CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES STUDIES ON FUNGUS INFECTION IN STRIPED CATFISH (Pangasianodon hypophthalmus) ON GROW - OUT STAGE By NGO THI MONG TRINH A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of science in Aquaculture Can Tho, January 2013 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES STUDIES ON INFECTION OF FUNGUS IN STRIPED CATFISH (Pangasianodon hypophthalmus) ON GROW - OUT STAGE By NGO THI MONG TRINH A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of science in Aquaculture Supervisor Dr PHAM MINH DUC Can Tho, January 2013 Acknowledgements I wish to express my deep appreciation and sincere gratitude to my advisor Dr Pham Minh Duc for his constant guidance I also thank for Mrs Dang Thuy Mai Thuy, Miss Nguyen Hoang Nhat Uyen who help devoted for me to anything in the laboratory to finish my thesis I wish to express my deep appreciation and sincere gratitude to all staff of Biology and Fisheries Diseases who give me good conditions to my thesis I am very grateful to my Advanced Aquaculture class to encourage and help me more things in that time when doing my study Finally, I want to thank my family that always to give me more advices and encouraging when I feel tired ABSTRACT This study was carried out the challenge test, the effects of salt and temperature on the growth of fungus, histopathology of the fungus on striped catfish (Pangasianodon hypophthalmus) The fungus which was used for the experiment was the isolation in the natural The total strains which isolating were 10 strains at An Giang province There were strains of fungi that were used for the challenge test that has designed with two series The infection rate is 42.2% for 105 of concentration, and 45.6% for the 106 of concentration The re-isolation result showed that the fungi which were injected on fish were mainly occurred in swimming bladder 17.2%-25.9%, kidney 10%-22.5%, and seldom in gills 0%-6.7% of 105, and 23.3%-34.5% in swimming bladder, 13.3%-23.8% in the kidney, and 0%-6.9% in gill of 106 for all strains The fungus can be grown differently when the temperature was different in the temperature test The result showed that the temperature was one of factor which infected to the growth of fungi colony The temperature which helped the fungi was good growth at 25oC-30oC, and the best growth was 28oC, and the fungi were lower growth at 20oC, and stopped growth at 35oC Beside that, the sodium chlorine was also infected to the growth of fungi The fungi were grown well on the GYA media with 0%-8% of sodium chlorine The histopathology was showed that the different between of healthy fish and fish which infected by fungi The healthy fish was not found the fungus on the sample of histopathology The mycelia were appeared on the fish which affected by fungal The result showed that was ensured the occurring of fungi on the striped catfish which isolated on the nature, and re-isolated on the challenge test CONTENTS Acknowledgements i Abstract ii Contents iii List of tables vi List of figures vii Chapter I: Introduction 1.1 Introduction 1.2 Objective 1.3 Research contents Chapter II: Literature review 2.1 Status of parasites disease 2.2 Disease caused by fungus 2.2.1 Fungus diseases on aquatic animal 2.2.2 Fungus diseases on fish 2.3 Methods using to study fungus Chapter III: Materials and Methods 3.1 Place and time study 3.2 Materials 3.2.1 Fish samples 3.2.2 Equipments 3.2.3 Medium and chemicals 3.3 Methods 10 3.3.1 Observation on wet-mount 10 3.3.2 Making the wet-mount 10 3.3.3 Isolation and identification 10 3.3.3.1 Isolation