The authors are grateful to Dr. Tak-Wah Wong and Mr. Zong-Jhe Hsieh for their support with experiments.

1. Photolysis system setup
<ol>
	<li>Mount six light-emitting diodes (LED) (DC 12 V) on the inside of an opaque plastic cup (8 cm x 7 cm) as shown in Figure 1 to establish a photolysis system31.</li>
	<li>Add reactants (see below) into the glass test tubes (13 mm in diameter and 100 mm in height) and secure the tubes at the top of the cup as shown in Figure 1. Place the experimental setup in a room with a steady temperature of 25 &#177; 3 &#176;C.</li>
	<li>Monitor and record the temperature of test units throughout the photolytic reactions by an infrared thermometer. 
	NOTE: Figure 2 shows the emission spectra for blue, green, red, and violet lights, as recorded using a calibrated UV-Vis optical spectrometer. The wavelengths corresponding to the absorbance maximum for blue, green, red, and violet LED lights used in the study are 465, 529, 632, and 405 nm, respectively. The LED chips can heat the apparatus during irradiation experiments. The whole photoreaction system in each experiment was, therefore, placed in a room where the temperature was kept constant at 25 &#177; 3 &#176;C.</li>
</ol>
2. The effect of light wavelength on the photolysis of FMN (Figure 3)
<ol>
	<li>Prepare a 0.1 mM FMN solution in 100 mM potassium phosphate buffer (PB) (pH 7.8). Expose FMN samples (3 mL each) to blue, green, red, or violet light at an intensity of 10 W/m2 for 5 min. Keep 3 mL of FMN solution in dark as a control.</li>
	<li>Record the absorbance of irradiated FMN samples in the 250-750 nm range using a UV/Visible spectrophotometer.</li>
</ol>
3. Nitro blue tetrazolium (NBT) reduction method to detect <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="font-size: 14px; margin: auto;" />&#160;(Figure 4).
NOTE: The NBT reduction method used here was slightly modified from the assay for riboflavin photoreaction. The NBT reduction method is used to evaluate the level of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> generated from FMN photolysis. The photochemically excited FMN is first reduced by L-methionine into a semiquinone, which then donates an electron to oxygen giving rise to <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" />31. The as-generated <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> reduces NBT to formazan, which has a characteristic absorbance at 560 nm32.
<ol>
	<li>Add 109.3 mg of L-methionine to 73.3 mL of PB (100 mM; pH 7.8). Vortex the solution to dissolve the L-methionine.</li>
	<li>After the L-methionine is fully dissolved, add 10 mg of NBT powder and 1.53 mL of 0.117 mM FMN to the solution. For each 1 mL of the reaction solution (i.e., a mixture of FMN, L-methionine, and NBT) applied, the final concentrations of FMN, methionine, and NBT are 2.4 x 10-6 M, 1.0 x 10-2 M, and 1.6 x 10-4 M, respectively.</li>
	<li>Expose the reaction solution (1 mL) to blue or violet LED light irradiation at 10 W/m2 for 1-5 min.</li>
	<li>Detect the <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> species (from the absorbance at 560 nm) produced by the photochemical reaction, which reduces NBT and produces formazan.</li>
</ol>
4. S. aureus viability after FMN photolysis (Figure 5)
<ol>
	<li>Transmit a colony of S. aureus(BCRC 10451) from a cultured plate into 10 mL of Lysogeny broth taken in a 15 mL screw-capped test tube. Culture in a shaker at 37 &#176;C for 16 h.</li>
	<li>Transfer 0.5 mL of the culture to a 1.5 mL centrifuge tube. Add sterilized water into the centrifuge tube to dilute the culture to an optical density of 0.5 at 600 nm (OD600) (~6 x 107 CFU/mL).</li>
