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-nitzs chia in natural populations is often difficult and time consuming because o f the need for detailed morphological observations, which often require sca nning or transmission electron microscopy, In earlier publications we descr ibed the development of large subunit ribosomal RNA. (LSU rRNA)-targeted fl uorescent 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 i dentification and quantification of target species in cultured as well as f ield samples. In this work we compared a variety of preservation techniques and assessed the stability of stored samples with respect to their reactiv ity towards the probes. Of the preservatives tested, a saline ethanol-based treatment gave the best results in terms of probes yielding a bright and u niform cell label. Culture samples treated with this fixative continued to react well with the probes for at least 6 weeks post-fixation whether store d in the preservative or dried post-preservation, with samples being kept a t either room temperature or -20 degrees C, Likewise, field samples contain ing a variety of diatoms and dinoflagellate species stored in the saline et hanol solution at room temperature were also stable for at least 4-6 weeks, reacting brilliantly towards a positive control probe. After prolonged sto rage, however, cell reactivity towards the probes diminished dramatically. Post-hybridization, samples stored at 4 degrees C were found to retain thei r fluorescence for at least 1 week. These results indicate a wider window o f opportunity for Pseudo-nitzschia analysis using whole cell hybridization than previously reported. Sample collection, preservation, and probing prot ocols optimized for Pseudo-nitzschia are also applicable to a wide range of phytoplankton species. The time required to execute the whole cell hybridi zation protocol was reduced by premixing probe with hybridization buffer. T he premixed probe solutions as well as fixative and wash solutions are all stable at room temperature for at least 6 weeks. Application of two differe nt species-specific probes, each labeled with a different fluorochrome, all owed detection of two species on a single filter. The Tatter could be adopt ed in the future to increase the rate of sample processing and decrease the cost of sample analysis.