Sybr green 1 dna

Cells were stained with the nucleic acid-binding dye SYBR Green I DNA (0.1% w/v stock; Life Technologies, Paisley, UK) and 1 mL of culture was filtered through a 0.2 μm black polycarbonate filter and then washed with 100 μL of sterile dd H₂O. The cells on the filter were enumerated using a Leica DMRP microscope equipped with epifluorescence, as previously described (Summers et al., 2013 (link)). The growth rate constant (k) for the log phase of growth was determined (Pirt, 1978 ).

Growth experiments were conducted with the isolates under conditions associated with fluvio-lacustrine systems on early Mars. The isolates were grown under anaerobic conditions, using a Mars simulation gas as the headspace (95.3% carbon dioxide, 2.7% nitrogen, 1.7% argon, 0.2% oxygen and 0.03% water vapour), 15 °C, circumneutral pH and 2% salinity. For these experiments, a minimal medium was used, which contained (g L⁻¹): 1 of NH₄Cl, 2 of Na-Lactate, 1 of Na-thioglycollate, 1 of ascorbic acid, 37 g NaCl and 13.25 g Na₂CO₃.
To monitor microbial growth and pH, 1 mL aliquots were aseptically removed after 1, 4, 7, 14, 21, 28 days. Cells were stained with the nucleic acid-binding dye SYBR Green I DNA (0.1% w/v stock; Life Technologies, Paisley, UK). One mL of culture was filtered through a 0.2 μm black polycarbonate filter and then washed with 100 μL of dd H₂O. Cells were enumerated using a Leica DMRP microscope equipped with epifluorescence, as previously described [62 (link)].