Archive for the ‘Hypochlorous Acid, Sodium Hypochlorite and Chlorine’ Category

Hypochlorous Acid as a Potential Wound Care Agent

October 11, 2013

Hypochlorous acid (HOCl), a major inorganic bactericidal compound of innate immunity, is effective against a broad range of microorganisms. Owing to its chemical nature, HOCl has never been used as a pharmaceutical drug for treating infection. In this article, we describe the chemical production, stabilization, and biological activity of a pharmaceutically useful formulation of HOCl.

Stabilized HOCl is in the form of a physiologically balanced solution in 0.5-1.0% saline at a pH range of 3.5 to 7.0. Chlorine species distribution in solution is a function of pH. In aqueous solution, HOCl is the predominant species at the pH range of 4 to 6. At pH values less than 3.5, the solution exists as a mixture of chlorine in aqueous phase, chlorine gas, trichloride (Cl3−), and HOCl. At pH greater than 5.5, sodium hypochlorite (NaOCl) starts to form and becomes the predominant species in the alkaline pH. To maintain HOCl solution in a stable form, maximize its antimicrobial activities, and minimize undesirable side products, the pH must be maintained at 5 to 5.5.

Using this stabilized form of HOCl, the potent antimicrobial activities of HOCl are demonstrated against a wide range of microorganisms. The in vitro cytotoxicity profile in L929 cells and the in vivo safety profile of HOCl in various animal models are described. On the basis of the antimicrobial activity and the lack of animal toxicity, it is predicted that stabilized HOCl has potential pharmaceutical applications in the control of soft tissue infection.  A remarkable feature of the immune system is its ability to launch an effective response against invading pathogens by deploying a group of highly reactive chemicals, including oxidized halogens, oxidizing radicals, and singlet oxygen.

The precursor of these reactive oxygen species (ROS) is the oxygen radical (O2), which is generated by specialized immune cells—neutrophils, eosinophils, mononuclear phagocytes, and B lymphocytes. Production of ROS in these cells is accompanied by a significant rise in oxygen consumption, a series of events collectively referred to as the oxidative burst. The primary enzyme responsible for ROS production is a mitochondrial-membrane–bound enzyme known as respiratory burst NADPH oxidase. Patients with chronic granulomatous disease have oxidase defective genes, which makes them susceptible to repeated infection.10,11 During a respiratory burst, neutrophils produce H2O2, which is converted to HOCl by the activity of the granule enzyme myeloperoxidase.

HOCl is known to be the major strong oxidant produced by neutrophils, and is a potent microbicidal agent within these cells. Experimentally, it has been estimated that 106 neutrophils stimulated in vitro can produce 0.1 μM HOCl. This quantity of HOCl can kill 1.5 × 107 Escherichia coli in less than 5 minutes. HOCl reacts readily with a range of biological molecules, particularly those with thiol, thiolether, heme proteins, and amino groups and may lead to tissue injury. Taurine, a nonessential amino acid naturally found at roughly 15 mM within neutrophils acts as a scavenger molecule for HOCl via the following mechanism, and effectively dampens the collateral damage to cellular macromolecules caused by HOCl.

To date, pure HOCl has not been developed as a commercial pharmaceutical formulation presumably because of the challenge of maintaining storage stability. In this article, we describe a method for the preparation and stabilization of a pure form of HOCl for potential use as a pharmaceutical agent. We show here that when compared to the commercially available disinfectants hydrogen peroxide and sodium hypochlorite (NaOCl), this formulation has improved in vitro antimicrobial activity and therapeutic index. Furthermore, we present data demonstrating an excellent safety profile for HOCL in animal toxicology studies. We believe the improved properties of our pure physiologically balanced stabilized form of HOCl may allow for its use in a clinical situation such as in the treatment or prevention of infection in burn or other wounds.

