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Abstract

Due to escalating cases of drug- resistant pathogens it is essential to know how to protect against possible infections. Hand washing and antibacterials are both popular means of prevention but it is important to know how just effective they are. Four hand washing methods were tested through colonial growth observation to determine how successful they were in removal of bacteria. Subsequent Gram staining analyses and morphological comparisons were performed to determine what species were present. In addition to hand bacteria, species determinations were conducted for a table surface and the human mouth. S. epidermis and M. luteus proved to be the most common bacteria discovered external to the body while E. coli and S. mutans were predominant in the mouth. Results of the hand washing test showed that a thorough scrubbing of the hands removes the most bacteria and even a quick wash can help significantly reduce bacterial numbers. Household antibacterial product analysis revealed Listerine did not kill a tested E. coli culture while Lysol did in a growth inhibition test. Taken as a whole, the studies reflect frequent and thorough hand washing along with use of certain antimicrobials can be effective measures in the fight against pathogenic bacteria.

Introduction

Americans are obsessed with germs. This is reflected by the fact that in recent years there has been tremendous expansion in the availability of antimicrobial products seen on store shelves. The growth rate in the United States for antibacterials has been around 4% since 2001 (De Guzman, 2006). The mention of E. coli alone can send mothers into a panic and reaching for anything declaring itself to be an antibacterial. The recent details of the possible devastation that could be caused by methicillin-resistant Staphylococcus aureus (MRSA) alone is enough to worry anyone with a just a basic knowledge of epidemiology. MRSA has been killing more people annually than AIDS and those numbers are steadily growing (Stein, 2007).

It is, of course, impossible to eliminate all the harmful germs around us in our day to day lives but steps can be taken to minimize contact with them. Hand washing and cleaning products are amongst the favorite and most effective methods of infection prevention (MFMER, 2007). An exploration will be made into the extent of microbial growth on unwashed hands then a comparison will be made to the same hands after varying degrees of washing with soap and water. It is hypothesized that a more thorough job of hand washing will result in less bacterial growth appearing on the test plates.

Additionally, determination of probable bacterial species present on not only the hands but also a common public table surface and the human mouth will be made through morphological observation and Gram staining. The most likely bacterial species to be observed on the hands will be S. epidermis and M. luteus while the mouth will presumptively produce E. coli and S. mutans colonies. The table surface will likely yield results similar to what is found on the hands.

Finally a test on the effectiveness of the two common household antimicrobials Listerine and Lysol will be carried out to determine if they are as effective as the public may understand them to be. It is believed the products will show some efficacy but not to the degree of complete sterilization which many people may perceive is their function.

Methods and Materials

Three separate tests were conducted during the course of the experiment. The first utilized both Luria Burtanii (LB) and Trypticase Soy Agar (TSA) media plates. These were divided into quadrants and labeled as unwashed, washed without towel, washed with towel, and scrubbed with towel. Using an aseptic technique, samples were taken from the thumb of a lab team member in a fashion which corresponded to the plate quadrants. After the samples for all quadrants on both plates were taken they were incubated at 37°C for one week. Hands were washed prior to sample collection according to the following guidelines:

  • Unwashed- sample taken without washing of hands
  • Washed without towel- ordinary soap was used for 10 seconds then the water faucet was turned off with wet hand then dried with a paper towel
  • Washed with towel- ordinary soap was used for 10 seconds then hands were dried before using a paper towel to turn off faucet
  • Scrubbed with towel- ordinary soap was used in concert with a scrub brush for one minute before hands were dried then faucet was turned off using paper towel

The second test utilized the LB, TSA and Eosin Methylene Blue (EMB) media plates. These were each divided into two sections and labeled as either table or mouth samples. A sterile cotton swab was dampened with sterile, distilled water and the excess was squeezed out against the side of the water container. The swab was used to take samples from the lab table and a lab team member’s mouth then dragged across the corresponding section on each plate using the aseptic method. After all samples were taken the plates were incubated for one week at 37°C.

The third test was used to test the effectiveness of two popular antiseptic products. Two TSA plates were divided into quadrants with positive and negative controls labeled diagonal from each other. The remaining two quadrants were labeled as Lysol and Listerine. A sterile swab was used to evenly spread E. coli bacterial culture across both plates. Ethanol-sterilized tweezers were used to place untreated paper disks into the sections marked as negative controls. A disk pretreated with vancomycin was placed in both sections marked as the positive control. Added to the other two quadrants were disks treated with their respective antiseptics. Tweezers were resterilized with ethanol between each disk placement. The two plates were also set aside in an incubator set at 37°C for a period of one week.

After the incubation period the plates were examined for bacterial growth. Observation of plate sections from the first two tests took into account the number of colonies (if countable) and their morphology. This data was recorded for later quantitative and qualitative comparisons. The third test measured the zone of inhibition around the test disks and was performed with a Vernier Caliper. The diameter of the circular area where no bacteria was able to grow around each disk was considered to be the zone of inhibition.

