characterization of bacteria isolated from silver pomfret (pampus argenteus euphrasen, 1788) cultured in nhatrang bay, vietnam

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES CHARACTERIZATION OF BACTERIA ISOLATED FROM SILVER POMFRET (Pampus argenteus Euphrasen, 1788) CULTURED IN NHATRANG BAY, VIETNAM By NGUYEN NHUT THANH A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Supervisor Ms. Tran Thi My Duyen Assoc. Prof. Dr. Dang Thi Hoang Oanh Can Tho, December 2013 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES CHARACTERIZATION OF BACTERIA ISOLATED FROM SILVER POMFRET (Pampus argenteus Euphrasen, 1788) CULTURED IN NHATRANG BAY, VIETNAM By NGUYEN NHUT THANH A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Supervisor Ms. Tran Thi My Duyen Assoc. Prof. Dr. Dang Thi Hoang Oanh Can Tho, December 2013 APPROVED BY SUPERVISOR The thesis “Characterization of bacteria isolated from silver pomfret (Pampus argenteus Euphrasen, 1788) cultured in Nhatrang bay, Vietnam” which edited and passed by the committee, was defended by Nguyen Nhut Thanh in 27/12/2013. Student sign Nguyen Nhut Thanh Supervisor sign Ass. Prof. Dang Thi Hoang Oanh ACKNOWLEDGEMENT First of all, I would like to express my deep and sincere gratitude to my supervisor, Dr. Dang Thi Hoang Oanh for her guidance throughout my degree program Many thanks are also given to all other doctors, lecturer of the college of aquaculture and fisheries, and especially to those of the department of aquatic biology and pathology for providing me with great working and learning conditions. I wish to thank all of my friends for helping me get through the difficult times, and for all the emotional support and encouragement, especially Vo Le Thanh Truc, Nguyen Trong Nghia, Au Thi Kim Ngoc, Bui Thi Diem My, Le Thanh Can. Finally, I would like to give thank my academic adviser, Dr Duong Thuy Yen, for her guiding and encouraged me, also my parents and all my family members for their love, understanding and support me more than four years in university. i ABSTRACT The purpose of this study was to isolate and characterize bacterial isolates which were recovered from diseased Silver pomfret cultured in Nha Trang Bay, Khanh Hoa province. A total of eights diseased speciments which were collected in October, 2012. Diseased fish displayed lethargic swimming, hemorrhagic on skin, tumor on the body and whitish granules in internal organs. Bacterial isolates were examined for morphology, selected biochemical characteristics as well as susceptibility to common used antibiotics in aquaculture. These isolates were identified as Photobacterium damselae, Vibrio parahaemolyticus, and Vibrio alginoticus, by using API 20E test kit. Result of antibiotic sensitivity tests showed that these isolates were completely resistance with amoxycilline, bicomarin, ampicillin; and they were all susceptible with norfloxacin, ciprofloxacin, florenicol and tetracycline. ii TABLES OF CONTENTS ACKNOWLEDGEMENT i ABSTRACT ii TABLES OF CONTENTS . iii LIST OF FIGURES . v LIST OF TABLES . vi CHAPTER INTRODUCTION . 1.1 Introduction 1.2 Research objective 1.3 Research activities CHAPTER LITERATURE REVIEW 2.1 Silver pomfret 2.2 Cause of diseases . 2.3 Some common bacterial diseases in brackish and marine fishes . 2.3.1 Vibriosis 2.3.2 Bacterial hemorrhagic septicaemia . 2.3.3 Streptococcosis 2.4 Some commonly used antibiotics . 2.4.1 Oxytetracycline 2.4.2 Ciprofloxacin . 2.4.3 Florfenicol . CHAPTER MATERIALS AND METHODS . 3.1 Time and sites of study . 3.2 Materials 3.3 Methods . 3.3.1 Fish sampling 3.3.2 Bacterial isolation 10 3.3.3 Bacterial identification . 10 3.3.4 Antimicrobial susceptibility testing 10 3.3.5 Data collection, calculation and analysis 11 CHAPTER 12 RESULT AND DISCUSSION 12 iii 4.1 Fish sampling and clinical signs . 12 4.2 Bacterial identification . 13 4.3 Antimicrobial susceptibility testing 15 CHAPTER CONCLUSIONS AND RECOMMENDATION 17 5.1 Conclusions 17 5.2 Recommendation . 