Investigating The Impact Sound Frequency Has on Escherichia coli Growth

Doris Huang – Life Science, Year 2

Abstract

Some variants of E. coli have detrimental effects on the human body (U.S. Food and Drug Administration, 2020). Due to poor sanitation, diseases are often more prevalent. By controlling the environment, it suggests a promising remedy for the ongoing health hazards. There are many limitations of how the environment can be controlled. However, audible sounds are a factor that is present everywhere and can be easily manipulated. Although audible sounds are an environmental factor that exists widely in the natural world, the interaction between biological materials and audible sounds is often overlooked in the literature. While some research suggests that sound may inhibit bacterial growth, the frequency that would best impede the growth of E. coli is still ambiguous. Therefore, the present experiment investigates the impact of three sound frequencies, 1,000 Hz, 6,000 Hz, and 18,000 Hz, on the growth of E. coli. The three treatment groups were placed into a sound chamber with a MP3 player. The most effective frequency was 6,000 Hz, where the average CFU/ml was less than half of the average control, suggesting a suppression in the growth rate of the E. coli. Potential future applications could include an alternative method for bacterial control, not restricted to only E. coli. These findings suggest a potential substitute for current methods of bacterial control. Additionally, in a world of increasing antibiotic resistance, alternative bacterial control methods must be researched.

Introduction

Although the majority of the strains of Escherichia coli (E. coli) are considered harmless, variants such as Shiga toxin-producing E. coli is among those that do pose harm towards humans (U.S. Food and Drug Administration, 2020). The transmission is typically through food: undercooked ground meats, raw milk and containment raw vegetables (World Health Organizations, 2018). Certain ranges for factors such as pH levels, temperature, water activity may either inhibit or allow the prosperity of the living organism. For Shiga toxin-producing E. coli for example, the optimum temperature is 37 °C. However, temperatures ranging from 7 °C to 50 °C will still allow the bacteria to grow (World Health Organizations, 2018). Although many of the environmental factors are thoroughly researched such as manipulations with pH levels on the impact of bacterial growth (Ratzke and Gore, 2018), one factor has only a surface level of research that is not typically discussed: sound frequency. According to Shaobin, et al. (2010), although audible sounds is an environmental factor that exists widely in the natural world, the interaction between biological materials and audible sounds is often overlooked. To the author’s knowledge, there have only been minor efforts to seek the correlation between audible sounds and microbes, despite being an environmental factor that is prominent in the natural world.  Understanding the impact of audible sounds on bacterial growth may open a new branch of studies, allowing for the manipulation of environmental factors to mitigate the spread of various bacterial diseases.

Gu, et al. (2016), investigated the manipulation of various acoustic parameters and studied the biological effects, including intracellular macromolecular synthesis and cellular morphology, on the growth of E. coli K-12. The E. coli K-12 was exposed to different conditions: sound frequencies of 250-16,000 Hz with sound intensity of 80dB and sound power level of 55 dB; sound intensities of 0-100 dB with sound frequency 8,000 Hz and sound power level of 55 dB; and sound power level 55-63 dB with 8,000 Hz and 80 dB. The results displayed that sound created a mechanical stress, as they found that as sound intensified bacterial growth was slower. However, the mechanism of how sound effects microorganism growth remains ambiguous. Conversely, Gu, et al (2010) conducted research to investigate the response of E. coli cells under environmental stresses such as salt and sound stresses. The results indicated that audible sounds could contribute significantly to the formation of colonies. Audible sound treatment appeared to promote growth even with the inhibitory effect of salt stress on E. coli growth.

Ku, et al. (2021) explored the effects of music on the motility of bacteria, specifically E. coli. The music used within this study was “Flight of The Bumblebee”: a classical music with varying tempos and frequencies. The experiment was quantified by two approaches: indirect motility assay and in-situ monitoring on the swimming behaviour of E. coli under an optical microscope. In both methods, the results indicated that with varying frequencies and tempos from the music, the motility of the E. coli was affected to different extents. In general, with music that had higher frequency and faster tempo, the motility could be further enhanced. Lower frequencies and slower tempo also had enhanced average motility compared to the control. The findings indicate a new analytical approach for the research of the motility of E. coli.