	<li>Transfer 0.5 mL of the culture to a 1.5 mL centrifuge tube, centrifuge at 14,000 x g for 10 min and decant the supernatant to obtain a cell pellet.</li>
	<li>Add 1 mL of FMN-buffered solution (30, 60, and 120 &#181;M FMN in PB) to the cell pellets obtained as in step 4.3, and vortex. For irradiated control, add 1 mL of PB alone.</li>
	<li>Transfer 1 mL each of viable bacterial cell solutions containing 30 &#181;M FMN and PB alone into glass tubes, and irradiate them with violet light at 10 W/m2 for 30 min.</li>
	<li>Transfer 1 mL each of viable bacterial cell solutions containing 30, 60, and 120 &#181;M FMN and PB alone into glass tubes and irradiate them with blue light at 20 W/m2 for 120 min. Set up another glass tube with 1 mL of viable bacterial cell solution containing 120 &#181;M FMN and irradiate it with blue light at 20 W/m2 for 60 min.</li>
	<li>Set up test tubes as described in steps 4.5 and 4.6 and cover them with thick aluminum foils. These tubes serve as dark controls.</li>
	<li>Keep the irradiation chamber (as well as the dark controls) in a cold room at 9 &#177; 1 &#176;C during the 30-120 min irradiation period. 
	NOTE: Heat released by LED lights cannot be ignored as the LED chips placed inside the cup can heat the photoreaction system during irradiation experiments. The experiments were, therefore, conducted in a cold room maintained at 9 &#177; 1 &#176;C.</li>
	<li>After irradiation, transfer 0.2 mL from each of the reaction solutions onto a Luria agar (LA) plate. Spread the bacteria over the plate by an L-shaped glass rod and incubate overnight at 37 &#176;C.</li>
	<li>Calculate the viable plate count and the inactivation rates of S. aureus after overnight growth. 
	NOTE: The inactivation rate of S. aureus is calculated as the percentage reduction, which is equal to [1 -I / D] &#215;100%, where I and D denote, respectively, the number of CFUs in the irradiated sample and dark control. The percentage reduction is defined as a negative value of the inactivation rate.</li>
</ol>
5. Detection of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="font-size: 14px; margin: auto;" />&#160;in S. aureus (Figure 6)
<ol>
	<li>Prepare the S. aureus samples as described in step 4.</li>
	<li>Dilute the bacterial density of the samples with sterilized water to an optical density of 0.5 at 600 nm (OD600, ~6 x 106 CFU/mL). Transfer 0.5 mL of the culture to a 1.5 mL centrifuge tube. Centrifuge at 14,000 x g for 10 min and decant the supernatant to obtain a cell pellet.</li>
	<li>Add 0.1093 g of L-methionine, 0.1 g of NBT, and 25 mL of FMN (400, 240, or 120 &#181;M) to 75 mL of PB. For each 1 mL of the reactant applied, the final concentrations of FMN, methionine, and NBT are 100 (60 or 30) x 10-6 M, 7.3 x 10-3 M, and 1.2 x 10-3 M, respectively.</li>
	<li>Add 1 mL of each reactant solution (i.e., with varying FMN concentrations) to the cell pellets obtained as in step 5.2. Irradiate the solutions by violet light at 10 W/m2 for 10 min.</li>
	<li>Centrifuge the mixture at 14,000 x g for 10 min and decant the supernatant. Resuspend the pellet in 1 mL of dimethyl sulfoxide (DMSO) to extract the reduced NBT. Detect the produced <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> species from the absorbance at 560 nm.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Express data as mean &#177; standard deviation (SD) of three independent tests.</li>
	<li>Apply a homoscedastic two-sample t-test to evaluate whether two measurements are different. p-values &#60; 0.05 are considered statistically significant.</li>
</ol>