17 REFERENCE 18 Appendix 1.Some biochemical tests used in bacterial identification . 20 Appendix 2. API 20E kit results . 22 Appendix 3. The diameters of inhibition zone of the antibiotic susceptible test 23 iv LIST OF FIGURES Figure 4.1 Fish sampled Figure 4.2 Internal of diseased fish Figure 4.3 Gram staining, O/F test and bacterial colonies on NA Figure 4.4 API 20E identification test result of Photobacterium damselae Figure 4.5 API 20E identification test result of Vibrio parahaemolyticus Figure 4.6 API 20E identification test result of Vibrio alginoticus Figure 4.7 API 20E identification test result of Aeromonas hydrophyla v LIST OF TABLES Table 4.1 The percentages of distinct strains Table 4.2 Susceptibility pattern of bacterial species vi CHAPTER INTRODUCTION 1.1 Introduction With more than 3200 kilometers of coastline and large area of water surface, Vietnam has great conditions to develop aquaculture (both fresh and marine aquaculture). In fact, aquaculture in Vietnam is developing year by year, especially farmed stripped catfish (Pangasianodon hypophthalmus) and penaeid shrimp (black tiger and white leg shrimp), Vietnam was ranked the world’s third of aquaculture (FAO 2010). Besides, marine aquaculture is contributed an important part for aquaculture with high value species such as sea bass, cobia (Rachycentron Canadum), grouper (Serranidae), 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. Silver pomfret is easy to find in Northern gulf and central southern of Vietnam, in the past, people mainly catch silver pomfret in the wild. In recent years, people started to culture silver pomfret in cages with intensive model. In this system, fish are stocked at high density, usually over feeding. This can make the fish get stress and diseases, especially this can lead to disease spread out easily on the sea to wild fish, or the others cages. Bacterial diseases have been reported as one of the worst problems causing up to 100% mortality of cultured fishes (Bui Quang Te, 2008). This is the new specie, there is a few research about silver pomfret are conducted on over the world. Antibiotics are known as the useful treatment for the bacterial disease, but can make bad result if not use at correct ways or dosage. Bacterial resistance often happens and cause treatment failure. People prefer using higher dosages of antibiotics when the previous dosages not have effect; they hope to heal illness completely, but residues in flesh product are not favorable to consumers. In addition, resistance characteristic could readily and quickly spread out in bacterial populations (Kumarasamy et al., 2010). 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. Thus, this thesis “Characterization of bacteria isolated from silver pomfret (Stromateoides argenteus Euphrasen, 1788) cultured in Nhatrang bay, Vietnam” is carried out to provide more information about disease on this species. 2.4.2 Ciprofloxacin Ciprofloxacin is the main metabolite of Enrofloxacin and is active against a broad spectrum of aerobic Gram (-) bacteria, including enteric pathogens such as Pseudomonas and Serratia marcescens. It is also active against Gram (+) pathogens, even when these bacteria have developed resistance to other antibiotics, such as penicillin (Wen et al., 2007). It is not active against anaerobic bacteria and may be used occasionally, in combination with other antibacterial agents, for the treatment of mycobacterial infections. The antibacterial effects of ciprofloxacin arise from its inhibition of Topoisomerase IV and bacterial DNA gyrase, which act by cleaving the DNA of the bacterial chromosome and rejoining the ends once a superhelix is formed (Banerjee et al., 2007). When these enzymes are inhibited, bacterial cell multiplication is interrupted. 