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

Chlorine is a potent, rapidly acting, short-term disin-
fectant. However, because chlorine is a gas, it has little
practical use. Instead, many compounds that release chlo-
rine find wide application, including hypochlorite and
organic and inorganic chloramines. Sodium hypochlorite
(NaOCl) is one of the most frequently used chloro-releasing
agents used in the field of chemical disinfection; hypo-
chlorous acid (HClO) is the main active ingredient of most
household bleaches and cleansers. HClO has a broad
antimicrobial spectrum covering bacteria, mycobacteria,
bacterial spores, viruses, algae, and protozoa. In human
medicine, hypochlorite solutions diluted in water or saline
have been used for vaginal, bladder, and urethral irriga-
tions, control of athlete’s foot and as infection prophylaxis
in the management of burns [1].
* Corresponding author at: David W. Ramey, PO Box 16883, Encino,
CA 91416.
E-mail address: ponydoc@pacbell.net (D.W. Ramey).
horse owners for the treatment of wounds in horses. In the
last few years, an over-the-counter hydrochlorous acid
preparation has been made available (Vetericyn VF Liquid,
Innovacyn, Inc, Rialto, CA). Similar products are marketed
as a medical device in the human market, but there has
been no approval granted for the veterinary product, by the
US Food and Drug Administration (FDA), as there are no
current requirements by the FDA for animal device clear-
ance [2].
Healing of wounds is thought to be enhanced by
decreasing the bacterial burden; however, the need for
antimicrobial treatments in wound management is not
consistently supported by experimental evidence. Never-
theless, countless topical agents have been used in an effort
to prevent wound infection and decrease surface contam-
ination. Unfortunately, the majority of such preparations
may be locally toxic and have limited to no proven effec-
tiveness in enhancing wound healing [3].
The first objective of this study was to evaluate the
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,
Escherichia coli and Staphylococcus aureus, in vitro. The
second objective of the study was to compare the com-
mercial product to that of a homemade solution (HClO,
0.012%).
2. Materials and Methods
One bottle of the commercially available aqueous
preparation was purchased at a retail tack and pet supply
store. The preparation consists of electrolyzed water (H2O,
99.816%), hypochlorous acid (HClO, 0.011%), sodium chlo-
ride (NaCl, 0.023%), and sodium phosphate (NaH2PO/
NaH2PO4, 0.015%). The pH of the buffered solution was 6.9.
In an effort to duplicate the concentration of HClO in the
commercial preparation, the amounts of necessary in-
gredients (distilled water and bleach) were calculated. A
homemade solution of equivalent concentration of chloride
ion was thus made by adding 1.5 mL of “concentrated”
bleach solution (8.25% Nalco) to 1 L of distilled water,
resulting in a concentration of 99.988% H2O and 0.012%
HClO. Because of concerns about possible instability of the
homemade bleach and/or water preparation, the home-
made preparation was held in a plastic bottle for 1 week
before testing. The pH of the unbuffered solution was 8.9,
measured after 1 week of storage.
Tests on the commercial and homemade solutions were
conducted at the California Animal Health and Food Safety
Laboratory, San Bernardino, CA, as follows:
2.1. Trial 1
Pure 18- to 24-hour-old E. coli and S. aureus cultures
growing on a sheep blood agar plates with well-isolated
colonies were used to prepare a bacterial suspension. The
turbidity of the bacterial suspension was adjusted to a 0.5
McFarland standard; approximately 108 colony forming
units per milliliter (CFU/mL). One microliters of each bac-
terial suspension was mixed thoroughly with 1 mL of
commercial solution. Similarly, 1 mL of each bacterial sus-
pension was mixed with 1 mL of water (control solution).
The mixtures were then allowed to sit for 1 hour at room
temperature and then streaked on to blood agar plates. The
plates were then incubated in 3%–7% CO2 for 20–24 hours,
and results were recorded as growth present or absent.
2.2. Trial 2
Pure 18- to 24-hour-old E. coli and S. aureus cultures
growing on a sheep blood agar plates with well-isolated
colonies were used to prepare six tubes of bacterial sus-
pension. The turbidity of the bacterial suspension was
adjusted to a 0.5 McFarland standard; approximately
108 CFU/mL. The tubes were centrifuged at 5,000 rpm for
10 minutes, and the supernatant was decanted. Five hun-
dred microliters and 1,000 mL of the commercial solution
were added to the tubes containing pellets of E. coli and S.
aureus. Similarly, 500 mL of water (control solution) was
added to the bacterial pellets in each of the remaining two
tubes. The tubes were then rigorously mixed and allowed
to sit for 1 hour at room temperature. After 1-hour
incubation, the bacterial suspensions were streaked on to
blood agar plates. The plates were then incubated in 3%–7%
CO2 for 20–24 hours, and results were recorded as growth
present or absent.
2.3. Trial 3
Trial 3 was the same as trial 2 except homemade solu-
tion was used instead of the commercial antibacterial
solution.
3. Results
In trial 1 (commercial solution), no growth of either
bacteria was seen in test solutions containing the com-
mercial HClO product after 24 and 48 hours of incubation.
Bacterial growth was seen in all control samples.
In trial 2 (commercial solution), no growth of E. coli was
seen at either 500 or 1,000 mL. No growth of S. aureus was
seen at 1,000 mL, but growth was present at 500 mL. Bac-
terial growth was present on all control samples.
