Enumerating algal viruses by flow cytometry
FCM Sample Collection .
Fix samples of virus and TE buffer ( i . e . , for background noise ) as described below .
Transfer 600 µL of culture sample to a sterile 1.2 mL cryovial tube .
Add 6 µL 25 % glutaraldehyde ( 0.25 % final concentration ) and gently vortex to mix .
Aliquot 200 µL to two additional cryovials ( i . e . , triplicate samples ) .
Transfer 3 x 1 mL of 0.02 - µm - filtered TE buffer to triplicate sterile 1.2 mL cryovials .
Add 10 µL 25 % glut to each tube and gently vortex to mix .
Snap cryovials into cryocanes and incubate at 4°C for 30 minutes in the dark .
Flash freeze in liquid nitrogen .
Store at - 80°C until analysis .
Virus Dilution and Counting
Dilute and stain a sample aliquot for viruses immediately after thawing .
Store thawed sample on ice .
An aliquot can be run for host cell counts while the diluted virus sample is incubating .
Dilute sample in 0.02 - µm - filtered 1X TE buffer according to the Dilution Table .
NOTE : For a new sample , prepare multiple dilutions to test and determine the most appropriate dilution factor .
Stagger these dilutions / staining by ~ 5 minutes to allow for sample loading and back - flushing between dilution tests .
Prepare a 1.7 mL microcentrifuge tube for staining by adding 2.5 µL of 100X SYBR Green I nucleic acid stain ( this can be pre - aliquoted and kept in the dark at room temperature ) .
Add 497.5 µL of the diluted sample to the staining tube ( 0.5X SYBR Green I final conc ) and mix by pipetting and gentle vortexing .
Incubate the diluted / stained sample for 15 minutes in the dark at room temperature .
After incubation , transfer 480 µL of the stained sample to a 5 mL round bottom tube .
Add 10 µL 3° Grn and 10 µL 4° YG beads to the 5 mL tube .
Run immediately on HIGH voltage settings : Trigger = 520 , TFSC = 40 , PFSC = 27 , SSC = 40 , 520 = 40 , 572 = 40 , 692 = 40 .
Load sample for 2 min , run for 2 - 4 min ( depending on concentration ) , and back - flush for 2 min .
Host Cell Counts .
Run an unstained aliquot of sample for host cell counts while the diluted virus sample is incubating .
Prepare a 5 mL round bottom tube with 10 µL 3° Grn and 10 µL 4° YG beads .
Depending on the host cell concentration , add 50 or 100 µL sample to the 5 mL tube .
Bring volume up to 500 µL by adding 430 or 380 µL 1X PBS to the 5 mL tube .
Run immediately on LOW voltage settings : Trigger = TFSC , TFSC = 27 , PFSC = 19 , SSC = 40 , 520 = 40 , 572 = 40 , 692 = 40 .
Load sample for 2 min , run for 2 - 4 min ( depending on concentration ) , and back - flush for 2 min .