2.4.3 Florfenicol This fluorinated antibiotic, derived from thiamphenicol, is a potent and broadly acting bacteriostatic agent. It is effective in the treatment of infections caused by Pasteurella piscicida, Aeromonas salmonicida, Vibrio anguillarum, and Edwardsiella tarda. Its chemicalstructure is very similar to that of chloramphenicol, and florfenicol is effective against bacteria that have developed the ability to deactivate other drugs, such as thiamphenicol and chloramphenicol. Pharmacokinetically, florfenicol use has been reported among some species of fish such as Atlantic salmon (Salmo salar), in which a bioavailability of more than 95% is present, exhibiting a good distribution among all of the organs and tissues. Its halflife in fish is less than 15 h (Yanong & Curtis, 2005). However, published information for shrimp is scarce, meaning that the kinetic behavior of this compound among these crustaceans has not yet been completely elucidated. CHAPTER MATERIALS AND METHODS 3.1 Time and sites of study - Time: January to June, 2013. - Locations: Fish were sampled at Nhatrang bay, Vietnam. - Place for conducting experiments: Department of Aquatic Pathology, College of Aquaculture and Fisheries, Cantho University. 3.2 Materials The media, chemicals, antibiotics and consumables used for this study are listed below: - Nutrient agar (NA) Tryotic soy agar (TSA). Crystal violet (color Index No.42555) Ammonium oxalate Iodine Potassium iodide Safranin (Color Index No.50240) Chemicals for biochemical tests: O/F, motility, Indole, Oxidase, Catalase… Antibiotics: enflorxacin (ENR/5g), florfenicol (FFC/30g,), amoxycilline (25g), norfloxacin (5g), ciprofloxacin (30g), trim/sulfa (1:19) (25g), tetracyline (30g), neomycin (30g), ampicilline (35g), doxycyline (30g). 3.3 Methods 3.3.1 Fish sampling Silver pomfret samples are collected from culture cages in Nhatrang bay, Nhatrang. Samples of 2-3 diseased fish are collected from each cage, then sampled on-cages. The fish have some clinical sign such as infected with water fleas, tumor on the body, the body cavity and internal organs are bleeding, internal organs has white spots. After that, keep the samples to transport to the laboratory of the College of Aquaculture and Fisheries, Cantho University for analysis. 3.3.2 Bacterial isolation Fish samples were first put on clean trays for observing. They were disinfected with 70 alcohols, and then carefully dissected to avoid damaging internal organs, and reduce the risk of contamination. Internal signs of fish were also observed and noted. Bacterial samples from liver, kidney, spleen, and brain were inoculated on nutrient agar plates (Merck) supplemented with 1.5% sodium chloride to acquire the salinity of 15‰ and incubated at 280C. After 24 hours of incubation, bacterial growth was checked, and representative bacterial colonies were sub-cultured for purity. 3.3.3 Bacterial identification Pure culture of bacterial isolates after being obtained were used in primary tests, including Gram staining, motility, oxidase, catalase, oxidative-fermentative, O/129 tests, following the method of Frerichs and Millar (1993), and Buller (2004). Detailed procedures for each specific test are shown in Appendix 3. Bacterial strains were fully identified by using API 20E test kit, following the instruction of the suppliers. 3.3.4 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, 2006). Five strains of bacteria which are identified used for this experiment. Incubating loops are used to take 1-2 colonies from pure culture, and put into bottle with about 30ml sterile BHI to vortex at 200 rounds/minutes for 24 hours. Bacterial solution is now transferred into 50ml falcon tube for centrifugation (4000 rounds/minute for 15 minutes). The upper solution part is eliminated, and bacteria are washed 2-3 times under sterile saline solution. After the final centrifugation, and elimination of the upper part, 25ml of saline solution is added, and the solution is well mixed. The density of bacteria is determined, using spectrophotometer (wavelength = 600nm). Bacterial solution is then diluted to the approximate concentration of 1-2 x 108 colony-forming units (CFU)/ml. The standardized bacterial suspension is evenly spread on the surface of the agar plates with a cotton swab. The surface of the medium is let to dry for 3-5 minutes to allow for the absorption of excess moisture. Antibiotic disks are placed on the surface of the inoculated and dried plate with sterile forceps, and lightly pressed down to ensure complete contact between the disk and the 10 agar surface. Position disks such that the minimum center-center distance is 24mm and no closer than 10-15mm from the edge of the petri dish. A maximum of six disks may be placed in a 9-cm petri dish and 12 disks on a 150mm plate. The zones of inhibition are observed after 24 hours of incubation at 280C. The zone of inhibition is the point at which no growth is visible to the unaided eye. Compare the diameter of the zone of inhibition of the test isolates with those in the chart of interpretative standard for veterinary pathogen. 3.3.5 Data collection, calculation and analysis Data was analysis by using Microsoft Excel 2007. 11 CHAPTER RESULT AND DISCUSSION 4.1 Fish sampling and clinical signs Fish were sampled at Nhatrang bay, in December 2012. From each cage, both fish with normal external appearance and diseased fish with abnormal swimming, severe ulcers and/or hemorrhage on skin were sampled (Figure 4.1). Some were strong infected with water fleas. A B Figure 4.1 Fish sampled A. Severe tumor B. Hemorrhage of body silver pomfret 12 The internal organs of healthy fish were showed normal display, while diseased fish were having bleeding body cavity and internal organs, we could also observed white spots on internal organs (Figure 4.2). Figure 4.2 Internal of diseased fish, A. Hemorrhage of body cavity and internal organs. B. White spot on internal organs 4.2 Bacterial identification Bacteria were isolated from the sampled by using nutrient agar media until they were pure. These bacteria were used for identification step. After undergoing some basic biochemical test based on the principles of Cowan and Steels (Barrow & Feltham 1993) and using API 20E system (Bio Merieux, France), isolates were identified as four species including Photobacterium damselae, Vibrio parahaemolyticus, and Vibrio alginoticus. The percentage of identity are shown in table 4.1 Table 4.1 The percentages of distinct strains Strains Identity (%) Photobacterium damselae 99.99 Vibrio parahaemolyticus 99.5 Vibrio alginoticus 93.4 No. of strains 1 Morphological, physiological and biochemical characteristics of those species are indicated in table 4.1. The details are described as follows: 13 Figure 4.3 Gram staining, O/F test and bacterial colonies on NA Photobacterium damselae CT 1-1G There are two strains were identified as Photobacterium damselae from silver pomfret, the colonies size was 2-3 mm after days of incubating on NA agar. The Gram-staining gave negative results but positive in O/F test, catalase and oxydase test. And the results of API 20E test are shown in figure 4.4 Figure 4.4 API 20E identification test result of Photobacterium damselae Vibrio parahaemolyticus CT2-2KU With Vibrio parahaemolyticus, after 24 hours of incubating, we observed that the colonies were circular, entire and low convex. The color was creamed on nutrient agar. The surface of the colonies was smooth and shiny. The Gram-staining had negative result, O/F test, catalase and oxydase test of this strain gave positive. The details of API 20E test are shown below Figure 4.5 API 20E identification test result of Vibrio parahaemolyticus 14 Vibrio alginoticus CT2-3KU This is strain Vibrio, we observed that it has the same characteristics as V. parahaemolyticus. A clear difference between the two biochemical species is found in the ability of sucrose fermentation which is negative for V. parahaemolyticus while it is