method 10 3.3.3.2 Identification method 11 3.4 Examination the effect of temperature and sodium chlorine on fungus 11 3.4.1 The effect of sodium chlorine 11 3.4.2 The effect of temperature 11 3.5 The challenge test 11 3.5.1 Collecting zoospores method 11 3.5.2 The challenge experiment 12 3.6 Examine the histopathology 12 Chapter IV: Results and Discussions 13 4.1 Clinical sign 13 4.2 Isolation and Identification 14 4.2.1 Observation on the wet-mount 14 4.2.2 Isolation 14 4.2.3 Identification 14 4.3 The effect of sodium chlorine on growth of fungus 17 4.4 The effect of temperature on growth of fungus 18 4.5 The challenge test 19 4.5.1 The clinical sign 19 4.5.2 The challenge test result 19 4.6 Histopathology 22 Chapter V: Conclusions and Recommendations 24 References 25 Appendices 28 LIST OF TABLES Table 4.1: The average of colony diameter of fungus in sodium chlorine test 18 Table 4.2: The average of colony diameter of fungus in temperature examination 19 Table 4.3: The infection rate of Fusarium sp on striped catfish 20 Table 4.4: The occurring frequency of Fusarium sp on striped catfish 20 LIST OF FIGURES Figure 4.1: The healthy fish and fungus disease fish in pond culture 13 Figure 4.2: Morphology characteristic of Fusarium sp 16 Figure 4.3: The characteristic of fish which using in the challenge test 21 Figure 4.4: The histopathology 23 CHAPTER I INTRODUCTION 1.1 Introduction Striped catfish (Pangasianodon hypophthalmus) is an importance species which is a high economy value, however, the farmers culture it with high density, and it is easy to make their fish get diseases There are many studies to search what kind of diseases which can infect to catfish General research is about pathology, the results that are catfish can be infected by bacterial, virus, parasites, or fungi Tu Thanh Dung, et al (2002) was found the bacteria disease on catfish that is very dangerous disease because it make more risk for farmers That bacteria is Edwarsiella ictaluri, it infect on liver, kidney of catfish and make many death fish Beside that, they also research to find the way to treat this disease There are many studies to search about this kind of bacteria, and show way how to know how many Edwarsiella ictaluri are infected on catfish (Nguyen Ngoc Dung and Dang Thi Hoang Oanh, 2010) Beside that, Nguyen Thi Thu Hang and Dang Thi Hoanh Oanh (2011) also found that catfish can be infected by Microsporidia; it is a group of parasite On the other hand, catfish can get disease from fungus, but there is no more research to study about that There is some research to find-out how fungi infect on aquatic animals In 2004, Kanit Chukanhom and Kishio Haitai were isolated freshwater fungi from eggs of common carp (Cyprinus carpio) in Thailand They were isolated some kind of fungus: Saprolegnia diclina, and Allomyces arbuscula Fungi can be caused disease for many species of fish such as: Snakehead (Channa striata), Climbing perch (Anabas testudineus) On study of Nguyen Thi Thuy Hang (2011), finding about the fungi which cause disease on Channa triata in fingerling stage with purposing is identify and put the forward to solve problem In 2010, Tran Ngoc Tuan and Pham Minh Duc have the study on fungus which infected on climbing perch That study focus on biology characteristic of fungal 4.6 Histopathology The histopathology was help this study to assert that the differences between healthy fish and fungus disease fish The fungus was not appearing on the swimming bladder of healthy fish In the reverse, the fungus was occurring on the swimming bladder of fish which