The filter was then mounted on an aluminium stub, sputter coated

with gold/palladium using a Cressington 208 HR High Resolution Sputter Coater, and observed with a Hitachi S-4700 field emission scanning electron microscope. Cells isolated from the surrounding sediment were pre-fixed for transmission electron microscopy (TEM) using 4% (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (SCB) (pH 7.2) with the addition of 0.3 M sorbitol. Roxadustat purchase The pre-fixed cells were washed in 0.2 M SCB (pH 7.2) three times and embedded in 2% of low melting temperature agarose and post-fixed in 1% (w/v) osmium tetroxide in 0.2 M SCB (pH 7.2) at room temperature for 1 hr, before being dehydrated through a graded series of ethanol and 100% acetone. The dehydrated cells were then infiltrated with acetone-Epon 812 resin mixtures and 100% Epon 812 resin. Ultra-thin serial sections were collected on copper Formvar-coated slot grids, stained with 2% (w/v) uranyl acetate and lead citrate, Hydroxychloroquine nmr and observed using a Hitachi H7600 electron microscope. DNA extraction, PCR amplification, alignment and phylogenetic analysis Genomic DNA was extracted using the MasterPure Complete DNA and RNA purification Kit (Epicentre, WI, USA) from 30 cells that were individually isolated and washed three times in sterile seawater

(i.e., “”isolate 1″”). This procedure was repeated three months later on a different sample of 30 individually isolated cells (i.e., Immune system “”isolate 2″”). Polymerase chain reactions (PCR) were performed using PuRe Taq Ready-To-Go PCR beads kit (GE Healthcare, Buckinghamshire,

UK). Nearly the entire eukaryotic SSU rDNA gene was amplified from each isolate using the eukaryotic universal primers 5′- TGATCCTTCTGCAGGTTCACCTAC-3′ [49] and 5′-GCGCTACCTGGTTGATCCTGCCAGT-3′ [50]. PCR amplifications consisted of an initial denaturing period (95°C for 3 min), 35 cycles of denaturing (93°C for 45 s), annealing (5 cycles at 45°C and 30 cycles at 55°C, for 45 s), extension (72°C for 2 min), and a final extension period (72°C for 5 min). The amplified DNA fragments were purified from agarose gels using UltraClean 15 DNA Purification Kit (MO Bio, CA, USA), and subsequently cloned into the TOPO TA Cloning Kit (Invitrogen, CA, USA). Two clones of the eukaryotic SSU rRNA gene, from each of the two isolates (i.e., four clones in total), were sequenced with the ABI Big-Dye reaction mix using the vector primers and internal primers oriented in both directions. The new sequences were screened with BLAST, identified by molecular phylogenetic analysis, and deposited into GenBank: HM004353, HM004354. The SSU rDNA sequences from B.