positive for V. alginolyticus (Shinoda, 2011) Figure 4.6 API 20E identification test result of Vibrio alginoticus 4.3 Antimicrobial susceptibility testing All strains were tested for their susceptibility with twelve commonly used antibiotics in aquaculture. From the table below, all strains of bacteria were complete resistance with amoxycilline, ampicilline, and bicomarin. They all were very sensitive to norfloxacin, ciprofloxacin, florenicol and tetracycline in which florenicol showed largest inhibition zone. With trim/sulfa, it was only susceptible with photobacterium damselae, the remaining strains were just intermediate inhibition. Photobacterium damselae was the strains which had highest number of resistance antibiotics (6/12 antibiotics). The other three had from three to four number of resistance antibiotic. Vibrio alginoticus was the most sensitive species to antibiotics (7/12 antibiotics) Table 4.2 Susceptibility pattern of bacterial species Antibiotics Amoxycilline Erythromycin Norfloxacin Ciprofloxacin Bicomarin Florfenicol Photobacterium damselae R R S S R S Aeromonas hydrophila R I S S R S 15 Vibrio parahaemolyticus R I S S R S Vibrio alginoticus R S S S R S Trim/sulfa R I Tetracyline S S Neomycin R R Ampicilline R R Doxycyline S S Enrofloxacine I S Note: S – susceptible, R –Resistant, I–Intermediate I S I R S S I S I R S S With these results of antibiotic susceptibility test, recommend the antibiotics should use for treating the bacterial disease of silver pomfret such as florfenicol. Do not abuse the antibiotics to avoid the antibiotics resistance of bacteria. Some antibiotics like amoxycilline, Bicomarin, ampicillin should not use because of wasting of money. Also considering about the withdraw time of the antibiotic when use. Enfrofloxacine had good results, could be use for treating bacterial disease but it had been ban in aquaculture field. 16 CHAPTER CONCLUSIONS AND RECOMMENDATION 5.1 Conclusions Based on basic biochemical characters and API 20E tests, three species of bacteria, which were isolated from silver pomfret were identified as Photobacterium damselae, Vibrio parahaemolyticus, and Vibrio alginoticus. All of these isolates were complete resistant to amoxycilline, bicomarin, ampicilline. The most susceptible antibiotic were florfenicol and tetracycline. 5.2 Recommendation - Some antibiotics such as tetracycline, doxycyline could be used for treat bacterial disease in silver pomfret. However, care should be taken concerning doses and mode of application. - Carry out more researches to determine pathogenesis of the isolated bacterial isolates to get more information. 17 REFERENCE 1. Austin, B., and D. Austin, 2007. Bacterial Fish Pathogens – Diseases of farmed and wild fish, Fourth edition. Praxis Publishing.United Kingdom. 552pp 2. Bùi Kim Tùng, 2001. Thuốc kháng sinh. Sở Khoa Học Công Nghệ Môi Trường tỉnh Bà Rịa-Vũng Tàu. 225 trang. 3. Bùi Quang Tể, 2006. Bệnh học thủy sản. Viện nghiên cứu nuôi trồng thủy sản 1. 439 trang. 4. Bùi Thị Tho, 2003. Thuốc kháng sinh nguyên tắc sử dụng thuốc chăn nuôi. NXB Hà Nội. 323 trang. 5. Bullock, G.L., 1981. Streptococcal infections of fishes. US Fish & Wildlife Publications. Washington, D.C. Fish Disease Leaflet 63. pp. 6. Bullock, G.L. and R.L. Herman, 1985. Eswardsiella infections of fishes. US Fish & Wildlife Publications. Washington, D.C. Fish Disease Leaflet 71. pp 7. Clinical and Laboratory Standards Institute. Methods for Broth Dilution Susceptibility Testing of Bacteria Isolated From Aquatic Animals; Proposed Guideline.CLSI document M49-P [ISBN 1-56238-577-1]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005 8. Davis P, Wheeler A (1985) The occurrence of Pampus argenteus (Euphrasen, 1788) in the North Sea. J Fish Biol 26:105–109 9. Đặng Thị Hoàng Oanh, 2007. Giáo trình nguyên lý kỹ thuật chuẩn đoán bệnh thủy sản. Khoa Thủy Sản. Đại học Cần Thơ. 87 trang. 