Loh, Karapetian and Gan

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Bactericidal Effectivities of Zinc, Copper, Silver, and Stainless Steel on Staphylococcus
aureus.
Sean Loh, Natalie Karapetian, and Wei Gan.
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
With antibiotic resistant bacteria on the rise, alternative methods of sterilization, such as
those utilizing the oligodynamic effect, are being researched with the aim of reducing the
spread of disease without fostering antibiotic resistant bacteria, which is of particular
importance to hospitals. The investigators predicted zinc would exhibit the highest level of
antibacterial activity and that zinc, copper and silver would all be more antibacterial than
stainless steel. Cultures of Staphylococcus aureus were grown on nutrient agar while
exposed to samples of the four different metals for 24 hours (n=10). The zone of inhibition
was measured to determine which metal was the most effective in inhibiting bacterial
growth. Of the four metals tested, zinc (7.343 mm ± 0.232, 𝐱̅ ± SEM) was shown to
statistically have a significant zone of inhibition (p=0.00, ANOVA; p<0.05, Bonferroni posthoc) in comparison to stainless steel (0.166 mm ± 0.071, 𝐱̅ ± SEM), copper (0.229 mm ±
0.075, 𝐱̅ ± SEM), and silver (0.350 mm ± 0.010, 𝐱̅ ± SEM). The investigators concluded that
zinc was the only bactericidal metal due to the highly oxidative nature of zinc, thereby
contributing to a strong oligodynamic effect.
Introduction
The primary objective of the proposed project is to determine which materials best inhibit or terminate
Staphylococcus aureus growth via the oligodynamic effect. In consideration of the ease in which bacteria pass from
surface to surface and the increasing commonality of antibiotic resistant bacteria, it is important to determine which
types of surfaces are most effective in preventing bacterial contamination, especially for use in medical facilities.
This knowledge will aid in the development of more sanitary surface options to be used in the construction of
commonly touched surfaces such as door knobs, countertops, and bathroom fixtures to aid in the combat against the
spread of bacterial diseases.
Certain metals exhibit bactericidal properties in low concentrations, known as the oligodynamic effect, via
mechanisms of oxidation-reduction reactions and intracellular accumulation of ions (Kraemer, 1905 and Miyano et
al., 2010). Metal ions are transported into the cell through the cell membrane, where the ions accumulate and
become toxic to the cell by slowing or terminating cellular metabolism (Rentz et al., 2003 and Khalid et al., 2014).
Additionally, oxidation-reduction reactions between metal ions and molecules in the bacterial cell walls can cause
the metal ions to bind to the cellular proteins, rendering the protein non-functional and causing the cell wall to
rupture (Rentz, 2003 and Miyano, 2010). While current understanding of the oligodynamic effect is primarily
focused around research done on metallic solutions, the investigators of this project aim to lend insight into the
bactericidal action of solid metals to be applied on fixtures such as doorknobs. Similar studies found copper and
silver to exhibit high antibacterial properties, leading the investigators of this project to expect that copper and silver
metals will be effective in inhibiting the growth of Staphylococcus aureus (Heidenau et al., 2005 and Miyano,
2010). However, the highly oxidative characteristics of zinc lead the project investigators to expect that zinc metal
will exhibit the highest level of bactericidal activity while zinc, copper, and silver will all be more antibacterial than
stainless steel (control).
Materials and Methods
Pieces of copper, silver, and zinc, and stainless steel were obtained. On 28 October 2015, ten pieces of each
metal type were cut out to the dimensions 1.0 cm by 0.7 cm using tin snips and the surface of piece of metal was
sanded with 220 grit sand paper and then autoclaved. Using aseptic technique, 150 µl of Staphylococcus aureus
inoculum was then dispensed onto each of the 10 Petri dishes with a P200 micropipette and spread with a cotton
swab to create a bacterial lawn on the surface of each plate of agar. One piece of each type of sterilized metal was
then pressed into each corner of the agar with sterilized forceps. The lid was then replaced, inverted, and incubated
in an air incubator at 37°C. After 24 hours of incubation, the Petri dishes were moved to a refrigerator. The zone of
inhibition from the edge of each of the four sides of each metal piece was measured for all metal samples in all Petri
dishes on 2 November 2015. An average zone of inhibition was then recorded for each of the 10 samples of each
metal by averaging the zone of inhibition measurements for the 4 edges of each sample. Measurements for samples
that had fallen from the agar during the incubation period were labeled β€œF” for fallen and the data for that sample
was not used in calculations.
Results
Mean Zone of Inhibition (mm)
An analysis of variance (ANOVA) was run on the measured average zone of inhibition for the metal
samples of each metal type. According to the results of the ANOVA, the p-value was reported to be 0.00, thereby
indicating a significant difference between the zones of inhibition for the different types of metal. The Bonferroni
correction post-hoc test was run to reveal a p-value of less than 0.05 for all comparisons tests that included zinc but
p-values greater than 0.05 for all comparisons that did not include zinc. This indicates that zinc was the only
significantly antibacterial metal tested and thereby exhibited higher bactericidal activity than stainless steel
(control), copper, and silver (Figure 1).
