BiologyOther

The Effect of Sponge Material on the Growth of Escherichia coli

ABSTRACT

The purpose of this experiment was to determine the antimicrobial effectiveness of copper in the aqueous conditions of a damp sponge and to compare copper’s antimicrobial effectiveness to that of common sponge materials currently on the market, including untreated-cellulose, odor-resistant, and triclosan-treated sponges. If copper sponges could replace triclosan-treated sponges, this could limit the harmful effects of triclosan on humans and the environment. A small cylindrical piece of each sponge type was submerged in distilled water, then placed in the center of an agar plate inoculated with E. coli K-12 bacteria. The plates were incubated at 37 degrees Celsius. After six days, the number of E. coli colonies on each plate were counted. Descriptive and statistical (one-way ANOVA) analyses were performed on the raw data. There was a statistically significant relationship between the type of sponge material and the number of E.coli colonies grown (p=0.0009). The pairwise comparisons showed a statistically significant difference between the untreated-cellulose sponge and all other sponge types (p<0.05). However, odor-resistant, triclosan-treated, and copper sponges showed no statistically significant difference in the number of E. coli colonies grown (p>0.05). The results demonstrate that copper has antibacterial properties in the aqueous conditions of a damp copper sponge and that its antibacterial effectiveness is equivalent to odor-resistant and triclosan-treated sponges. This suggests that copper could potentially replace triclosan in sponges and reduce the harmful effects of triclosan on humans and the environment.

INTRODUCTION

Copper possesses antimicrobial properties on dry solid surfaces. These effects are enjoying both a resurgence of usage and media attention as they are being put to use in new settings, including touch surfaces in hospitals, to prevent the spread of infection[1]. Currently, a synthetic, broad-spectrum antimicrobial agent called triclosan is used in many consumer products such as household sponges, clothing and toothpaste. Being broad-spectrum, it targets a wide range of microorganisms as opposed to a single group of microorganisms. Triclosan has known risks to humans and the environment. The purpose of this experiment was to determine the antibacterial effectiveness of copper under aqueous conditions and to determine whether copper could be an effective and safe replacement for triclosan as an antimicrobial agent under the aqueous conditions of a copper sponge.

Kitchen sponges can promote bacterial growth due to their warm, moist environment and the presence of residual human food. As a result, the use of sponges often leads to the spread of bacteria in the kitchen[2]. A previous study has shown that copper has an antimicrobial effect on organisms in an aqueous solution[3]. Another study showed that dissolved copper ions play a role in the killing of bacteria[4]. Learning more about the antimicrobial effect of copper in aqueous conditions could lead to improved sanitary conditions in household kitchens, food service industries, hospitals, and schools.

Triclosan has been detected in human urine, including that of children and pregnant women. In children, an association has been found between the presence of urinary triclosan and allergies[5]. In pregnant women, researchers have found a positive correlation between the use of triclosan-containing products and triclosan concentration in maternal urine[6]. It is desirable to find a valid replacement for triclosan because triclosan can harm the human body and pollute the environment[7]. The overuse of triclosan may lead to antibiotic resistance among bacteria. Under laboratory conditions, bacteria including E. coli have been shown to acquire resistance to triclosan after serial exposure to increasing concentrations of triclosan[8].

It is hypothesized that if copper has antibacterial properties in a damp copper sponge, then a damp copper sponge will inhibit bacterial growth more than an untreated-cellulose sponge and to the same degree as damp sponge types containing chemical antimicrobial agents. To determine this, the antimicrobial effectiveness of a copper sponge will be tested and compared to the antimicrobial effectiveness of different common sponge types currently on the market (untreated-cellulose, odor-resistant, and triclosan-treated).

