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 ) .