The average zone of inhibition for the Petri dishes containing Staphylococcus aureus and stainless steel
(control) was 0.166 mm ± 0.071 (mean ± SEM, n=8), average zone of inhibition with zinc was 7.343 mm ± 0.232
(mean ± SEM, n=10), average zone of inhibition with copper was 0.229 mm ± 0.075 (mean ± SEM, n=7), and the
average zone of inhibition with silver was 0.350 mm ± 0.010 (mean ± SEM, n=10).
*
8
7
6
5
4
3
2
1
0
Stainless Steel
(control)
Zinc
Copper
Silver
Metal Type
Figure 1: Mean zone of inhibition (mm) for each type of metal tested. Mean values for stainless steel, zinc, copper and silver are 0.166 mm ±
0.071, 7.343 mm ± 0.232, 0.229 mm ± 0.075, and 0.350 mm ± 0.010, respectively. Analysis of variance (ANOVA) indicated p=0.00 and
Bonferroni post-hoc test indicated p < 0.5 for zinc but p > 0.5 for all other metals tested, signifying zinc is the only significantly antibacterial
metal, as indicated by the asterisks. Error bars are mean ± SEM.
Discussion
The results of our experiment were consistent with our hypothesis to a limited degree. Experimental data
suggests that zinc is the most antibacterial metal of those tested, as predicted by our hypothesis. However the data
produced by this experiment also suggests silver and copper lack the bactericidal properties found in other studies
(Heidenau et al., 2005 and Miyano, 2010), as the results of this experiment did not indicate that copper and silver
are any more antibacterial than stainless steel. The average measurement of the zone of inhibition of metals showed
that zinc was had the greatest antimicrobial properties, which was theorized was due to zinc’s higher rate of
oxidation when compared to the other metals being tested (Figure 1). It should be noted that while the results
indicate that zinc has greater antibacterial properties than more common materials, there have been reported cases
and research done on people who have allergies to zinc oxide. Reactions to zinc oxide range can anywhere from
mild swelling at the point of contact to breathing difficulties (Hiroshi et al., 2006). However, such allergies are also
extremely rare and are found in less than one percent of the population (Achou et al., 2010). The lower rates of
oxidation of the other metals tested may have resulted is a weaker oligodynamic effect compared to zinc, as
evidenced by the low p-value calculated. This is consistent with previous research that have shown easily oxidized
metals to have greater antibacterial properties than more conventional materials used in construction (Kraemer, 1905
and Miyano et al., 2010).
One of the fastest growing concerns in modern hospitals is how to contain the spread of bacteria and
diseases in hospitals. With so many diseases in such close proximity, being able to minimize human exposure is a
top priority. One of the ways that bacteria can travel is through contact with infected surfaces. The rise of antibioticresistant bacteria such as methicillin-resistant Staphylococcus aureus (Ayliffe, 1997) as well as the ease of
transferring bacteria from surface to surface makes research into more effective antimicrobial surfaces highly
important. The purpose of this experiment was to determine which of four different metallic compounds was the
most effective at inhibiting microbial growth on its surface. The results of this research can result in more sanitary
work conditions, particularly in places like hospitals.
Literature Cited
Achou, A., Ines, BA., Faika, BM., Fethi, M., Habib, K., Nabil, T., Saber, D. 2012. Generalized allergy due to zinc in
insulin treated with zinc-free insulin. Acta Diabetoligica. 49 (3): 239-241.
Ayliffe, G.A.J. 1997. The progressive intercontinental spread of Methicilin-Resistant Staphylococcus aureus.
Clinical Infectious Diseases 24: S74-S79.
Hiroshi, S., Kazuo, K., Tadamachi, S., Yanagi, T., Yoko, Y. 2005. Macrophage migration inhibitory factor in zincallergy systemic contact dermatitis. Cytokine 35 (5/6): 270-274.
Heidenau, F., W. Mittelmeier, R. Detsch, M. Haenle, F. Stenzel, G. Ziegler, H. Gollwitzer. 2005. A novel
antibacterial titania coating: Metal ion toxicity and in vitro surface colonization. Journal of Material Science:
Materials in Medicine 16: 883-888.
Khalid, Quthami, Bassam O. AlJohny, Milton Wainwright. 2014. Antibacterial effects of pure metals on clinically
important bacteria growing in planktonic cultures and biofilms. African Journal of Microbiology Research 8 (10):
1080-1088.
Kraemer, Henry. 1905. The Oligodynamic Action of Copper Foil on Certain Intestinal Organisms. American
Philosophical Society 44 (179): 51-65.
Miyano, Yasuyuki, Koyama Kunihiro, Sreekumari Kurissery, Nandakumar Kanavillil, Yoshiro Sato, Yasushi
Kikuchi. 2010. Antibacterial properties of nine pure metals: A laboratory study using Staphylococcus aureus and
Escherichia coli. Biofouling 26 (7):851-858.
Rentz, Eric. 2003. Viral Pathogens and Severe Acute Respiratory Syndrome: Oligodynamic Ag + for Direct Immune
Intervention. Journal of Nutritional and Environmental Medicine 13 (2): 109-118.
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