The effect of alcohol concentration on the growth of Micrococcus luteus and Pseudomonas aeruginosa

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1 The effect of alcohol concentration on the growth of Micrococcus luteus and Pseudomonas aeruginosa Matthew Andrade, Maneet Dhillon, Vineet Gupta, Vivian Lac, Noeline Rasanayagam, Shagana Visuvanathan, and Jialin Wen Department of Biology, The University of Western Ontario, London, ON, Canada N6A 5B7 INTRODUCTION: Antibacterial products and disinfectants are used incessantly and deemed essential in today s society. Antibacterial products inhibit the growth and reproduction of bacteria, essentially killing them. They are used domestically to disinfect homes from harmful bacteria and in medical clinics and hospitals where cleanliness and a bacteriafree environment are necessary for the health of patients. However, antibacterial use has become a controversial issue in the scientific world; its use has countless risks associated with it. Infants consistently exposed to antibacterial agents may experience an abnormal maturation of the immune system because it has not been exposed to necessary bacteria to allow maturation, leading to a possible rise in allergies among children (Levy 2001). Furthermore, extensive use of antibacterial products in domestic and health care facilities may actually lead to bacterial resistance to certain agents and cross-resistance to antibiotics may be a result (McDonnell and Russell 1999). Although there are many risks associated with the use of antibacterial products, they continue to be popular, which leads to the question: How effective are antibacterial products at eliminating bacteria truly? Are products with higher concentrations of antibacterial agents more effective? These particular questions were explored in this experiment, where the experimental bacteria, Micrococcus luteus (Schroeter) Cohn and Pseudomonas aeruginosa (Schroeter) Migula, were exposed to Purell and various concentrations of alcohol. P.aeruginosa is commonly found under nails, while M.luteus is found on flora of human skin (Baron 1996). Moreover, both bacteria are also customarily found on household items and in dust (Madigan and Martiniko. 2005). Due to the nature of the environments they commonly inhabit, P.aeruginosa and M.luteus are ideal bacteria for this experiment because they are the target of many antibacterial products. Alcohol and Purell (alcoholic hand sanitizer) were the antibacterial agents chosen because alcohol is a key ingredient in many antibacterial products available in the market today. Objective: To determine the effect of Purell and various concentrations of alcohol on the diameters of the zones of inhibition of Micrococcus luteus and Pseudomonas aeruginosa.

2 Hypothesis: The different alcohol concentrations will have an effect on the diameter on the zones of inhibition of Micrococcus luteus and Pseudomonas aeruginosa. Prediction: The diameters of the zones of inhibition of Micrococcus luteus and Pseudomonas aeruginosa will be larger when treated with higher alcohol concentrations when compared to the diameter of the zones of inhibition of the control (0% alcohol). METHODS: Samples of M. luteus and P. aeruginosa were vortexed with LB broth in Eppendorf tubes and the mixture was spread onto 30 agar plates per bacterium. Each agar plate had a well cut out in the center with a diameter of 6.3 mm and was filled with one of five treatments: deionized water (control), 40% alcohol, 50% alcohol, 65% alcohol and Purell hand sanitizer (65% ethanol content). Each treatment had six replicates. The plates were incubated in the dark at 27 C for hours. The diameters of the zones of inhibition were measured in millimetres using Vernier Calipers. STATISTICAL ANALYSIS: Data in this study were analysed by a one-way analysis of variance (ANOVA) to compare the effects of each treatment on P. aeruginosa and M. luteus. Additionally, Tukey s HSD test was conducted for each bacterium to identify any differences among the means (IBM SPSS Statistics ).

3 Fig. 1. Effect of various alcohol concentrations on the mean ± SD of diameter of zone of inhibition (mm) of Pseudomas aeruginosa and Micrococcus luteus. Means followed by the same letter within each variable are not significantly different (P<0.05) according to Tukey's HSD test. RESULTS: In general, at higher concentrations of alcohol, the diameters of the zones of inhibition of P. aeruginosa and M. luteus were greater. As alcohol concentration increased, a significant increase (F = , P<0.001) in the diameters of the zones of inhibition of P. aeruginosa occurred (Figure 1a). Likewise, with increased alcohol concentration, a significant increase (F = , P<0.001) also occurred in the case of M. luteus (Figure 1b). For both bacterial species, the diameters of the zones of inhibition were only significantly greater than the control (0% alcohol) when alcohol concentration was 65% (either the 65% solution or Purell treatment) (Figure 1a and 1b). There was no significant difference between the effect of 65% Purell and 65% alcohol solution on diameters of the zones of inhibition of P. aeruginosa (Figure 1a) and M. luteus (Figure 1b). DISCUSSION: The results of the experiment support the hypothesis that higher concentrations of alcohol have an effect on the diameters of the zones of inhibition. However, our prediction was not supported because although the Purell and the 65% alcohol solution were significantly more effective than the control, the 40% and 50% alcohol concentrations were not significantly different from the control. Alcohol acts as an antibacterial agent by disrupting the cell membrane. It is necessary to use antibacterial agents with alcohol concentrations ranging from 60%- 80% in order to effectively eliminate bacteria (Patel and Crank 2008). Exposure to sublethal concentrations (below 60%) will confer resistance to bacterial species and higher concentrations (above 80%) will evaporate too quickly (D Antonio et al. 2010). Purell

4 and the 65% alcohol were within the lethal range but the lower concentrations were not potent enough to make a significant difference in comparison to the control. There have been some concerns with alcohol-based antibacterial products, particularly hand sanitizers, in regards to child safety as ingestion of small amounts can lead to intoxication and additionally, the products are highly flammable. However, nonalcohol based hand sanitizers are not as effectively biocidal. On the other hand, some have long-lasting effects whereas alcohol-based hand rubs evaporate within seconds and leave hands feeling dry with prolonged use. Additional research into naturally forming biocides that are highly effective may provide us with safer alternatives. CONCLUSION: An alcohol concentration of 65% was most effective against bacteria while the lower concentrations were not significantly different from the control (0% alcohol).

5 REFERENCES: Baron S (ed.) (1996). Medical microbiology, (4th edn). University of Texas Medical Branch, Galveston, TX D Antonio NN, Rihs JD, Stout JE and Yu VL (2010). Revisiting the hand wipe versus gel rub debate: Is a higher-ethanol content hand wipe more effective than an ethanol gel rub? Am J Infect Control 38: IBM SPSS Statistics 21 (2013). Ontario Universities Student SPSS Version 21.0 [computer program]. IBM Corp., Armonk, NY Levy SB (2001). Antibacterial household products: cause for concern. Emerg Infect Dis 7: Madigan J and Martiniko MJ (2005). Brock s biology of microorganisms, (11th edn). Lebanon, IN McDonnell G and Russell AD (1999). Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 12: Patel GP and Crank CW (2005). Antimicrobial resistance in gram-positive and gramnegative bacteria [online]. Internet Continuing Education 9. Available from ACKNOWLEDGEMENTS: The authors thank Mark Possmayer and Ruth Jakobs for their comments and suggestions, Jeni Duro, Sriya Peiris, and Domonique Lam for their assistance in carrying out the experiment, Professor Krajnyk for guidance in the experimental process, and The University of Western Ontario for providing the necessary resources.