Hypochlorous acid was prepared in 154 mM NaCl by acidifying reagent-grade NaOCl to the pH range of 3.5 to 4.0 with dilute HCl. A Beckman pH meter was used to accurately measure the final pH values. The concentration of active total chlorine species in solution expressed as [HOCl]T (where [HOCl]T = [HOCl] + [Cl2] + [Cl3−] + [OCl−]) in 0.9% saline was determined by converting all the active chlorine species to OCl− with 0.1 M NaOH and measuring the concentration of OCl−. The concentration of OCl− was determined spectrophotometrically at 292 nm (ε = 362 M− 1 cm− 1)15 with an Agilent 8453 UV-visible spectrophotometer.

All microorganisms used in these studies were purchased from the American Type Culture Collection (ATCC), grown and propagated according to the recommendations for each strain by ATCC. Bacterial cells were harvested at stationary phase and concentrations were determined by 10-fold dilution as direct colony count. To prepare inoculum, bacteria were diluted in sterile saline before use to minimize the effect of broth on HOCl.

A modification of the National Committee Consensus on Laboratory standardized protocol “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically” was used in these studies. Sterile 0.9% saline at pH 3.5 to 4.0 (vehicle) was used as the diluent. Use of such a diluent allows for the determination of the intrinsic activity of HOCl in the absence of any interfering molecules. Specifically, each test article is diluted using 2-fold serial dilution in acid-washed glass tubes to give a range of concentrations from approximately 2 to 0.002 mM in a final volume of 1 mL. Each dilution is inoculated with 5 × 105 CFU/mL test bacteria and co-incubations were carried out at room temperature for 60 minutes. At 60 minutes post-treatment, 0.1 mL of each reaction was immediately transferred into pre-labeled 1.5-mL microfuge tubes containing 0.9 mL Dey and Engley (D/E) neutralizer broth (Hardy Diagnostic, Santa Maria, CA). Minimum bactericidal concentration (MBC) is determined by plating 0.1 mL of each sample onto an agar plate. Plates were then incubated overnight at 35°C, and examined for colony growth. The concentration at which there was a complete absence of colony growth is determined to be the MBC. Comparative MBC results provide estimates of the susceptibility of various test articles against test organisms.

For time kill studies, 5 mL of test article at an approximate MBC concentration was inoculated with approximately 106 CFU/mL of each test organism and incubated for 0, 5, 10, 15, 20, 30, 60, and 90 minutes at room temperature. For each time point, 0.1 mL was transferred into 0.9 mL of D/E neutralizer broth and 0.1 mL of this mixture was plated and incubated as previously described.

L929 (ATCC CCL-1, NCTC clone 929) is a connective-tissue cell line derived from normal subcutaneous areolar and adipose tissue of a 100-day-old male C3H mouse. L929 cells were purchased from ATCC and propagated according to supplier’s recommendations. These cells were then seeded at 1.5 × 104 cells per well in 96-well plates and incubated overnight at 37°C. On the day of testing, growth medium was aspirated from each well, and 30 μL fresh medium was added per well. Test articles were diluted by 2-fold serial dilution using 154 mM saline at the desired pH for each test article. Following that, 170 μL of each dilution was added to each well for a total volume of 200 μL per well. After 60 minutes’ exposure at 37°C, test articles were replaced with 200 μL of fresh tissue culture media and incubated for 24 hours at 37°C. Cell viability was determined by addition of WST-8 (Dojindo, Japan) reagent and the absorption at 450 nm read spectro-photometrically. Orange-red formazan, which is produced by live cells, is a direct measure of cell viability in this assay.

The therapeutic Index of an antimicrobial agent is defined as the ratio of the concentration to achieve 50% cell toxicity (CT50) to MBC.

