Plating Prochlorococcus and Synechococcus strains in top agarose for plaque assays
Prepare base plates using 0.4 % GIBCO LM agarose ( 0.4g agarose / 100 ml ) .
Make up plates using filtered , autoclaved 75 % Sargasso Sea Water ( SSW ) .
Add appropriate agarose , then microwave to a boil .
Add appropriate nutrients to bring levels up to media of choice ( SN , Pro99 ? )
Vortex / shake to mix nutrients thoroughly and asceptically pour plates in hood .
Allow plates to sit overnight to solidify before adding top agarose .
Prepare your cyanophage dilutions ahead of plating cells in top agarose by using media for dilutions in 5 ml Falcon tubes .
Prepare top agarose as 0.5 % GIBCO .
Low - Melt agarose .
Use filtered , autoclaved 75 % SSW to make up plates .
Add appropriate agarose ( 0.5 g / 100 ml SSW ) and microwave as above .
Allow to cool in a pre - heated water bath to ~ 29°C ( solidifies ~ 24 - 28°C ) .
Asceptically add filter sterilized nutrients to bring level of top agarose nutrients to desired media of choice .
Add appropriate volume of cells to top agarose , vortex to thoroughly mix and plate immediately .
Inoculate a liquid culture using the same dilution chosen for the plates as an indicator of when the plates might turn green ( within a few days of each other ) .
Incubate all plates overnight at ~ 2 - 5 uE light without parafilming the plates to allow the top agarose to solidify completely .
After the o / n incubation parafilm the plates ( humidity control ) and place at the appropriate light levels
