Impact of the cold shock phenomenon on quantification of intracellular metabolites in bacteria.

In the present work the effect of quenching on quantification of intracellular metabolites in Corynebacterium glutamicum was investigated. C. glutamicum showed a high sensitivity to cold shock. Quenching of the cells by -50 degrees C buffered methanol prior to cell separation and extraction led to drastically reduced concentrations for free intracellular amino acids compared to those for nonquenched filtration. As demonstrated for glutamate and glutamine, this was clearly due to a more than 90% loss of these compounds from the cell interior into the medium during quenching. With lower methanol concentration in the quenching solution the metabolic losses were significantly lower but still amounted to about 30%. Due to the fact that quenching with ice-cold NaCl (0.9%) also resulted in significantly lower pool sizes for intracellular amino acids, a basic cold shock phenomenon is most likely the reason for the observed effects. The results clearly demonstrate that quenching combined with cell separation for concentration of the cells and removal of the medium is not applicable for intracellular metabolite analysis in C. glutamicum. Sampling by quick filtration without quenching allows complete cell separation and authentic quantification of intracellular metabolite pools exhibiting time constants significantly larger than sampling time.