High Intensity Blue Light (405-470 nm) Sterilization of Surface Bacteria

with Riboflavin (Vitamin B2)

By: Ava Chaikin

As Partial Completion of the Science Research II course

June 2023
Massapequa High School

Teacher: Dr. Paul Hesleitner

Abstract:
This project seeks to determine a new mode of bacterial sterilization predominantly with
high intensity blue light. This study proposes the limited use of ultraviolet light due to potential
health implications. Applications discussed include creating an iRobot to sterilize nursing homes,
facilities that handle food, and hospitals. The furtherance of this application is predicated on the
success of the principle that when bacteria is treated with Riboflavin, Vitamin B2, as a
photosensitizer and illuminated with intense visible light the bacteria would be killed and the
surface sterilized. To determine the validity of this notion, surface bacteria was cultured from
locations around Massapequa High School. A 0.01M aqueous Riboflavin solution was made
using powdered Riboflavin. The Riboflavin solution was sprayed onto the surface bacteria; the
bacteria was put under the blue light for a period of time and then placed back in the incubator.
The cultures were then counted to compare with the control plates to determine how many
bacteria were eliminated. The data collected suggested that the bacteria was not sterilized by this
procedure. In the future, suspension cultures can be utilized in an attempt to distribute the
photosensitizer more thoroughly. Different wavelengths of light and photosensitizers can be
utilized to determine if they would catalyze sterilization. Most crucially, more trials can be done
with the above procedure to mitigate the potential for human error in this trial. In summation,
this is an ongoing study and more research is needed to conclude this potential sterilization
method as not possible.
Introduction:
A vast array of past experiments have been conducted to determine the effectiveness of
light sterilization. Amid various journal articles it has been determined that light sterilization
with Ultra Violet (UV) light is effective. That said, UV light can prove to be detrimental to
humans. As noted by the American Cancer Society, UV light can cause premature aging of the
skin, liver spots, weaken the immune system, and increase risk of obtaining melanoma. The
alternative would be using diffused UV light or blue light in an attempt to sterilize the bacteria
whilst still ensuring the safety of people who are around the sterilization mechanism—which is
discussed in project goals (RIKEN, 2022). Blue light was utilized due to the fact that it has a
wavelength relatively close to that of UV light but is safer when humans are exposed to it.
Studies previously conducted with regards to this topic have looked into niche uses for
the proposed sterilization mechanism. For instance, one research team looked into the
sterilization of the bacteria strand, Propionibacterium acnes, with blue light to reduce acne in
young adults and combat antibiotic resistant bacteria (Ashjenazi et al., 2002). Similarly, years
later a research team studied the effectiveness of blue light sterilization for infectious diseases
with Propionibacterium acnes and Helicobacter pylori (Dai et al., 2012). Another study sought
to determine the effect of light emitting diodes (LEDs) of various wavelengths and illumination
temperatures on foodborne pathogens (Ghate et al., 2013). Though this topic has been studied for
years, each journal article lacks the diversity needed to create a sterilization mode that would be
beneficial on a large scale (e.g. in hospitals or nursing homes). These topics often study specific
strains of bacteria which may lack practicality in large scale facilities seeing that they would
require a system that can sterilize most strains of gram-negative surface bacteria.
Photosensitizers utilized often seem to be left unspecified, and it often takes an extensive amount
of time to sterilize the bacteria. In an attempt to differentiate this experiment from the previous,
the bacteria that is being sterilized was cultured from real surfaces; if bacteria were sterilized, it
can be suggested that the procedure followed is veristle enough to sterilize many strands of
bacteria. Further, a photosensitizer will be utilized to aid in the sterilization of the bacteria.
The photosensitizer utilized for this project is called Riboflavin—or Vitamin B2.
Riboflavin was utilized as it has no safety implications, is readily available, and was noted in
various articles as an efficient photosensitizer. Though vitamin B2 has various health benefits
such as aiding in metabolic processes (conversion of carbohydrates into glucose and
metabolizing fats and proteins), it can also be used in an attempt to aid in the sterilization of
bacteria: specifically antibiotic resistant bacteria (Mount Sinai, 2023). Photosensitizers can be
simply defined as a chemical that absorbs light energy. This is often used in antibacterial
photodynamic therapy (APDT); “a process utilizing light and light sensitive agents … and is
usually applied in an oxygen-rich environment” (Ghorbani et al., 2018). To do this, the photons
of light are absorbed into the photosensitizer and passed on to the surrounding molecules.
Oxidative molecules are formed, killing bacterias’ organic substances. Unlike antibiotics, this
versatile process allows for this to sterilize a favorable amount of bacteria species given the
correct photosensitizer. This is depicted in Figure 1.
Project Goal or Hypothesis:

Figure 1: Diagram showing the

process of APDT and depicting how
If surface bacteria is sprayed with a 0.01M solution of the photosensitizer, Riboflavin, and
placed under high intensity blue light (405-470 nm) for 2 minutes, then there will be fewer
colonies on the experimental plate than the control plate (not sprayed with Riboflavin or placed
under blue light). With the data to support this hypothesis, the chief aim of this project is to
sterilize (gram negative) bacteria efficiently in attempt to create a “vacuum” or iRobot that can
safely (without ultraviolet light) sterilize bacteria on the floors of facilities where sterilization is
crucial (e.g. nursing homes, restaurants, and potentially hospitals).

Methods & Materials:

The materials used for this experiment are as follows:
➢ Petri Dishes with Agar
○ These were used to grow surface bacteria to be used as control and experimental
plates.
➢ Incubator
○ The incubator was utilized to keep the surface bacteria (in the agar plates) at a
temperature at which they would go efficiently.
➢ Swabs
○ Swabs were used to pick up bacteria from the desired surface (locker room
bathroom sink) and transfer it to the aforementioned agar plates.
➢ Parifilm
○ Parifilm was utilized to seal agar plates.
➢ 0.01M Riboflavin Solution (Riboflavin and Water)
○ Riboflavin solution was sprayed on the experimental plate to aid in the
sterilization of bacteria via the process mentioned in the introduction.
➢ Spray Bottle
○ The spray bottle holds the Riboflavin solution to ensure a controlled amount of
the solution was sprayed on the bacteria.
➢ Sharpie Marker
○ The Sharpie was used to label the agar plates to keep track of the experimental
plate.
➢ 250mL Beaker
○ The beaker was utilized to measure the correct amount of water for the Riboflavin
solution.
➢ Digital Balance
○ The balance was used to mass the correct amount of Riboflavin.
➢ 405-470 nm Blue Light
○ The blue light was used as the first attempt to sterilize surface bacteria.

The following variables and methods implemented during the experiment:

Independent Variable- Utilizing Riboflavin in conjunction with the blue light.
Dependent Variable- Bacterial growth or sterilization
Constants- Constants include temperature of the incubator, source of bacteria samples, blue light
wavelength, and time in between taking the samples and counting colonies.
Control Group- The group lacking Riboflavin was the control group.
Experimental Group- The group with Riboflavin and blue light was the experimental group.

The following procedure was used to conduct the experiment:

Step 1-Bacteria Collection:
1.1- Gather agar plates, swabs, and a Sharpie marker.
1.2- Open the swabs.
1.3- Take one swab out of the package.
1.4- Swab for surface bacteria.
1.5- Take the swab from step 1.4, and swab the entire agar plate.
1.6- Label the agar plate with the location of bacteria (from step 1.4) and “control” or
“experiment” depending on if it will be subject to the blue light or not.
1.7- Close the agar plate.
1.8- Seal the agar plate with parafilm.
1.9- Put the agar plate into the incubator for two days.
1.10- Repeat steps 1.1-1.9 for the experimental plate.

Step 2-Make Riboflavin Solution

2.1- Measure water in a 250 mL beaker.
2.2- Measure Riboflavin on a digital balance.
2.3- Place the Riboflavin in the beaker.
2.4- Stir the solution.
2.5- Place the solution in the spray bottle.
*If the solution is not used immediately, place the spray bottle in the refrigerator.

Step 3-Sterilize the Bacteria

3.1- Remove the bacteria from the incubator.
3.2- Take pictures of the bacteria for comparison (see Figure 2).
3.3- Spray Riboflavin on the agar plate until every colony is covered.
3.4- Place the agar plate under the blue light.
3.5- Leave the Riboflavin on the bacteria for five (5) minutes.
3.6- Turn on the blue light.
3.7- Leave the agar plate under the blue light for two (2) minutes.
3.8- Turn off the blue light after the two minutes are up.
3.9- Place the agar plate in the incubator for two days.

Step 4-Data Collection

4.1- Remove the bacteria from the incubator.
4.2- Take pictures of the bacteria for comparison (see Figure 3).
4.3- Count the colonies on the control plate.
4.4- Record that value on a spreadsheet.
4.5- Count the colonies on the experimental plate.
4.6- Record that value on the same spreadsheet from step 4.3.

