growing peptide chain (Chambers. cited by Austin and Austin.2 Enrofloxacin Enrofloxacin was developed as an antimicrobial agent during the 1980s for exclusive use in veterinary medicine and has proven to be effective in the treatment of bacterial diseases that affect aquaculture organisms. It is primarily 6 . Globally. 2. 2001). 2..1 Oxytetracycline Oxytetracycline is widely employed to treat bacterial infections in aquaculture farms. Darwish and Ismaiel.. ricksettsias. norfloxacin. favoring its absorption and availability. quinolones. gentamicin. this is not only wasting of money but also make opportunities for bacterial resistance. and tiamulin are used (Holmstrom et al.. 2006). 1996. mycoplasmas. 2. 2007). it needs to pass through the external membrane via passive diffusion through the OmpF and OmpC pores.. 2004). Jara. 2005. and through the cytoplasm membrane via an energy dependent process (Jara. 2000.iniae (Darwishet al. It belongs to the tetracycline group. sulfamethazine. In order for oxytetracycline to interact with its target site. florfenicol. and others (Gómez-Gil et al.amoxicillin in controlling S. 2002. Wang et al. perfloxacin. Prescott et al. The residual of antibiotic in fish (aquatic species) could affect to human health... which possess determinants for resistance to this class of antibiotics. People usually take over dosages for sure that they treat the disease completely.. Reed et al. SotoRodríguez et al.. thereby impeding the bonding of aminoacyl-tRNA (aminoacyl transfer RNA) to the A-site of the ribosome. and enrofloxacin (Roque et al. 2003). which exerts antimicrobial action against both Gram (-) and (+) bacteria. 2001. The antibiotics most frequently used in aquaculture to combat bacterial diseases include oxytetracycline. 2007). by bonding directly to the S7 protein of the 30S subunit of the bacterial ribosome.

even when these bacteria have developed resistance to other antibiotics.. tilapia (Oreochromis niloticus). and European seabass (Dicentrarchus labrax) (Intorre et al.. inhibiting DNA synthesis. which act by cleaving the DNA of the bacterial chromosome and rejoining the ends once a superhelix is formed (Banerjee et al.3 Ciprofloxacin Ciprofloxacin is the main metabolite of Enrofloxacin and is active against a broad spectrum of aerobic Gram (-) bacteria. which is inhibited by enrofloxacin. 2006). bacterial cell multiplication is interrupted. black shrimp (Penaeus monodon). The information related to this antibiotic for the most widely grown shrimp species such as Litopenaeus vannamei is scarce. 2008. Chinese shrimp (Penaeus chinensis). It is also active against Gram (+) pathogens. such as Mexico. 2007). such as penicillin (Wen et al. 2. The mechanism of enrofloxacin acts at the level of the cellular nucleus. as well as the potential for extra-label use in the United States provides a need for efficient methods to monitor food supplies for the presence of fluoroquinolones residues (Schneider et al. such as crab (Scylla serrata). Wen et al. Xu et al..4. None of the fluoroquinolones included enrofloxacin is approved for use in shrimp in the United States.lipophilic and has a low molecular weight. It is not active against anaerobic bacteria and may be used occasionally. because each organism possesses a different metabolism. but pharmacokinetic studies on enrofloxacin have been carried out using other species. During the multiplication phase of the bacteria...4. including enteric pathogens such as Pseudomonas and Serratia marcescens. It is important to note that the pharmacokinetic results for enrofloxacin obtained for these species should not be extrapolated to other aquatic species. favoring tissue penetration. the DNA folds and unfolds alternately. 2. 2007. When these enzymes are inhibited. 2007). 2002). thus causing the bacteriocidal effect (Williams et al. causing a collapse of bacterial metabolism and preventing the genetic information from being copied.. and the cultivation conditions may have a significant influence over the kinetic behavior displayed by the antibiotic. Tu et al. 2005). in combination with other antibacterial agents.4 Florfenicol 7 . This process is controlled by the enzyme DNA gyrase. The antibacterial effects of ciprofloxacin arise from its inhibition of Topoisomerase IV and bacterial DNA gyrase. 2000... Their potential use in other countries. for the treatment of mycobacterial infections.

