Carotenoid producing bacteria from Antarctica

Study Extent

Sampling

Method steps

1. Approximately 100 mg of each sample was suspended in 900 μl of sterile NaCl 0.9% (w/v) solution, serially diluted and plated on adequate media. The isolation medium for samples of organic matter, sediments and ice water was Tryptic Soy Agar (TSA, Sigma Aldrich), and for sea water was TSA complemented with 20 g/L of sea salts (Sigma). Plates were incubated at 10 °C for 7–10 days, and colored colonies were selected for strain isolation by streak-plating technique. Once purity was verified, strains were conserved at −80 °C on glass beads with 20% glycerol in Tryptic Soy Broth (TSB, Oxoid) and sea salts when needed. Strains were characterized by colony and cell morphology, Gram staining and pigment composition.
2. Genomic DNA extraction was performed with a commercial kit according manufacturer’s instructions (Genomic DNA Purification Kit, Thermo Fisher). Amplification of the 16S rRNA gene fragments were done in a Palm-1870 Cycler TM (Corbett Research UK Ltd) as follows: initial denaturation 3 min at 95 °C, then 35 cycles of 45 s at 94 °C, 45 s at 58 °C, 60 s 72 °C, and a final extension step 9 min at 72 °C. Reaction mixtures contained: 2.5 U polymerase (Mango Taq, Bioline), 10 μL of buffer solution, 2.5 μL of 50 mM MgCl2, and 2.5 μL each of forward primer 27 F (5´-AGAGTTTGATC MTGGCTCAG-3´) and reverse primer 1492R (5´-TACGGYTACC TTGTTACGACTT-3´), genomic DNA, and water to 50 μL final volume. The PCR products were analyzed by electrophoresis with 1% agarose gels. DNA sequencing was carried out by Macrogen Inc. (Korea) using universal primers.