Journal of Equine Veterinary Science 35 (2015) 161–164

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Journal of Equine Veterinary Science

journal homepage: www.j-evs.com

Short Communication

Commercial and Homemade Extremely Dilute

Hypochlorous Acid Solutions Are Bactericidal Against
Staphylococcus aureus and Escherichia coli In Vitro
David W. Ramey a, *, Hailu Kinde b
a
Ramey Equine, PO Box 16883, Encino, CA 91416
b
CAHFS San Bernardino Branch, School of Veterinary Medicine, University of California Davis, San Bernardino, CA

a r t i c l e i n f o a b s t r a c t

Article history: A commercially available extremely low concentration of hypochlorous acid solution
Received 19 August 2014 (HClO, 0.011%) sold over the counter for wound care in horses completely inhibits growth
Received in revised form 26 November 2014 of Staphylococcus aureus and Escherichia coli in vitro when in solution. A homemade
Accepted 3 December 2014
solution of equivalent concentration of hypochlorous acid (HClO, 0.012%) equally inhibits
Available online 10 December 2014
the two organisms tested, at a much lower cost. Such solutions may have wide potential
applications in equine medicine.
Keywords:
Ó 2015 Elsevier Inc. All rights reserved.
Hypochlorous acid
Wound
Wound care
Disinfectant
Bacteriology
Cytotoxicity

1. Introduction Many over-the-counter products are available for use by

horse owners for the treatment of wounds in horses. In the
Chlorine is a potent, rapidly acting, short-term disin- last few years, an over-the-counter hydrochlorous acid
fectant. However, because chlorine is a gas, it has little preparation has been made available (Vetericyn VF Liquid,
practical use. Instead, many compounds that release chlo- Innovacyn, Inc, Rialto, CA). Similar products are marketed
rine find wide application, including hypochlorite and as a medical device in the human market, but there has
organic and inorganic chloramines. Sodium hypochlorite been no approval granted for the veterinary product, by the
(NaOCl) is one of the most frequently used chloro-releasing US Food and Drug Administration (FDA), as there are no
agents used in the field of chemical disinfection; hypo- current requirements by the FDA for animal device clear-
chlorous acid (HClO) is the main active ingredient of most ance [2].
household bleaches and cleansers. HClO has a broad Healing of wounds is thought to be enhanced by
antimicrobial spectrum covering bacteria, mycobacteria, decreasing the bacterial burden; however, the need for
bacterial spores, viruses, algae, and protozoa. In human antimicrobial treatments in wound management is not
medicine, hypochlorite solutions diluted in water or saline consistently supported by experimental evidence. Never-
have been used for vaginal, bladder, and urethral irriga- theless, countless topical agents have been used in an effort
tions, control of athlete’s foot and as infection prophylaxis to prevent wound infection and decrease surface contam-
in the management of burns [1]. ination. Unfortunately, the majority of such preparations
may be locally toxic and have limited to no proven effec-
* Corresponding author at: David W. Ramey, PO Box 16883, Encino,
tiveness in enhancing wound healing [3].
CA 91416. The first objective of this study was to evaluate the
E-mail address: [email protected] (D.W. Ramey). antibacterial effect of a low concentration of hypochlorous

0737-0806/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jevs.2014.12.004
162 D.W. Ramey, H. Kinde / Journal of Equine Veterinary Science 35 (2015) 161–164

