Improved methodology for identification of protists and microalgae from plankton samples preserved in Lugol's iodine solution: combining microscopic analysis with single-cell PCR.

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

SUBMITTER: Auinger BM 

PROVIDER: S-EPMC2293146 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature