Sampling techniques for extended soil microbiological studies: a review and a proposal for unified guidelines

Soil is a complex and dynamic system that harbors an immense diversity of microorganisms essential to ecosystem functioning, agricultural productivity, and global biogeochemical cycles. Recent advances in molecular techniques have significantly improved our ability to study soil microbial communities. However, the lack of standardized sampling protocols remains a critical limitation, hindering data comparability and reproducibility across studies. This review synthesizes current methodologies in soil sampling for microbiological analysis, identifying key inconsistencies in sampling timing, site selection, depth, and sample handling. To date, no previous work has systematically compared these aspects. Temporal variability in microbial communities is often neglected, with few studies providing clear justification for sampling times or transparent, georeferenced site information. The use of geospatial tools and photographic documentation can significantly improve site characterization and enhance reproducibility. Composite sampling is commonly used to reduce spatial heterogeneity, yet protocols differ in subsample number, plot size, and litter treatment. Aseptic techniques and proper tool sanitation are essential to minimize contamination, especially when transitioning between sites. Most studies focus on the top 20 cm of soil due to high microbial activity, but depth-specific strategies are needed to capture vertical variability. Furthermore, appropriate sample transport and storage, typically on ice and at –20 °C, are vital for preserving microbial integrity and DNA quality. Moving forward, it is essential that research efforts prioritize the development of harmonized, globally accepted sampling protocols. Such standardization will strengthen the reliability and reproducibility of soil microbiological assessments at large spatial scales, laying the groundwork for globally comparable datasets and more robust ecological insights.