Aims: To investigate critical electrical and biological factors governing the efficacy of pulsed light (PL) for the in vitro inactivation of bacteria isolated from the clinical environment. Development of this alternative PL decontamination approach is timely, as the incidence of health care-related infections remains unacceptably high. Methods and Results: Predetermined cell numbers of clinically relevant Gram-positive and Gram-negative bacteria were inoculated separately on agar plates and were flashed with £60 pulses of broad-spectrum light under varying operating conditions, and their inactivation measured. Significant differences in inactivation largely occurred depending on the level of the applied lamp discharge energy (range 3AE2-20 J per pulse), the amount of pulsing applied (range 0-60 pulses) and the distance between light source and treatment surface (range 8-20 cm) used. Greater decontamination levels were achieved using a combination of higher lamp discharge energies, increased number of pulses and shorter distances between treatment surface and the xenon light source. Levels of microbial sensitivity also varied depending on the population type, size and age of cultures treated. Production of pigment pyocynanin and alginate slime in mucoid strains of Pseudomonas aeruginosa afforded some protection against lethal action of PL; however, this was evident only by using a combination of reduced amount of pulsing at the lower lamp discharge energies tested. A clear pattern was observed where Gram-positive bacterial pathogens were more resistant to cidal effects of PL compared to Gram negatives. While negligible photoreactivation of PL-treated bacterial strains occurred after full pulsing regimes at the different lamp discharge energies tested, some repair was evident when using a combination of reduced pulsing at the lower lamp discharge energies. Strains harbouring genes for multiple resistances to antibiotics were not significantly more resistant to PL treatments. Slight temperature rises (£4AE2°C) were measured on agar surfaces after extended pulsing at higher lamp discharge energies. Presence of organic matter on treatment surface did not significantly affect PL decontamination efficacy, nor did growth of PL-treated bacteria on selective agar diminish survival compared to similarly treated bacteria inoculated and enumerated on nonselective agar plates. Conclusions: Critical inter-related factors affecting the effective and repeatable in vitro decontamination performance of PL were identified during this study that will aid further development of this athermal process technology for applications in health care and in industry. Very rapid reductions (c. 7 log 10 CFU cm )2

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Aims:  To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal injury in food‐borne bacteria exposed to pulsed‐plasma gas discharges (PPGD). Methods and Results:  The fluorescent redox probe 5‐cyano‐2,3‐ditolyl tetrazolium chloride (CTC) was used for enumerating actively respiring cells of Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella enterica serovar Typhimurium that were suspended in sterile water at 4°C and exposed to separate PPGD and heat treatments. While there was good agreement between use of respiratory staining (RS) and direct‐selective agar plate counting (PC) for enumerating untreated bacteria, there were c. 1 and 3 log‐unit differences in surviving cell numbers per millilitre for test organisms subjected to PPGD and heat treatments respectively, when enumerated by these different viability indicators. PPGD‐treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. Conclusions:  Use of this RS method revealed that substantial subpopulations of test bacteria rendered incapable of forming colonies by separate PPGD and heat treatments may remain metabolically active. Significance and Impact of the Study:  Use of this RS method offers interesting perspectives on assessing established and novel microbial inactivation methods, and may also provide a better understanding of mechanisms involved in microbial inactivation induced by high‐intensity PPGD treatments.