infected by fungus According to Solangi and Lightner (1975), the Fusarium sp was infected on white shrimp (Penaeus setiferus) and brown shrimp (Penaeus aztecus) They showed that fungus was AA attacked on the gill lamella of two species in the histopathology samples with PAS staining method The single macroconidium was occurring after 4hrs to inject on the white shrimp, and 8hrs to inject on the brown shrimp On the study of Sosa et al., 2007, the researchers showed that the mycelia of Aphanomyces invadans fungus on fresh water fish which lived in estuarine in Florida when they observed on the histopathology samples that stained with periodic acid Schiff’s – hematocylin – metanil yellow The study indicated the free hyphae in the muscle of fish with no granulomatous response, and the granulomas surrounding hyphae According to Tuan (2010), the colonies granulomas of fungus also occurred on the muscle of climbing perch (Anabas testudineus) in all of fish which injected with fungal conidia On the study of Duc et al (2010), they were also found that the two genus of fungi Plectosporium oratosquillae and Acremonium sp that caused black spot on the gill filaments of kuruma prawn (Penaeus japonicus) by the histopathological examination They were using the Grocott-H&E staining method to reveal the numerous hyphae in the gill filaments at day 34 of NJM 0662, and day of NJM 0672 Necrosis of muscles and the encapsulation of hyphae were observed around the injection site on the prawn at day 45 or day 10 after inoculation The hyphae were not appearing in the control group of prawn B C D Figure 4.4: The histopathology (A) and (B): the swimming bladder of fish C C A B A without infected by fungus; (C): the numerous hyphae of fungus which infecting on fish (40X, showing by arrow); (D): the hyphae with septum in the swimming bladder of fish (100X, showing by arrow) H&E staining CHAPTER V CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions Fusarium sp was the fungi which isolated on the striped catfish (Pangasianodon hypophthalmus) in pond culture The fungi can cause diseases on the striped catfish (Pangasianodon hypophthalmus); it was only causing the infection rate from 37.90% to 48.10% for 105 of fungus conidia, and from 41.40% to 48.30% for 106 of fungus conidia The colonies of fungus can be infected of concentration of sodium chlorine on GYA medium, and temperature The average diameter of colony was from 2.89 cm to 5.61 cm for 0%-8% of concentration of sodium chlorine; and 2.21 – 5.50 cm for 20oC-30oC of temperature It depends on the difference of sodium chlorine concentration or temperature value, the average of fungus diameter was different The mycelia of fungi were occurring in swimming bladder because of Fusarium sp infected Therefore, the histopathological examinations of that organ show the mycelia which were clear with the H&E staining method 5.2 Recommendations Should be collected more fish samples which to evaluate exactly the infection ability of fungal on striped catfish (Pangasianodon hypophthalmus) The challenge test which should does more times with many difference concentrations of conidia to have exactly confirming about causing disease ability of fungus REFERENCES Alderman, and D.J Polglase, 1985 Fusarium tabacium (Beyma) Gams as a gill parasite in the crayfish, Austropotamobius pallipes Journal of Fish Diseases 8: 249-252 Altinok, I, and I Kurt, 2003 Molecular diagnosis of fish diseases: a review Turkish Journal of Fisheries and Aquatic Sciences 3:131-138 Atskins, S.D and I.M Clark, 2004 Fungal molecular diagnostics: a mini review