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Background  Quenching in cold buffered methanol at −40 °C has long been the preferred method for sub-second inactivation of cell metabolism during metabolic fingerprinting. However, methanol is known to cause intracellular metabolite leakage of microbial cells, making the distinction between intra- and extracellular metabolites in microbial systems challenging. In this paper, we tested three quenching protocols proposed for microbial cultures: fast filtration, cold buffered methanol and cold glycerol saline.    Results  Our results clearly showed that cold glycerol saline quenching resulted in the best recovery of intracellular metabolites in  Lactobacillus paracasei  subsp.  paracasei  ( L. paracasei ). Membrane integrity assayed by propidium iodide revealed that approximately 10 % of the  L. paracasei  cell membranes were damaged by contact with the cold buffered methanol solution, whilst cold glycerol saline quenching led to minimal cell damage. Due to the nature of the  L. paracasei  culture, fast filtration  took several minutes, which is far from ideal for metabolites with high intracellular turnover rates.    Conclusion  The implementation of a reliable, reproducible quenching method is essential within the metabolomics community. Cold glycerol saline prevented leakage of intracellular metabolites, and, thus, allowed more accurate determinations of intracellular metabolite levels.

Metabolic fingerprinting of Lactobacillus paracasei: the optimal quenching strategy