acid solution (HClO, 0.011%) against two common bacteria, incubation, the bacterial suspensions were streaked on to
Escherichia coli and Staphylococcus aureus, in vitro. The blood agar plates. The plates were then incubated in 3%–7%
second objective of the study was to compare the com- CO2 for 20–24 hours, and results were recorded as growth
mercial product to that of a homemade solution (HClO, present or absent.
0.012%).
2.3. Trial 3
2. Materials and Methods
Trial 3 was the same as trial 2 except homemade solu-
One bottle of the commercially available aqueous tion was used instead of the commercial antibacterial
preparation was purchased at a retail tack and pet supply solution.
store. The preparation consists of electrolyzed water (H2O,
99.816%), hypochlorous acid (HClO, 0.011%), sodium chlo- 3. Results
ride (NaCl, 0.023%), and sodium phosphate (NaH2PO/
NaH2PO4, 0.015%). The pH of the buffered solution was 6.9. In trial 1 (commercial solution), no growth of either
In an effort to duplicate the concentration of HClO in the bacteria was seen in test solutions containing the com-
commercial preparation, the amounts of necessary in- mercial HClO product after 24 and 48 hours of incubation.
gredients (distilled water and bleach) were calculated. A Bacterial growth was seen in all control samples.
homemade solution of equivalent concentration of chloride In trial 2 (commercial solution), no growth of E. coli was
ion was thus made by adding 1.5 mL of “concentrated” seen at either 500 or 1,000 mL. No growth of S. aureus was
bleach solution (8.25% Nalco) to 1 L of distilled water, seen at 1,000 mL, but growth was present at 500 mL. Bac-
resulting in a concentration of 99.988% H2O and 0.012% terial growth was present on all control samples.
HClO. Because of concerns about possible instability of the In trial 3 (homemade solution), no growth of either
homemade bleach and/or water preparation, the home- bacteria was seen at either 500 or 1,000 mL. Bacterial
made preparation was held in a plastic bottle for 1 week growth was present in all control samples.
before testing. The pH of the unbuffered solution was 8.9,
measured after 1 week of storage. 4. Discussion
Tests on the commercial and homemade solutions were
conducted at the California Animal Health and Food Safety Hypochlorous acid has been recognized as a microbicide
Laboratory, San Bernardino, CA, as follows: since at least 1915 [4]. Dakin’s solution has been recognized
as a wound disinfectant for nearly a century and has been
2.1. Trial 1 shown to kill pathogenic microorganisms with minimal
cytotoxicity [5]; however, its concentration, 0.5% HClO [6],
Pure 18- to 24-hour-old E. coli and S. aureus cultures is much higher than that of the tested solutions. HClO is a
growing on a sheep blood agar plates with well-isolated potent oxidant, and the bactericidal action appears to be
colonies were used to prepare a bacterial suspension. The primarily related to the destruction of cellular electron
turbidity of the bacterial suspension was adjusted to a 0.5 transport chains and the adenine nucleotide pool and
McFarland standard; approximately 108 colony forming abolition of adenosine triphoshate production [7]. Immune
units per milliliter (CFU/mL). One microliters of each bac- cells such as neutrophils, macrophages, and eosinophils
terial suspension was mixed thoroughly with 1 mL of produce reactive oxygen species when confronted with
commercial solution. Similarly, 1 mL of each bacterial sus- infectious agents, so in a sense, there is biological precedent
pension was mixed with 1 mL of water (control solution). for its application [8].
The mixtures were then allowed to sit for 1 hour at room It has not been established that HClO solutions enhance
temperature and then streaked on to blood agar plates. The wound healing; however, the possible cytotoxicity of HClO
plates were then incubated in 3%–7% CO2 for 20–24 hours, solutions has been investigated on human dermal fibro-
and results were recorded as growth present or absent. blasts in vitro [9]. At concentrations of 0.1% and upward, the
compound has a profound cytotoxic effect. At concentra-
2.2. Trial 2 tions of 0.01% dose-dependent mitochondrial dysfunction
is observed, and cell survival increases with decreasing
Pure 18- to 24-hour-old E. coli and S. aureus cultures concentrations of the chemical. Dakin’s solution at con-
growing on a sheep blood agar plates with well-isolated centrations of 0.5%, 0.125%, and 10-fold serial dilutions
colonies were used to prepare six tubes of bacterial sus- from 0.25% to 0.00025% have been evaluated, with most
pension. The turbidity of the bacterial suspension was concentrations being shown to be detrimental to macro-
adjusted to a 0.5 McFarland standard; approximately phage viability and function [10]. The concentration of
108 CFU/mL. The tubes were centrifuged at 5,000 rpm for hypochlorous acid in the tested solutions was 0.011%, a
10 minutes, and the supernatant was decanted. Five hun- concentration at which cell toxicity of between 20% and
dred microliters and 1,000 mL of the commercial solution 75% was seen in human fibroblasts. At a concentration of
were added to the tubes containing pellets of E. coli and S. 0.01% HClO, there is almost total depletion of cellular
aureus. Similarly, 500 mL of water (control solution) was adenosine triphoshate in the fibroblasts; however, this
added to the bacterial pellets in each of the remaining two appears to be at least partially reversible due at least in part
tubes. The tubes were then rigorously mixed and allowed to the relatively short-term action of HClO [11]. (Interest-
to sit for 1 hour at room temperature. After 1-hour ingly, hypochlorite concentrations ranging from 0.005% to
 D.W. Ramey, H. Kinde / Journal of Equine Veterinary Science 35 (2015) 161–164 163

0.00025% caused proliferation and expansion of human 5. Conclusions

dermal fibroblast lines in vitro.) However, in another study,
solutions of water and sodium hypochlorite in a stabilized This study showed that single samples of extremely
sodium chloride solution were also been demonstrated to dilute solutions of HClO are bactericidal in vitro for two
have antibacterial properties, but without obvious cyto- common equine pathogens. The study assumes that the
toxic effects [12]. commercial samples are consistent; if manufacturing
Although production details of the commercial product standards are not consistent, the results may differ be-
were not disclosed, electrolyzed water is the primary tween individual bottles of the product. Given the low cost
component. The electrolysis of tap water produces a solu- and easy availability of ingredients, as well as myriad uses
tion of sodium hypochlorite and is most likely the source of in human medicine, it would seem that there are ample
the hypochlorous acid contained in the solution. Electro- reasons for investigation of the use of such extremely
lyzed water is produced by the electrolysis (i.e., using a direct dilute solutions of hypochlorous acid in equine medicine,
current to drive an otherwise nonspontaneous chemical including, but not limited to, areas such as in wound
reaction) of ordinary tap water containing sodium chloride. care, dentistry, and in the treatment of endometritis.
Electrolyzed water itself has been shown to inactivate E. coli, Investigations into safety issues pertaining to such solu-
Salmonella enteritidis, and Listeria monocytogenes in vitro tions, for example, potential cytotoxicity, should be per-
[13], increased the survival of Sprague-Dawley rats in a formed, as well.
third-degree burn wound model [14], and assisted in the
healing of chronic venous leg ulcers in people [15].
Sodium phosphate is often used for pH control in
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