10. Fontana, R., G.L. Cascio, M. Ligozzi, O. Friscia, T. Oldoni and the Italian Epidemiological Observatory Collaborative Group, 2001. Antimicrobial susceptibility of respiratory isolates of Enterobacteriaceae and Staphylococus aureus in Italy: Incidence and Trends over the period 1997-1999. Published online. Italia. Eur J Clin Microbiol Infect Dis. 20. 854–863. 11. Frerichs, G.N. and S.D. Millar, 1993. Manual for the isolation and identification of fish bacterial pathogens. Institute of Aquaculture. Scotland. 60 pp. 12. Geert Huys, 2002. Antibiotic susceptibility testing of aquaculture associated bacteria with the dics diffusion method. Standard Operating Procedure (SOP). 13. Huys, G., K. Bartie, M. Cnockaert, D.T.T. Oanh, N.T. Phuong, T. Somsiri, S. Chinabut, F.M. Yusoff, M. Shariff, M. Giacomini, A. Teale, J. Swings, 2006. Biodiversity of chloramphenicol-resistant mesophilic heterotrophs from Southeast Asian aquaculture environments. Research in Microbiology. 158: 228-235. 18 14. Inglis, V, R.J. Roberts and N.R Bromage, 1994. Bacterial disease of fish. Institute of aquaculture. The University Press. Cambrige. 59-79. 15. Kumarasamy, K. K., M. A. Toleman, T. R. Walsh, J. Bagaria, F. Butt, R. Balakrishnan, U. Chaudhary, M. Doumith, C. G. Giske, S. Irfan, P. Krishnan, A. V. Kumar, S. Maharjan, S. Mushtaq, T. Noorie, D. L Paterson, A. Pearson, C. Perry, R. Pike, B. Rao, U. Ray, J. B. Sarma, M. Sharma, E. Sheridan, M. A. Thirunarayan, J. Turton, S. Upadhyay, M. Warner, W. Welfare, D. M. Livermore, N. Woodford, 2010. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. 16. Nguyen Dai Duong, 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, A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture 17. Plumb, J.A., 1999. Health maintenance and principle microbial diseases of cultured fishes. First edition. Iowa State University Press / Ames. 328 pp. 18. Noga, E. J., 2000. Fish disease: Diagnosis and Treatment. Second edition. Blackwell publishing. Iowa State University. 299 pp. 19. Serrano, P.H., 2005. Responsible use of antibiotics in aquaculture. FAO fisheries technical paper. 469. 97 pp. 20. Shinoda S., 2011. Sixty Years from the Discovery of Vibrio parahaemolyticus and Some Recollections. Biocontrol Science, 16: 129-137. 21. Tài liệu hướng dẫn thực tập giáo trình chuyên môn Bệnh Học Thủy Sản năm 2007. Khoa Thủy Sản. Đại học Cần Thơ. 22. Tran Huu Tinh, 2012, Antimicrobial susceptibility testing of bacterial agents isolated fromasian seabass (Latescalcarifer), A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture 23. Từ Thanh Dung, Đặng Thị Hoàng Oanh Trần Thị Tuyết Hoa, 2005. Giáo trình bệnh học thủy sản. Khoa Thủy Sản. Đại học Cần Thơ. 123 trang. 24. Trần Hữu Tài, 2011. Hiện trạng bệnh tình hình sử dụng thuốc hóa chất nuôi cá lóc nông hộ tỉnh Hậu Giang. Luận văn tốt nghiệp Đại Học. Khoa Thủy Sản. Đại học Cần Thơ. 25. Woo, P. T. K, and D. W. Bruno. 1998, Fish Diseases and Disorders, Volume 3.Viral, Bacterial, and Fungal Infections.CABI Publishing, United Kingdom. 19 Appendix 1.Some biochemical tests used in bacterial identification 1. Plate inoculation procedure for culture and purification of bacteria - Inoculate sample on a small segment of the surface of the culture medium. Flame inoculation loop until red hot, allow to cool and touch loop on edge of uninoculated area of medium to ensure coolness. Spread part of sample over about ¼ of the plate by making 3-4 parallel streaks with the loop. Repeat streaking procedure as shown, flaming and cooling the loop between each sequence. Label underside (not lid) of plate and incubate. 