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A chemical (ethanol; formic acid; acetonitrile) protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters), indicated that the method could inactivate  Escherichia coli  MRE 162 and  Klebsiella pneumoniae  ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent,  Bacillus anthracis  UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent  B .  anthracis  Vollum vegetative cells and spores ranging in concentration from 10 6 -10 8 cfu per extract.  B .  anthracis  was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts  B .  anthracis  was only recovered from extracts that had undergone prolonged Luria (L)-broth (7 day) and L-agar plate (a further 7 days) incubations. We hypothesise that the recovery of  B .  anthracis  in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable  B .  anthracis .

Observations on the Inactivation Efficacy of a MALDI-TOF MS Chemical Extraction Method on Bacillus anthracis Vegetative Cells and Spores