The authors declare no conflicts of interest.

A photosensitizer increases the photochemical reaction of chemical compounds to generate ROS. Pathogenic microorganisms can be inactivated by light irradiation in the presence of photosensitizers. This study determines the aPDI of S. aureus due to ROS generated by violet light irradiation of an exogenous photosensitizer, FMN.
As shown in Figure 3, for FMN, the absorbance at 444 nm is reduced significantly after 5 min of irradiation using violet or blue light and there is no significant change in the FMN absorbance under irradiation with green or red light. NBT reduction is used to determine the formation of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> via a charge-transfer process10,33. A previous study by the authors used FMN under blue, green, or red-light irradiation to determine the formation of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> using NBT reduction. Blue light irradiation produces an enormous amount of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> whereas the photolytic efficiency of green and red light is less than 3% of that achieved by blue light10. These results show that a charge-transfer process is produced when FMN is exposed to blue or violet light irradiation and that there is no significant change upon irradiation by green or red light.
A previous study by the authors34 showed that the mechanism for FMN photolysis can be described with primary FMN photolytic reactions (Equation 1):
<img alt="Equation 2" src="/files/ftp_upload/63531/63531eq02.jpg" style="margin: auto;" /> (1),
where 1FMN* and 3FMN* are the singlet and triplet states of the excited FMN, respectively. The photolytic reaction mechanisms for these photoinduced FMN are possibly initiated by a triplet&#8722;triplet annihilation or a triplet-ground state quenching process35,36,37,38. The ground state of FMN (FMN0) is an electron donor that provides an electron to 3FMN* and generates the semi-reduced (FMN&#8226;-) and semi-oxidized (FMN&#8226;+) forms, as shown in Equation 2
<img alt="Equation 3" src="/files/ftp_upload/63531/63531eq03.jpg" style="margin: auto;" /> (2)
The photodegradation pathways for FMN result from photosensitization. FMN is promoted to the electronically excited state, FMN&#8226;, via a photolytic reaction and then, a neutral FMN is reduced when FMN&#8226; loses an electron, as shown in Equations 3-8 below:
<img alt="Equation 4" src="/files/ftp_upload/63531/63531eq04.jpg" style="margin: auto;" /> (3)
<img alt="Equation 5" src="/files/ftp_upload/63531/63531eq05.jpg" style="margin: auto;" /> (4)
<img alt="Equation 6" src="/files/ftp_upload/63531/63531eq06.jpg" style="margin: auto;" /> (5),
where FMN&#8226;+ is a radical cation species.
The interaction of FMN&#8226;- with O2 (3&#931;g-) produces the reactive superoxide radical, <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" />, followed by the hydroperoxyl radical, HOO&#8226;, which is generated in the reaction between <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> and a proton and eventually leads to the degradation of FMN0:
<img alt="Equation 7" src="/files/ftp_upload/63531/63531eq07.jpg" style="margin: auto;" /> (6)
<img alt="Equation 8" src="/files/ftp_upload/63531/63531eq08.jpg" style="margin: auto;" /> (7)
<img alt="Equation 9" src="/files/ftp_upload/63531/63531eq09.jpg" style="margin: auto;" /> (8),
where FMN&#8226;- is a radical anion.
In the present study, in the absence of FMN, the inactivation rate in the case of S. aureus is 53% under violet light irradiation at 10 W/m2 for 30 min and 26% under blue light irradiation at 20 W/m2 for 120 min, suggesting that blue light irradiation deactivates fewer bacteria than violet light (Figure 5). It has been reported earlier that irradiation at wavelengths longer than 430 nm without an exogenous photosensitizer does not inactivate S. aureus cells, although porphyrins in S. aureus have a maximum absorbance at 405 &#177; 5 nm and cause bacterial inactivation&#160;after irradiation39.