METHOD

A total of 20 sterile agar plates were obtained and organized into four groups of five plates. The groups were labelled as “untreated”, “odor-resistant”, “triclosan”, and “copper”. The plates were labelled with the trial number (1 – 5) for each independent variable level. A bacterial spreader and sterile transfer pipette were used to inoculate each of the twenty sterile agar plates with a lawn of E. coli K-12 bacteria. A pure cellulose sponge, an odor-resistant sponge, a triclosan-treated sponge, and a copper sponge were obtained. From each sponge type, five cylindrical samples were cut out with a circular base diameter of approximately one centimeter. Sterile forceps were used to submerge each cylindrical sponge sample in distilled water for about two seconds. The cylindrical sponge sample was then removed from the distilled water and placed with the cylindrical base firmly contacting the corresponding bacteria-inoculated agar plate. The lids were quickly placed on each agar plate and sealed using tape. Each agar plate was inverted and placed in the incubator at 37 degrees C. Every 24 hours, each plate was removed to assess colony growth. After six days (144 hours), the plates were removed from the incubator and the number of E. coli colonies grown on each plate was counted and recorded.

This experiment used live E. coli K-12 bacteria so there was a risk of spreading the bacteria. Certain strains of E. coli bacteria can cause severe/bloody diarrhea, anemia, kidney failure, and/or infections, including urinary tract infections. As a safety precaution, aseptic technique was utilized at all times during the course of the experiment. Aseptic technique involved the sterilization of forceps, media, filter disks, an L-shaped glass rod, and culture dishes before use. Measures were also taken to prevent subsequent contamination by contact with non-sterile objects. Work surfaces were washed with a 10% bleach solution after experimentation. Aseptic technique was used to pour media, transfer cultures, and grow the bacteria. All petri dishes were sealed immediately after inoculation and remained sealed throughout the experiment. All cultures were disposed of by autoclaving at 121 degrees Celsius and 15 psi for 15 minutes, then disposed of in a biohazard bag. Hands were washed with antibacterial soap prior to and after experimentation. All glassware, forceps, and other equipment used were sterilized in an autoclave upon completion of the experimentation. All experimentation was completed in the school’s microbiology laboratory under the supervision of a teacher who was a qualified scientist.

Descriptive statistics (mean, median, and range) were calculated for each type of sponge and statistical analysis was performed on the raw data using a one way ANOVA test. This test compares the means, or averages, of each group of data and determines whether the differences in those means are statistically significant.

RESULTS

Data Presentation

Table 1: Descriptive Statistics for Number of E. coli Colonies Grown

 

Untreated

Odor-Resistant

Triclosan-Treated

Copper

Mean

312

25.8

0.2

4.4

Median

273

7

0

0

Range

557

99

1

11

 

Figure 1: The Average Number of E. Coli Colonies Grown for each Type of Sponge

Figure 2: Box-and-Whisker Plot of Data Distribution

As shown in Table 1 and Figure 1, the use of a triclosan-treated sponge resulted in the fewest mean number of E. coli colonies grown (0.2). The use of a copper sponge resulted in the second-fewest E. coli colonies grown (4.4). The use of an odor-resistant sponge resulted in the second-most E. coli colonies grown (25.8), while the use of an untreated-cellulose sponge resulted in the most E. coli colonies grown (312).

The untreated-cellulose sponge had the greatest median (273), the odor-resistant sponge had the second-highest median (7), and the triclosan-treated sponge and copper sponge both had the lowest median of (0). This reflects the fact that sponges which contain antibacterial substances greatly reduce the number of bacteria colonies that grow. Because the untreated-cellulose sponge contains no antibacterial substances, it did not reduce the number of E. coli colonies that grew.

According to Table 1 and Figure 2, the use of the untreated-cellulose sponge resulted in the greatest range (557), the odor-resistant sponge resulted in the next greatest range (99), the copper sponge resulted in the second-lowest range (11), and the triclosan-treated sponge resulted in the lowest range (1).

Final growth of E. coli after six days of incubation at 37°C.