Further analysis of four bacterial samples from the first two tests was performed by utilizing the Gram staining technique. To begin this procedure, one drop of distilled water was placed in the center of a clean slide before a sample from a single bacterial colony was suspended in it. The sample was heat-fixed to the slide by passing it through a Bunsen burner flame and allowed to cool. After the slide was treated with crystal violet dye and allowed to sit for one minute, it was rinsed and iodine was applied to fix the dye. A second rinsing was done before using an ethanol alcohol decolorizer and rinsing again. A safranin counterstain was applied to the slide and let sit for one minute before a final rinse in water was performed. This procedure was performed for all four samples before viewing under a 40X objective.

Results

While it was difficult to determine exact numbers of colonies on all of the media plates, general visual inspections were sufficient in determining the effects of washing one’s hands; as the degree of hand washing increased the bacterial presence decreased. Scrubbing in conjugation with a paper towel resulted in no detectable amounts of M. luteus and an extreme reduction of S. epidermis on both the TSA and LB plates. One E. coli colony was observed on the LB unwashed hand sample and the most minimal washing procedure resulted in no further colony growths. The LB plate yielded enough numerical data to be used to develop a graphical representation illustrating the effectiveness of each of the hand washing methods (Figure 1.0) where a clear and direct relationship can be seen.

Figure 1.0- Effects of Handwashing on Bacterial Colony Growth

Figure 1.0- Samples from the thumb were taken on an LB media plate after varying degrees of handwashing then incubated for one week at 37 degrees Celsius. The resulting colonies were counted and classified by morphology.

The cultured samples from the lab table only showed results on the LB plate which were seven small, yellow colonies. The mouth samples produced a medium sized white colony on the LB plate and two of the same morphological type on the TSA plate. The EMB mouth sample showed many very small shiny green colonies indicating Gram- negative bacteria.

The results of the Gram stain procedure showed all of the hand bacteria tested were Gram-positive. These were also all noted to be cocci, many of which formed chains and clusters. The mouth sample displayed what appeared to be a mixture of both Gram- positive and negative cells.

The test for effectiveness of antimicrobial products showed zero growth retardation for both controls as well as the Listerine sample. Lysol was able to produce a zone of inhibition measuring 9mm on plate 1 and 10mm on plate 2.

Discussion

After considering the morphological and quantitative data from the hand washing experiments, it is evident that a more thorough job done while washing one’s hands results in a decreased presence of bacterial growth. A total elimination of E. coli was seen when a quick towel-free washing was performed but this should not be seen as a totally reliable method for complete E. coli removal as only one colony of that bacterial species was detected from the unwashed hand.

Surprisingly, the table samples did not yield any bacterial growth on either the EMB or TSA plates and only a minimal presence of what was thought to be M. luteus on the LB plate. The mouth sample had more pronounced results showing growth of what was likely E. coli which is common to the mouth. This was expected since the warm, moist environment of the mouth is conducive to bacterial growth (Kononen, 2000).

Further analysis of three hand washing samples showed all of the bacteria were Gram-positive and tended to form cluster and chains. This morphology was consistent with the hypothesis that there were S. epidermis and M. luteus colonies present. The mouth sample displayed Gram- negative results which supported the morphological evidence for the presence of E. coli. The mouth also contained Gram- positive bacteria which were not visually detected on the TSA or EMB plates. This was thought to be S. mutans which is known to be prevalent in the human mouth (Kononen, 2000).

The zone of inhibition test showed Lysol is indeed successful when used to prevent or kill E. coli growth. Listerine did not show positive results but the experiment only tested against one type of Gram- negative bacteria. There are many other forms of potentially harmful microbes it may be effective against. Because the positive control failed, it was thought perhaps the culture used may have developed a resistance to vancomycin which under normal conditions would prove to be effectual. The failure of the positive control also leaves open the possibility of Listerine being successful in killing other strains of E. coli.

The Centers for Disease Control and Prevention in Atlanta acknowledge hand washing as the most significant prevention method against the spread of pathogens (CDC, 2005). This simple procedure is often not done with the thoroughness needed for significant microorganism removal or neglected altogether. Based upon the results of this study it is clear that even minimal hand washing can be an effective tool in bacterial removal. Use of antimicrobial products in conjunction with keeping a clean environment further adds to prevention of pathogenic infection. It has been widely acknowledged these measures can be effective against even “superbugs” such as the dreaded MRSA (Tomich, 2007). While there is no way to completely isolate oneself from microbes, there are simple everyday measures that can be taken to minimalize exposure to possible pathogens.

Works Cited

Centers for Disease Control and Prevention (CDC). (2005, September). Safe Water System & Handwashing Guide for Healthcare Workers. Retrieved October 18, 2007, from CDC Web site: http://www.cdc.gov/safewater/publications_pages/fact_sheets/SWSTrainingGuidNurses.pdf

De Guzman, D. (2006). SDA: Scrubbing antimicrobial myths. Chemical Market Reporter , 269 (4), 31.

Kononen, E. (2000). Development of oral bacterial flora in young children. Annals of Medecine , 32 (2), 107-112.

Mayo Foundation for Medical Education and Research (MFMER). (2007, October 16). MayoClinic.com. Retrieved October 17, 2007, from Hand washing: An easy way to prevent infection: http://www.mayoclinic.com-Hand Washing: A Simple Way to Prevent Infection

Stein, R. (2007, October 17). Drug-Resistant Staph Germ’s Toll Is Higher Than Thought. The Washington Post , p. A01.

Tomich, N. (2007). Preventing MRSA. Modern Healthcare , 37 (27), 24.