The influence of experimental conditions on the final result of photoinhibition of Staphylococcus aureus

Abstract Background By and large, phototherapies are a promising approach to promote inactivation of microorganisms using light exposure, providing an effective alternative to control multidrug-resistant bacterial infections. Considering this, intercomparison between experiments is vitally important. Most experiments are performed using multiwell plates in which the volume of the culture medium is not standardized. In such cases, light attenuation and the distance it travels, which also depends on the volume and vessel geometry, can lead to different results. This study investigated how the different volumes imply different depths that light will have to travel through in this volume and the relation of this parameter with the result that will be obtained. Methods Staphylococcus aureus was exposed to 460 nm light with 50, 100 and 200 J/cm 2 , in 100, 200, 500 and 1000 μL of inocula in a 24-well plate to investigate pure light inactivation. Results The literature suggests that fluence is the most important light parameter to obtain a high eradication of microbial cells in phototherapies. Our results show evidence that different geometrical configurations, taking into account the volume of the vessels, clearly affect the in vitro results, risking misinterpretation of dosimetry studies. Effects, such as dose distribution and decantation, are discussed throughout the paper. Conclusion The outcome strongly depends on the volume and vessel geometry used. This study aims to encourage the standardization of phototherapies in vitro in general.