Inactivation of microbicidal active halogen compounds by sodium thiosulphate and histidine/methionine for time-kill assays

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Rapid inactivation of antimicrobial test agents after exact incubation times with microorganisms is required in time-kill assays. Sodium thiosulphate and a combination of methionine and histidine were compared for neutralisation of active halogen compounds.Test oxidants were mixed with surplus sodium thiosulphate (3%–6%) or histidine/methionine (1% each) in phosphate-buffered saline and incubated for different times, followed by addition of Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa at 1000CFU/ml. After further incubation, quantitative cultures were performed.Thiosulphate did not sufficiently inactivate chlorine and bromine compounds, indicated by a 10-fold (S. aureus) up to >100-fold (E. coli, P. aeruginosa) reduction of CFU. This was particularly true for high concentrations of the oxidants of about 50mM, for highly reactive agents (HOCl and bromamine T) more than for chloramine T and N-chlorotaurine, and for short pre-incubation times before addition of the bacteria. By contrast, histidine/methionine proved to be suitable for chloramines and bromamine T and for low concentrations of HOCl (0.07%). HOCl at 0.7% could neither be inactivated completely by thiosulphate nor by histidine/methionine. In contrast to chlorine and bromine compounds, iodine was neutralized by thiosulphate, but not by histidine/methionine.Histidine/methionine is superior to inactivate chlorine and bromine and should replace sodium thiosulphate at least in killing tests with high concentrations of these disinfectants. Inclusion of a short reaction time (maximum one minute) of test oxidant and neutralising substance before addition of bacteria is decisive in inactivation tests to obtain reliable results.