Materials:
1. Potassium citrate (1%), per liter: 10 g K3C6H5O7•H2O (Fisher); 1.44 g Na2HPO47H2O (Fisher); 0.24 g KH2PO4 (Fisher); pH 7
2. Branson 185 Sonifier (Branson Ultrasonics Corporation, Danbury, CT)
3. Low speed swing-out benchtop centrifuge
4. Ice bucket
5. 50 ml conical sterile polypropylene centrifuge tubes
Methods:
1. Determine, using standard techniques, the dry weight and moisture content of the soil sample.
2. Place a five gram dry weight soil sample into a 50 ml conical centrifuge tube and add 15 ml of 1% K citrate extraction buffer and mix with brief vortexing.
3. Place sample on ice for 20 to 30 minutes.
4. Sonicate samples on ice in 3 one minute intervals with one minute rest on ice in between sonications. Use a Branson model S-450a sonicator with a 1/2’’ externally threaded disrupter + 3/4’’ and a 1/8’’ tapered microtip at a duty cycle setting of 30% and an output control setting of 3.
5. Centrifuge soil buffer mixture at 3000 X g for 30 minutes at 4° C in a low speed table top centrifuge.
6. Carefully remove the supernatant and filter through a 0.22 m Sterivex (Millipore, Corp.) filter. The filtered supernatant contains the extracted virus particles.
7. If the sample is to be used for microscopic observation of virus particles then the sample should be placed into a cryovial, snap frozen in liquid N2 and stored at –80°C until preparation for microscopy.