Ocular irritation, skin sensitization, and wound toxicology studies were performed. A preliminary (non–good laboratory practice [non-GLP]) study with a development formulation of HOCl (2.5 mM; 0.013% w/v) was carried out at the Brookdale Eye Clinic (K. Najafi, MD, unpublished data). Dutch pigmented rabbits received either 5% ophthalmic povidone-iodine (Betadine) (15 eyes) or the development formulation (15 eyes). Each eye received 0.1 mL of solution every 8 hours for a total of 72 hours and observations were made periodically during this time. The effect of the development formulation was compared to 5% ophthalmic-grade Betadine.
A GLP ocular irritation study (NAMSA, Toledo, OH) was designed to determine the potential for ocular irritation following a single instillation in the rabbit. New Zealand White rabbits (5 per group) were used. Hypochlorous acid (NVC-101) was instilled in the right eye at concentrations of 0.01%, 0.03%, and 0.1% w/v (pH 3.5). The left eyes were used as the controls and were untreated, vehicle (saline) or positive control treated. In all cases, the volume used was 0.1 mL, which was placed into the lower conjunctival sac. Evaluations for irritation were made at 24, 48, and 72 hours. At 24 hours, the cornea was examined using fluorescein stain. GLP repeat-dose wound toxicity studies (Charles River, Spencerville, OH) were designed to provide maximum exposure to full-thickness wounds in rats and mini-pigs. Wounds were treated with NVC-101 at concentrations of 0.01%, 0.03%, and 0.1% w/v (pH 3.5). The test material was applied to the wounded area directly using soaked gauze. The treated site was covered for approximately 24 hours per day for 28 days. Wounds achieving 75% closure were kept open by abrasion. Parameters used to assess systemic toxicity included clinical signs (including observations of the site), body weights, food intake, clinical chemistries (blood and urine), hematology, organ weights, and gross and microscopic tissue evaluations.

The molar percentage of each species in physiologically balanced HOCl solution is a function of pH. A low pH and high [Cl−] favors the formation of Cl2. Once Cl2 is formed in the aqueous phase, it migrates into the headspace. The transfer of Cl2 from the solution to the headspace of the container results in a decrease in active chlorine concentration in solution. Therefore, the degassing of Cl2 becomes a major path for loss of HOCl in an open system (nonsealed). This is a potential problem for clinical use of HOCl. To stabilize the physiologically balanced HOCl solution, minimizing the formation of Cl2 is essential.

Stabilized HOCl demonstrates broad-spectrum antimicrobial activity at concentrations ranging from 0.1 to 2.8 μg/mL.

Minimum bactericidal concentration (μg/mL) of HOCl for a broad spectrum of microorganisms tested at room temperature for 60 min
The exception is Aspergillus niger, where a higher concentartion of HOCl (86.6 μg/mL) was required for effective killing of the organism under the same assay conditions. Time kill is an in vitro measure of how fast a given antimicrobial can kill test bacteria. The rate of kill by stabilized HOCl was first demonstrated at the MBC values for each microorganism using an inoculum size of 1 × 106 mL−1 for each test bacteria. The majority of test organisms were killed (>99.99%) within the first 2 minutes of exposure. Among the bacterial species tested, Streptococcus pyogenes 49399 was the only exception, which required approximately 10 minutes of exposure for effective killing, under the same assay conditions. The killing rate of stabilized HOCl with NaOCl and H2O2 was then determined against 3 specific test organisms—E. coli 25922, P. aeruginosa 27853, and S. aureus 29213—at room temperature for a total of 90 miniutes.

Comparative time kill studies of HOCl, NaOCl, and H2O2 against 3 test organisms at room temperature for a total of 90 min. It is worth mentioning that all these time kill studies were also performed with an inoculum size of 1 × 107/mL for each test bacteria.

Comparative MBC (μM) of HOCl, NaOCl, and H2O2 tested against 3 organisms at room temperature for 60 min. As the results show, HOCl at its MBC values for different test organisms (5.6–12.5 μM) was able to kill all 3 test bacteria in less than 1 minute, with no significant bacterial killing effect from its excipient, saline at pH 4.0. However, the kill time for NaOCl at MBC values 10 to 50 μM varied from 5 to 15 minutes for the same 3 test organisms. In contrast, H2O2was only able to kill P. aeruginosa 27853 at 7500 μM in about 10 minutes, but did not kill S. aureus 29213 at its highest concentration tested (20,000 μM) even up to 90 minutes’ exposure time under the same assay conditions.