Data Analysis

Table 2: One-Way ANOVA for the Number of E. coli Colonies Grown

Overall P-Value:

0.0009

Pairwise Comparisons:

Sponge Type 1

Sponge Type 2

P-Value

Odor-Resistant

Triclosan

4.33

Odor-Resistant

Untreated

0.0055

Odor-Resistant

Copper

4.59

Triclosan

Untreated

0.0026

Triclosan

Copper

5.72

Copper

Untreated

0.0029

Results of the one-way ANOVA (Table 2) show that the type of material in a sponge has a statistically significant effect on the number of E. coli colonies grown (p=0.0009). The pairwise comparisons show that only the mean number of E. coli colonies grown on the plates of the untreated-cellulose sponge showed a statistically significant difference compared to the other three types of sponges (p < 0.05). There was no statistically significant difference among the mean number of E. coli colonies grown with the odor-resistant, triclosan-treated, and copper sponges (p > 0.05).

DISCUSSION

It was hypothesized that if copper has antibacterial properties in aqueous conditions, then a damp copper sponge will inhibit bacterial growth more than a damp untreated-cellulose sponge, and to the same degree as other damp sponge types containing chemical antimicrobial agents. Both parts of the hypothesis were supported by the data. Except for the untreated-cellulose sponge, the odor-resistant, triclosan-treated and copper sponge types showed no statistical difference in antibacterial effect when compared to one another. The results are supported by scientific knowledge and research because odor-resistant chemicals, triclosan, and copper are known antimicrobial substances, while there are no known antimicrobial substances in untreated-cellulose sponges.

The results of this experiment can be applied to real life when selecting different types of sponges to use in household and commercial settings. The Environmental Protection Agency (EPA) considers triclosan to be detrimental to the environment and human health, so replacing triclosan-treated sponges with copper sponges would be beneficial because it could prevent contamination of the environment from triclosan, and would reduce the risk of human poisoning from triclosan. Regarding potential safety issues of copper, trace amounts of copper are found in drinking water; however, this is regulated by the EPA and at concentrations below 1.3 mg/L, copper is not likely to be harmful[9]. It is known that consuming high amounts of copper in drinking water can cause vomiting, diarrhea, stomach cramps, nausea, and possible liver or kidney damage[10]; however, the use of a copper sponge would not be expected to affect drinking water. To date, copper sponges are not readily available to the average consumer. This may be due to the fact that chemical antibacterial products are already widely marketed and to lack of knowledge about the effectiveness of copper sponges. The information from this experiment could dramatically change the market for copper sponges and lead merchants to offer copper sponges in their stores, which do not carry the potentially harmful effects of triclosan-treated sponges.

Some of the raw data for the independent variable levels displayed high ranges. This may be due to an experimental error related to the fact that counting bacterial colonies using the quadrant method is not very precise; one plate may contain many small colonies, while another plate may contain only a few large colonies. This may also be explained by uneven inoculation of the initial lawn of E. coli bacteria at the start of the experiment.

Procedural improvements could include measuring zones of inhibition rather than counting bacterial colonies, comparing sponges of a consistent base material, and ensuring that cylindrical sponge samples are of uniform size by having a more precise cutting method and/or measuring sample mass. This experiment could be expanded by weaving pure copper and copper alloy filament/thread into untreated-cellulose sponges to test whether or not such an application could be used in the manufacture of kitchen sponges. Another possible expansion could be to cut very thin slices of copper sponge to the size of filter paper disks (rather than the thick pieces of sponge that were used); the experiment could then be repeated measuring zones of inhibition. Finally, various formulations of copper, such as copper colloid, could be substituted for triclosan or other antibiotic agents used in products such as acne medicine.

CONCLUSIONS

  1. Copper is an effective antimicrobial agent in aqueous conditions.
  2. Copper is equally as effective as triclosan as an antimicrobial agent in a damp sponge.
  3. Copper could potentially serve as a replacement for triclosan as an antimicrobial agent in sponges.