A previous study by the present authors showed that E. coli and S. aureus are deactivated by DNA cleavage that is induced by the formation of through photolysis of riboflavin or FMN10,24,40. The respective inactivation rates for S. aureus are 97% and 96%, respectively, using 120 &#181;M FMN under 20 W/m2 blue light irradiation (energy dose of 14.4 J/cm2) for 120 min and 30 &#181;M FMN under 10 W/m2 violet light irradiation (energy dose of 1.8 J/cm2) for 30 min (Figure 5). Therefore, violet light irradiation is more effective in deactivating S. aureus as low-energy doses of violet light can deactivate S. aureus in the presence of lower FMN concentrations and shorter illumination periods as compared with blue light. Although the extent of FMN photolysis achieved by violet light is higher compared with blue light (Figure 3), the average photolytic effects of FMN on NBT reduction under blue and violet light irradiation are similar (Figure 4).
E. coli is a common Gram-negative bacterium. Riboflavin or FMN photolysis by blue light irradiation inactivates E. coli via ROS10,24,41 with an inactivation rate of 96% when using FMN10,24. S. aureus, on the other hand, is a Gram-positive bacterium, which is effectively inactivated by FMN photolysis under violet light irradiation, as demonstrated here (Figure 5).&#160;Therefore, FMN photolytic reaction under blue or violet light irradiation can produce ROS and inactivate both Gram-negative and Gram-positive bacteria.
In the absence of FMN, however, little <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> is produced in S. aureus that is exposed to violet light irradiation (Figure 6); therefore, endogenous photosensitizers only slightly decrease the viability of S. aureus under violet light irradiation, compared to the effect in the presence of exogenous photosensitizer such as FMN. Under the same conditions, FMN that is exposed to violet light irradiation decreases the viability of S. aureus to result in a 96% inactivation rate, which is much higher than that for endogenous intracellular photosensitizers alone. The production of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> in FMN-treated S. aureus under violet light irradiation has significantly increased, as shown in Figure 6. These results show that FMN when exposed to violet light irradiation causes an elevated level of bacterial inactivation.
FMN can achieve a photo-excited state when irradiated using UV1,2,21,22,23, blue10,24, and violet light31; however, the cytotoxicity that is caused by UV radiation and high-intensity short-wavelength&#160;irradiation must be considered42. On the other hand, red and green radiations with longer wavelengths do not produce significant amounts of ROS during FMN photolysis10. Therefore, the ROS-induced aPDI of S. aureus requires irradiating FMN with violet light that has an intermediate wavelength. The spectral width at half height (W1/2) for violet light must be narrow to avoid any overlap with the UV absorption and inhibit risk due to UV radiation31. The micronutrient, FMN, is as such harmless, being an essential vitamin for the human body. Using an appropriate photosensitizer such as FMN, low-energy violet light irradiation can suppress pathogenic bacteria, providing a safe and effective means of the sanitary process. In addition to the photolytic reaction of FMN on S. aureus, other issues deserve further investigation, such as the phototoxicity of FMN on microbes, arising from the stress caused by <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> , which can be studied using an RNA-seq technique along with qPCR.
FMN photolysis via violet light irradiation leads to photochemical oxidation by which ROS is generated and in turn enhances the inactivation of pathogenic bacteria. The use of violet light is considered a critical step in FMN photodecomposition. The limitation of the current technique is that the wavelength interval of violet light must be quite narrow to avoid the risk due to UV irradiation. The current method has the potential for future medical applications, such as infection treatment of surface skin along with deep subcutaneous tissues or muscles by inserting an optical fiber to direct the violet light to the infected areas. Photochemical treatment using FMN is therefore a safe and simple way to ensure effective hygiene practices.

