antimicrobial susceptibility of edwardsiella tarda isolated from clown knifefish (chitala chitala)

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES ANTIMICROBIAL SUSCEPTIBILITY OF Edwardsiella tarda ISOLATED FROM CLOWN KNIFEFISH (Chitala chitala) By NGUYEN MINH THUAT A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Can Tho, December 2013 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES ANTIMICROBIAL SUSCEPTIBILITY OF Edwardsiella tarda ISOLATED FROM CLOWN KNIFEFISH (Chitala chitala) By NGUYEN MINH THUAT A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Aquaculture Supervisor Dr. TU THANH DUNG Can Tho, December 2013 APPROVEMENT The thesis “Antimicrobial susceptibility of Edwardsiella tarda isolated from clown knifefish (Chitala chitala)” defended by Nguyen Minh Thuat, which was edited and passed by the committee on 27-12-2013. Sign of Supervisor Sign of Student Dr. Tu Thanh Dung Nguyen Minh Thuat i ACKNOWLEDGEMENT First of all, I would like to express my sincere gratitude to my supervisor, Dr. Tu Thanh Dung for her enthusiastic guidance, advice, and encouragement during the thesis implementation. Many thanks are also given to all other staffs of the College of Aquaculture and Fisheries, and especially to those of the Department of Aquatic Pathology for providing the great working and learning conditions. Special thanks to many of my friends, especially Nguyen Khanh Linh, Pham Dang Hoa Hiep, Nguyen Bao Trung, and Tran Thi My Han for their kindly help throughout the experimental period. Thanks for my academic advisor, Dr. Duong Thuy Yen, who was guiding and giving me a lot of useful advices over the last four years. Above all, I am grateful to my parents and other members in my family for their greatly support and encouragement during studying period. Nguyen Minh Thuat ii ABSTRACT This study was conducted to access the antimicrobial susceptibility of Edwardsiella tarda isolated from clown knifefish (Chitala chitala) cultured in the Mekong Delta, Vietnam. Fish samples were collected in Haugiang and Dongthap provinces. A total number of 17 bacterial isolates were obtained from diseased fishes in different commercial farms. Naturally infected clown knifefish showed clinical signs of petechial hemorrhages on body, fins and the mandible. Internally, ascites, hepatomegaly and splenomegaly were also found. The conventional and rapid identification system were used to identify these isolates as Edwardsiella tarda. All of E. tarda isolates were tested with 16 different antibiotics by using disk diffusion method. The results showed that most of isolates were sensitive to amoxicillin/clavulanic acid, florfenicol, cefotaxime, doxycycline, cefalexin, and cefazolin. However, most of E. tarda isolates were highly resistant against sulfamethoxazole/trimethoprim, norfloxacin, enrofloxacin, oxytetracycline, ampicillin, rifampicin, and novobiocin. Especially, in this study, all of isolates showed multiantibiotic resistance. Besides, three different antibiotics were also used to determine minimal inhibitory concentration (MIC) by using dilution method. iii TABLE OF CONTENTS Acknowledgement . ii Abstract . iii Table of contents . iv List of tables . vi List of figures . vii List of abbreviations viii Chapter 1: Introduction 1.1 General introduction . 1.2 Research objectives 1.3 Research contents Chapter 2: Literature review 2.1 Clown knifefish (Chitala Chitala) . 2.2 Common disease on clown knifefish 2.3 Edwardsiella tarda infection . 2.4 Antimicrobial used in aquaculture 2.5 Common antibiotic groups and modes of action . 2.5.1 Beta-lactams . 2.5.2 Tetracyclines 2.5.3 Phenicols 2.5.4 Quinolones and fluoroquinolones 10 2.5.5 Sulfonamides 10 2.5.6 Aminoglycosides 10 2.6 Antimicrobial resistance 11 2.7 Antimicrobial susceptibility testing of E. tarda . 12 Chapter 3: Materials and methods 14 3.1 Time and places . 14 3.2 Materials 14 3.3 Methods . 14 iv 3.3.1 Fish sampling . 14 3.3.2 Bacterial isolation . 