Bacterial headspace sampler for real-time volatilome monitoring

The bacterial volatilome is rich in information that can be exploited for metabolic investigations and infectious disease diagnostics, including identification and, potentially, rapid antimicrobial susceptibility testing. Robust and reliable headspace sampling is critical for the capture of bacterial emissions. Here, we introduce a standardized bacterial headspace sampling technique for reproducible and real-time analysis of bacterial growth plates. The system is based on an inert volume sampler for the accumulation and triggered extraction of trace-level microbial volatile organic compounds (mVOCs) from inoculated samples under controlled temperature and relative humidity. Integrated sample dilution is achieved using high-resolution mass flow controllers to meet the dynamic range requirements of various analytical tools, for instance, mass spectrometers and chemical sensors. A built-in analyte gas standard enables calibration between measurements. The sampling characteristics are assessed for key mVOCs (acetaldehyde, acetone, benzene, and ethanol) within relevant concentration ranges, down to parts-per-billion by volume (ppb) levels. In vitro tests with Escherichia coli confirm accurate capture of mVOC dynamics reflecting physiological changes during bacterial growth, as monitored by soft-ionization mass spectrometry over 24 hours. This setup offers a standardized approach compatible with diverse analytical tools for exploring the microbial volatilome.