such as thiamphenicol and chloramphenicol. and florfenicol is effective against bacteria that have developed the ability to deactivate other drugs.This fluorinated antibiotic. Vibrio anguillarum. is a potent and broadly acting bacteriostatic agent. Pharmacokinetically. Its chemicalstructure is very similar to that of chloramphenicol. exhibiting a good distribution among all of the organs and tissues. in which a bioavailability of more than 95% is present. Its halflife in fish is less than 15 h (Yanong & Curtis. It is effective in the treatment of infections caused by Pasteurella piscicida. and Edwardsiella tarda. 8 . 2005). derived from thiamphenicol. However. published information for shrimp is scarce. florfenicol use has been reported among some species of fish such as Atlantic salmon (Salmo salar). meaning that the kinetic behavior of this compound among these crustaceans has not yet been completely elucidated. Aeromonas salmonicida.

and reduce the risk of contamination. Vietnam.2 Materials The media. Cantho University for analysis. 3. Cantho University. motility.42555) Ammonium oxalate Iodine Potassium iodide Safranin (Color Index No.50240) Chemicals for biochemical tests: O/F.1 Time and sites of study - Time: January to June . 3. chemicals.CHAPTER 3 MATERIALS AND METHODS 3. Oxidase. Crystal violet (color Index No.3 Methods 3. 3. antibiotics and consumables used for this study are listed below: - Nutrient agar (NA) Tryotic soy agar (TSA).3.2 Bacterial isolation Fish samples were first put on clean trays for observing and recording of external signs into a sampling sheet as shown Appendix 2. Samples of 2-3 diseased fish are collected from each cage. College of Aquaculture and Fisheries.3. Internal signs of fishwere also observed and noted. Indole. 9 . and then carefully dissected to avoid damaging internal organs. Nhatrang. then sampled on-cages and keep the samples to transport to the laboratory of the College of Aquaculture and Fisheries. They were disinfected with 700 alcohols. 2013. Locations: Fish are sampled at Nhatrang bay. Catalase… Antibiotics: Enflorxacin (ENR/5g).1 Fish sampling Silver pomfret samples are collected from culture cages in Nhatrang bay. Florfenicol (FFC/30g. Place for conducting experiments: Department of Aquatic Biology and Pathology.).

2006). 3. and bacteria are washed 2-3 times under sterile saline solution. brain. 25ml of saline solution is added. Detailed procedures for each specific test are shown in Appendix 3. and representative bacterial colonies were sub-cultured for purity.3. The upper solution part is eliminated. and put into bottle with about 30ml sterile BHI to vortex at 200 rounds/minutes for 24 hours.and incubated at 280C. Position disks such that the minimum center-center distance is 24mm and no closer than 10-15mm from the edge of the petri dish. catalase. following the method of Frerichs and Millar (1993). following the instruction of the suppliers. Bacterial solution is now transferred into 50ml falcon tube for centrifugation (4000 rounds/minute for 15 minutes). Incubating loops are used to take 1-2 colonies from pure culture. and Buller (2004). 10 . kidney. A maximum of six disks may be placed in a 9-cm petri dish and 12 disks on a 150mm plate. spleen. The density of bacteria is determined. Besides that.4 Antimicrobial susceptibility testing The susceptibility patterns of the identified isolates were made by using disk diffusion method described in Clinical and Laboratory Standards Institute document M2-A09 (CLSI. and eyes were inoculated on agar plates already supplemented with sodium chloride to acquire the salinity of 15‰. PCR technique also use to indentify the bacteria. After the final centrifugation. and elimination of the upper part. and the solution is well mixed. including Gram staining.3 Bacterial identification Pure culture of bacterial isolates after being obtained were used in primary tests. The surface of the medium is letto dry for 3-5 minutes to allow for the absorption of excess moisture. oxidative-fermentative. Bacterial solution is then diluted to the approximate concentration of 1-2 x 108 colony-forming units (CFU)/ml. 3. oxidase.3. bacterial growth was checked.Bacterial strains were fully identified by using API 20E. using spectrophotometer (wavelength = 600nm). and lightly pressed down to ensure complete contact between the disk and the agar surface.Bacterial samples from liver. The standardized bacterial suspension is evenly spreadon the surface of the agar plates with a cotton swab. motility. Antibiotic disks are placed on the surface of the inoculated and dried plate with sterile forceps. O/129 tests. After 24 hours of incubation.

