On detection of Pseudo-nitzschia (Bacillariophyceae) species using whole cell hybridization:: Sample fixation and stability

Some species within the genus Pseudo-nitzschia H. Peragallo are associated with production of domoic acid, the agent responsible for amnesic shellfish poisoning (ASP), Identification and enumeration of particular Pseudo-nitzschia in natural populations is often difficult and time consuming because of the need for detailed morphological observations, which often require scanning or transmission electron microscopy, In earlier publications we described the development of large subunit ribosomal RNA. (LSU rRNA)-targeted fluorescent DNA probes for discriminating among a variety of Pseudo-nitzschia species collected from Monterey Bay, California, Probes are applied using whole cell hybridization and a custom filtration manifold, enabling rapid identification and quantification of target species in cultured as well as field samples. In this work we compared a variety of preservation techniques and assessed the stability of stored samples with respect to their reactivity towards the probes. Of the preservatives tested, a saline ethanol-based treatment gave the best results in terms of probes yielding a bright and uniform cell label. Culture samples treated with this fixative continued to react well with the probes for at least 6 weeks post-fixation whether stored in the preservative or dried post-preservation, with samples being kept at either room temperature or -20 degrees C, Likewise, field samples containing a variety of diatoms and dinoflagellate species stored in the saline ethanol solution at room temperature were also stable for at least 4-6 weeks, reacting brilliantly towards a positive control probe. After prolonged storage, however, cell reactivity towards the probes diminished dramatically. Post-hybridization, samples stored at 4 degrees C were found to retain their fluorescence for at least 1 week. These results indicate a wider window of opportunity for Pseudo-nitzschia analysis using whole cell hybridization than previously reported. Sample collection, preservation, and probing protocols optimized for Pseudo-nitzschia are also applicable to a wide range of phytoplankton species. The time required to execute the whole cell hybridization protocol was reduced by premixing probe with hybridization buffer. The premixed probe solutions as well as fixative and wash solutions are all stable at room temperature for at least 6 weeks. Application of two different species-specific probes, each labeled with a different fluorochrome, allowed detection of two species on a single filter. The Tatter could be adopted in the future to increase the rate of sample processing and decrease the cost of sample analysis.