protocol_200.txt

Isolation of Haloviruses from Natural Waters
Salt lake samples are collected from hypersaline waters, which typically range from 15% w/v total salt, up to saturation (ca 35%).
Samples are collected in sterile 5–10 mL vessels and may be stored for several weeks at room temperature.
In the laboratory, cells and cellular debris are removed by centrifugation (5,000g, 10 min, room temperature).
The supernatant is then screened directly for viruses by plaque assay.
The choice of host strains depends on the experimental objectives, and includes well-characterized members of the Halobacteriaceae, such as Hbt.
salinarum (host for ΦH and several others) or Har.
hispanica (host for SH1, His1, His2, and others), or natural isolates from the same source, such as Hrr.
coriense (host for HF2).
Base and overlay plates (90 mm) are made with MGM (see guidelines), solidified using 15% w/v agar.
A range of salt water concentrations should be examined, because salt concentration seems to greatly affect the size and clarity of plaques.
Plates can be stored indefinitely at 4°C (wrapped in plastic to prevent dessication), but should be warmed to room temperature or warmer for use.
For virus isolation, 100–500 µl of the cleared water sample is mixed with 150 µl of exponentially growing host cells.
Then, 3–4 mL of molten (50°C) top-layer MGM (with 0.7% w/v agar) is added, and the solution mixed gently and poured evenly over the plate.
After setting on a level surface for 5–10 min, plates are incubated aerobically, inverted in airtight containers at 30°C and 37°C for 1–4 d, and checked every day for plaques.
Any visible plaques are picked using sterile glass Pasteur pipettes, or sterile plastic micropipette tips.
These agar plugs are then transferred to tubes containing 500 µl of halovirus diluent: 2.47 M, NaCl; 90 mM, MgCl2; 90 mM, MgSO4; 60 mM, KCl; 3 mM, CaCl2; 10 mM, Tris-HCl, pH 7.5;
They are then vortexed to homogenize the sample.
These suspensions are then replaqued on overlay plates to purify the isolates and to eliminate “false plaques” caused by artifacts in the agar overlay or contaminants in the water sample.