000 1. Dec. Meterials Nutrient agar (Merck) Nutrient broth (Merck) API 20 E (BioMeuriex.000 3. Compare the diameter of the zone of inhibition of the test isolates with those in the chart of interpretative standard for veterinary pathogen. TIME TABLE Time table of the research proposal Month No Research activities 1 Proposal preparation and defense 2 Title of research activities 1 3 Title of research activities 2 4 Data analysis and writing 5 Thesis defense April June July August Sept.905.630.The zones of inhibition are observed after 24 hours of incubation at 280C.200 3. France) Cost 1.240 242.560 Total 10.000 Student’s sign Muller hinton agar (Merck) Stationary Supervisor’s sign 11 .124.098.000. COST ESTIMATION No. The zone of inhibition is the point at which no growth is visible to the unaided eye.

1993. M. Streptococcal infections of fishes. 552pp 2. Warner. 158: 228-235. Yusoff. Friscia. Cascio. 2001. L Paterson. D.L.L. Kumar. Balakrishnan. 2006. 2006. V. Fish Disease Leaflet 71. Thuốc kháng sinh. K. Kumarasamy.L. G. Roberts and N. Bartie.R Bromage. Praxis Publishing. NXB Hà Nội. Khoa Thủy Sản. R. D. The University Press. Geert Huys. 15. Sở Khoa Học Công Nghệ và Môi Trường tỉnh Bà Rịa-Vũng Tàu. D. F.. F.. Giacomini.T. M. G. Austin. 14. Welfare. Eur J Clin Microbiol Infect Dis. Chinabut. and R. Davis P. Biodiversity of chloramphenicol-resistant mesophilic heterotrophs from Southeast Asian aquaculture environments. 3. S. Published online. Giske. Viện nghiên cứu nuôi trồng thủy sản 1. Chaudhary. Bùi Kim Tùng. S. Fontana. A. S. Wheeler A (1985) The occurrence of Pampus argenteus (Euphrasen. Walsh. C. J. Italia. Research in Microbiology. Thirunarayan. Đặng Thị Hoàng Oanh. U. Rao. M. V. M. Fourth edition. and D. Butt.C. G. Sheridan. Institute of aquaculture. Upadhyay. N. Standard Operating Procedure (SOP). Millar.CLSI document M49-P [ISBN 1-56238-577-1]. Austin. P. M. Bùi Thị Tho. D. 439 trang. Maharjan. S. T. 5. W. 60 pp. Thuốc kháng sinh và nguyên tắc sử dụng thuốc trong chăn nuôi. 1981. U. 1788) in the North Sea. Pearson. 2001. Inglis. Clinical and Laboratory Standards Institute. Shariff. 854–863. Proposed Guideline. G. B. Sarma. S.J. A. Swings. B. Sharma. Wayne. J.United Kingdom..C.T. 12. R. O.N. 1985. Pike. Teale. 20. Antimicrobial susceptibility of respiratory isolates of Enterobacteriaceae and Staphylococus aureus in Italy: Incidence and Trends over the period 1997-1999. Toleman. Institute of Aquaculture. Ligozzi. Mushtaq.D. M. Fish Disease Leaflet 63. Pennsylvania 19087-1898 USA. Bagaria. M. 4.T. Bacterial disease of fish. 10. M. Doumith. Noorie. 11.. Suite 1400. E.. Oldoni and the Italian Epidemiological Observatory Collaborative Group. Manual for the isolation and identification of fish bacterial pathogens. T. 323 trang. Cnockaert. Irfan. 2007. Perry. Phuong.L.M. B. 87 trang. 6 pp 7. 13. Washington. Clinical and Laboratory Standards Institute. 59-79. Turton. A. J. Scotland. Antibiotic susceptibility testing of aquaculture associated bacteria with the dics diffusion method. 2003. Bacterial Fish Pathogens – Diseases of farmed and wild fish. US Fish & Wildlife Publications. Ray. M. Frerichs. Bullock. and S. R. Đại học Cần Thơ. Bùi Quang Tể. 1994. A. J. Cambrige. A. M. Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals. 2002. T. J Fish Biol 26:105–109 9. US Fish & Wildlife Publications. K. Herman. Bullock. R. 940 West Valley Road. 2007. 6. Somsiri. T. Bệnh học thủy sản. 2005 8. Washington. Krishnan. K. G. C. Giáo trình nguyên lý và kỹ thuật chuẩn đoán bệnh thủy sản. 7 pp. R. Huys. Livermore. Oanh. Eswardsiella infections of fishes. G. D. 12 . 225 trang.REFERENCE 1.