The relative cell toxicity of HOCl, NaOCl, and H2O2 was assessed following a standard method used to examine the cytotoxicity of liquid disinfectants. This toxicity assay utilizes an established adherent cell line, L929, and the end point is relative cell viability measured by addition of WST-8 (Dojindo, Japan) colorimetric reagent. Orange-red formazan, which is produced by live cells, is a direct measure of cell viability in this assay. Cytotoxicity was measured using 2-fold dilutions of HOCl, NaOCl, and H2O2 as compared to untreated or vehicle-treated control L929 cells. The CT50 was calculated for each test article. The CT50 values for HOCl (15–25 μg/mL) and NaOCl (38–42 μg/mL) were reproducible and closely matched published results for NaOCl.However, the CT50 values for H2O2 were more variable (5–35 μg/mL), probably due to the chemical instability of H2O2 under these assay conditions.

Relative cell toxicity of hypochlorous acid (HOCl; pH 4.0), hypochlorite (OCl−; pH 10.5), and hydrogen peroxide (H2O2; pH 7.0) on L929 cells. Cytotoxicity measured in a cell proliferation assay is expressed as the concentration (μg/mL) . The therapeutic indices for HOCl, NaOCl, and H2O2 were assessed using L929 cells and 3 clinically relevant bacterial strains—E. coli 25922, P. aeruginosa 27853, and S. aureus 29213. Relative therapeutic index of hypochlorous acid (HOCl; pH 4.0), hypochlorite (OCl−; pH 10.5), and hydrogen peroxide (H2O2; pH 7.0). The value for stabilized HOCl is approximately 98-fold higher than that for H2O2 for the gram-negative bacterium E. coli 25922, and more than 1000-fold higher than H2O2 for gram-positive organisms like S. aureus 29213.

Stabilized HOCl is reactive, and therefore is not persistent. To evaluate its potential toxicity, several well-established animal models were used. Stabilized HOCl was found to be nonirritating in (rabbit eye) and non-sensitizing in (guinea pig) animal models. No ocular irritation was observed following the instillation of a development formulation (0.013% HOCl) into the eyes of Dutch pigmented rabbits every 8 hours for 72 hours (data not shown). Stabilized HOCl at concentrations of 0.01%, 0.03%, and 0.10% w/v in a standard Buehler-design dermal sensitization study in guinea pigs showed no evidence of dermal reaction. Similarly, 28-day toxicity studies in full-thickness wounded rats and mini-pigs with daily application of stabilized HOCl at 0.01%, 0.03%, and 0.1% w/v together with a 24-hour occluded dressing showed no evidence of systemic toxicity. Microscopic examination of the wound area showed the expected signs of wounding and subsequent wound repair.

The germicidal properties of HOCl have been well reported. Hypochlorous acid is widely used as a disinfectant, for example, in sanitizing wash solutions and swimming pools. In these applications, the reactive chemical is formed in solution by the addition of chlorine to water. Similarly, HOCl is used to treat drinking water and is formed following addition of chlorine gas or NaOCl. Between pH levels of 3 and 6, the predominant species is HOCl. At higher pH, hypochlorite ion (OCl−) is formed, whereas at lower pH, the solution exists as a mixture of chlorine (Cl2) in solution, chlorine gas in the headspace, and HOCl. The control of this reaction has been utilized in industrial practices to optimize the availability of the active antimicrobial, HOCl.
Stabilized HOCl is a dilute solution of HOCl in 150 mM (0.9%) sodium chloride at an unbuffered pH of 3.5. The solution is stored in inert sealed containers designed for maximum product stability. In a pH range of 3 to 6 the predominant species is HOCl. At pH values greater than 5.5, hypochlorite ion (OCl−) is formed, and around pH 7.5 (the pKa of HOCl of the chlorine species in solution is at 50/50% mixture [HOCl/OCl−]). As the pH increases from 9.5, the concentration of OCl− in solution reaches its maximum level, becoming 100% hypochlorite (also referred to as bleach). However, on the acidic side at pH less than 4, the solution exists as a mixture of chlorine (Cl2) in aqueous phase, chlorine gas in the headspace, trichloride (Cl3−), and HOCl. At pH less than 3, an appreciable amount of Cl2 gas forms, which may cause the rapid loss of all active chlorine in an open container. To keep the solution stable and maintain its desired activity, the pH of the solution should remain between 5 and 5.5 and the solution should be stored in a tightly sealed container. For the first time, we have been able to determine these conditions to stabilize HOCl and to assess its biological properties as a pharmaceutical product.