Riboflavin-5&#8242;-phosphate (FMN) is formed by phosphorylation at the riboflavin 5&#8242;-position of the ribityl side-chain and is required by all flavoproteins for numerous cellular processes to generate energy. It also plays the role of vitamin for some functions in the human body1. FMN is approximately 200 times more soluble in water than riboflavin2.
The antibacterial photodynamic inactivation (aPDI) of bacteria is an efficient way to control resistance to bacteria3,4 because it does not depend on the mode of bacterial resistance. Clinically, aPDI is used to treat soft tissue infections in order to decrease infection of nosocomial skin due to multi-resistant bacteria5,6,7,8,9. aPDI also produces cell death by generating reactive oxygen species (ROS). ROS, such as superoxide radicals (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" />), singlet oxygen, hydroxyl radicals (&#8226;OH), and peroxyl radicals, are free radicals or molecules that contain reactive oxygen10,11,12 and are normally reactive13. Similar to DNA damage that is caused by ROS, membrane peroxidation and destruction of endothelial cells are also adverse biochemical reactions that are attributed to ROS in cells12.
The use of aPDI for pathogenic bacteria&#160;involves a visible or UV light source to inactivate microorganisms in the presence of chemical compounds, such as methylthioninium chloride14, PEI-ce6 conjugate15, porphyrin16, titanium dioxide17, toluidine blue O18, and zinc oxide nanoparticles19. Toluidine blue O and methylene blue are phenothiazinium dyes and methylene blue has toxic properties. Zinc oxide nanoparticles and UV irradiation are linked to adverse health and environmental effects. As such, the exploitation of a reliable, secure, and simple photosensitizer via photolysis under visible irradiation deserves further study.
The micronutrient, riboflavin or FMN, is not toxic and is indeed used for food manufacturing or feeding20. Both FMN and riboflavin are highly sensitive to light irradiation2. Under UV1,2,21,22,23 and blue light irradiation10,24, these two compounds achieve an excited state. The activated riboflavin or FMN that is produced by photolysis is promoted to its triplet state and ROS are generated simultaneously2,25. Kumar et al. reported that riboflavin activated by UV light selectively causes increased injury to the guanine moiety of DNA in pathogenic microorganisms26. Under irradiation by UV light, photodynamically activated riboflavin is demonstrated to promote the generation of 8-OH-dG, which is a biomarker for oxidative stress, in double-stranded DNA27. It is reported that S. aureus and E. coli are deactivated by ROS during riboflavin or FMN photolysis10,24,28. A previous study by the authors showed that the photolytic reactions involving riboflavin and FMN reduce crystal violet, a triarylmethane dye and an antibacterial agent that generates <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" />, and eliminate most of the antimicrobial capability of crystal violet28. When flavin adenine dinucleotide or FMN is irradiated by blue light, the resulting ROS produce apoptosis in HeLa cells for their poisoning in vitro29. Using photochemical treatment in the presence of riboflavin, Cui et al. inactivated lymphocytes by inhibiting their proliferation and cytokine production30.
The photolysis of riboflavin is used for the inactivation of blood pathogen by UV10,24, but blood components can be impaired under UV light irradiation30. It is also reported that platelets exposed to UV progressively enhance the performance of the activation markers P-selectin and LIMP-CD63 on their membranes. The cytotoxicity of UV and high-intensity irradiation needs to be investigated and a photosensitizer that is uncomplicated and safe during an FMN photoreaction involving visible light would be of great use.
Light of shorter wavelengths has more energy and is much more likely to cause severe damage to cells. However, in the presence of a suitable photosensitizer, irradiation with low-intensity violet light can inhibit pathogenic microorganisms. The photosensitization and the generation of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> by FMN when irradiated with violet light is thus important to study, in order to determine the pathway by which ROS from FMN photolysis increases the inactivation of bacteria.
Antimicrobial control is a common issue and the development of new antibiotics frequently takes decades. After irradiation with violet light, photo&#173;inactivation that is intermediated by FMN can annihilate environmental pathogenic bacteria. This study presents an effective antimicrobial protocol in vitro using violet light for driving FMN photolysis and thus generating <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> for aPDI. The microbial viability of S. aureus is used to determine the feasibility of FMN-induced aPDI.