The research cited illustrates that the growth rate and motility of E. coli is impacted by sound frequency. When exposed to various sound frequencies bacteria growth rate has been reported to be either slowed or enhanced. Understanding the threshold at which frequency impedes bacterial growth could allow for sound treatment to be utilized for the control of bacterial growth. With increasing antibiotic resistance, it is crucial to for alternative methods for bacterial control and treatments to be understood (Centers for Disease Control and Prevention, 2024). However, information such as what frequency would best mitigate the growth of E. coli is still ambiguous. The present experiment will investigate the impact of three sound frequencies, 1,000 Hz, 6,000 Hz, and 18,000 Hz, on the growth of E. coli. 1,000 Hz is the sound level in which E. coli is exposed to in the environment; 6,000 Hz would indicate stress towards the bacteria and 16,000 Hz would indicate more stress towards the E. coli. With the 16,000 Hz experiment, it’ll reveal the trajectory of the growth of E. coli.

Materials and Methods

Two sound chambers were constructed using acoustic foams (Foneso) with two plastic containers (Dollarama). Four sheets of acoustic foam were used to minimize the sound leakage from the plastic containers. For two sheets of acoustic foam, the spiked sides were cut to maximize the area to place the MP3 player (Leguwu) and two Petri dishes containing E. coli. The sound chamber was sound proofed using sheets of acoustic foam. Frequencies of 6,000 Hz, 1,000 Hz and 16,000 Hz were transferred to an MP3 player. LB Agar was melted using the microwave on power 7 for three minutes. The lid was loose for venting to occur. The Petri dishes were labelled and prepared. When the agar was cooled and solidified, the lid was closed.  Centrifuge tube racks and four centrifuge tubes labeled numbers one to four were prepared for the 1 in 1000 dilution of the E. coli. Using a graduated cylinder and balance, 10 ml of water and 0.18 g of sodium chloride were mixed in a 250 ml beaker to create a saline solution. A micropipette was used to pipette 1.5 ml of the saline solution into the centrifuge tube #1. Three E. coli bacterial colonies were picked up from a source E. coli Petri dish using three inoculating loops. All three inoculating loops were swirled in centrifuge tube #1. Using a micropipette, 100 µl from tube #1 was added to tube #2, which contained 900 µl of saline creating a 1 in 10 dilution. To prepare the 1 in 100 dilution, 100 µl from tube #2 was added to 900 µl of saline in tube #3. Finally, the process was repeated for tube #4, which created a 1 in 1000 dilution.

A micropipette was used to pipette 0.3 ml from tube #4 onto the Petri dishes. The L spreader was used to evenly spread the E. coli on the Petri dish. This process was repeated five times until all the Petri dishes were plated. The two control plates were directly placed in the incubator. Two Petri dishes labelled with the same frequency were placed in the same sound chamber with the MP3 player playing the relevant frequency. The two sound chambers, with the lid shut, were then placed in the incubator with the MP3 player working (Figure 1). Data was collected after one week and the process was repeated until two trials of each of the three sound frequencies were complete. The data was collected through the method of colony-forming units per milliliter (CFU/ml). CFU/ml is calculated through counting the number of individual colonies on the Petri dishes, then multiplying the reciprocal of the dilution factor (1000 in this case) and finally accounting for the volume of culture that was plated. The values obtained per each sound frequency in one week was the average of 2-4 Petri dishes within the same week. For each sound frequency, there are two repetitions throughout the four weeks this experiment was conducted.  

Figure 1: Sound chamber where bacteria was exposed to the sound frequencies via MP3 player

Results

In Table 1, n represents the total number of samples used to calculate the mean: n=8 for control, n=6 for 16,000 Hz, but n=4 for 1,000 Hz and 6,000 Hz treatment groups.

Table 1: Average CFU/ml Values and Standard Deviation of Each Sound Frequencies and Control

A standard deviation error bar is displayed within Figure 2. The highest averaged CFU/ml recorded was from the control with 2.27E+07 CFU/ml. The average CFU/ml for the three sound frequencies displayed values much lower than the average CFU/ml for the control. The average CFU/ml for 1,000 Hz, 6,000 Hz, and 16,000 Hz were 1.23E+07, 8.40E+06, and 1.17E+07 respectively.