The biological effect of HOCl on bacteria has been extensively studied. HOCl has broad-spectrum antimicrobial activity and is able to kill microorganisms very rapidly. Respiratory loss in bacterial cell membrane as a result of an irreversible reaction of HOCl with sulfur- and heme-containing membrane enzymes and structural proteins lead to cell death and non-viability.

Topical antiseptics with a long history of use, such as NaOCl (Dakins’ solution), hydrogen peroxide, acetic acid, and povidone-iodine remain in widespread use today. These antimicrobial agents used at typical concentrations are cytotoxic and impede wound healing, and so are now discouraged by some experts for use on chronic ulcers. Stabilized HOCL is a low-concentration, acidified, unbuffered solution of HOCl in saline. Under the conditions of the formulation, the active ingredient is primarily HOCl in equilibrium with a small amount of dissolved chlorine. The studies presented here have shown that stabilized HOCl exhibits rapid, concentration-dependent activity against a wide variety of gram-negative and gram-positive bacteria, yeast, and fungal pathogens, as long as the narrow effective pH range is maintained. In vivo, HOCl is produced intracellularly in abundance in response to phagocytosis of pathogens by neutrophils and plays an important role in the destruction of pathogens.

HOCl, the active ingredient has rapid and broad-spectrum antimicrobial activity against clinically relevant microorganisms in vitro and in vivo. Although vegetative bacteria are more susceptible to HOCL than endospore-forming bacteria and fungi, HOCL is fully capable of inactivating all groups of gram-negative and gram-positive bacteria, yeast, and fungi, including S. aureus, methicillin-resistant S. aureus, vancomycin-resistant E. faecium and Bacillis anthracis spores . HOCL has been shown to be nonirritating and non-sensitizing in animal models. There was no evidence of ocular irritation following a single instillation of NVC-101 in the eyes of New Zealand White rabbits at concentrations of 0.01%, 0.03%, and 0.1% w/v. No ocular irritation was observed following the instillation of a development formulation in the eyes of Dutch pigmented rabbits every 8 hours for 72 hours (data not shown). HOCL at concentrations of 0.01%, 0.03%, and 0.1% w/v in a standard Buehler-design dermal sensitization study in guinea pigs showed no evidence of dermal reaction. The active ingredient is reactive, and therefore is not persistent. Its persistence of antimicrobial properties has not yet been tested in the in vivo wound environment. Thus, absorption and systemic toxicity are expected to be insignificant. Therefore, in the 28-day wound toxicity studies in rats and mini-pigs with daily application of HOCL at 0.01%, 0.03%, and 0.1% w/v, with 24-hour occluded dressing, there was no evidence of systemic toxicity. Furthermore, microscopic examination of the wound area showed the expected signs of wounding and subsequent wound repair.

