Infectious Pancreatic Necrosis Virus (IPNV) enumeration through epifluorescence microscopy: technical aspects
Maria Soledad Pavlov1 · Yoanna Eissler*1 · Juan Carlos Espinoza1 · David E. Garland2 · Juan Kuznar1
Investigación y Gestión de Recursos Naturales, Facultad de Ciencias,
Universidad de Valparaíso, Valparaíso, Chile
*Corresponding author: firstname.lastname@example.org
Financial support: Innova Chile/CORFO, grant 05 CT6IPD-22 (2008-2011) and Universidad de Valparaíso, Chile, grant DIPUV 11/2007.
Keywords: digital image analysis, fluorescence microscope methods, immunofluorescence, Infectious Pancreatic Necrosis Virus (IPNV), SYBR-Green I.
A method for counting Infectious Pancreatic Necrosis Virus (IPNV) through epifluorescence microscopy was analyzed in detail. Image processing and statistic considerations are included. The particle size of viruses was compared in different experimental conditions such as the staining of the virus with SYBR-Green I or with antibodies for specific fluorescence labeling of viral proteins. The type of surface used as mounting support was assayed as well. The results indicated that the most suitable method involves the mounting of the viral-containing suspension on a membrane filter followed by the staining with a monoclonal antibody specific for a viral protein combined with a FITC (fluorescein isothiocyanate)-conjugated secondary antibody.