Results:
Bacteria was cultured from the girls’ locker room bathroom at Massapequa High School.
As seen in Figure 2, cultures were successful. Riboflavin was made and applied in accordance
with method steps 2, 3.3 and 3.5 respectively. Light was first broadly spread across the plate; in
the future, it will be concentrated to a particular colony. After failure to accurately determine if
bacteria had been (to any degree) sterilized, bacteria was transferred from the blue light plate to
an additional agar plate (see figure 4). This was in an attempt to determine if bacteria was able to
reproduce (i.e. if the bacteria had not grown on the plate in Figure 4 but did grow on the control
plate, it could be determined that the bacteria had been sterilized).

Figure 2: Bacteria Figure 3: Bacteria after

Figure 4: This is a photograph of an

attempt at seeing if any bacteria was

Analysis:
As depicted in Figures 2, cultured bacteria did grow successfully. As seen in Figure 3, no
evident change was seen in the bacteria. Figure 4 also shows the lack of sterilization as bacteria
was still grown after being transferred from the sterilized plate to additional agar plates. This
suggests that the bacteria was not sterilized when blue light was shown on the plate with
Riboflavin (or without Riboflavin).

Conclusions:
To restate, the hypothesis for this experiment was the following: If surface bacteria is
sprayed with a 0.01M solution of the photosensitizer, Riboflavin, and placed under high intensity
blue light (405-470 nm) for 2 minutes, then there will be fewer colonies on the experimental
plate than the control plate (not sprayed with Riboflavin or placed under blue light). With the
data collected from the above trial, this hypothesis must be rejected seeing that no sterilization
was evident after being treated with a 0.01M solution of Riboflavin and placed under high
intensity blue light with the preceding wavelength. That said, this is an ongoing study; it requires
more data to reject the hypothesis with certainty.
Future research includes utilizing different wavelengths of light (such as a red light) with
Riboflavin as a photosensitizer. Interestingly, Vera Bärenfaller and her co-authors collected data
supporting that red light should be able to sterilize some times of surface bacteria suggesting this
wavelength should be further studied (Bärenfaller et al., 2016). Comparably, a recent study
evaluated the impacts of violet, blue, green, and red light regarding the sterilization of P.
fluorescens as planktonic cells, individual cells, and biofilms (Angarano et al., 2020).
Additionally, various other photosensitizers could be tested to determine their effectiveness
regarding bacteria sterilization. Moreover, by utilizing suspension cultures rather than agar plates
in the future, the Riboflavin may be able to more properly be absorbed by bacteria allowing for
sterilization. In furtherance, determining the shortest amount of time the light needs to be on the
bacteria is important to determine the necessary speed of the vacuum. It is also important to
determine if the bacteria is gram positive or gram negative; with this information, a greater
understanding of the cell membrane could be established allowing for the potential to expand on
the photosensitizers used. Ideally, gram negative bacteria would be sterilized and they are more
commonly harmful to people and are more resistant to antibiotics. Sterilization has become a
more pressing concern with the Covid-19 pandemic and individuals becoming more aware of
antibiotic resistant bacteria; with this, creating systems of bacteria sterilization that is effective
and mitigates harm to humans is necessary.
 References

Angarano, V., Akkermans, S., Smet, C., Chieffi, A., & Van Impe, J. F.M. (2020,

November). The potential of violet, blue, green and red light for the inactivation of P.

fluorescens as planktonic cells, individual cells on a surface and biofilms. Food and

Bioproducts Processing, 124, 184-195. Science Direct.

https://www.sciencedirect.com/science/article/abs/pii/S0960308520304818

Ashkenazi, Malik, Harth, & Nitzan. (n.d.). “Eradication of Propionibacterium Acnes by

Its Endogenic Porphyrins after Illumination with High Intensity Blue Light.” FEMS

Immunology and Medical Microbiology. U.S. National Library of Medicine.

https://pubmed.ncbi.nlm.nih.gov/12589953/#:~:text=Eradication%20of%20P.,75%20J

%20cm(%2D2)

Bache, Maclean, MacGregor, Anderson, Gettinby, Coia, & Taggart. (n.d.). “Clinical

Studies of the High-Intensity Narrow-Spectrum Light Environmental Decontamination

System (Hins-Light Eds), for Continuous Disinfection in the Burn Unit Inpatient and

Outpatient Settings.” Burns : Journal of the International Society for Burn Injuries. U.S.

