Viruses from Sulfolobus and close relatives
For each sample to be collected, one anaerobic collection vessel (Fig 1A in guidelines) is prepared.
A small amount (ca. 50–220 mg) of elemental sulfur (e.g., Riedel-deHaën) is placed in an anaerobic tube.
0.1 mL of a 2% resazurin solution and 0.1 mL of water saturated with H2S are added.
The air in the tube is displaced with CO2 and N2 by the Hungate technique and the tube is stoppered (Hungate et al. 1966).
A cap is placed on the tube and the assemblage autoclaved.
Liquid and wet sediment samples are collected from turbid terrestrial hot springs with high temperature > 70˚C and low pH < 4.
Samples are collected in sterile 50-mL conical flasks at the end of an extendible pole with a clamp (see Fig. 1A in guidelines).
After most of the sediment is allowed to settle, the pH of the liquid is carefully adjusted to ca. 5.5 with solid CaCO3 by slow addition and stirring.
Once the pH is adjusted the sample is transferred to a pre-prepared anaerobic tube using a syringe (see above and Fig. 1A in guidelines).
If the resazurin indicator changes to pink, drops of H2S-saturated water are added until the sample clears.
Samples can be maintained for up to 2 weeks at room temperature before enrichment.
Samples collected either in anaerobic tubes or filled centrifuge tubes are diluted 1:50 or 1:100 in Sulfolobus growth medium* (Zillig et al. 1994).
Incubate samples at 80˚C with shaking (150 rpm) for up to 2 weeks.
The medium was buffered with 0.7 g glycine per liter and the pH was adjusted to pH 3–3.5 with 1:2 diluted sulfuric acid.
For long-term 80˚C growth, our favorite bath liquid is PEG 400, which is a noncorrosive, nontoxic, water soluble compound that does not evaporate (see Fig. 1B in guidelines).
When growth is detected by either an increase in turbidity or production of a characteristic “damp sock” odor (W. Zillig pers. comm.), samples are plated on Gelrite® plates (see below and Fig. lC in guidelines), rediluted 1:50, and screened for VLP production by a spot-onlawn assay (see below and Fig. 1D in guidelines) or electron microscopy (see below and Fig. 2 in guidelines).
The second round of enrichment culture is also plated and screened for virus production.
Plates are made by slowly adding 6–10 grams/L Gelrite (Kelco) to Sulfolobus media (see above) and boiling until dissolved.
Calcium (Ca(NO3)2) and magnesium (MgCl2) are added to a final concentration of 1.5 and 5 mM, respectively, to stabilize the gel.
Before the gel solidifies, ca. 25 mL is poured into standard (90 mm) Petri plates with cams.
After the Gelrite solidifies, plates can be stored at 4˚C indefinitely.
Approximately 0.1 mL, from undiluted to 10-3, of enrichment cultures are spread on Gelrite plates in the presence or absence of 0.5 mL 0.2% Gelrite dissolved in Sulfolobus medium.
Plates are incubated inverted in airtight moist containers at 75–80˚C for approximately 1 week before colonies appear (Fig. 1C in guidelines).
