Improved Methodology for Identification of Protists and Microalgae from Plankton Samples Preserved in Lugol's Iodine Solution: Combining Microscopic Analysis with Single-Cell PCR

@article{citeulike:2836886, abstract = {Here we introduce a method for quantitative analysis of planktonic protists and microalgae from preserved field samples combining morphological and small-subunit (SSU) rRNA gene sequence analysis. We linked a microscopic screening with PCR of single cells using field samples preserved with Lugol's iodine solution. Cells possessing a rigid cell wall were incubated with Viscozyme and subsequently with proteinase K for cell disruption; this was unnecessary for fragile cells. The addition of sodium thiosulfate to the PCR tube considerably decreased the inhibiting effect of the fixative (iodine) on the PCR and thus allowed for successful single-cell PCR even of long DNA fragments (up to as many as 3,000 base pairs). We further applied the protocol to investigate the dominant SSU rRNA genotypes in distinct flagellate morphospecies originating from different samples. We hypothesized that despite the morphological similarity, protist morphospecies in different habitats or sampled during different seasons are represented by different genotypes. Our results indicate species-specific differences: the two species Ochromonas sp. and Dinobryon divergens were represented by several different genotypes each, and for the latter species, the dominating genotype differed with habitat. In contrast, Dinobryon pediforme, Dinobryon bavaricum, and Synura sphagnicola were exclusively represented by a single genotype each, and the respective genotype was the same in different samples. In summary, our results highlight the significance of molecular variation within protist morphospecies. 10.1128/AEM.01803-07}, author = {Auinger, Barbara M. and Pfandl, Karin and Boenigk, Jens }, citeulike-article-id = {2836886}, doi = {http://dx.doi.org/10.1128/AEM.01803-07}, journal = {Appl. Environ. Microbiol.}, keywords = {18s, eukaryote, lugol, pcr, single\_cell}, month = {April}, number = {8}, pages = {2505--2510}, posted-at = {2008-07-02 15:25:12}, priority = {3}, title = {Improved Methodology for Identification of Protists and Microalgae from Plankton Samples Preserved in Lugol's Iodine Solution: Combining Microscopic Analysis with Single-Cell PCR}, url = {http://dx.doi.org/10.1128/AEM.01803-07}, volume = {74}, year = {2008} }

TY - JOUR ID - citeulike:2836886 L3 - citeulike-article-id:2836886 TI - Improved Methodology for Identification of Protists and Microalgae from Plankton Samples Preserved in Lugol's Iodine Solution: Combining Microscopic Analysis with Single-Cell PCR JF - Appl. Environ. Microbiol. VL - 74 IS - 8 SP - 2505 EP - 2510 N2 - Here we introduce a method for quantitative analysis of planktonic protists and microalgae from preserved field samples combining morphological and small-subunit (SSU) rRNA gene sequence analysis. We linked a microscopic screening with PCR of single cells using field samples preserved with Lugol's iodine solution. Cells possessing a rigid cell wall were incubated with Viscozyme and subsequently with proteinase K for cell disruption; this was unnecessary for fragile cells. The addition of sodium thiosulfate to the PCR tube considerably decreased the inhibiting effect of the fixative (iodine) on the PCR and thus allowed for successful single-cell PCR even of long DNA fragments (up to as many as 3,000 base pairs). We further applied the protocol to investigate the dominant SSU rRNA genotypes in distinct flagellate morphospecies originating from different samples. We hypothesized that despite the morphological similarity, protist morphospecies in different habitats or sampled during different seasons are represented by different genotypes. Our results indicate species-specific differences: the two species Ochromonas sp. and Dinobryon divergens were represented by several different genotypes each, and for the latter species, the dominating genotype differed with habitat. In contrast, Dinobryon pediforme, Dinobryon bavaricum, and Synura sphagnicola were exclusively represented by a single genotype each, and the respective genotype was the same in different samples. In summary, our results highlight the significance of molecular variation within protist morphospecies. 10.1128/AEM.01803-07 KW - 18s KW - eukaryote KW - lugol KW - pcr KW - single_cell AU - Auinger, Barbara AU - Pfandl, Karin AU - Boenigk, Jens PY - 2008/04/15/ UR - http://dx.doi.org/10.1128/AEM.01803-07 ER -