Aims Fast and reliable methods for the early detection and identification of micro-organism are of high interest. In addition to established methods, direct mass spectrometrybased analysis of volatile compounds (VCs) emitted by micro-organisms has recently been shown to allow species differentiation. Thus, a large number of pathogenic Gram-negative bacteria, which comprised Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Proteus vulgaris and Serratia marcescens, were subjected to headspace VC composition analysis using direct mass spectrometry in a low sample volume that allows for automation. Methods and Results Ion-molecule reactionmass spectrometry (IMR-MS) was applied to headspace analysis of the above bacterial samples incubated at 37 degrees C starting with 102CFUml-1. Measurements of sample VC composition were performed at 4, 8 and 24h. Microbial growth was detected in all samples after 8h. After 24h, species-specific mass spectra were obtained allowing differentiation between bacterial species. Conclusions IMR-MS provided rapid growth detection and identification of micro-organisms using a cumulative end-point model with a short analysis time of 3min per sample. Significance and impact of the study Following further validation, the presented method of bacterial sample headspace VC analysis has the potential to be used for bacteria differentiation.