A microscopic approach to investigate bacteria under in situ conditions in sea-ice samples

Microbial populations and activity within sea ice have been well described based on bulk measurements from melted sea-ice samples. However, melting destroys the micro-environments within the ice matrix and does not allow for examination of microbial populations at a spatial scale relevant to the organism. Here, we describe the development of a new method allowing for microscopic observations of bacteria localized within the three-dimensional network of brine inclusions in sea ice under in situ conditions. Conventional bacterial staining procedures, using the DNA-specific fluorescent stain DAPI, epifluorescence microscopy and image analysis, were adapted to examine bacteria and their associations with various surfaces within microtomed sections of sea ice at temperatures from -2degrees to 15degreesC. The utility and sensitivity of the method were demonstrated by analyzing artificial sea-ice preparations of decimal dilutions of a known bacterial culture. When applied to natural, particle-rich sea ice, the method allowed distinction between bacteria and particles at high magnification. At lower magnifications, observations of bacteria could be combined with those of other organisms and with morphology and particle content of the pore space. The method described here may ultimately aid in discerning constraints on microbial life at extremely low temperatures.