<table><tbody><tr><td>Blue, green and red LED lights</td><td>Vita LED Technologies Co., Tainan, Taiwan</td><td></td><td>DC 12 V 5050</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma-Aldrich, St. Louis, MO</td><td>190186</td><td></td></tr><tr><td>Infrared thermometer</td><td>Raytek Co. Santa Cruz, CA</td><td>MT4</td><td></td></tr><tr><td>LB broth</td><td>Difco Co., NJ</td><td></td><td></td></tr><tr><td>L-Methionine</td><td>Sigma-Aldrich, St. Louis, MO</td><td>1.05707</td><td></td></tr><tr><td>NBT</td><td>Bio Basic, Inc. Markham, Ontario, Canada</td><td></td><td></td></tr><tr><td>Power supply</td><td>China tech Co., New Taipei City, Taiwan</td><td>YP30-3-2</td><td></td></tr><tr><td>Riboflavin 5&amp;prime;-phosphate</td><td>Sigma-Aldrich, St. Louis, MO</td><td>R7774</td><td></td></tr><tr><td>RNase</td><td>New England BioLabs, Inc. Ipswich, MA</td><td></td><td></td></tr><tr><td>Solar power meter</td><td>Tenmars Electronics Co., Taipei, Taiwan</td><td>TM-207</td><td></td></tr><tr><td>Staphylococcus aureus subsp. aureus</td><td>Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan</td><td>10451</td><td></td></tr><tr><td>UV-Vis optical spectrometer</td><td>Ocean Optics, Dunedin, FL</td><td>USB4000</td><td></td></tr><tr><td>UV-Vis spectrophotometer</td><td>Hitachi High-Tech Science Corporation,Tokyo, Japan</td><td>U-2900</td><td></td></tr><tr><td>Violet LED</td><td>Long-hui Electronic Co., LTD, Dongguan, China</td><td></td><td></td></tr></tbody></table>

