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 potential
development of sanitizer-resistant 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 over multiple generations. Petri dishes of
nutrient agar were divided into separate sections for plain, original formula Purell
sanitizer, aloe-scented sanitizer, and sterile water before being inoculated with cultures of
Serratia marcescens. 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
incubated (????) for 48 hours before being plated again. The procedure was repeated for a
total of three generations. The single factor ANOVA test suggested there is a significant
difference between generations for plain and aloe Purell (what exactly is the significant
difference between? Zone of inhibition?) (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 that bacteria build a resistance to Purell of both the plain and aloe variety
both the plain and aloe variety of Purell hand sanitizer. More Future generations may
reveal the extent to which bacteria can build a resistance to the sanitizers.
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 when 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 well-
known sanitizer brand, has both a plain, 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 Purell, though
more so for the plain Purell. (This sentence sounds
kind of casual, how about something like… It is
hypothesized that there will be a significant
difference in zone of inhibition radius between
subsequent generations for both the aloe-scented and
plain Purell; however, this difference is expected to
be greater in the plain Purell. To be honest, not sure
you even need the last part. There’s not really a test
that I know of to tell if there is a significant
difference between the differences between the two;
so I’m not sure how you would test that.)
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
Results
The mean radius of the zone of inhibition for
the first generation of plain Purell was 7.45mm ±
0.78 (n=10, ± S.E.M.). The mean radius for the last
generation of plain Purell was 0.95mm ± 0.23 (n=10,
± S.E.M.). An ANOVA run using data from all
generations of plain Purell 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 aloe Purell was 6.95mm ± 0.45
(n=10, ± S.E.M.). The mean radius for the last
generation of aloe Purell was 1.53mm ± 0.26 (n=10,
± S.E.M.). An ANOVA run using data from all
generations of aloe Purell 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 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).
9.00
Average Zone of Inhibition
Radius (mm)
rich broth (Criterion lot # : 07037) were prepared and
autoclaved for three hours (is this correct? Ours only
autoclaved for about 30 min.). 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 10 µL drop of sterile water
on top of a sterile paper chad which was then placed
on the inoculated agar (slightly inconsistent to use a
chad for one and not for the other two). All of the
measurements were done using a calibrated
micropipette and sterile aseptic 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 (how did you
define where the initial spot was when the chads were
not used?) 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 (lawn spread?). For each new
generation, the zone of inhibition was measured and a
new sample of bacteria was collected using the same
techniques as before. 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 Purell,
and aloe-scented Purell groups for each generation
and an ANOVA was run on the data to compare each
group. (In my option, this last paragraph is redundant
when you will be explaining that in the results.)
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 zone of inhibition would become smaller
over successive generations. There is was a statistical
difference between the first and last 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 ETtOH 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.
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
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.
The paper was looking at the selection for ethyl alcohol resistant genes in Serratia marcescens. This was done by
using both regular Purell and aloe Purell hand sanitizers (as well as water as a control), as the use of hand sanitizers are very
popular. A culture of Serratia marcescens was obtained and lawn spread onto nutrient agar plates. 10 samples of each group
had 10 microliters micropipetted onto one section each of agar plates that were divided into thirds. They were incubated for 48
hours and the radius of the zone of inhibition was measured. Then, bacterial from the edge of the zone of inhibition that was
not killed was transferred to nutrient broth and allowed to grow. Then 2 subsequent generations of the bacteria were spread
onto agar plates and grown in this fashion. The results showed that in both sanitizer groups there was a significant decrease in
the zones of inhibition, suggesting that the bacteria was gaining resistance to the sanitizer. This decrease was not seen in water,
the control. Also, the effectiveness of the two types of sanitizer was not statistically different.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current
state of knowledge.
I personally think the paper was excellent. It definitely seemed like it had been looked over and edited
several times before it was submitted. It was surely not a first draft. I think the results and data analysis was
outstanding; I hardly had to mark anything. Also, the graph looks very nice, in my opinion. It looks like something
which would be in an official journal article, which I like, and since these will be printed in black and white, it is nice
that it is already black and white. Everything else was very good, as well. As stated, it seemed like a final draft.
However, I believe that the discussion should maybe reiterate why this study is important. For example,
the fact that hand sanitizer is being used all the time, which may cause bacteria to gain resistance. You did relate it
to past studies and give potential ideas for future studies, which is good, but you did not reiterate the importance
of your study. Similarly, in the introduction, it may be interesting to explain why we should be worried about
bacteria becoming resistant to sanitizer.
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and
minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.
Key:
Text typed in blue is comments.
Highlighted text is removed.
Red text is additional words or changed content.
All issues were addressed directly in the paper, using the aforementioned key. It was a very good paper,
thanks for making my job easy!
This paper was a final version
This paper was a rough draft
Recommendation
 This paper should be published as is
This paper should be published with revision (This paper should make a few minor changes, but
otherwise it is almost ready for publishing)
 This paper should not be published