16 3.3.3 Bacterial identification 16 3.3.4 Disk diffusion method . 17 3.3.5 Minimal inhibitory concentration (MIC) test 18 Chapter 4: Results and discussion . 19 4.1 Fish sampling and bacterial isolation . 19 4.2 Bacterial identification . 21 4.3 Antimicrobial susceptibility testing . 22 4.4 Minimal inhibitory concentration (MIC) 29 Chapter 5: Conclusions and recommendations 31 5.1 Conclusions 31 5.2 Recommendations 31 References . 32 Appendix 1: Major antimicrobial drugs used in aquaculture 37 Appendix List of antimicrobials and drugs are banned 38 Appendix List of antimicrobials and drugs are limited 49 Appendix 4: Fish disease diagnosis form . 40 Appendix 5: Some biochemical tests used in bacterial identification 41 Appendix 6: Bacterial identification scheme 45 Appendix 7: List of solvents and diluents . 45 Appendix 8: Preparation of culture dilution series . 46 Appendix 9: Species of fish infected with Edwardsiella tarda 47 Appendix 10: Biochemical characteristics of E. tarda . 49 Appendix 11: Zone diameter and MIC interpretive criteria . 50 Appendix 12: Some types of multi-antibiotics resistance . 50 Appendix 13: Sensitivity test of 17 isolates of E. tarda . 51 Appendix 14: Sample collection information . 52 v LIST OF TABLES Table 4.1: Sample collection sites and number of bacterial isolates 19 Table 4.2: Primary tests of E. tarda . 21 Table 4.3: The number and percentages of susceptible, intermediate and resistant E. tarda isolates . 23 Table 4.4: The MIC values (µg/ml) of antibiotics with E. tarda isolates 29 vi LIST OF FIGURES Figure 3.1: The political map of Haugiang province . 15 Figure 3.2: The political map of Hongngu district . 15 Figure 4.1: External and internal signs of diseased fish 20 Figure 4.2: Colonies morphology on TSA media and gram staining of E. tarda 20 Figure 4.3: Percentages of susceptible, intermediate and resistant isolates 23 Figure 4.4: Sensitivity of E. tarda with NOR (1), KZ (2), DO (3), ENR (4) . 24 Figure 4.5: The percentage of isolate with multi-antibiotics resistance 27 Figure 4.6: The MIC value of enrofloxacin (isolate EHG4) (16µg/ml) . 29 vii LIST OF ABBREVIATIONS AMC Amoxicillin/clavulanic acid, AMP Ampicillin, BHIA Brain Heart Infusion Agar, CFU Colony forming unit, CLSI Clinical and Laboratory Standards Institute, CTX Cefotaxime, DO Doxycycline, ENR Enrofloxacin, FDA Food and Drug Administration, FFC Florfenicol, KZ Cefazolin, OT Oxytetracycline, MHA Mueller-Hinton Agar, MIC Minimal inhibitory concentration, NAFQAD National Agro-Forestry-Fisheries Quality Assurance Department, NOR Norfloxacin , NV Novobiocin, PBPs Penicillin binding proteins, RD Rifampicin, S Streptomycin, SXT Trimethoprim+sulfamethoxazole, TE Tetracycline, TSA Tryptic soya agar, UB Flumequine, VASEP Vietnam Association of Seafood Exporters and Producers. viii Appendix 2: List of chemicals and antibiotics banned for use in fishery production and trading (VASEP, 2012) No 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Chemicals, antibiotics Aristolochia spp and by products Chloramphenicol Chloroform Chlorpromazine Colchicine Dapsone Dimetridazole Metronidazole Nitrofuran (including Furazolidone) Ronidazole Green Malachite (Malachite) Ipronidazole Nitroimidazoles others Clenbuterol Diethylstilbestrol (DES) Glycopeptides Trichlorfon (Dipterex) Gentian Violet (Crystal violet) Fluoroquinolones Trifluralin Cypermethrin Deltamethrin Enrofloxacin 38 Applied for Animal feed, veterinary medicines, chemicals, environmental treatment substances, disinfectants, preservation subtances, hand cream used in seed production, aquatic animal and amphibians culture, fishery services, preservation and proceesing activities Appendix 3: List of chemicals and antibiotics restricted for use in fishery production and trading (VASEP, 2012) No 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Chemicals, antibiotics Amoxicillin Ampicillin Benzylpenicillin Cloxacillin Dicloxacillin Oxacillin Oxolinic Acid Colistin Diflubenzuron Teflubenzuron Emamectin Erythromycine Tilmicosin