J Appl Genet 45(1): 3-15 Book, 2002 Status of Pangasiid aquaculture in Vietnam Report prepared by the MRC Fisheries Programme at the request of Technical Advisory Body on Fisheries Management in the Lower Mekong Basin 16pp Burns, C D., M E Berrigan and G E Henderson, 1979 Fusarium sp infections is the freshwater prawn Macrobachium rosenbergii (De Man) Aquaculture 16: 193198 Chukahom, K., and Hatai, K., 2004 Freshwater fungi from eggs of the common carp (Cyprinus carpio) in Thailand Mycoscience 45:42-48 Diler, Ö and Y Bolat, 2001 Isolation of Acremonium sp from crayfish, Astacus leptodactylus in Egirdir Lake Bull Eur Ass Fish Pathology 21: 164-168 Duc, P M, 2009 Studies on fungal infection on mantis shrimp Oratosquilla oratoria due to anamorphic fungi PhD Thesis Nippon Veterinary and Animal Science University Duc, P M., Phuong, N T., Tuan, T N., 2010 Tong quan benh nam tren dong vat thuy san Tap chi khoa hoc Khoa Thuy San Truong Dai Hoc Can Tho Duc, P.M, and K Hatai, 2009 Pathogenictiy of anamorphic fungi Plectosporium oratosquillae and Acremonium sp to mantis shrimp, Oratosquilla oratoria Fish Pathology 44: 81-85 Duc, P.M., K Hatai, O Kurata, K Tensha, U Yoshitaka, T Yaguchi, S I Udagawa, 2009 Fungal infection of mantis shrimp, Oratosquilla oratoria caused by two anamorphic fungi found in Japan Mycopathology 167: 229-247 Ferguson, H W., Turnbull, J F., Thompson, K., Dung, T T., and Crumlish, M., 2001 Bacllary necrosis in farmed Pangasianodon hypophthalmus (Sauvage) from Mekong Delta, Vietnam Journal of Fish Diseases 24: 509-513 Gams, W., H A van der Aa, A.J van der Plaats-Niterink, R.A Samson and J.A Stalpers, 1980 CBS course of mycology Centraalbureau voor Schimmelcultures Baarn (The Netherlands), Institute of the Royal Netherlands, Academy of Science and Letters, Amsterdam-Zuid 109pp Hang, N T T., and Oanh, D T H., 2011 Ket qua nghien cuu buoc dau ve benh gao tren ca tra (Pangasianodon hypophthalmus) Ky yeu Hoi Nghi Khoa Hoc Thuy San lan 262-269 Hatai, K and S Egusa, 1979 Studies on the pathogenic fungus of mycotic granualomatosis-III Development of the medium for MG-fungus Fish Pathology 13(3): 147-152 (Japanese) Hatai, K., D Roza and T Nakayama, 2000 Identification of lower fungi isolated from larvae of mangrove crab, Scylla serrata, in Indonrsia Mycoscience 41: 565-572 Hatai, K., S Kida, S Udagawa, 1986 Fusarium oxysporum in red sea beam, Pagrus sp Journal Wildlife Diseasea 22: 570-571 Hemanand, T., N M Meitei, P K Bandyopadhyay and A K Mitra, 2008 A new species of Henneguya, a gill parasite of freshwater fish Anabas testudineus (Bloch) affected with ulcerative disease syndrome from Manipur, Idian Türkiye Parazitologi Dergisi 32(1): 82-85 Ho, W and W Ko, 1997 A Simple method for obtaining single spore isolates of fungi Bot Bull Acard Sin 38: 41-44 Huong, P T., Nam, N T., Dung, T T., and Tuan, N A., 2011 Su khang khang sinh cua vi khuan Edwardsiella ictaluri va Aeromonas hydrophila gay benh tren ca tra (Pangasianodon hypophthalmus) o dong bang song Cuu Long Ky yeu Hoi Nghi Khoa Hoc Thuy San lan 250-261 Hussein, M M A and K Hatai, 2002 Pathogenicity of Saprolegnia specises associated with outbreaks of salmonid saprolegniosis in Japan Fisheries science 68: 1067-1072 Hussein, M M A and K Hatai, 1999 Sprolegnia salmonis sp nov isolated from sockeye salmon, Onchhynchus nerka Mycoscience 40: 387-391 Johnson, R A., J Zabrecky, Y Kiryu and J D Shields, 2004 Infection experiments with Aphanomyces invadans in four species of estuarine fish Journal of Fish Diseases 27: 287-295 Kanchanakhan, S., U Saduakdee and S Areerat, 1999 Virus isolation