Figure 2:  Average CFU/ml Values for Tested Sound Frequencies and Control

Discussion

Based on the results from Figure 2, the E. coli exposed to sound frequencies had less CFU/ml compared to the control. The average CFU/ml varied between the three sound frequencies: however, not as anticipated. The frequency that showcased the biggest reduction in CFU/ml compared to the control was the 6,000 Hz with an average CFU/ml of 8.40E+06 compared to the average control value of 2.27E+07. Although through past research it has been suggested that sound frequency can mitigate the growth of E. coli, the results of the present study do not directly display a correlation of increased frequency resulting in decreased bacterial growth rate. The results obtained by 16,000 Hz doesn’t align with the trend and results that the studies mentioned observed.

In the first week of the experiment the vortex was not used. However, for the weeks that followed, a vortex was used to thoroughly mix the bacterial suspension. This inconsistency may have resulted in the outlier values of the control and possibly the 6,000 Hz treatment. In fact, in that test week 6,000 Hz and the control yielded lower CFU/ml (Appendix A). In the future, the experimental methods should be kept consistent.

The higher than anticipated CFU/ml in the 16,000 Hz group obtained from Figure 2 as mentioned above may be because of the uneven distribution of the exposure of the sound frequency to the bacteria. As shown in Figure 1, the MP3 player is placed on the side and may be muffled by the acoustic foam due to the limited space. A further limitation that occurred during the experiment was the battery life of the MP3 player. Based on the average battery life of the MP3 player, it is believed that the bacteria were exposed to the sound frequencies for 8-10 hours, contributing experimenter errors. To avoid these issues in future experiments, it is recommended to learn to code Arduino. Not only would this be cost efficient, but the Arduino battery life would be much longer, ranging from a few days to a few weeks (Arduino, 2025).

The first trial for the 6,000 Hz was contaminated and more dilution was necessary to accurately count the bacterial colonies. Therefore, after the first trial the dilution was changed from 1 in 10 to 1 in 1000. However, due to the strenuous work in counting individual bacterial colonies into the thousandths, it is recommended for future experiments to have a 1 in 10,000 dilution for both accuracy and ease of counting. Additionally, due to space constraints and time constraints, only two Petri dishes per trial and two repetitions were able to be conducted for each sound frequency. Future experiments should include an upgraded sound chamber with sufficient space to allow three Petri dishes to fit and ensure to have three trials done.

The most effective frequency was the 6,000 Hz, where the average CFU/ml was less than half of the average control, suggesting a suppression in the growth rate of the E. coli. Potential future applications could include an alternative method for bacterial control, not restricted to only E. coli. With the rise of antibiotics resistance, these findings suggest a potential substitute for current methods of bacterial control. Future studies should pivot away from E. coli and explore a broader spectrum of bacteria and the bacteria’s responses towards different sound frequencies.

References

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Gu, S., Zhang, Y., & Wu, Y. (2016). Effects of sound exposure on the growth and intracellular macromolecular synthesis of E. colik-12. PeerJ, 4, e1920. https://doi.org/10.7717/peerj.1920

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Ku, H.-N., Lin, W.-F., Peng, H.-L., & Yew, T.-R. (2021). In-situ monitoring the effect of acoustic vibration in the form of music on the motility of Escherichia coli. Applied Acoustics, 172, 107620. https://doi.org/10.1016/j.apacoust.2020.107620

Liu, Y., Yin, Q., Luo, Y., Huang, Z., Cheng, Q., Zhang, W., Zhou, B., Zhou, Y., & Ma, Z. (2023). Manipulation with sound and vibration: A review on the micromanipulation system based on sub-MHz acoustic waves. Ultrasonics Sonochemistry, 96, 106441–106441. https://doi.org/10.1016/j.ultsonch.2023.106441

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Ratzke, C., & Gore, J. (2018). Modifying and reacting to the environmental pH can drive bacterial interactions. PLOS Biology, 16(3), e2004248. https://doi.org/10.1371/journal.pbio.2004248

Appendix

Appendix A– Average CFU/ml for Each Individual Trial