inactivation of pathogens via visible-light photolysis of riboflavin-5&#8242;-phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Effect of light wavelength on FMN 
The absorbance spectra of 0.1 mM FMN that is irradiated using blue, green, red, and violet LEDs are shown in Figure 3. There are two peaks for FMN (372 nm and 444 nm) for the dark control. Green and red light have no effect because changes in the spectra are insignificant. On the other hand, the respective absorbance of FMN at 444 nm is reduced by about 19% and 34%, respectively,&#160;after blue and violet light irradiation at 10 W/m2 for 5 min, suggesting that irradiation with blue/violet light increases FMN photolysis.
Detection of <img alt="Equation 10" src="/files/ftp_upload/63531/63531eq10.jpg" style="margin: auto;" /> from FMN that is irradiated using blue or violet light 
NBT reduction was used to determine the formation of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" />32. FMN is highly sensitive to visible and UV light even for short periods of radiation. <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> is formed from the intermediate photochemical reactions during the photolysis of FMN. Figure 4 shows that the photochemical effect of FMN on NBT reduction depends on the irradiation time, as <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> is formed from FMN exposed to blue or violet light irradiation in a time-dependent manner. The average photolytic effect of blue light and violet light is, however, similar as shown in Figure 4.
Effect of FMN photolysis on S. aureus viability 
The effect of FMN exposed to violet or blue light irradiation on S. aureus viability was determined. FMN photolysis using blue or violet light irradiation results in significant inactivation of S. aureus. The greater the concentration of FMN, the higher the inactivation rate of S. aureus that is exposed to blue light irradiation at 20 W/m2 for 120 min as shown in Figure 5A. Inactivation rates of 26%, 39%, 43%, and 97% were achieved for S. aureus using 0, 30, 60, and 120 &#181;M FMN, respectively, under 20 W/m2 blue light irradiation for 120 min. As shown in Figure 5A, there is no significant difference (p-value = 0.20) in the inactivation rate for S. aureus using blue light irradiation with either 60 &#181;M FMN at 20 W/m2 for 120 min (energy dose, 14.4 J/cm2) or 120 &#181;M FMN at 20 W/m2 for 60 min (energy dose, 7.2 J/cm2). On the other hand, as shown in Figure 5B, inactivation rates of 53% and 96% were achieved for S. aureus without and with 30 &#181;M FMN, respectively, under violet light irradiation at 10 W/m2 for 30 min (energy dose of 1.8 J/cm2). Therefore, FMN exposed to blue or violet light irradiation has a greater effect on the viability of S. aureus by increasing the bacterial inactivation. FMN treated with violet light irradiation is more efficient in S. aureus inactivation.
Detection of <img alt="Equation 10" src="/files/ftp_upload/63531/63531eq10.jpg" style="margin: auto;" /> in FMN-treated S. aureus under violet light irradiation 
The production of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> in FMN-treated S. aureus under violet light irradiation was determined using the NBT reduction method. The absorbance value at 560 nm was used to evaluate the extent of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> production. The photochemical effect of FMN on <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> generation in S. aureus is proportional to the FMN concentration (Figure 6). The 560 nm absorbance values for S. aureus without FMN treatment are 0.31 and 0.07 under 10 W/m2 violet light irradiation for 10 min and in the dark, respectively (Figure 6). This suggests that little <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> was produced in untreated S. aureus after violet light irradiation. The respective 560 nm absorbance values for S. aureus treated with 100 &#181;M FMN are 1.36 and 0.08 under 10 W/m2 violet light irradiation for 10 min and in the dark, respectively. Therefore, the photochemical effect on NBT reduction in FMN-treated S. aureus increases under violet irradiation,&#160;as <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> is produced abundantly.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63531/63531fig01.jpg" /> 
Figure 1: Schematic representation of the photoreaction system. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63531/63531fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63531/63531fig02.jpg" /> 
Figure 2: The emission spectra of blue, green, red, and violet LED lights in this study. The emission maxima for the blue, green, red, and violet lights are at 465, 529, 632, and 405 nm, respectively, and the spectral widths at half-height (W1/2) are 26, 31, 14, and 12 nm, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63531/63531fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63531/63531fig03.jpg" /> 
Figure 3: Absorbance spectra of FMN treated with LEDs of various wavelengths at 10 W/m2 for 5 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63531/63531fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63531/63531fig04.jpg" /> 
Figure 4: NBT reduction to determine the effect of blue and violet light irradiation at 10 W/m2 for 1-5 min on FMN photolysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63531/63531fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63531/63531fig05.jpg" /> 
Figure 5: Effect of FMN photolysis on S. aureus viability. The percentage reduction is defined as a negative value of the inactivation rate for S. aureus treated with (A) FMN under blue light irradiation at 20 W/m2 for 120 min and (B) FMN under violet light irradiation at 10 W/m2 for 30 min. FMN concentrations and irradiation times are indicated at the top of the figure. Note that there is no significant difference (p = 0.20) in the inactivation rate for S. aureus using 60 &#181;M FMN at 20 W/m2 blue light irradiation for 120 min or 120 &#181;M FMN at 20 W/m2 blue light irradiation for 60 min (A). Results are expressed as mean &#177; SD, where n = 3. *p &#60; 0.05; **p &#60; 0.01; ****p &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63531/63531fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63531/63531fig06.jpg" /> 
Figure 6: Effect of FMN concentration on <img alt="Equation 10" src="/files/ftp_upload/63531/63531eq10.jpg" style="margin: auto;" /> generation in S. aureus subjected to violet light irradiation at 10 W/m2 for 10 min. Overall, little <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> is produced in S. aureus in the absence of FMN although violet light irradiation slightly increases <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> in the irradiated sample (PB (Violet light)) relative to dark control (PB (Dark)). Results are expressed as mean &#177; SD, where n = 3. * p &#60; 0.05; ** p &#60; 0.01; **** p &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63531/63531fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5â€²-Phosphate at JoVE.com