from epizootic ulcerative syndrome diseased fishes Asian Fisheries Science 12: 327-335 Khoa, L V and K Hatai, 2005 First case of Fusarium oxysporum infection in culture Kuruma Prawn Penaeus japonicus in Japan Fish Pathology 40: 195-196 Khoa, L V, K Hatai, A Yasa and K Sawada, 2005 Morphologgy and molecular phylogeny of Fusarium solani from Kuruma prawn Penaeus japonicus with black gills Fish Pathology 40: 103-109 Khoa, L V., K Hatai, and T Aoki, 2004 Fusarium incarnatum isolated from black tiger shrimp Penaeus monodon Fabricius, with black gill disease cultured in Vietnam Journal of Fish Diseases 27: 507-515 Kitacharoen, N and K Hatai, 1997 Aphanomyces frigidophilus sp nov from eggs of Japan char, Salvelinus leucomaenis Mycoscience 38: 135-140 Kitacharoen, N., K Hatai, A Yamamoto, 1997 Aquatic fungi developing on eggs of salmonids Journal of aquatic animal health 9: 314-316 Lilley, J.H and S Chinabut, 1999 DNA-based studies on Aphanomyces invadans, the fungal pathogen of epizootic ulcerative syndrome (EUS) DNA-based molecular diagnostic techniques FAO Fisheries Technical Paper 395: 83-87 Marin, S., V Sanchis, N Magan, 1995 Water activity, temperature, and pH effects on growth of Fusarium moniliforme and Fusarium proliferatum isolated from maize Can Journal Microbiol 41(12): 1063-1070 Mobashir A Solangi and Donald V Lightner, 1975 Cellular inflammatory response of Penaeus aztecus and P setiferus to the pathogenic fungus, Fusarium sp., isolated from California brown shrimp (P californiensis) Journal of inventerbrate pathology 27: 77-86 Nam, N T., Huong, P T., Phuong, T D., and Dung, T T., 2010 Nghien cuu su da khang thuoc cua vi khuan Edwardsiella ictaluri gay benh tren ca tra (Pangasianodon hypophthalmus) Tap chi khoa hoc 14b: 200-210 Neish, G A., and Hughes, G C., 1980 Disease of fishes, Book 6, Fungal Diseases of Fishes T W F Publications, Neptune, New Jersey 159p Rhoobunjongde, W., K Hatai, S Wada and S Kubota, 1991 Fusarium moniliforme isolated from gills of kuruma prawn (Penaeus japonicus) with black gill disease Nippon Siusan Gakkaishi 57:629-635 Truong Thi Ho, Nontawith Areechon, Prapansak Srisapoome, and Songsri Mahasawasde, 2008 Identification and Antibiotic sensitivity test of bacteria isolated from Tra catfish (Pangasianodon hypophthalmus) cultured in pond in Vietnam Kasetsart J (Nat Sci.) 42: 54-60 APPENDICES Appendix 1: The strains of fungi which isolated on the fish samples Ordinal number Date Number of fish Place Strains of fungal 05/9/2012 An Giang AG1202, AG1205 19/09/2012 An Giang 5/10/2012 15 An Giang 27/10/2012 An Giang AG1204 AG1201, AG1208, AG1210 AG1203, AG1206 10/11/2012 10 An Giang AG1207, AG1201 16/11/2021 15 An Giang 28/11/2012 10 An Giang AG1204, AG1205 AG1201, AG1202, AG1209 Appendix 2: The steps to culture the fungi on lame (Riddles’s method) Step Contents Cutting small part of GYA media Moving the agar to the lame that put in the V shape of class rod, below that was the paper with some water, all of it to place on the Petri dish Culturing the fungi on the place of agar Putting the lamella on the agar Incubating it at 28OC in the incubator in days Moving the lamella to the other lame with one drop of cotton blue Placing it in some hours and using the paste to fix it Appendix 3: The steps to collect the zoospores for challenge test Step Contents Culturing the fungi on the GYA media at 28oC in days to get pure culture Adding 10 ml of sterile saline solution (0.85% NaCl) to the surface of fungal colonies, and breaking it by sterile loop to get conidia Filtering the conidia through two sterile layer of filter