Studies on the mechanism of the photosensitized inactivation of E. coli and reactivation phenomenon

In order to find a more satisfactory interpretation of the phenomenon of photosensitized inactivation of bacteria, studies were performed under various experimental conditions on methylene blue and E. coli. In summary the findings are as follow:— 1. The dye is absorbed by the bacteria according to the Langmuir isotherm and can be removed by ionic substitutions; the dye binding to the bacteria is predominantly ionic; the dye-bacteria complex produces a new absorption peak in the 610 mµ wave length region, and the action spectrum corresponds to the spectral absorption of the dye-bacteria complex. 2. There is an optimum dye concentration range for the photosensitized inactivation. 3. Photosensitized inactivation of bacteria can take place both in the frozen and liquid states and the presence of oxygen is essential to the inactivation process. 4. Hydrogen peroxide, formed by reoxidation of the reduced methylene blue, does not inactivate bacteria. 5. Following the photosensitized inactivation, E. coli lose their ability to reduce the methylene blue in the presence of various hydrogen donors, suggesting that enzymes are involved in the inactivation process. 6. Bacteria inactivated by photosensitization can be reactivated by prolonged storage after irradiation; the recovery rate increases with increasing temperature (maximum 37°), and is also influenced by the presence of various hydrogen donors. In view of collected experimental data, the basic reaction mechanisms are analyzed in photosensitized inactivation. The first step of the reaction seems to be excitation of the dye-bacteria, or dye-bacteria oxygen complex, by a photon which produces an activated complex. In such a state, molecular oxygen is capable of producing an oxidizing reaction, which results in the inactivation of the bacteria. Some aspects of the detailed reactions taking place at the cell surface are discussed.