 

REFERENCES

[1] Sun, L. H. 2015. “The bacteria-fighting super element making a return to hospitals: Copper.” Washington Post, September 20, 2015. https://www.washingtonpost.com/national/health-science/the-bacteria-fighting-super-element-making-a-return-to-hospitals-copper/2015/09/20/19251704-5beb-11e5-8e9e-dce8a2A2a679_story.html

[2] Erdogrul, Ö, & Erbilir, F. n.d. “Microorganisms in Kitchen Sponges.” Internet Journal of Food Safety. V.6:17-22. http://www.internetjfs.org/articles/ijfsv6-4.pdf

[3] Cervantes, H. I., MD, Álvarez, J. A., MD, Muñoz, J. M., MD, Arreguín, V., MD, Mosqueda, J. L., MD, & Macías, A. E., MD. 2013. “Antimicrobial activity of copper against organisms in aqueous solution: A case for copper-based water pipelines in hospitals?” American Journal of Infection Control. 41, no. 12: e115-e118. https://doi.org/10.1016/j.ajic.2013.03.309

[4] Molteni, C., Abicht, H. K., & Solioz, M. 2010. “Killing of Bacteria by Copper Surfaces Involves Dissolved Copper.” Applied and Environmental Microbiology. 76, no. 12: 4099-4101. https://doi.org/10.1128/AEM.00424-10

[5] Bertelsen, R. J., Engel, S. M., Jusko, T. A., Calafat, A. M., Hoppin, J. A., London, S. J., Eggesbø, M., Aase, H., Zeiner, P., Reichborn-Kjennerud, T., Knudsen, G. P., Guidry, V. T., & Longnecker, M. P. 2014. “Reliability of triclosan measures in repeated urine samples from Norwegian pregnant women.” Journal of Exposure Science & Environmental Epidemiology. 24, 517-521 https://doi.org/10.1038/jes.2013.95

[6] Weiss, L., Arbuckle, T.E., Fisher, M., Ramsay, T., Mallick, R., Hauser, R., LeBlanc, A., Walker, M., Dumas, P., Lang, C. 2015. “Temporal variability and sources of triclosan exposure in pregnancy.” International Journal of Hygiene and Environmental Health. 218, no. 6: 507-513. https://doi.org/10.1016/j.ijheh.2015.04.003

[7] Williams, R. M. 2006. “Triclosan – a controversial antibacterial.” Townsend Letter for Doctors and Patients. Beyond Pesticides, May. https://www.beyondpesticides.org/assets/media/documents/antibacterial/triclosan-williams..pdf

[8] The Alliance for the Prudent Use of Antibiotics (APUA). 2011. “Triclosan.” Personal Home. Clorox Company, January. http://www.tufts.edu/med/apua/consumers/personal_home_21_4240495089.pdf

[9] Environmental Protection Agency. n.d. “National Primary Drinking Water Regulations.” Accessed February 15, 2016. https://www.epa.gov/ground-water-and-drinking-water/national-primary-drinking-water-regulations#main-content

[10] Minnesota Department of Health. n.d. “Copper in Drinking Water Health Effects and How to Reduce Exposure.” Accessed February 15, 2016. http://www.health.state.mn.us/divs/eh/water/factsheet/com/copper.html

 

APPENDIX

http://ecx.images-amazon.com/images/I/51UyyEwPfOL._SX522_.jpg pink sponge.jpg

Untreated-Cellulose sponge Odor-Resistant sponge

Quickie Roller Mop Refill with Antimicrobial Microban https://s.yimg.com/fz/api/res/1.2/o1FF0UaP7cq5ngwT3DQbsw--/YXBwaWQ9c3JjaGRkO2g9MzMwO3E9OTU7dz0zMzA-/http://thumbnail.image.rakuten.co.jp/@0_mall/imaster/cabinet/kenko207/e208727h_l.jpg?_ex=330x330

Triclosan-treated Sponge Copper Sponge

 

IMG_0656.jpgIMG_5387.jpg

Actual sponges and cylindrical sponge samples used; plate set up

 

About the Author

Kathleen Love is 17 years old and attends Yorktown High School in Arlington, Virginia. She has won several awards for her science work and plans to study environmental science in college. Her other hobbies include playing the flute and piano, running, and rowing on the Potomac River in Washington, D.C.

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