paper Determining the number of conidia by Hemacytometer Determining the concentration of conidia with 2.5 x 105 and 2.5 x 106 to inject on the fish Appendix 4: The effect of sodium chlorine (0%) on growth of fungi on GYA media (cm) Fungi strains Day Day Day Average SD Average SD Average SD AG1201 AG1204 AG1205 2.77 2.75 2.95 2.82 0.11 3.28 3.32 3.51 3.37 0.12 3.35 3.44 3.64 3.48 0.15 3.31 4.12 4.15 3.86 0.48 3.4 3.23 3.2 3.28 0.11 3.45 3.36 3.38 3.40 0.05 4.46 4.46 4.25 4.39 0.12 4.51 4.52 4.45 4.49 0.04 4.63 4.65 4.58 4.62 0.04 Appendix 5: The effect of sodium chlorine (2%) on growth of fungi on GYA media (cm) Fungi strains Day Average SD Average SD Average SD Day Day AG1201 AG1204 AG1205 3.49 3.58 3.76 3.61 0.14 3.52 3.60 3.79 3.64 0.14 3.64 3.69 3.72 3.68 0.04 5.51 5.6 5.43 5.51 0.09 5.64 5.69 5.55 5.63 0.07 5.69 5.74 5.63 5.69 0.06 4.2 4.35 4.29 4.28 0.08 4.25 4.42 4.34 4.34 0.09 4.32 4.47 4.38 4.39 0.08 Appendix 6: The effect of sodium chlorine (4%) on growth of fungi on GYA media (cm) Fungi strains Day Day Day Average SD Average SD Average SD AG1201 AG1204 AG1205 4.39 4.13 4.13 4.22 0.15 4.41 4.16 4.15 4.24 0.15 4.52 4.23 4.26 4.34 0.16 5.35 5.27 5.17 5.26 0.09 5.38 5.31 5.21 5.3 0.09 5.41 5.36 5.29 5.35 0.06 4.72 4.65 4.73 4.70 0.04 4.81 4.72 4.81 4.78 0.05 4.85 4.81 4.89 4.85 0.04 Appendix 7: The effect of sodium chlorine (6%) on growth of fungi on GYA media (cm) Fungi strains Day Day Day Average SD Average SD Average SD AG1201 AG1204 AG1205 3.14 3.19 3.27 3.20 0.07 3.16 3.22 3.30 3.23 0.07 3.22 3.28 3.35 3.28 0.07 3.34 3.25 3.23 3.27 0.06 3.41 3.32 3.31 3.35 0.06 3.48 3.39 3.38 3.42 0.06 5.17 5.23 5.25 5.22 0.04 5.21 5.29 5.31 5.27 0.05 5.28 5.32 5.38 5.33 0.05 Appendix 8: The effect of sodium chlorine (8%) on growth of fungi on GYA media (cm) Fungi strains Day Day Day Average SD Average SD AG1201 AG1204 AG1205 2.76 2.79 2.87 2.81 0.06 2.80 2.91 2.90 2.87 0.06 2.96 3.02 3.01 4.96 4.78 4.84 4.86 0.09 5.01 4.81 4.91 4.91 0.10 5.12 4.89 5.01 5.35 5.4 5.41 5.39 0.03 5.39 5.43 5.46 5.43 0.04 5.43 5.46 5.48 3.00 0.03 Average SD 5.01 0.12 5.46 0.03 Appendix 9: The effect of temperature on growth of fungi at 20oC (cm) Strain Day Average SD Day Average SD Day Average SD AG1201 2.14 2.18 2.18 2.17 0.02 2.18 2.23 2.24 2.22 0.03 2.21 2.26 2.28 2.25 0.04 AG1204 2.24 2.21 2.26 2.24 0.03 2.27 2.25 2.29 2.27 0.02 2.31 2.28 2.31 2.3 0.02 AG1205 2.45 2.52 2.52 2.50 0.04 2.48 2.58 2.59 2.55 0.06 2.51 2.61 2.62 2.58 0.06 Appendix 10: The effect of temperature on growth of fungi at 25oC (cm) Strain Day Average SD Day Average SD Day Average SD AG1201 3.84 3.76 3.79 3.80 0.04 4.05 4.1 4.13 4.09 0.04 4.25 4.19 4.21 4.22 0.03 AG1204 4.25 4.16 4.14 4.18 0.06 4.31 4.24 4.23 4.26 0.04 4.39 4.29 4.31 4.33 0.05 AG1205 5.45 5.37 5.43 5.42 0.04 5.52 5.44 5.56 5.51 0.06 5.59 5.51 5.64 5.58 0.07 Appendix 11: The effect of temperature on growth of fungi at 28oC (cm) Strain Day Average SD Day Average SD Day Average SD AG1201 4.37 4.31 4.29 4.32 0.04 4.41 4.38 4.34 4.38 0.04 4.46 4.42 4.38 4.42 0.04 AG1204 4.15 4.26 4.22 4.21 0.06 4.21 4.3 4.28 4.26 0.05 4.29 4.37 4.32 4.33 0.04 AG1205 4.46 4.46 4.25 4.39 0.12 4.5 4.52 4.31 4.44 0.12 4.52 4.57 4.35 4.48 0.12 Appendix 12: The effect of temperature on growth of fungi at 30oC (cm) Strain Day Average SD Day Average SD Day Average SD AG1201 3.25 3.36 3.28 3.30 0.06 3.31 3.42 3.35 3.36 0.06 3.38 3.49 3.41 3.43 0.06 AG1204 3.87 4.16 3.75 3.93 0.21 3.89 4.2 3.86 3.98 0.19 3.91 4.24 3.89 4.01 0.20 AG1205 3.62 3.58 3.55 3.58 0.04 3.68 3.62 3.59 3.63 0.05 3.71 3.65 3.63 3.66 0.04 Appendix 13: The procedure of histopathogy Step Contents Fixing sample The samples are fixed in NPF solution (NaH2PO4: 4.4g; Na2HPO4: 6.5g; Formalin: 100ml; DW: 900ml) for 24 hours Conducting clean under tap water for 10 minutes Then transferring to the 70% alcohol for preservation and handling of samples Trimming and orienting The samples trimmed and oriented for suitable size Cutting samples with a thickness of 5mm Putting the sample into catseet and processing Sample processing procedures Sample processing procedure is done on Tissue processing (MICROM, STP 120-2) Procedure is presented in Table 3.1 Casting blocks The sample is placed and positioned into a fixed mold of stainless steel Putting the molten paraffin at 65°C (paraffin: beeswax 7:3 ratio) on mold for fixed Filling mold by paraffin and put casseet with symbol of sample on mold Solidifying paraffin by placing in the refrigerator then separating paraffin blocks from the mold Cutting samples and putting the sample to lame Using Microtome machine to cut the sample, the thickness of slice is µm Putting it in a basin of warm water (45-50°C) for deparaffination Putting one sight of lame in a basin of warm water at an angle of 45 º and lift slowly Drying the slide at 45 ºC-50ºC Staining samples Samples were stained by the process of Harris's Haematoxylin & Eosin Pasting lamelle to lame Using Entellan colloid to coat on the sample and paste lamelle on sample Dripping a small drop of colloid on the sample Putting lamelle tilted 45º and in contact with drop of colloid Putting lame down slowly to avoid air bubbles Reading results Observing the sample under a microscope at magnification 40X and 100X (drip oil) Identifying hyphae and spores on the samples and taking photos Appendix 14: The procedure for processing sample Chemicals Time Alcohol 80º 15 minutes Alcohol 95º 15 minutes Alcohol 95º 20 minutes Alcohol 100º 20 minutes Alcohol 100º 30 minutes Alcohol 100º 30 minutes Xylen 40 minutes Xylen 20 minutes Xylen 30 minutes Paraffin:Xylene (1:1) 40 minutes Paraffin: Beeswax (1:1) 50 minutes Paraffin:Beeswax(7:3) 60 minutes Appendix 15: The procedure for staining samples (Harris’s Haematoxylin & Eosin) Chemicals Time Xylen minutes Xylen minutes Alcohol 100º minutes Alcohol 100º minutes Alcohol 90º minute Alcohol 80º minute Alcohol 70º minutes Tap water 30 seconds Haematoxylin minutes Running TW 15 minutes Eosin minutes Tap water 10 seconds Alcohol 80º minutes Alcohol 95º minutes Alcohol 100º minutes Alcohol 100º minutes Xylen minutes Xylen minutes [...]...Study on fungus in Vietnam is not more, that is the reason why the research Studies on fungus infection on striped Catfish (Pangasianodon hypophthalmus) in grow – out stage was done 1.2 Objective of study The aim is to describe isolation and identification of causative agents of fungal infection found in the striped catfish (Pangasianodon hypophthalmus) in grow- out stage Beside that,... be bringing some things new to the studying about the disease on striped catfish 4.3 The effect of sodium chlorine on growth of fungi Concentration of sodium chlorine infected on fungus which grew on the GYA medium in 6 days That was showed on the table 4.1 Each of fungus was different growth such as: AG1201 was growth best in 4% of concentration of sodium chlorine, and lowest in 8% of concentration... fungi which isolated on the striped catfish (Pangasianodon hypophthalmus) in pond culture The fungi can cause diseases on the striped catfish (Pangasianodon hypophthalmus); it was only causing the infection rate from 37.90% to 48.10% for 105 of fungus conidia, and from 41.40% to 48.30% for 106 of