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

inactivation-pathogens-via-visible-light-photolysis-riboflavin-5

Nanyang Technological University

Research

JoVE Journal

Bioengineering

1.8K Views.  Ming Chuan University. Dit protocol maakt gebruik van zichtbare violette lichtbestraling om flavine mononucleotide fotolyse te induceren. Dit produceert een grote hoeveelheid vrije radicalen, die pathogene bacteriën zoals Staphylococcus aureus en E.coli remmen. We hebben flavine mononucleotide getest als een geschikte fotosensitizer.In dit protocol gebruiken we zichtbaar violet licht dat veilig is en geen dure apparatuur vereist. Deze methode kan worden gebruikt voor de therapie van gewonde huid of geïnfec...

Video: Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5′-Phosphate

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

Immunology and Infection

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. Products are routinely stored at 22 &#176;C on an agitating shaker, a condition that can promote bacterial growth. Although the total number of bacteria believed to be introduced into a platelet product is extremely low, these bacteria can multiply to a very high titer prior to transfusion, potentially resulting in serious adverse events. The aim of this study was to evaluate a riboflavin based pathogen reduction process against a panel of bacteria that have been identified as common contaminants of platelet products. This panel included the following organisms: S. epidermidis, S. aureus, S. mitis, S. pyogenes, S. marcescens, Y. enterocolitica, B. neotomae, B. cereus, E. coli, P. aeruginosa and K. pneumoniae. Each platelet unit was inoculated with a high bacterial load and samples were removed both before and after treatment. A colony forming assay, using an end point dilution scheme, was used to determine the pre-treatment and post-treatment bacterial titers. Log reduction was calculated by subtracting the post-treatment titer from the pre-treatment titer. The following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805;5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%). The results from this study suggest the process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass optical fiber inserted into the electron column above the objective lens polepiece, and a laser, an adjustable light source, was used to fabricate the device. After the illuminator has been calibrated using an external measuring system, it allows one to adjust the intensity of the lighting to the needs of the observed process. This lighting system was utilized to image antimicrobial photodynamic therapy phenomena, which are currently the subject of intense research. The sample was prepared by spotting a suspension of bacteria on a carbon, graphene, or silicon nitride substrate, blotting the excess solution, spotting the photosensitizer solution, blotting the excess liquid again, and then assembling the liquid cell with a second substrate or graphene film. The process of the imaging experiment itself includes choosing the right place for observation with the use of low magnification and a minimum dose of electrons, and then cyclical activation of the light source to capture subsequent images at specified intervals with the minimum amount of electrons necessary. The electron dose of each exposure and the time and intensity of lighting used need to be carefully recorded due to the complexity of the observed phenomena as, at the same time, the process is both light- and electron-driven. After the actual experiment is performed, additional control observations must be made, in which the same doses of electrons are used but without additional light influence and smaller doses of electrons are used for higher doses of light. This makes it possible to distinguish light-induced microstructural effects from those caused by electrons in both the fields of life and materials science.

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The present protocol describes transmission electron microscope (TEM) modifications with a light illumination system, the fabrication of liquid cells, and in situ TEM observations of light-induced interactions between bacterial cells and a photosensitizer. The sample preparation methods, electron beam damage, and imaging are also discussed.

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass ...

Engineering

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

Biochemistry

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

Biology

Analyzing Petite Colony Formation and Drug Resistance in Candida glabrata

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (&#8805;99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

Author Spotlight: Photodynamic Therapy as a Novel Approach to Induce Petite Colonies in Drug&#45;Resistant Candida for Antifungal Research

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

A Riboflavin and UV Light Treatment of Platelet Products to Eliminate Bacteria

Source: Keil, S. D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52820">Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination.</a> J. Vis. Exp. (2015).
This video demonstrates the riboflavin and UV light-based pathogen reduction process against high-titer bacterial contamination in human platelet products. This process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

1. Platelet Product Preparation

	Collect a standard apheresis human platelet product at an accredited blood bank. The platelet product must meet the ...

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5′-Phosphate

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