Prophage induction in marine Synechococcus
Filter each sample through a 0.2 - µm filter to a volume of approximately 5 mL to remove most of the ambient viruses .
Add virus - free ( 0.02 - µm filtered ) water prepared from the same sample and reduce the volume a second time .
Return the retentate to its original volume by adding virus - free seawater , dividing into aliquots , and incubating with and without inducing agent .
Amend treated samples with the inducing agent mitomycin C at a concentration of 1 µg / mL or with the inducing agent of choice .
Employ the most probable number ( MPN ) method to enumerate the cyanophage population ( Suttle and Chan 1994 ) .
Prepare a one - to five - dilution series of the environmental or prophage induction treatment sample using 96 - well microtiter plates ( Costar , Corning Inc . ) .
Freshly dilute a susceptible Synechococcus host 1 : 10 and place in each well .
Prepare control plates similarly using sterile SN media in the first column of wells .
Prepare three replicate treatment and control plates from each site .
Incubate the plates until good growth of the host organism is evident ( 10–14 days ) .
Score wells as positive for virus if lysis of the host organism is evident as a well clearing .
Calculate viral abundance for each plate using an MPN program ( Hurley and Roscoe 1983 ) .
Evaluate treatment and control cyanophage and Synechococcus counts by paired t test between samples using Minitab statistical software .
Perform comparison of induction results and environmental parameters using linear regression and X2 analysis , also using Minitab .