Tylosin Florfenicol Lincomycine Neomycine Paromomycin Spectinomycin Chlortetracycline Oxytetracycline Tetracycline Sulfonamide (kinds) Trimethoprim Ormetoprim Tricainemethanesulfonate Danofloxacin Difloxacin Ciprofloxacin Sarafloxacin Flumequine 39 MRLs (ppb) 50 50 50 300 300 300 100 150 1000 500 100 200 50 100 1000 100 500 500 300 100 100 100 100 50 50 15-330 100 300 100 30 600 Appendix 4: Fish disease diagnosis form Date:________________________________ Case No.:___________________ Name:_______________________________ Phone:_____________________ Address:__________________________________________________________ _________________________________________________________________ HISTORY Freshwater:_____ Marine:______ System size: ____________ l/ha Species affected:___________________________________________________ Species in system:__________________________________________________ No. fish in system:______________________ % fish affected: _____________ Average fish size:_______________________ g/cm Age(s) of affected fish: ______________________________________________ When morbidity started:_________________ When mortality started:_______ When morbidity ended:__________________ When mortality ended:________ How long has the system been set up?______ Temperature:_______________ Types of life support present:_________________________________________ Water source:__________________ Water appearance (cloudy, colored):___________________________________ History of maintenance, including water exchange and water quality checks: _________________________________________________________________ Behavioral changes? Y-____N-____Describe:__________________________ Respiratory rate: (normal___, faster___, slower__):_____________________ Appearance of fish:_________________________________________________ _________________________________________________________________ Appetite (normal:___, less:___, more:__):_______________________________ Other clinical signs:_________________________________________________ 40 Appendix 5: Some biochemical tests used in bacterial identification Gram’s Stain Method:  Place a loopful of sterile saline onto a clean glass slide, using a sterile loop.  Use a sterile loop to evenly spread a small amount of bacteria on the slide.  Naturally dry the slide.  Use forceps to hold the slide with the film upwards and slowly pass through a flame three times to fix the film.  Let the slide cool, and place it in a staining dish.  Leave the slide immersing in the crystal violet solution for about minute.  Wash off the residual stain with water, and then immerse the slide in iodine for minute.  Gently mix the slide in alcohol/acetone mixture for 10 seconds.  Carefully wash the slide with water and then let it dry.  Place the slide in Saffranin solution for minutes.  Use water to wash the slide, let it dry, and clean its bottom.  Observe the stained slide under the microscope at x40 objective, and then x100 objective with oil immersion.  Gram positive bacteria have the color of blue/purple, while Gram negative bacteria are red/pink when being observed under the microscope. Motility Test:  Place Vaseline on the four corners of a coverslip.  Put the coverslip, Vaseline up, on the bench.  Place a loopful of sterile saline on the coverslip, using a sterile loop.  Sterilize the loop again, and take a tiny amount of bacteria to gently spread it out in the saline. 41  Gently lower a microscope slide onto the Vaseline mounds without allowing the slide to touch the drop of culture.  Quickly, but gently, invert the slide so that the drop is hanging from the coverslip.  Adjust the lens to see bacterial motility. Oxidase Test:  Place the tip of the oxidase stick onto a bacterial colony, and observe the stick for about 30 second to see if there are any changes on color. The appearance of blue/purple indicates a positive result. Catalase test:  Place a small amount of a bacterial colony (18 to 24 hours old) on a clean glass slide.  