Direct evidence that free-living bacteria can form small patches in the water column has been unavailable, despite long-standing hypotheses, because traditional sampling methods destroy the aggregations. The recently developed spatial information preservation (SIP) method sufficiently preserves the two-dimensional, relative spatial distribution of aquatic microorganisms to resolve patches on the scale of tens to hundreds of micrometers (nanoscale patchiness). We collected samples from lake water using the SIP method. Nanoscale patches were not found in several unman- ipulated lake samples. However, we were able to stimulate patch formation by filtering out organisms larger than bacteria and adding freeze-thawed diatoms. Patchiness developed within 3 h and then dispersed by 16 h after the addition of the algae. Patches did not form in an unfiltered sample that was rotated. The potential for free-living aquatic bacteria to occur in patches has important implications for aquatic microbiology.