Up-dating the Cholodny method using PET films to sample microbial communities in soil

The aim of this work was to investigate the use of PET (polyethylene terephtalate) films as a modern development of Cholodny’s glass slides, to enable microscopy and molecular-based analysis of soil communities where spatial detail at the scale of microbial habitats is essential to understand microbial associations and interactions in this complex environment. Methods. Classical microbiological methods; attachment assay; surface tension measurements; molecular techniques: DNA extraction, PCR; confocal laser scanning microscopy (CLSM); micro- focus X-ray computed tomography (μCT). Results. We first show, using the model soil and rhizosphere bacteria Pseudomonas fluorescens SBW25 and P. putida KT2440, that bacteria are able to attach and detach from PET films, and that pre-conditioning with a filtered soil suspension improved the levels of attachment. Bacteria attached to the films were viable and could develop substantial biofilms. PET films buried in soil were rapidly colonised by microorganisms which could be investigated by CLSM and recovered onto agar plates. Secondly, we demonstrate that μCT can be used to non-destructively visualise soil aggregate contact points and pore spaces across the surface of PET films buried in soil. Conclusions. PET films are a successful development of Cholodny’s glass slides and can be used to sample soil communities in which bacterial adherence, growth, biofilm and community development can be investigated. The use of these films with μCT imaging in soil will enable a better understanding of soil micro-habitats and the spatially-explicit nature of microbial interactions in this complex environment.