Add one to two drops of 3% hydrogen peroxide.  Record observations: positive if rapid bubble formation and negative if no bubble formation Glucose Oxidation-Fermentation (O-F) Test:  Prepare two sterile O-F tubes  Sterilize the inoculating loop and let it cool  Use the tip of a sterile straight wire to pick up an inoculum of a pure growth from a culture plate, and make a single stab into the agar tube.  Inoculate the second O-F tube, and aseptically cover the agar with 1mm layer of liquid paraffin.  Incubate both tubes for up to days, observe and read the results: Open tube Green Closed tube Green Results No reaction on glucose Blue at top Yellow Yellow Green Green Yellow Alkaline reaction Oxidative Fermentative 42 API 20E test kit:  Put a little of water into the tray of the test kit for keeping moisture during incubation in incubator  Using sterile inoculating loop to take a few colonies and put in 5ml distilled water or sterile saline, and mix them  Using sterile pipette to draw bacteria into the cells of the test  Put sterile paraffin into cells: ADH, LDC, ODC, H2S, and URE  Incubate in the incubator at 28-30oC and read the results after 24 hours  Reading table is shown in the following table: 43 44 Appendix 6: Identification scheme for the identification of bacterial genera that is important in aquaculture (Ruangpan and Tendencia, 2004) Appendix 7: List of solvents and diluents needed for the preparation of stock solutions of antimicrobial agents (CLSI, M100-S22E, 2012) Antimicrobial agents Ampicillin Enrofloxacin Tetracycline Solvent Diluent Phosphate buffer, pH 6.0, 0.1 Distilled water mol/L 1/2 volume of water, then mol/L Distilled water NaOH dropwise to dissolve Distilled water Distilled water 45 Appendix 8: Preparation of culture dilution series per isolate Tube Final concentration (µg/ml) Antimicrobial solution Bacterial solution 1024 2ml (stock solution) 2ml 512 2ml (1024µg/ml) 2ml 256 2ml (512µg/ml) 2ml 128 2ml (256µg/ml) 2ml 64 2ml (128µg/ml) 2ml 32 2ml (64µg/ml) 2ml 16 2ml (32µg/ml) 2ml 2ml (16µg/ml) 2ml 2ml (8µg/ml) 2ml 10 2ml (4µg/ml) 2ml 11 2ml (2µg/ml) 2ml 12 0.5 2ml (1µg/ml) 2ml 13 0.25 2ml (0.5µg/ml) 2ml 46 Appendix 9: Species of fish infected with Edwardsiella tarda (Evan et al, cited by Woo and Bruno, 2011) Fish species 1. Angel fish (Pterophyllum scalare) 2. Atlantic salmon (Salmo salar) 3. Australian eel (Anguilla reinhardti) 4. Black skirt tetra (Gymnocorymbus temetz) 5. Three-spot/blue gourmi (Trichogaster trichopterus) 6. Barramundi (Asian sea bass) (Lates calcarifer) 7. Blue tilapia (Tilapia aureus) 8. Brook trout (Salvelinus fontinalis) 9. Brown trout (Salmo trutta trutta) 10. Brown bullhead (Ictalurus nebulosus) 11. Carp (Labeo rohita) 12. Channel catfish (Ictalurus punctatus) 13. Chinook salmon (Oncorhynchus tshawytscha) 14. Climbing perch (Anabas testudineus) 15. Coloured (common) carp (Cyprinus carpio) 16. Common (Indian) carp (Catla catla) 17. Crimson sea bream (Evynnis japonica) 18. Emerald shiner (Notropis atherinoides) 19. European eel (Anguilla anguilla) 20. European sea bass (Dicentrarchus labrax) 21. Gillhead sea bream (Sparus aurata) 22. Glowlight tetra (Hemigrammus erythrozonus) 23. Goldfish (Carassius auratus) 24. Grass carp (Ctenopharyngodon idella) Habitat Continent Country A Europe Belgium References (s) Vandepitte et al. (1983) W N. America Canada Martin (1984) W, E Australia Australia A NR NR A, E Asia Singapore Eaves et al. (1990) Bullock and Mc Craren (1989) Dixon and Contreras (1992); Ling et al. (2001) C Australia Australia E Asia Taiwan C N. America Canada E N. America USA NR NR NR C Asia India C N. America USA Uhland et al. (2000) Meyer and Bullock (1973) Bullock and Mc Craren (1989) Swain and Nayak (2003) Meyer and Bullock (1973) C, W N. America USA Amandi et al. (1982) C Asia India Sahoo et al. (2000) C Asia India C Asia India Sae-Oui et al. (1984) Swain and Nayak (2003) C Asia Japan NR NR NR C/W Europe Norway/ Spain Kusuda et al. (1977) Bullock and Mc Craren (1989) Taksdal et al. (1989); Alcaide