97 pp. Volume 3. Woo. Fish Diseases and Disorders. J. Từ Thanh Dung. Responsible use of antibiotics in aquaculture. 23. 16. 123 trang. Iowa State University. 20. Emergence of a new antibiotic resistance mechanism in India. 2012. and Fungal Infections. A thesis submitted in partial fulfillment of the requirements forthe degree of Bachelor of Aquaculture 22. Bacterial. 469. 2000. and the UK: a molecular. Khoa Thủy Sản. 2012 Flofenicol and enrofloxacin resistance in heterotrophic bacteria isolated from snake head fish (chana striatus) and climbing perch (anabes tedtudineus) farms in the Mekong river delta. Hiện trạng bệnh và tình hình sử dụng thuốc và hóa chất trong nuôi cá lóc ở nông hộ tại tỉnh Hậu Giang. United Kingdom. 24. E. 1998. Giáo trình bệnh học thủy sản. T. Antimicrobial susceptibility testing of bacterial agents isolated fromasian seabass (Latescalcarifer). P. Nguyen Dai Duong. 2005. J. 2005.. A thesis submitted in partial fulfillment of the requirements forthe degree of Bachelor of Aquaculture 17. 1999. Trần Hữu Tài. Plumb. Đại học Cần Thơ.A. Second edition. 18. 328 pp.. Serrano. Blackwell publishing. Luận văn tốt nghiệp Đại Học. biological. Khoa Thủy Sản. K. Fish disease: Diagnosis and Treatment. Khoa Thủy Sản. FAO fisheries technical paper. 21. 2010. Noga. First edition. 19. Tran Huu Tin.CABI Publishing. P. 13 .. Tài liệu hướng dẫn thực tập giáo trình chuyên môn Bệnh Học Thủy Sản 1 năm 2007. W.H. Đại học Cần Thơ.Viral. Đặng Thị Hoàng Oanh và Trần Thị Tuyết Hoa. Bruno. and D. 299 pp. and epidemiological study. Đại học Cần Thơ. Pakistan.N. 2011. Woodford. Iowa State University Press / Ames. Health maintenance and principle microbial diseases of cultured fishes.

