The Effectiveness of Different Antibacterials on Killing Bacteria

Angelica Cervantes UC Davis Chapter Mentor: Miguel Macias Gonzalez

Abstract In everyday life, people use antibacterial products to sanitize their homes. Some of the common active ingredients in household cleaning products are thymol, lactic acid, sodium hypochlorite, and ethanol (e.g. active ingredient in some hand sanitizers). The purpose of our experiment was to determine if antibacterial products lost effectiveness in killing common bacteria in everyday environments after repeated use. This is an important environmental issue because if these products are losing effectiveness, then our common living areas are not being properly sanitized and this could lead to more illnesses caused by the bacteria. The hypothesis in this study is that thymol will lose its effectiveness to kill bacteria after repeated treatments because it originates from natural herbal compounds that are constantly exposed to bacteria; therefore some bacteria may already have some tolerance to it. Bacteria from our hands were cultivated in a nutrient rich media and treated with four different antibacterial products, which include the active ingredients of thymol, lactic acid, sodium hypochlorite, and ethanol (e.g. hand sanitizer), respectively. The treatments were applied by soaking a stack of ten circular filter papers with a seven millimeter diameter on a recently inoculated plate. The repeated measures were made by cultivating bacteria from the previously treated plate into a new plate and treating it with the same active ingredient, from which it originated. The kill zone was measured after allowing the bacteria to grow for 48 hours. After two repeated treatments, our results showed that some antibacterial products’ kill zones decreased. Thymol’s kill zone showed a higher decreasing trend than the other antibacterial products. The experiment is still in progress, two more repeated measures of the kill zone will be taken. So far, the results

indicate that there are differences in the effectiveness of killing bacteria after repeated treatments between the active ingredients. The reasons for these differences between the active ingredients could be due to their mode of action, which has been supported by the data so far. To prevent the buildup of tolerance it is suggested to change the cleaning product with a different active ingredient after a period of time.

Introduction

In everyday life, people use antibacterial products to sanitize their homes. Some of the common active ingredients in household cleaning products are thymol, lactic acid, sodium hypochlorite, and ethanol (e.g. active ingredient in some hand sanitizers). These active ingredients are used to target common household bacteria such as Staphylococcus spp., Streptococcus spp., Bacillus spp., and E. coli. The project studies the effectiveness of different antibacterial products after repeated use. This is an important environmental issue because if these products are losing effectiveness, then our common living areas are not being properly sanitized and this could lead to more illnesses caused by the bacteria. In the project, it will be studied if the repeated use of antibacterial products lose their effectiveness on killing bacteria after constant use. This project consists of testing the efficiencies of different antibacterial products after repeated use. It will be interesting to compare different antibacterial with different active ingredients (

the chemicals that creates the effect of the product), and try to see if it makes a difference to change products every once in a while. If an antibacterial is used repeatedly, then its effectiveness will decrease.

Experimental Method

The following are the materials used:16 Petridishes with rich media for bacteria culture, disposable inoculation loops, 65% ethyl alcohol, Thymol, Lactic Acid, and 10% Bleach, Circles of filter paper and a hole punch, Latex gloves and q-tips, A horizontal laminar flow hood. Procedures: Note: Wear latex gloves while working with bacteria. 1) Take a humid q-tip and rub it against the area where the bacteria will be harvested from. 2) With the same q-tip rub it on a petridish with the rich media. 3) Put the petridish in the dark in room temperature for three days. 4) Once the colony of bacteria has grown use a disposable inoculation loop to harvest bacteria from a single colony and spread it to a new petridish, and repeat step 3. 5) Number three new petridishes 1 to 3 and trace the area where the bacteria will be applied, label each traced segment with the treatment that will be applied in that segment. 6) Stack 10 filter papers and hole punch them to create small circles of 10 stacked filter papers. 7) Dip the small stacks of filter paper in step 6 and dip them in the desired anti-bacterial product for 10 seconds. 8) Place the soaked small stack of filter paper and plate it in the respective area of the petridish with applied bacteria, avoid keeping the petridish open once the treatment is applied to prevent the filter paper from drying out before it has an effect on the bacteria.

9) Do step 7 through 8 for all treatments. 10) Allow the closed petridishes to stand still for 10 minutes to allow the antibacterial to spread. 11) Invert the petridishes up-side-down and place them in a dark place at room temperature for one day or two. 12) Measure the kill zone and record data. 13) Repeat the procedures from 5 to 12 three more times, but plate bacteria from petridish one to the new petridish one. Transfer bacteria from treatment 1 to the new treatment 1 (e.g. use bacteria treated with bleach form petridish one to transfer to the new petridish one in the new bleach treatment). For the last treatment use four petridishes and label with one antibacterial treatment. 14)Trace an area in each petridish which will be plated with bacteria and label each area with the treatment that will be applied. 15)Take bacteria from petridishes 1 to 3 from one treatment to transfer onto its respective petridish from step 14. 16)Follow steps 6 to 12.

