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Diluent
Eagle's Minimum Essential Medium (Eagle's MEM)
This can be formulated according to standard tissue culture protocols or, alternatively, supplied complete as Minimum Essential Medium Eagle (HEPES Modification), from Sigma Ltd (500 ml).
Disinfectant solution
0.4% FAM in tap water, made up daily,
or
0.2% citric acid in tap water.
Preparation of test samples
Sera may arrive as whole bloods in vacutainers. To separate the serum, either leave overnight at +4°C and tip off into bottles or centrifuge the tubes in a bench centrifuge at approximately 600g (1-3000 rpm) for 5-10 minutes. In the latter case, pour off the serum directly into a bottle or use a pipette. Always ensure contamination from less tightly packed loose red blood cells is kept to a minimum. Label each serum.
For testing, sera (and reference control sera) are diluted 1:4 in diluent and heat inactivated at 56°C for 30-45 minutes. Samples are held at 4°C until required.
If sera are to be kept for some time before testing they should be stored undiluted at -20°C.
Tissue culture
Appropriate cell cultures, grown as monolayers in tubes or flasks, should be prepared as required in a dedicated tissue culture facility. Description of tissue culture methodology and procedures is beyond the scope of this module. Any suitably sensitive cell line may be used. At the WRL for FMD IBRS-2 cells are used as a routine although BHK cells are an alternative. It may be necessary to passage the virus several times on the selected cell line to achieve a suitable level of adaptation.
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