4. Repeat streaking procedure as shown. Lower a microscope slide onto the Vaseline and press lightly to ensure a good seal. Apply iodine solution for 1 min. 2. Wash with water. allow to cool and touch loop on edge of uninoculated area of medium to ensure coolness. 3. drain and/or blot dry and examine.blue/purple Gram-negative organisms – pink/red. Catalase Test 14 . Label underside (not lid) of plate and incubate. Decolorise with alcohol/acetone (95% :5%) until no more violet color emanates from the smear Wash thoroughly with water. Wash with water. Carefully invert the slide and attached coverslip and examine under the microscope. Place a loopful of liquid culture on the centre of the coverslip within the vaseline ring. Motility - - Ring the outside edge of a coverslip with Vaseline. A motile organism is one which actively moves to change its position relative to other organisms present. Gram-staining method: - Apply ammonium oxalate/crystal violet solution to heat-fixed smear for 1 min. Apply safranin solution for 2 min. Flame inoculation loop until red hot.APPENDIX 1. Focus first onto the edge of the hanging drop with a low-power objectives before progressing through higher power objectives. flaming and cooling the loop between each sequence. Spread part of sample over about ¼ of the plate by making 3-4 parallel streaks with the loop. Plate inoculation procedure for culture and purification of bacteria - Inoculate sample on a small segment of the surface of the culture medium. Tip off iodine solution. Gram-positive organisms. True motility is non-random and must not be confused with vibratory Brownian movement or convection currents.

8 15 8g 10g 4 ml 1000 ml . 7. Result Oxidative Fermentative No reaction Open tube Yellow (+) Yellow (+) Blue/Green (-) Covered tube Green (-) Yellow (+) Green (-) Note: Negative (-) has to wait until 7 days. 6. The ability to ultilize sugar as a sole carbon source are characteristic for different bacteria within the Enterobacteriaceae. Overlay the medium in one tub with sterile liquid paraffin to a depth of 1 cm. Scrape a sample of bacterial culture off the plate with a glass rod or platinum wire and transfer to a drop of 3% H2O2 on a clean glass slide. Sugar Fermentation There are many different sugars can be metabolized by bacteria. 5. The test organism (grown on a media free from glucose and nitrate) is removed with a platinum wire of glass rod and smeared across the surface of wet paper.- A plate of nutrient agar is streaked and incubated at the optimum temperature for 24 hr (or longer if required to see good growth). Oxidase Test - Wet a small piece of filter paper with oxidase reagent. The standard carbohydrate broth base consists of nutrient broth supplemented with a bromthymol blue (as a pH indicator) Nutrient broth Sugar 1. vibrios and aeromonads. A positive reaction is shown by the development of a dark purplecolor within 10-30 sec. A positive test is the almost immediate production of gas bubbles. Glucose Oxidation-Fermentation (O-F) Test - Inoculate two tubes by stabbing with needle carrying bacteria.6% bromthymol blue Distilled water Adjust pH to 6. Examine daily for up ti 7days.

Repeat one more time. PCR procedure Tissue of liver store in 1. Motioning reversal 25 times. Protein will settle down into one small layer between the two phases.Dispense the 1% sugar medium into screw capped tube. eliminate ethanol and grin in 600µl Lysis buffer (0. reversal and centrifugal 13000 cycles in 15 minutes.5µl Proteinase K (40mg/ml). 1mM EDTA.8% Triton. incubating at 370C in 15 minutes. 0. Centrifuge 13000 cycles in 10 minutes. 8.1M Tris-HCl). Put 50µl TE buffer (10mM Tris. 0. 40µl SDS 10% and 2.001 EDTA.5M NaCl.5µl RNase (2mg/ml). Add 600µl cool Isopropanol and centrifuge 13000 cycles in 10 minutes. Hand tightly to wash DNA. Reversal and dry the tube in few minutes. pH = 7) into DNA tube and store in 40C. release the solution above and add 600µl cool alcohol 70%. motioning reversal 25 times and incubating solution at 370C in 30 minutes. 1% SDS.5ml tube. 0. Carefully take away alcohol above. Continue adding to 600µl Chloroform – Isoamyl (24:1). DNA will settle down to the bottom. Add 2. - - - - 16 . 5 ml per tube. Autoclave the medium at 100oC for 10 minutes. Carefully suck the solution above into another 1. Put 600µl Phenol – Chloroform – Isoamyl (25:24:1) and reversal the tube to wash DNA.5ml tube with ethanol.