Quantification of toxic and toxin-producing cyanobacterial cells by RING-FISH in combination with flow cytometry
Limnol. Oceanogr. Methods 9:67-73 (2011) | DOI: 10.4319/lom.2010.9.67
ABSTRACT: Blooms of potential toxic cyanobacteria increased in number and extent during the last decades. Species like Microcystis aeruginosa - due to their ability to produce toxins - are a fundamental threat for human and animal lives in drinking and recreational waters. To prevent detrimental effects, it is vital to quantify cyanobacterial cells that are toxic (i.e., possess toxin genes) and toxin-producing in these waters. Therefore, we applied and modified the method RING-FISH (recognition of individual genes fluorescence in situ hybridization) targeting the microcystin-synthetase gene D or its transcript, respectively. Using two toxin-producing cyanobacterial strains as well as two nontoxic controls, we compared RING-FISH with real-time quantitative PCR (qPCR), finding high agreement of both methods. RING-FISH is easy to apply for epifluorescence microscopy and yields valuable information on the genetic toxic potential and toxicity of cyanobacteria populations. In combination with flow cytometry unicellular toxic and toxin-producing cyanobacteria, e.g., M. aeruginosa, can be quantified by RING-FISH in a high throughput manner. Complemented with fluorescence activated cell sorting (FACS) toxic and toxin-producing cells can be retrieved from complex population for further molecular analyses. In contrast to qPCR, our RING-FISH protocol largely circumvents PCR biases and quantifies cells possessing or expressing certain functions independent of the copy number of the targeted gene. Furthermore, RING-FISH is easily adaptable for the detection and quantification of functional genes of other prokaryotic and eukaryotic cells.