fungus conidia The colonies of fungus can be infected of concentration of sodium chlorine on GYA medium, and... laboratory condition to check the infection ability of that fungus in four weeks Fish which injected with macroconidia was isolated three organs: swimming bladder, kidney, and gill to check the occurring of fungus The fungus was only appearing on the treatments group, was not have the fungus on the control group The result shows that the infection rate was 0% on the non- treatment group, and the infection rate... macroconidia of fungus with cotton blue staining (arrows showing) 4.2 Isolation and identification 4.2.1 Observation on the wet-mount The sampling was checked the parasite, the occurring of the fungus that was infected on the fish before isolating the fungus Parasite has infected on the fish, but it was only 2-3 individuals per lame That parasite which attacked on striped catfish was Trichodina spp... also determines the probability to cause disease of fungus which is the basic to prevent the fungus disease on striped catfish 1.3 Research contents of study o To isolate and identify fungal infection in striped catfish disease o To examine the effects temperature and sodium chlorine on the growth of fungus strains o To observe histological characteristics of striped catfish with fungus infection CHAPTER... appearing in the control group of prawn B C D Figure 4.4: The histopathology (A) and (B): the swimming bladder of fish C C A B A without infected by fungus; (C): the numerous hyphae of fungus which infecting on fish (40X, showing by arrow); (D): the hyphae with septum in the swimming bladder of fish (100X, showing by arrow) H&E staining CHAPTER V CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions Fusarium... staining with cotton blue (100X; showing of arrow); (E): The stalk of fungus with cotton blue staining (40X; showing of arrow); (F): The stalk of fungus with cotton blue staining (100X, showing of arrow) Following that morphology characteristic and the type of colony of the fungus, and combine with the identification system of de Hoog et al (2000), the genus of Fusarium sp which isolated on striped catfish. .. culture the fungus This study was also used some the chemicals which was saline solution using to treat the specimen before isolation; cotton blue using to stain the sample to find out the mycelium on the fish or observing the characteristic of mycelium Some drugs were also using to inhibit the other agents to grow on the culture of fungus: ampicillin, streptomycin 3.3 Methods 3.3.1 Observation on wet-mount... japonicus was infected by Fusarium sp to cause the black spots on the gills In 1974, Johnson has observed the infection of Fusarium sp on the pink shrimp (P durorarum) in the laboratory in Texas, the research was only showed that the infection of fungi, did not give the mortalities rate However, that was also given one of the important information for the next researches on the marine aquatic species In American, ... AQUACULTURE AND FISHERIES STUDIES ON INFECTION OF FUNGUS IN STRIPED CATFISH (Pangasianodon hypophthalmus) ON GROW - OUT STAGE By NGO THI MONG TRINH A thesis submitted in partial fulfillment of... will be bringing some things new to the studying about the disease on striped catfish 4.3 The effect of sodium chlorine on growth of fungi Concentration of sodium chlorine infected on fungus which... Studies on fungus infection on striped Catfish (Pangasianodon hypophthalmus) in grow – out stage was done 1.2 Objective of study The aim is to describe isolation and identification of causative