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Principles of fluorescent wavelength–based cell discrimination

One of the serious issues for fluorescence-based cell enumeration in geological samples (e.g., sediment and rock) is to distinguish few microbial cells in the forest of nonspecific background fluorescence signals from minerals. Morono et al. (2009) reported that using SYBR Green I fluorescent dye, SYBR Green I–stained cells (hereafter, SYBR-stained cells) are distinguishable from nonbiological background signals from SYBR-stainable particulate matter (SYBR-SPAM) based on the difference in fluorescent wavelength pattern (i.e., most fluorescent spectra from SYBR-SPAM shift to longer wavelengths than those from SYBR-stained cells). Based on the fluorescence characteristic of SYBR Green I stain, it is possible to discriminate cell-derived fluorescence signals from the background by processing green- and red-filtered fluorescent intensity of microscopic images (Morono et al., 2009). Although this method is independent of human variation in cell recognition, it is still necessary to aseptically prepare the membrane filter for image acquisition and processing. Therefore, even using the automated-robotic slide loader system (Morono and Inagaki, 2010), onboard difficulties with potential contamination and unbiased high-throughput cell counting are still present, such as

  • Filter preparation requires very careful and aseptic handling, which is in most cases very difficult on ships and take considerable human effort and time for sample processing;

  • Deployment of the highly sensitive robotic microscopic system on every microbiological expedition is difficult; and

  • Maintaining stable and constant operation of automatic z-focus adjustment on a continuously moving ship is hard.

Although the image-based automated cell count method has been useful and robust for shore-based microbial cell counting, an innovative onboard high-throughput technique for filtration- and eye-independent cell count for geologic samples is required.

Flow cytometry is a powerful tool for identifying and enumerating fluorescence-labeled cells, based on cell size, fluorescent intensity, and wavelength. It is commonly used for medical sciences and aquatic microbial ecology. The application of FCM to subseafloor life has great potential for high-throughput and statistically reliable cell counts onboard because discrimination of cell-derived SYBR fluorescence from SYBR-SPAM is essentially the same as with the image-based technique and thus should be possible using FCM. However, it is necessary to separate cells effectively from most nonbiological particles (e.g., minerals) and prepare SYBR-stained cell suspensions. During Expedition 329, the cell detachment protocols of Kallmeyer et al. (2008) and Morono et al. (2009) were modified and a standardized FCM cell counting method was developed for various types of open ocean sediment using cored samples from the South Pacific Gyre.