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 ) .
