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Pathogenesis of Duck Plague. Ii. Standardization of Primary Cell Culture Technique for Propagating Duck Plague Virus

M.R. ISLAM
Department of Pathology, Bangladesh Agricultural University, Mymensingh

Abstract/ Executive Summary:

A suitable protocol and optimum conditions for the preparation of primary duck embryo fibroblast (DEF) cell culture have been standardized.

Keywords: Cell culture, Duck embryo fibroblast cell, Duck plague virus

Location: Department of Pathology, Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Duck

Objectives:

To standardize the technique for preparation of primary duck embryo fibroblast (DEF) cell culture with an immediate goal of growing duck plague virus (DPV) in cell culture.

Methodology:

Duck eggs: Fertile duck eggs were obtained from Bangladesh Agricultural University Poultry Farm. Tissue culture medium: The working medium was prepared in double distilled water from a 10x concentrated stock of M 199 Medium (with Earle's salts, L-glutamine) and supplemented with 10% tryptose phosphate broth and 80 µg gentamycine and 100 iu nystatin per ml. The medium was buffered with 10 mM NaHC0₃and 14 mM HEPES to a pH of 7.2 to 7.4. Newborn calf serum was added just before use at the rate of 10% to the growth medium and 2% to the maintenance medium. Cell culture protocol: The DEF cell culture was prepared by cold trypsinization method following the procedure described by Freshney (1983) with some modifications. Briefly, under a strict aseptic condition, 12-days old duck embryos were chopped into small pieces, washed 3 times with Dulbecco's PBS-A and treated overnight with 0.05% trypsin at 4°C. In the next morning, most of the trypsin solution was discarded and the tissue pieces were incubated for 30 minutes at 37°C in a water bath. Then a small amount of cold growth medium was added to the tissues and the cells were disaggregated by gentle stirring for 1 5 minutes on a magnetic strirrer. The cell suspension was collected, number of cells were counted, adjusted to required concentration and dispensed to culture bottles. The cells were incubated at 38°C. Optimization of culture conditions: The optimum environment for the growth of DEF cells was created on the basis of published literature. However, the required concentration of calf serum in the growth medium and the optimum cell-seeding rate were determined experimentally. For this, different concentrations of calf serum including 2%,5%,10% and 15% and different cell seeding rate, such as 0.25, 0.5, 1, 2, and 3 million cellslml, were compared for their effects on cell growth. Staining of cell monolayers : The cells were normally examined live under a microscope. However, for more detailed examination the cells were grown on cover slip in a petridish. When confluent, the cell sheath was fixed in Bouin's fluid and stained with Harris' hematoxylin and eosin.

Source of Information: BAU Res. Prog. 7: 474-477; 1993

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

A suitable protocol and optimum conditions for the preparation of primary duck embryo fibroblast (DEF) cell culture have been standardized.

Knowledge Management: Report/Proceedings