Bacterial Resistance of Serratia marcescens to Hand Sanitizer
Parinaz Malakzadeh, Patrick Mahoney, and Paige Organ
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Overuse of hand sanitizers is a growing concern due to predictions of sanitizerresistant bacterial strains. Bacteria cultures exposed to antibacterial agents in sanitizers
over multiple generations are expected to undergo a significant shrinking of the zones of
inhibition. Before being inoculated with cultures of Serratia marcescens, petri dishes of
nutrient agar were divided into (three) separate sections(Semicolon) for plain, original
formula Purell sanitizer, aloe-scented (Purell) sanitizer, and sterile water before being
inoculated with cultures of Serratia marcescens (maybe move this part to the beginning of
sentence) After 48 hours of incubation, the zone of inhibition radii were measured and
recorded before samples of bacteria were taken from the zone of inhibition’s edge. These
samples were placed in separate test tubes of nutrient broth and autoclaved for 48 hours
before being plated again. The procedure was repeated for a total of three generations. The
single factor ANOVA test suggested there (was) is a significant difference between
generations for (original) plain and aloe Purell (p = 1.54x10-10 and 4.49x10-9, respectively).
There was no significant difference between generations for sterile water (p = 0.43). Thus,
the data suggest(s) that (Serratia marcescens) bacteria build a resistance to Purell of both
the plain (original) and aloe variety. More generations may reveal the extent to which
bacteria can build a resistance to the sanitizers.
Size 12 Font
Introduction
The marketing of hand sanitizer in the United States is a big business. Sales of hand sanitizer grew over
70% between 2007 and 2009, where the peak of hand sanitizer sales grew to over $300 million (Fottrell, 2013).
Hand sanitizers are a constant presence - from public bathrooms, to the checkout line at grocery stores. Due to its
portability, using hand sanitizer can be easier and more convenient than washing with soap and water. It can also be
more effective. In a controlled study comparing hand sanitizer to soap and water, hand sanitizer proved to be more
than twice as effective as soap and water (Liu et al., 2010).
It has been theorized that continued use of alcohol-based sanitizer could lead to strains of bacteria that are
resistant to the protein-denaturing effects of the sanitizer (Aiello et al., 2005). This resistance would be caused by
selective pressure being placed on the bacteria by the sanitizers. One study by Reynolds et al. (2006) has suggested
that scented hand sanitizers may be less effective due to additives; thus different additives to alcohol based hand
sanitizers may increase or decrease the effectiveness of the product. Purell, a common sanitizer brand, has both a(n)
plain,(no comma)original formula as well as an aloe-scented formula. It is hypothesized that there will be a
significant difference in zone of inhibition radius between generations for aloe-scented and plain (original) Purell,
though more so for the plain Purell. (there would be a greater difference in zone of inhibition radius between
generations of Serratia marcescens in original Purell compared to aloe-scented purell.)
(Maybe add more
information about the bacteria you are using.)
Methods and Materials
The experiment took place at Saddleback College, Mission Viejo in Room SM 244 and was conducted on
Mondays, Wednesdays, and Fridays from 4 November 2013 to 18 November 2013. A sample of Serratia
marcescens was obtained from the Saddleback Biology Department. One liter of nutrient agar (Criterion lot # :
11339) and one liter of nutrient rich broth (Criterion lot # : 07037) were prepared and autoclaved for three hours.
Using sterile Petri dishes, ten nutrient agar plates were prepared and inoculated with Serratia marcescens using the
lawn spread method. Each of the ten plates was divided into three equal sections using lines drawn on the base of the
plate. Each section was then treated with one of three methods: The first method was placing a 10 µL drop of plain,
original formula Purell hand sanitizer (70% ETOH by volume) directly onto the inoculated plate. The second
method was placing a 10 µL drop of aloe-scented Purell hand sanitizer (70% ETOH by volume) directly on the
plate. The third method was to place (placing a) 10 µL drop of sterile water on top of a sterile paper chad which was
then placed on the inoculated agar. All of the measurements were done using a calibrated micropipette and sterile
techniques. The plates were then placed in an incubator at 30 °C for 48 hours.
After the plates had incubated, the radii of the zone of inhibition around the spots were measured using a
ruler. The surviving bacteria from the inner edges of the zones of inhibition were then collected and placed into 30
separate test tubes of nutrient broth and kept in an incubator to grow for 48 hours. All test tubes were labeled with
both a number and group. The bacteria grown from these cultures were used to inoculate the next generation of Petri
dishes. Using a sterile cotton swab, the three sections of the Petri dishes were inoculated with bacteria grown from
each of the three respective groups. For each new generation the zone of inhibition was measured and a new sample
of bacteria was collected using the same techniques. Nutrient agar and broth was prepared as needed. This procedure
was repeated for a total of three generations.
The radius of the zone of inhibition was averaged for the control (sterile water), plain (original) Purell, and
aloe-scented Purell groups for each generation and an ANOVA was run on the data to compare each group.
Results
The mean radius of the zone of inhibition for the first generation of Serratia marcescens) of (in) plain
Purell was 7.45mm ± 0.78 (n=10, ± S.E.M.). The mean radius for the last generation (of Serratia marcescens) of (in)
plain Purell was 0.95mm ± 0.23 (n=10, ± S.E.M.). An ANOVA run using data from all generations of plain Purell
(bacteria) showed that there was a statistical difference between generations (p=1.54x10-10).