2. Gram-staining method: - Apply ammonium oxalate/crystal violet solution to heat-fixed smear for min. Wash with water. Apply iodine solution for min. Tip off iodine solution. Decolorise with alcohol/acetone (95% :5%) until no more violet color emanates from the smear Wash thoroughly with water. Apply safranin solution for min. Wash with water, drain and/or blot dry and examine. Gram-positive organisms- blue/purple Gram-negative organisms – pink/red. 3. Motility - - Ring the outside edge of a coverslip with Vaseline. Place a loopful of liquid culture on the centre of the coverslip within the vaseline ring. Lower a microscope slide onto the Vaseline and press lightly to ensure a good seal. Carefully invert the slide and attached coverslip and examine under the microscope. Focus first onto the edge of the hanging drop with a low-power objectives before progressing through higher power objectives. True motility is non-random and must not be confused with vibratory Brownian movement or convection currents. A motile organism is one which actively moves to change its position relative to other organisms present. 4. Catalase Test 20 - A plate of nutrient agar is streaked and incubated at the optimum temperature for 24 hours (or longer if required to see good growth). 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. A positive test is the almost immediate production of gas bubbles. 5. Oxidase Test - Wet a small piece of filter paper with oxidase reagent. 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 positive reaction is shown by the development of a dark purplecolor within 10-30 sec. 6. Glucose Oxidation-Fermentation (O-F) Test - Inoculate two tubes by stabbing with needle carrying bacteria. Overlay the medium in one tub with sterile liquid paraffin to a depth of cm. Examine daily for up ti 7days. Result Open tube Oxidative Yellow (+) Fermentative Yellow (+) No reaction Blue/Green (-) Note: Negative (-) has to wait until days. Covered tube Green (-) Yellow (+) Green (-) 7. Sugar Fermentation There are many different sugars can be metabolized by bacteria. The ability to ultilize sugar as a sole carbon source are characteristic for different bacteria within the Enterobacteriaceae, vibrios and aeromonads. The standard carbohydrate broth base consists of nutrient broth supplemented with a bromthymol blue (as a pH indicator) Nutrient broth Sugar 1.6% bromthymol blue Distilled water 8g 10g ml 1000 ml Adjust pH to 6.8 Dispense the 1% sugar medium into screw capped tube, ml per tube. Autoclave the medium at 100oC for 10 minutes. 21 Appendix 2. API 20E kit results Tests Photobacterium damselae CT 1-1G Photobacterium damselae CT 2-1KU V. Alginoticus CT 2-3KU V. parahaemolyticus CT 2-2KU A. hydrophila CT 2-1T ONPG - - - - - ADH + + - - - LDC - - + + + ODC - - + - - CIT - - + + + H2 S - - - - - URE + + - - - TDA - - - - - IND - - - + - VP + + - - - GEL - - + - + GLU + + + + + MAN - - + + + INO - - - - - SOR - - - - - RHA - - - - - SAC - - + - + MEL - - - - - AMY - - + + + ARA - - - + + 22 Appendix 3. The diameters of inhibition zone of the antibiotic susceptible test Antibiotics CT2-1_KU Photobacterium damselae CT2-1_T Aeromonas hydrophila CT1-1_G Photobacterium damselae CT2-2_KU Vibrio parahaemolyticus CT2-3_KU Vibrio alginoticus Amoxycilline Erythromycin Norfloxacin Ciprofloxacin Bicomarin Florfenicol Trim/sulfa Tetracyline Neomycin Ampicilline Doxycyline Enrofloxacine 0 18 18 25 22 0 18 15 12 18 16 25 15 22 0 18 17 13 15 19 20 15 23 11 20 17 15 24 21 26 15 23 13 23 22 17 24 26 22 13 25 13 25 26 23 [...]... The aim of this research is to investigate the bacterial pathogen which are isolated from silver pomfret (Pampus argenteus) in Nhatrang bay, then find out the antibiotic which is susceptible to these bacteria 1.3 Research activities This thesis focuses on the following contents: 1 Classification to species level of bacteria that was isolated from silver pomfret 2 Antibiotics susceptibility testing 2... treatment of mycobacterial infections The antibacterial effects of ciprofloxacin arise from its inhibition of Topoisomerase IV and bacterial DNA gyrase, which act by cleaving the DNA of the bacterial chromosome and rejoining the ends once a superhelix is formed (Banerjee et al., 2007) When these enzymes are inhibited, bacterial cell multiplication is