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Detection and enumeration of microbial life in marine subsurface environments provides primary information for deciphering the extent and habitability of Earth’s biosphere. Hence, accurate and precise knowledge of microbial cell abundance at high spatial resolution is a major challenge for the exploration of subsurface life. Cell-counting efforts during drilling expeditions typically depend on direct eye count of fluorescent dye–stained cells using epifluorescence microscopy. The improved cell extraction and filter preparation techniques of Expedition 329 provide reliable cell counts to levels as low as 1000 cells/cm3 of sediment. However, shipboard microscopic observation requires a great deal of time and effort in observing hundreds of microscopic fields of view per sample to produce statistically meaningful data. Processing the large number of samples necessary for such observation causes considerable difficulty given the limited time and manpower onboard. During Expedition 329, experiments with an alternative cell detection and enumeration method using flow cytometry (FCM) equipped with a 96-well autosampler system were conducted. Expedition 329 provided an unprecedented opportunity to test a variety of open-ocean sediment types in the South Pacific Gyre at depths ranging from the seafloor to basaltic basement.