TITLE: No Title
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[DESCRIPTION]
In plaque-assay based viral discovery many more plaques are often generated than can be examined at any one time, it is therefore of value to be able to archive plaques for future investigation. To address the potential for differences among viruses in tolerance to storage, this protocol provides large-scale archival approaches that include storage of picked plaques at both 4°C and -20°C or -80°C. We note however, that viruses differ in their sensitivity to storage conditions (Clark 1962 [1]; Fortier and Moineau 2009 [2]) and, though we describe methods that include multiple conditions, we highlight also that including additional approaches, such as storage in infected cells (Golec et al. 2011 [3]), is likely to increase total proportion of recovered viruses in large collections.



[1]  Clark, W.A. (1962). Comparison of Several Methods for Preserving Bacteriophages. Appl. Microbiol. 10, 466–471.
[2]  Fortier, L.-C., and Moineau, S. (2009). Phage Production and Maintenance of Stocks, Including Expected Stock Lifetimes. In Bacteriophages, M.R.J. Clokie, and A.M. Kropinski, eds. (Humana Press), pp. 203–219.  
[3] Golec, P., Dąbrowski, K., Hejnowicz, M.S., Gozdek, A., Łoś, J.M., Węgrzyn, G., Łobocka, M.B., and Łoś, M. (2011). A reliable method for storage of tailed phages. Journal of Microbiological Methods 84, 486–489.

[BEFORE_START]
Materials for plaque archival procedures

 Virus material: Agar overlay plates with plaques to be archived   Archival and filtration materials:
Sterile host growth media (250uL per plaque)
Sterile 50% glycerol solution (50:50 glycerol:host growth media; 125uL per plaque)

 When opting for centrifugation-based filtration:
96-well polypropylene microplates for collection and as filtration receivers (Greiner Bio-One #651261); requires two 96-well plates per every 48 plaques.
96-well filter-bottom plates, 0.22um (EMD Millipore MSGVS2210) or 0.45um (EMD Millipore MSHVS4510); requires one 96-well plate per every 48 plaques.
Centrifuge that can accommodate 96-well plates
Sterile adhesive 96-well plate aluminum foil covers

 When opting instead for vacuum-based filtration:
Users will need to ensure compatibility of selected manifold, filter-bottom plates, and receiver plates. The below items are an example of compatible components.
96-well polypropylene microplates for collection and as filtration receivers (Greiner Bio-One #651261); requires two 96-well plates per every 48 plaques.
96-well filter-bottom plates, 0.22um (EMD Millipore MSGVS2210) or 0.45um (EMD Millipore MSHVS4510); requires one 96-well plate per every 48 plaques.
MultiScreenHTS Vacuum Manifold for 96-well plate format (EMD Millipore MSVMHTS00)
Vacuum pump
Sterile adhesive 96-well plate aluminum foil covers
 
Cataloging spreadsheet: see template example document provided with this protocol - "Supplementary File 1" from the MethodsX publication.

[GUIDELINES]
The described procedures are of greatest utility when the number of plaques to be archived is on the order of several hundreds - and it is then optimal to divide the tasks of cataloging plaques and physically archiving them over three consecutive days, this is how the protocol is presented here.

[STEPS]
SECTION: Cataloging Plaques (Day 1)
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SECTION: Cataloging Plaques (Day 1)
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SECTION: Collecting Plaque Plugs (Day 2)
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SECTION: Collecting Plaque Plugs (Day 2)
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SECTION: Processing Plaque Plugs (Day 3)
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SECTION: Processing Plaque Plugs (Day 3)
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SECTION: Processing Plaque Plugs (Day 3)
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SECTION: Processing Plaque Plugs (Day 3)
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SECTION: Processing Plaque Plugs (Day 3)
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SECTION: Recovering viruses from archives (future)
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