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 CountingDilute 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.