## 3 Discussion
As can be seen in Figure 6, ROS production by MB remains at a similar level throughout the experiment. In the case of the mixture of MB and AuBNPs, the efficiency of oxidative species production increased compared to the control (the control was the bacterial suspension) by 29 \(\pm\) 3, 32 \(\pm\) 3, 35 \(\pm\) 3, and 41 \(\pm\) 3%, (using light doses of 31.5, 63, 126, and 189 J cm\({}^{-2}\)), respectively. Under the same experimental conditions, the mixture of MB and AuChNPs increased the formation of oxidative species by 34 \(\pm\) 4%, 41 \(\pm\) 4%, 46 \(\pm\) 4.0%, and 50 \(\pm\) 4% compared to the control. The obtained results clearly showed that the higher efficiency of aPDI in the presence of gold nanoparticles is the result of increased production of oxidizing compounds (probably hydroxyl radicals). The dark toxicity of MB+AuBNPs and MB+AuChNPs was also studied, and it was found that these mixtures containing nontoxic concentrations of MB and gold nanoparticles showed bactericidal activity. Finally, we proposed a simple photodidal procedure that involved triple exposure of the biofilm to laser light in the presence of a nontoxic concentration of MB alone (31.25 mg L\({}^{-1}\)), combined with the removal of dead bacteria that protected the deep cell layers from photosensitization. This method increased the efficiency of biofilm destruction compared to a single photosensitization in the presence of a nontoxic concentration of MB and a light dose  
Figure 6: Effectiveness of generating oxidative species by MB alone, MB+AuBNPs, and MB+CNNPs as photosensizers. The dichlorodihydrofluorescein diacetate as a fluorescent probe was used. Average \(\pm\)SD of three independent experiments is shown. of 189 J cm\({}^{-2}\), and this efficiency met the requirements of the ASM. It should be noted that washing the biofilm removed both dead and viable bacterial cells. The number of viable cells that were present in the biofilm washing water did not exceed 3 \(\pm\) 1 and 5 \(\pm\) 1% of the total number of viable biofilm-forming pathogens on the glass and stainless steel discs, respectively. The protocol of photodestruction of biofilms formed on glass and steel surfaces by _S. aureus_ MRSA isolates in the presence of MB at a concentration of 125 mg L\({}^{-1}\), and the procedure that involved triple exposure of the biofilm to laser light has been proven to be effective disinfection methods. These simple and inexpensive techniques can be used to disinfect a variety of small surfaces (from medical areas, fitness salons, spas, and beauty salons, to public places). It is worth noting that these procedures were more effective compared to the efficiency of removing biofilms from glass/steel discs using commercially available disinfectants (the product designed as1)was a mixture of ethyl alcohol and propan-1-ol; the product designed as2)was quaternary ammonium salts). The attractiveness of the methods of biofilm destruction proposed above may be further enhanced by the use of inexpensive LED technologies with the appropriate light length, which is readily available on the commercial market. Methylene blue has been used as a photosensitizing agent since the 1920s, and its administration has been approved by the U.S. Food and Drug Administration. Our understanding of bacterial biofilm formation has advanced significantly over the past 20 years, leading to the development of several (in addition to aPDI) promising biofilm destruction strategies. Conventional strategies (mentioned in the Introduction chapter) to control biofilm growth most often include cleaning and disinfection strategies such as physical (clean-in-place) and chemical (using sodium hypochlorite, hydrogen peroxide, ozone, and peracetic acid) methods. It has been shown that mechanical treatment (cleaning in place) cannot remove all bacterial cells, while chemical treatment only partially eliminates bacterial cells and inhibits biofilm formation [33; 34].