The mean radius of the zone of inhibition for the first generation of (Serratia marcescens) of (in) aloe
Purell was 6.95mm ± 0.45 (n=10, ± S.E.M.). The mean radius for the last generation (of Serratia marcescens) of (in)
of aloe Purell was 1.53mm ± 0.26 (n=10, ± S.E.M.). An ANOVA run using data from all generations of aloe Purell
(bacteria) showed that there was a statistical difference between generations (p=4.49x10-9).
The mean radius of the zone of inhibition for the first generation (bacteria) of the control group (Sterile
Water) was 0.10mm ± 0.015 (n=10, ± S.E.M.). The mean radius for the last generation of control group (Sterile
Water) was 0.08mm ± 0.028 (n=10, ± S.E.M.). An ANOVA run using data from all generations of sterile water
showed that there was not a statistical difference between generations (p=0.43).
Average Zone of Inhibition
Radius (mm)
9.00
8.00
Aloe Purell
7.00
Plain Purell
6.00
Sterile Water
5.00
4.00
3.00
2.00
1.00
0.00
Gen 1
Gen 2
Gen 3
Figure 1. Average zone of inhibition radius of each group per generation (n = 10). The average radius significantly differs
between generations for both aloe and plain Purell (p = 4.49x10-9 and p = 1.54x10-10 respectively, single factor ANOVA test).
There was no statistical difference between generations for sterile water (p = 0.43, single factor ANOVA test). Error bars are ±
S.E.M.
Discussion
The data collected supports the hypothesis that the ring of inhibition would become smaller over
generations. There is a statistical difference between generations with both the original Purell as well as the aloe
version. However, there was no statistical difference in the effectiveness of the original version versus the aloe
version of the Purell product. This is most likely due to there being equal amounts of the active ingredient (70
percent ETOH by volume) of. There was also no statistical difference between any of the generations of the control
(sterile water) group which also supports our hypothesis. The experiment could be continued for several more
generations to ensure the accuracy of the data as well as test the limits of the bacteria’s ability to resist the sanitizers.
The active ingredient in Purell, ethyl alcohol, works by denaturing bacterial proteins, leading to death
(Aiello et al., 2005). This may change the effectiveness of the product on different species, such as coliform
bacteria, as they have different proteins. This may also mean that buildup of sanitizer-resistant genes varies from
species to species as well. Pan et al. (2006) and Reynolds et al. (2006) conducted studies using sanitizers with mixed
peroxides and 33 percent isopropanol as their respective active ingredients and found that the effectiveness of each
product varied. Treatments of peroxides resulted in a resistance to the sanitizer, while the 33 percent isopropanol
was ineffective concentration to produce resistance. Thus the impact of sanitizers on bacteria cannot be determined
by one species or one type of sanitizer alone. It is suggested that additional tests using different species and
sanitizers with different active ingredients should be conducted.
Literature Cited
Aiello, Allison E., et al. 2005. “Antibacterial cleaning products and drug resistance.” Emerg Infect Dis 11 (10): p
1565-1570.
Fottrell, Quentin. "Hand Sanitizer Spread." Wall Street Journal. 15 Jan. 2013. Web.
Liu, Pengbo, et al. 2010. “Effectiveness of Liquid Soap and Hand Sanitizer Against Norwalk Virus on
Contaminated Hands”. Applied and Environmental Microbiology 76 (2): p 394.
Pan, Y., F. Breidt, and S. Kathariou. 2006. "Resistance of Listeria Monocytogenes Biofilms to Sanitizing Agents in
a Simulated Food Processing Environment." Applied and Environmental Microbiology 72 (12): p 771.
Reynolds, Scott A; Foster, Levy; Walker, Elane S. 2006. Journal of Environmental Health 69 (4): p 48, 51.
Need 10 references)
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s): Parinaz Malakzadeh, Patrick
Mahoney, and Paige Organ
Title: Bacterial Resistance of Serratia
marcescens to Hand Sanitizer
Summarize the paper succinctly and dispassionately. Do
not criticize here, just show that you understood the
paper.
Strengths: This paper has a good explanation on
how they performed their experiment and
explained how this test could show how
bacteria can become resistant in the real world.
They related their experiment to previous
experiment and connected them together well.
Overall, they seemed like they knew what they
were studying and understood their results and
why they got them.
Weaknesses: A little more information about
the bacteria they are using. A little more
explanations about the sterile water control
group. Maybe better transitions between
sentences.
Technical Criticism
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This experiment tested to see if there would
be a greater difference in zone of inhibition
radius between generations of Serratia
marcescens in original Purell compared to
aloe-scented purell. In this experiment, the
researchers placed the bacteria in an original
Purell sanitizer, an aloe scented sanitizer and
water. The zones of inhibition were
measured after bacteria were placed in an
incubator for 48 hours. They performed this
procedure for three generations of bacteria.
Their data supported their hypothesis that
the zone of inhibiton radius between the
bacteria would be different.
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General Comments
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weaknesses and whether they are serious, or
important to our current state of knowledge.
 This paper should be published with revision
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errors, and minor textual problems. It's not the
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manuscript.
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