FISH PATHOLOGY SECTION LABORATORY MANUAL Edited by Theodore R. Meyers, Ph.D. Special Publication No. 12 2nd Edition Alaska Department of Fish and Game Commercial Fisheries Division P.O. Box 25526 Juneau, Alaska 99802-5526 January 2000 Rev. 03/98 i TABLE OF CONTENTS (continued) TABLE OF CONTENTS PREFACE .............................................................................................................................v CHAPTER/TITLE Page .................................................................................................................... 1. Sample Collection and Submission.............................................................................1-1 to 1-8 I. Finfish Diagnostics....................................................................................................... 1-1 II. Finfish Bacteriology ..................................................................................................... 1-2 III. Virology ........................................................................................................................ 1-3 IV. Fluorescent Antibody Test (FAT) ................................................................................ 1-4 V. ELISA Sampling of Kidneys for the BKD Agent (see ELISA Chapter 9).................... 1-5 VI. Parasitology and General Necropsy ........................................................................... 1-5 VII. Histology ...................................................................................................................... 1-5 VIII. Sample Shipment Instructions..................................................................................... 1-6 2. Materials List for Sample Submission....................................................................2-1 to 2-2 3. Standard Necropsy Procedures for Finfish ...........................................................3-1 to 3-6 I. General Necropsy Procedure...................................................................................... 3-1 II. Staining Procedures .................................................................................................... 3-5 4. Bacteriology 4-1 to 4-24 ............................................................................................................ I. Media Preparation ....................................................................................................... 4-1 II. Media Formulations ..................................................................................................... 4-2 III. Stains and Reagents ................................................................................................... 4-6 IV. Test Descriptions ......................................................................................................... 4-7 V. Summary/Storage of Quality Control Organisms for Bacteriology Test Media........4-16 VI. Commercial Identification Systems ...........................................................................4-16 VII. Biochemical Characteristics of Common Bacterial Fish Pathogens ........................4-17 VIII. Bacterial Fish Diseases: Causative Agents and Signs............................................4-18 IX. References.................................................................................................................4-20 X. Appendices ................................................................................................................4-21 5. Virology and Cell Culture........................................................................................5-1 to 5-44 I. Suggested Tissue Types and Sample Sizes .............................................................. 5-1 II. Collecting Ovarian Fluid, Whole Fish, and Tissue Samples in the Field.................... 5-2 III. Maintenance of Stock Cell Lines-Passage of Confluent Cell Monolayers................. 5-3 IV. Cell Counting Using the Hemocytometer.................................................................... 5-5 V. Incubating Cell Lines ................................................................................................... 5-7 VI. Storing Tissue Culture Cells........................................................................................ 5-7 VII. Detecting and Avoiding Tissue Culture Contaminants ............................................... 5-8 VIII. Mycoplasma Screening of Continuous Cell Lines ...................................................... 5-9 IX. Processing Ovarian Fluid, Whole Fish, and Tissue Samples ..................................5-11 X. Cytopathic Effects (CPE) of Virus Infection in Tissue Culture Cells.........................5-13 XI. Plaque Assay.............................................................................................................5-14 XII. Quantal Assay` ..........................................................................................................5-18 XIII. Storing: Freezing and Thawing Virus Isolates ..........................................................5-21 XIV. IHNV Concentration in Water Samples.....................................................................5-22 XV. Alkaline Phosphatase Immunohistochemical Procedure (APIH) .............................5-24 XVI. Biotinylated DNA Probe.............................................................................................5-25 XVII. Plaque Reduction Serum Neutralization Assay........................................................5-31 XVIII.Fluorescent Antibody Staining....................................................................................5-34 Rev. 03/98 i TABLE OF CONTENTS (continued) CHAPTER/TITLE Page 5. Virology and Cell Culture (continued) XIX. Washing Glassware...................................................................................................5-34 XX. Media .......................................................................................................................5-35 XXI. Appendix ....................................................................................................................5-40 XXII. References..................................................................................................................5-40 XXIII. Glossary .....................................................................................................................5-42 6. Histology for Finfish and Shellfish........................................................................6-1 to 6-19 I. II. Fixation and Decalcification.......................................................................................6-10 III. Tissue Dehydration and Infiltration (all tissues) ........................................................6-12 IV. Embedding Tissues into Paraffin Blocks...................................................................6-12 V. Cutting Paraffin Blocks and Mounting Sections on Glass Slides .............................6-13 VI. Routine Staining of Paraffin Sections – Hematoxylin and Eosin..............................6-14 VII. References.................................................................................................................6-16 7. Transmission Electron Microscopy ........................................................................7-1 to 7-8 I. Fixation and Embedment of Tissues from Vertebrates .............................................. 7-1 II. Fixation and Embedment of Tissues from Marine Invertebrates................................ 7-2 III. Retrieval and Embedment of Cut Sections from Histological Slides.......................... 7-2 IV. Negative Staining of Virus Particles ............................................................................ 7-3 V. Staining Thick Sections for Light Microscopy ............................................................. 7-4 VI. Staining Thin Sections for TEM................................................................................... 7-4 VII. Reagents...................................................................................................................... 7-5 VIII. References................................................................................................................... 7-7 8. Fluorescent Antibody Staining for Bacteria and Viruses...................................8-1 to 8-12 I. Fluorescent Antibody Methods for Bacteria................................................................ 8-1 II. Microwell Fluorescent Antibody Test for IHNV ........................................................... 8-7 III. Ovarian Fluid Filtration FAT......................................................................................... 8-8 IV. Reagents....................................................................................................................8-10 V. References.................................................................................................................8-12 9. ELISA for the Detection of Antigen of Renibacterium salmoninarum .............9-1 to 9-30 I. Reagents...................................................................................................................... 9-1 II. III. ELISA Materials and Equipment ............................................................................... 9-9 IV. Raw Sample Preparation...........................................................................................9-12 V. ELISA Preparation and Performance........................................................................9-16 VI.