In trial 3 (homemade solution), no growth of either
bacteria was seen at either 500 or 1,000 mL. Bacterial
growth was present in all control samples.
4. Discussion
Hypochlorous acid has been recognized as a microbicide
since at least 1915 [4]. Dakin’s solution has been recognized
as a wound disinfectant for nearly a century and has been
shown to kill pathogenic microorganisms with minimal
cytotoxicity [5]; however, its concentration, 0.5% HClO [6],
is much higher than that of the tested solutions. HClO is a
potent oxidant, and the bactericidal action appears to be
primarily related to the destruction of cellular electron
transport chains and the adenine nucleotide pool and
abolition of adenosine triphoshate production [7]. Immune
cells such as neutrophils, macrophages, and eosinophils
produce reactive oxygen species when confronted with
infectious agents, so in a sense, there is biological precedent
for its application [8].
It has not been established that HClO solutions enhance
wound healing; however, the possible cytotoxicity of HClO
solutions has been investigated on human dermal fibro-
blasts in vitro [9]. At concentrations of 0.1% and upward, the
compound has a profound cytotoxic effect. At concentra-
tions of 0.01% dose-dependent mitochondrial dysfunction
is observed, and cell survival increases with decreasing
concentrations of the chemical. Dakin’s solution at con-
centrations of 0.5%, 0.125%, and 10-fold serial dilutions
from 0.25% to 0.00025% have been evaluated, with most
concentrations being shown to be detrimental to macro-
phage viability and function [10]. The concentration of
hypochlorous acid in the tested solutions was 0.011%, a
concentration at which cell toxicity of between 20% and
75% was seen in human fibroblasts. At a concentration of
0.01% HClO, there is almost total depletion of cellular
adenosine triphoshate in the fibroblasts; however, this
appears to be at least partially reversible due at least in part
to the relatively short-term action of HClO [11]. (Interest-
ingly, hypochlorite concentrations ranging from 0.005% to
 D.W. Ramey, H. Kinde / Journal of Equine Veterinary Science 35 (2015) 161–164
0.00025% caused proliferation and expansion of human
dermal fibroblast lines in vitro.) However, in another study,
solutions of water and sodium hypochlorite in a stabilized
sodium chloride solution were also been demonstrated to
have antibacterial properties, but without obvious cyto-
toxic effects [12].
Although production details of the commercial product
were not disclosed, electrolyzed water is the primary
component. The electrolysis of tap water produces a solu-
tion of sodium hypochlorite and is most likely the source of
the hypochlorous acid contained in the solution. Electro-
lyzed water is produced by the electrolysis (i.e., using a direct
current to drive an otherwise nonspontaneous chemical
reaction) of ordinary tap water containing sodium chloride.
Electrolyzed water itself has been shown to inactivate E. coli,
Salmonella enteritidis, and Listeria monocytogenes in vitro
[13], increased the survival of Sprague-Dawley rats in a
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
various solutions, and the pH of the commercial solution
was 6.9. The pH of the unbuffered homemade solution
was 8.9. The relevance of the pH of the solutions is
unknown; the topic of pH and wound healing is compli-
cated and the subject of ongoing research [16]. The control
solution (water) had a pH of 7.3. The pH environment of
chronic wounds has been recorded within the range of
7.15–8.9 [17].
Although the tonicity of the solutions was not
measured, all were, presumably, hypotonic. Rinsing with
hypotonic solutions appears to not be detrimental to
wounds. For example, there is no evidence that using tap
water to cleanse acute wounds in human adults increases
infection, and some evidence that it reduces it. However,
there is not strong evidence that cleansing wounds per se
increases healing or reduces infection [18].
One potential problem with HClO solutions is that they
are potentially rapidly neutralized in a wound environ-
ment. This may be one reason why second applications of
an HClO product have been shown to be more effective at
killing wound bacteria than a single application [9].
The stability of bleach and/or water solutions is a po-
tential concern. Hypochlorite solution concentration drops
rapidly when the pH is not stabilized, when pH levels fall,
and when solution is exposed to direct sunlight [19]. In this
study, no attempts were made to stabilize the homemade
solution. However, the nominal costs associated with prep-
aration of the homemade solution would seem to obviate
concerns about stability; if treatment with such a compound
were desired, it would be extremely cost effective to make
up solutions of bleach and water on an as-needed basis.
Storage of bleach solutions in closed containers, protection
from sunlight, and cold conditions have all been shown to
delay degradation of bleach and/or water solutions [20], and
such solutions to the degradation problem are very practical.
In this study, storage of the bleach and/or water solution for
1 week did not affect its bactericidal capability. Homemade
solutions of bleach and water are widely used in other ap-
plications. For example, such solutions have been shown to
improve water quality and reduce human diarrheal diseases
in developing countries [15].
163
5. Conclusions
This study showed that single samples of extremely
dilute solutions of HClO are bactericidal in vitro for two
common equine pathogens. The study assumes that the
commercial samples are consistent; if manufacturing
standards are not consistent, the results may differ be-
tween individual bottles of the product. Given the low cost
and easy availability of ingredients, as well as myriad uses
in human medicine, it would seem that there are ample
reasons for investigation of the use of such extremely
dilute solutions of hypochlorous acid in equine medicine,
including, but not limited to, areas such as in wound
care, dentistry, and in the treatment of endometritis.
Investigations into safety issues pertaining to such solu-
tions, for example, potential cytotoxicity, should be per-
formed, as well.
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