1) Inoculate flasks with NC64A Chlorella in MBBM (or Pbi in FES, SAG 241-80 in MBBM) and incubate for several days at 25C with continuous light and shaking.

2) Infect flasks of Chlorella with virus at an moi of 0.01 to 0.001. Incubate flasks for 48-72 hours at 25C with continuous light and shaking. This material is now termed "lysate".

3) Centrifuge lysate in the Sorvall GSA rotor at 5,000 rpm, 5 min, 4C. Discard pellets.

4) Add NP-40 to lysate supernatants to a final concentration of 1% (from a stock of 10% or 20%). This solubilizes the green pigment in the supernatant. If NP-40 is not available, Triton X-100 may be substituted at the same concentration as NP-40.

5) Centrifuge the lysate in the Beckman #19 rotor at 17,000 rpm, 50 min, 4C. Alternatively, centrifuge the lysate in Beckman Ti50.2 rotors (or #30 rotors) at 20,000 rpm, 60 min, 4C. Discard supernatants.

6) Resuspend virus pellets in a small volume of 50 mM Tris-HCl, pH 7.8 (approximately 1.0 mL per 100 mL of original lysate).

7) Layer the virus suspension onto 100-400 mg/mL linear sucrose density gradients made up in Beckman SW28 rotor tubes (layer approximately 4.0 mL per gradient). The gradients are equilibrated with 50 mM Tris-HCl, pH 7.8.

8) Centrifuge the gradients in Beckman SW28 rotors at 20,000 rpm, 20 min, 4C. The virus should be the major band about 1/2 to 2/3 deep in the gradient.

9) Remove virus bands from the gradients with sterile bent needles to Oak Ridge 30 mL polypropylene tubes. Split the virus from 3 gradients between 2 tubes. Dilute virus to tube volume with 50 mM Tris-HCl, pH 7.8. Centrifuge tubes in Beckman Ti50.2 rotors at 27,000 rpm, 3 hours, 4C. Discard supernatants.

10) Resuspend virus pellets in a small volume of 50 mM Tris-HCl, pH 7.8. Store virus at 4C.

1) Determine concentration (A260/mL) with a UV spectrophotometer.

2) Determine titer (PFU/mL) by plaque assay.

3) 1 A260 unit of PBCV-1 routinely yields 1.5-2.5 X 1010 PFU.

4) For critical work, a second purification through sucrose gradients may be necessary.