et al. (2006) C Europe Spain Blanch et al. (1997) E N. America USA Baya et al. (1997) A Asia Singapore NR NR NR E Asia Taiwan Chua (1996) Bullock and Mc Craren (1989) Bullock and Mc Craren (1989); Wang and 47 Iveson (1971) Wang and Wang (1997) 25. Japanese eel (Anguilla japonica) 26. Japanese (olive) flounder (Paralichthys olivaceus) 27. Banded knifefish (Gymnotus carapo) C C C Asia Japan Korea Taiwan Wang (1997) Hoshinae (1962); Egusa (1976) Kokuska (1973) Liu and Tsai (1980) C Asia Japan Nakatsugawa (1983) C Australia Sydney Zoo W W N. America Asia USA Japan Reddacliff et al. (1996) White et al. (1973); Francis-Floyd et al. (1993) Kusuda et al. (1976) A Asia Singapore Chua (1996) C Asia Japan C Asia Japan Africa Egypt Africa Africa Zaire Transvaal C Europe Turkey W, C N. America USA Kaige et al. (1986) Aoki and Kitao (1981); Miyashita (1984) Badran (1993); Saleh (2005) Van Damme and Vandepiite (1980) Bragg (1988) Benli and Yildiz (2004) Horenstein et al. (2004) C, E Australia New S. Wales Reddacliff et al. (1996) A Asia Singapore C C Asia S. America Japan Venezuela Humphrey et al. (1986) Yasunaga et al. (1982); Choi (1991); Sano et al. (2001) Clavijo et al. (2002) C Asia Thailand A Europe Czechoslovakia Supamataya (1988) Vladick et al. (1983); Humphrey et al. (1986) A Australia Sydney Zoo Reddacliff et al. (1996) 41. Spotted snakehead (Channa punctata) C Asia India 42. Striped bass (Morone saxatilis) W N. America USA C, E Europe Spain Kumar et al. (2007) Herman and Bullock (1986); Baya et al. (1997) Nougayrede et al. (1994); Baya et al. (1997); Castro et al. (2006) C Asia Japan Yasunaga et al. (1982) 28. Largemouth bass (Microteprus salmoides) 29. Mullet (Mugil cephalus) 30. Malawi blue cichlid (Maylandia zebra) 31. Mozambique tilapia (Tilapia mossambicus) C 32. Nile tilapia (Oreochromic niloticus) W W 33. Oyster toadfish (Opsanus tau) 34. Rainbow trout (Oncorhynchus mykiss) 35. Rosy barb (Puntius conchonius) 36. Red sea bream (Pagrus major) 37. Red tilapia tetrahybrids 38. Sand goby (Oxyeleotris marmoratus) 39. Siamese fighting fish (Betta splendens) 40. Spotted bonytongue (Scleropages leichardu) 43.Turbot (Scophthalmus maximus; Psetta maxima) 44. Yellow tail (Seriola quinqueradiata) 48 45. Walking catfish (Clarias batrachus) 46. Wels catfish (Silurus glanis) 47. Zebrafish (Danio reiro) C Asia India Sahoo et al. (1998) E A, E Europe N. America France USA Caruso et al. (2002) Pressley et al. (2005) Appendix 10: Biochemical characteristics testing of E. tarda by using API 20E test kit salt tolerance test Test ONPG ADH DC ODC |CIT| H2S URE TDA IND |VP| |GEL| GLU MAN INO SOR RHA SAC MEL AMY ARA 0% Nacl 1% Nacl 2% Nacl 3% Nacl E.tarda EDT1 EHG4 + + + + + + + + + + + + + + + + + + + + 49 E.tarda ( Buller, 2004) + W + + + + + + + + Appendix 11: Zone diameter (mm) and MIC interpretive criteria (µg/ml) (CLSI, M100-S22E, 2012) MIC criteria (µg/ml) Zone diameter (mm) 10 11 12 13 14 15 16 Antibiotics Abr (µg) S I R S I R Ampicillin Cefazolin Rifapicin Nofloxacin Amoxycilin Oxytetracylin Novobiocin Flumequin Tetracycline Flofenicol Streptomycin Cephalexin Cefotaxime Doxycicline Enrofloxacin AMP 10 >=17 14-16 =23 20-22 =20 15-19 =17 14-16 =18 14-17 =20 16-19 =21 16-20 =16 11--15 =15 12--14 =20 17--19 =15 12--14 =20 16-19 =26 23--25 =14 11--13 =23 17--22 =16 11 --15 [...]... their antimicrobial susceptibility 1.2 Research objectives The research was conducted to investigate the antimicrobial susceptibility of E tarda isolated from clown knifefish (Chitala Chitala) and find out the most effective antibiotics for treatment 1.3 Research contents  Isolation and identification of Edwardsiella tarda isolates  Investigation of antimicrobial susceptibility of isolated E tarda. .. lowest MIC value (1 mg/l) to control the growth of E tarda In 2012, in the study about distribution of MIC value of antibiotics against Edwardsiella tarda isolated from olive flounder (Paralichthys olivaceus) was conducted by Kim et al., the minimum inhibitory concentration (MIC) of eight antibiotics against 49 strains of Edwardsiella tarda isolated from olive flounder (Paralichthys olivaceus) was... biochemical characteristics of E tarda isolated from clown knifefish were completely similar to reference E tarda isolate (Buller, 2004) and most of isolates from different hosts mentioned above However, there were some differences from strains isolated from Japanese flounder which was negative for citrate and positive for succrose utilization Disease caused by E tarda was fisrt isolated on eels (Anguilla... of reddening of abdomen, ascites and hemorrhagic internal organs After isolation and identification, the authors 3 concluded that Aeromonas hydrophila was the causative agent of haemorrhagic disease in clown knifefish 2.3 Edwardsiella tarda infection Edwardsiella tarda is one of the members of genus Edwardsiella and family Enterobacteriaceae which has 20 genera and more than 100 species of falcultatively... tarda and determination of minimal inhibitory concentration (MIC) 2 CHAPTER 2 LITERATURE REVIEW 2.1 Clown knifefish (Chitala Chitala) Clown knifefish is a fish of genus Chitala and order Osteoglossiformes with different common names: Chital (Bangladesh); Humped featherback (English); clown knifefish (Fishbase) The body is elongated and strongly compressed laterally Dorsal profile is highly convex Scales... 20 females, with a mean age of 48.20 years Nearly all of E tarda were isolated from extraintestinal sources, especially pus and urine and most of them were subsequently found to be nosocomial-acquired infections Forty one patients were cured of the infection Three cases died from bacteremia and serious underlying diseases In 2001, a series of 11 cases of extraintestinal E tarda infection is presented;... antimicrobial agent cannot exert an effect (Coyle, 2005) 11 2.7 Antimicrobial susceptibility testing of E tarda In the research ―Comparative in vitro activities of selected antimicrobial agents against Edwardsiella tarda was done by Reinhardt et al (1985), 29 strains of E tarda were susceptible to clinical achievable concentrations of ampicillin, chloramphenicol, tetracycline, trimethoprim, sulfamethoxazole,... changed to culture the other species and clown knifefish (Chitala chitala) is one of the favorable species for farmer’s selection Because of high economic value and easy consumption, clown knifefish has become an important cultured species in the Mekong Delta Besides, they are also favourable ornamental fish due to black spot on the flanks body In Vietnam, clown knifefish have been cultured in some provinces... identification Bacterial isolates were identified by following the method of Frerich and Millar (1993) and using API 20E test kit The primary tests of these bacteria were displayed in Table 4.2 Table 4.2: Primary tests of E tarda isolated from clown knifefish and the reference isolate (Buller, 2004) Gram Motility Oxidase Catalase O/F E tarda + + +/+ E tarda (Buller, 2004) + + +/+ Basing on the colony morphology... disease on clown knikefish At the present, the research on clown knifefish is limited and not significant However, in 2012, Tu Thanh Dung and Tran Thi My Han performed the ―Study of agent causing haemorrhagic disease on clown knifefish Fish samples were collected from 17 commercial clown knifefish farms in the Mekong Delta provinces: Haugiang, Dongthap and Cantho Diseased fish showed gross signs of reddening . red disease of eels (Egusa, 19 76) . Edwardsiella tarda was first isolated in Japan by Hoshinae (1 962 ) with the name of Paracolabacterium anguillimortiferum. In 1 965 , Ewing et al described as. 14 3.3 Methods 14 v 3.3.1 Fish sampling 14 3.3.2 Bacterial isolation 16 3.3.3 Bacterial identification 16 3.3.4 Disk diffusion method 17 3.3.5 Minimal inhibitory concentration (MIC). identification 41 Appendix 6: Bacterial identification scheme 45 Appendix 7: List of solvents and diluents 45 Appendix 8: Preparation of culture dilution series 46 Appendix 9: Species of fish