Results & Analysis

This is the graph that shows the results of the first 4 trials.

This graph shows the result of the 5th trial

The data in figure 1 demonstrates that as the treatments are repeated the kill zone decreases. The treatment with the largest kill zone is 10% bleach followed by Lysol, 7th Generation and 62% ethanol (active ingredient in some hand sanitizers). The data in figure 2 demonstrates the effect of the antibacterial products on bacteria that has been repeatedly treated with one antibacterial. Bacteria that have been repeatedly treated with 10% bleach have the largest kill zone with 10% bleach, but its kill zone has decreased from the previous treatment (compare kill zone value between figure 1 and 2). Bacteria that have been repeatedly treated with Lysol showed the largest kill zone with Lysol. Bacteria that have been repeatedly treated with 7th Generation have no kill zone with 7th Generation but do have a kill zone with any other antibacterial. The bacteria that have been repeatedly treated with 62% ethanol have the smallest kill zone with the 62% ethanol treatment

Discussion & Conclusion

The project was of great interest because it was very fascinating to think about how, in everyday life, we use these antibacterial products. We use these products to sanitize our homes and our surroundings and it sparked our interest to find out if these products really do kill the 99.9% of the bacteria they say they do or if after some time, the products would become less efficient at killing the bacteria. Some of the questions that came up were that maybe the antibacterial product was not provided enough time to spread well on the petridishes. It is probable that our results were what they were because some antibacterial products could act faster than others. A solution to this would be to allow the antibacterial to spread for a longer period of time and then measure its kill zone. A complication that came up during the project was that at first, the kill zone was not visible. The problem was that the antibacterial products dried out too quickly. To fix this, instead of only using one filter paper, we stacked ten filter papers and then soaked it in the solution. This way the antibacterial didn’t dry too fast and we were able to see results after 24 hours. This research shows which antibacterial product is the most effective at creating a kill zone and it shows how after repeated use, there is a decreasing trend of efficiency in effect. After doing repeated treatments on bacteria that is commonly found in our environment, the results show differences in the effectiveness of killing bacteria after repeated treatments between the active ingredients. The data demonstrates that the antibacterial

product with the most efficiency was 10% Bleach (whose active ingredient is sodium hypochlorite), followed by Lysol (whose active ingredient is lactic acid), then 7th generation (whose active ingredient is thymol), and the antibacterial with the least efficiency in killing bacteria was 62% ethanol (which is the active ingredient in most common hand sanitizers). One of the reasons why 10% bleach was the most efficient at creating a kill zone was that it may act faster than the rest, and due to its mode of action. The active ingredient in bleach is known to denature proteins which makes them lose their function Thymol was one of the products with the least efficiency in killing bacteria,. Thymol is known to prevent bacterial growth by interrupting glucose uptake and lactate production meaning that it may target specific enzymes that could acquire resistance by simple mutations. To prevent the buildup of tolerance, we changed the cleaning product with a different active ingredient after a period of time and our results showed the 7th Generation bacteria had a zero kill zone for 7th generation but had a kill zone for 62% ethanol which was not a very strong antibacterial in throughout the whole experiment and that in the 62% ethanol bacteria the 7th Generation had a good size kill zone which also did not had such large kill zones throughout the experiment. This shows that it is good to change antibacterial once in a while to prevent the build up of tolerance. There was not always clear results, however, and the reason for this could have been human error and it is possible that there are experimental errors such as not allowing enough time for the antibacterial to spread. If I were to do this experiment again, I would let the antibacterial spread for a longer period of time and record my results. I would check that if by allowing more time for the antibacterial to spread, if there would be more noticeable results in the kill zone of each bacteria.

Acknowledgements

Special thanks to Dr. Richard W. Michelmore for providing the lab space and material, and to Keri Cavanaugh and Manjula Govindarajulu helping in preparing the petridishes. I would also like to thank my mentor, Miguel, for all the patience and assistance in getting the project completed.

Appendices Raw data for both graphs:

10% Bleach Lysol 7th generation 62% EtOH

10% bleach Lysol 7th Generation 62% EtOH

1 10.66666667 4.666666667

2 8.777777778 2.666666667

3 7.333333 0.777778

4 6.666666667 0.666666667

3.666666667 0

0.444444444 1

0.111111 0

0.055555556 0.222222222

10% bleach 4 0.33 4.3 3

Lysol 1.3 0.83 1.3 1

7th Generation 0 0 0 2

62% EtOH 0 0 1 0.5