MPN (Most Probable Number) assay for infectivity of algal viruses
Virus Dilution Series.
Label a series of 5 mL round bottom tubes from 10-1 to 10-10.
Aliquot 1.8 mL culture media to each tube.
Dilute 200 µL virus sample into the “10-1” tube and vortex to mix.
Use a clean pipette tip to transfer 200 µL from the “10-1” tube to the “10-2” tube and vortex to mix.
Repeat serial dilution to 10-10.
Transfer 500 µL virus sample to a sterile 1.2 mL cryovial (to preserve for FCM counts).
In the chemical hood, add 5 µL 25% glutaraldehyde (0.25% final concentration) and gently vortex to mix.
Aliquot 250 µL to a duplicate cryovial and snap cryovials into cryocanes.
Incubate at 4°C for 30 minutes in the dark.
Flash freeze in liquid nitrogen and store at -80°C until analysis.
Plate Setup.
Pour remaining culture medium into a sterile sample reservoir.
Use a multichannel pipette to add 50 µL medium to all wells in Column 9 (“Control”) on all plates.
Discard unused medium and fill sample reservoir with host culture.
Add 150 µL host cells to all wells in Columns 1-9 on all plates.
Discard remaining culture.
Pour “10-10” viral dilution into new sterile sample reservoir.
Use a multichannel pipette to add 50 µL 10-10-diluted virus sample to all wells in Column 8 on all replicate plates.
Discard remaining 10-10-diluted virus sample and pour “10-9” viral dilution into the same sample reservoir.
Use the same pipette tips to add 50 µL 10-9-diluted virus sample to all wells in Column 7 on all plates.
Repeat additions of diluted virus samples from most dilute to most concentrated (moving from right to left across the microplates).
After final 10-3-diluted virus sample is added to plates, measure optical density for T0 in plate reader (as described below) and incubate (unstacked to prevent shading) at standard growth conditions for ~2 weeks, measuring growth every few days.
Data Collection.
Turn on the Molecular Devices SpectraMax 340PC plate reader.
Log into the attached computer and open the SoftMax Pro 6 software.
Open or create a new “Basic Endpoint” protocol file (.spr).
Rename the experiment appropriately and configure a plate with the following settings:Read Type: Endpoint Wavelength: 750 nm Plate Type: 96-well standard clear bottom Read Area: All Path check: Calibration on Shake: Once for 3 sec.
More settings: Column priority.
Add “New plates” as needed so there is one for each replicate plate in your assay (settings will copy to these new plates within the same experiment).
If the SpectraMax doesn’t automatically connect, click on the instrument icon in the top left corner and manually select it from the menu.
Select the plate to be read in the software, and place the corresponding plate on the plate reader drawer (with Column 1 closest to the instrument).
Remove the plate lid and select “Read” to read the plate.
Read each plate individually, and copy the data (must right-click to do this) into an Excel spreadsheet.
Create a new data file (.sda) from the same master protocol for each time point.
Visually inspect plates and record observations.
The “Dilution Factor” is the dilution ratio used for inoculating the wells of that column.
The “Volume” is the volume of the dilution added to each well in that column.
Dilution Factor: Use results from a subset of the serial dilutions such that all host wells were lysed in the most concentrated of the dilutions (i.e., 24 of 24 wells were positive for viral activity) and all host wells were healthy in the most dilute of the dilutions (i.e., 0 of 24 wells were positive for viral activity).