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 .
