Fecal microbiota transplantation is a compelling treatment for recurrent 
Clostridium difficile infections, with potential applications against other 
diseases associated with changes in gut microbiota. But variability in fecal 
bacterial communities-believed to be the therapeutic agent-can complicate or 
undermine treatment efficacy. To understand the effects of transplant 
preparation methods on living fecal microbial communities, we applied a 
DNA-sequencing method (PMA-seq) that uses propidium monoazide (PMA) to 
differentiate between living and dead fecal microbes, and we created an analysis 
pipeline to identify individual bacteria that change in abundance between 
samples. We found that oxygen exposure degraded fecal bacterial communities, 
whereas freeze-thaw cycles and lag time between donor defecation and transplant 
preparation had much smaller effects. Notably, the abundance of Faecalibacterium 
prausnitzii-an anti-inflammatory commensal bacterium whose absence is linked to 
inflammatory bowel disease-decreased with oxygen exposure. Our results indicate 
that some current practices for preparing microbiota transplant material 
adversely affect living fecal microbial content and highlight PMA-seq as a 
valuable tool to inform best practices and evaluate the suitability of clinical 
fecal material.