Analyzing Microorganisms in Environmental Samples Using Stable Isotope Probing with H218O

INTRODUCTION When analyzing environmental samples of microorganisms by stable isotope probing (SIP), labeling the DNA with H 2 18 O, instead of organic or nitrogenous compounds, offers important advantages because water cannot be used as an energy, carbon, or nitrogen source. As a result, addition of the label is unlikely to influence microbial growth rates in soil directly, and microbial communities can be exposed to the label for long periods of time because they are not exposed to abnormally high substrate concentrations. Because all organisms incorporate water into their DNA, performing SIP with H 2 18 O is a method for identifying microorganisms that have grown during incubation with H 2 18 O, as well as microorganisms that have not grown (i.e., did not incorporate the label) but survived the incubation. This protocol describes the use of SIP with H 2 18 O to study soil microorganisms.