National Library of Medicine. https://pubmed.ncbi.nlm.nih.gov/22103991/

Bärenfaller, Clausen, Sculean, & Eick. (n.d.). “Effect of Photoactivated Disinfection

Using Light in the Blue Spectrum.” Journal of Photochemistry and Photobiology, B,

Biology. U.S. National Library of Medicine. https://pubmed.ncbi.nlm.nih.gov/26994334/

Buchovec. (2017, May 9). “Inactivation of Gram (−) Bacteria

Salmonella Enterica by Chlorophyllin-Based Photosensitization:

Mechanism of Action and New Strategies to Enhance the Inactivation

Efficiency.” Journal of Photochemistry and Photobiology B: Biology. Elsevier.

https://www.sciencedirect.com/science/article/pii/S1011134417302762

Deshpande, & Ajay. (2018, April). “Efficacy and Safety Evaluation of High-Density

Intense Pulsed Light in the Treatment of Grades II and IV Acne Vulgaris as Monotherapy

in Dark-Skinned Women of Child Bearing Age.” The Journal of Clinical and Aesthetic

Dermatology. U.S. National Library of Medicine.

“Does UV Rradiation Cause Cancer?: American Cancer Society.” Does UV Rradiation

Cause Cancer? (n.d.). American Cancer Society. https://www.cancer.org/healthy/cancer-

causes/radiation-exposure/uv-radiation.html#:~:text=Exposure%20to%20UV%20rays

%20can,to%20become%20inflamed%20or%20burned

“Far-Ultraviolet Led Can Kill Bacteria and Viruses Efficiently without Harming

Humans.” (2022, October 4). Phys.org, Phys.org. https://phys.org/news/2022-10-far-

ultraviolet-bacteria-viruses-efficiently-humans.html

Ghate, Zhou, Yang, Khoo, Yoon, & Yuk. (n.d.). “Antibacterial Effect of Light Emitting

Diodes of Visible Wavelengths on Selected Foodborne Pathogens at Different

Illumination Temperatures.” International Journal of Food Microbiology. U.S. National

Library of Medicine. https://pubmed.ncbi.nlm.nih.gov/24026011/

Ghorbani, J. (2018, December 31). “Photosensitizers in Antibacterial Photodynamic

Therapy: An Overview.” Laser Therapy, U.S. National Library of Medicine.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513767/

Gomelsky, & Hoff. (n.d.). “Light Helps Bacteria Make Important Lifestyle Decisions.”

Trends in Microbiology. U.S. National Library of Medicine.

https://pubmed.ncbi.nlm.nih.gov/21664820/

Gupta, Murray, Vrahas, Tegos, & Hamblin. (n.d.). “Blue Light for Infectious Diseases:
Propionibacterium Acnes, Helicobacter Pylori, and beyond?” Drug Resistance Updates :

Reviews and Commentaries in Antimicrobial and Anticancer Chemotherapy. U.S.

National Library of Medicine. https://pubmed.ncbi.nlm.nih.gov/22846406/

Jo, M. (2022, May 23). “Milliwatt-Power Far-UVC Algan Leds on Sapphire Substrates.”

AIP Publishing. AIP Publishing LLCAIP Publishing.

https://aip.scitation.org/doi/pdf/10.1063/5.0088454

“Journal of Food Protection.” (n.d.). Journal of Food Protection. Elsevier.

https://meridian.allenpress.com/jfp/article/67/5/1027/169877/Inactivation-of-

Staphylococcus-aureus-by-Pulsed-UV.

Kim, Min-Jeong, & Hyun-Gyun Yuk. (2017, February 15). “Antibacterial Mechanism of

405-Nanometer Light-Emitting Diode against Salmonella at Refrigeration Temperature.”

Applied and Environmental Microbiology. U.S. National Library of Medicine.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311417/

Kwon, H. (2012, June 15). “Effect of 635 Nm Irradiation on High Glucose-Boosted

Inflammatory Responses in LPS-Induced MC3T3-E1 Cells - Lasers in Medical Science.”

SpringerLink. Springer-Verlag. https://link.springer.com/article/10.1007/s10103-012-

1122-3

Maclean, M. (2009, April). “Inactivation of Bacterial Pathogens Following Exposure to

Light from a 405-Nanometer Light-Emitting Diode Array.” Applied and Environmental

Microbiology. U.S. National Library of Medicine.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2663198/

Shimoda, H. (2021, June 17). “Efficacy of 265-Nm Ultraviolet Light in Inactivating

Infectious SARS-COV-2.” Journal of Photochemistry and Photobiology. Elsevier.

https://www.sciencedirect.com/science/article/pii/S266646902100035X

Vermeulen, Keeler, Nandakumar, & Leung. (n.d.). “The Bactericidal Effect of Ultraviolet

and Visible Light on Escherichia Coli.” Biotechnology and Bioengineering. U.S. National

Library of Medicine. https://pubmed.ncbi.nlm.nih.gov/17680675/#:~:text=Radiation

%20at%20265%20nm%20in,mJ%2Fcm(2)

“Vitamin B2 (Riboflavin).” (n.d.). Mount Sinai Health System.

https://www.mountsinai.org/health-library/supplement/vitamin-b2-riboflavin