interrupted 2.4.3 Florfenicol This fluorinated antibiotic,... the salinity of 15‰ and incubated at 280C After 24 hours of incubation, bacterial growth was checked, and representative bacterial colonies were sub -cultured for purity 3.3.3 Bacterial identification Pure culture of bacterial isolates after being obtained were used in primary tests, including Gram staining, motility, oxidase, catalase, oxidative-fermentative, O/129 tests, following the method of Frerichs... should use for treating the bacterial disease of silver pomfret such as florfenicol Do not abuse the antibiotics to avoid the antibiotics resistance of bacteria Some antibiotics like amoxycilline, Bicomarin, ampicillin should not use because of wasting of money Also considering about the withdraw time of the antibiotic when use Enfrofloxacine had good results, could be use for treating bacterial disease... possess determinants for resistance to this class of antibiotics Oxytetracycline is a bacteriostatic antibiotic that exerts its antimicrobial effect against protein synthesis, by bonding directly to the S7 protein of the 30S subunit of the bacterial ribosome, thereby impeding the bonding of aminoacyl-tRNA (aminoacyl transfer RNA) to the A-site of the ribosome This prevents the addition of amino acids to... petri dish A maximum of six disks may be placed in a 9-cm petri dish and 12 disks on a 150mm plate The zones of inhibition are observed after 24 hours of incubation at 280C The zone of inhibition is the point at which no growth is visible to the unaided eye Compare the diameter of the zone of inhibition of the test isolates with those in the chart of interpretative standard for veterinary pathogen 3.3.5... Hemorrhage of body silver pomfret 12 The internal organs of healthy fish were showed normal display, while diseased fish were having bleeding body cavity and internal organs, we could also observed white spots on internal organs (Figure 4.2) Figure 4.2 Internal of diseased fish, A Hemorrhage of body cavity and internal organs B White spot on internal organs 4.2 Bacterial identification Bacteria were isolated. .. tilapias than from marine fish such as flounders and sardines (Kusada and Salati, 1999, cited by Roberts, 2012).Fish infected by this species often get damaged brain, exophthalmia, surface and internal 5 More importantly, fish pathogen S.iniae can cause disease in human hemorrhaging (Austin and Austin, 2007) This bacterial agent can be treated with fluoroquinolone compound, enrofloxacin (Stoffregenet... florfenicol and tetracycline 5.2 Recommendation - Some antibiotics such as tetracycline, doxycyline could be used for treat bacterial disease in silver pomfret However, care should be taken concerning doses and mode of application - Carry out more researches to determine pathogenesis of the isolated bacterial isolates to get more information 17 REFERENCE 1 Austin, B., and D Austin, 2007 Bacterial Fish Pathogens... Antimicrobial susceptibility testing All strains were tested for their susceptibility with twelve commonly used antibiotics in aquaculture From the table below, all strains of bacteria were complete resistance with amoxycilline, ampicilline, and bicomarin They all were very sensitive to norfloxacin, ciprofloxacin, florenicol and tetracycline in which florenicol showed largest inhibition zone With trim/sulfa, . fishes 4 2. 3.1 Vibriosis 4 2. 3 .2 Bacterial hemorrhagic septicaemia 5 2. 3.3 Streptococcosis 5 2. 4 Some commonly used antibiotics 6 2. 4.1 Oxytetracycline 7 2. 4 .2 Ciprofloxacin 8 2. 4.3 Florfenicol. INTRODUCTION 1 1.1 Introduction 1 1 .2 Research objective 2 1.3 Research activities 2 CHAPTER 2 LITERATURE REVIEW 3 2. 1 Silver pomfret 3 2. 2 Cause of diseases 3 2. 3 Some common bacterial diseases. 17 5.1 Conclusions 17 5 .2 Recommendation 17 REFERENCE 18 Appendix 1.Some biochemical tests used in bacterial identification 20 Appendix 2. API 20 E kit results 22 Appendix 3. The diameters