Heggers and colleagues have investigated the toxic effects of various concentrations of NaOCl (at pH 7.5, this was actually a 50:50 mixture of NaOCl and HOCl) in vitro and in vivo in the rat incision model. Concentrations used in previous studies were often quite high and although they had antimicrobial properties, they also exhibited some local toxicity that was not desirable. Heggers et al. conducted their experiments in the range of concentrations they expected to be active but not toxic to the cells or detrimental to wound healing. The concentrations evaluated were 0.25%, 0.025%, and 0.0125% w/v in the in vitro studies and 0.25% and 0.025% w/v in the in vivo studies. Ten clinical isolates were used in the in vitro studies (both gram-positive and gram-negative species). The bactericidal potential of the 3 concentrations was determined. All concentrations killed gram-positive bacteria within 30 minutes, but the lowest concentration did not kill gram-negative bacteria. Mouse fibroblasts were exposed to various concentrations of NaOCl for 10-, 20-, or 30-minute intervals. These cells remained viable except at the highest concentration, where cell death by 10 minutes was noted. In the incision rat model, 3 (2.5-cm) full-thickness wounds were created on each animal. The incisions were closed and the covered gauze was saturated every 4 hours with the NaOCl or saline. Subsets of animals were sacrificed on days 3, 7, and 14. Tissue sections were collected. Breaking strength was measured (force required to rupture the scar, in kilograms). The values for the breaking strength were higher as a function of duration, but the treated and control groups were not different. This study concluded that the concentration of 0.025% retains its bactericidal property without causing injury to the fibroblast cells.

Dakins’ solution (NaOCl) has been used as an antimicrobial for decades. A study to assess the bactericidal activity and toxicity of 0.5% and 0.1% NaOCl was undertaken. Only the toxicity portion of the study is discussed here. The insult to guinea pig skin was assessed following application of 0.5% solution of NaOCl buffered to a pH of 7.49 for up to 2 weeks (soaked gauze resoaked every 8 hours). The animals were sacrificed on day 1, 4, 7, or 14. The hair was removed from the skin before application but the skin was intact. The application of 0.5% solution resulted in basal cell toxicity (15% decrease in viability after 2 weeks of treatment), and so a lower concentration of 0.1% solution was evaluated (pH 7.4). This lower concentration did not result in toxicity to the basal cells. Control and treated skin sites were similar when the microscopic morphology was evaluated. Epidermal hyperplasia and an inflammatory influx were noted in the treated animals at 2 weeks. The authors concluded that the solutions were therapeutic candidates for thermal injury. It is important to note that at pH 7.4, these solutions will have approximately equimolar quantities of HOCl and NaOCl.

In the present comparative studies, we have demonstrated that H2O2 and hypochlorite (NaOCl) are effective against certain bacteria (more effective against gram negatives, but not gram positives). However, those effective antimicrobial concentration ranges begin to correlate with higher cytotoxicity on mammalian cells, as compared tostabilized HOCL. This antiseptic profile of NaOCl resemble some of the over-the-counter antiseptics: silver nitrates or silver ions, Betadine, and acetic acid (data not shown). Moreover, there are other antiseptics that are less toxic to mammalian cells but at the same time have lower antimicrobial activity (ethanol, hydrogen peroxide, and 5% mafenide acetate solution) as compared toHOCL. The Department of Health and Human Services discourages the use of commonly used antiseptic solutions to treat wound infection in general and chronic nonhealing wounds in particular because, for reasons mentioned above, their uses are contraindicated. Therefore data presented in this study should help in selecting safer antimicrobial agents for wound disinfection, irrigation, and dressing. This reevaluation of accumulated evidence is intended as a basis to help practitioners make informed decisions for choosing the appropriate topical antimicrobial for wound care management.

As the development of bacterial resistance to antibiotics continues and controversy regarding the use of topical antiseptics persists, the need for research and development of new classes of antimicrobial agents that are safe and broadly effective and have low toxicity and low propensity to induce antimicrobial resistance becomes inevitably critical. Currently, the use of broad-spectrum topical antibiotics to treat wounds that are failing to heal or those at risk for getting infected is not recommended by the Department of Health and Human Services. These recommendations are based on the following reasons: antibiotics may cause allergic reactions; especially when applied topically may have lower tissue distribution; greater effect on endogenous microflora (disturbance of the normal commensal microflora); induce resistance; and eventually have reduced therapeutic efficacy. By the same token, antiseptics are not encouraged because of their higher toxicity, potential development of resistance (like antibiotics), and, more important, direct impact on wound healing process.

Efficacy of topical antimicrobial agents in the management of serious infections, for example, biofilm- and catheter-related wounds, particularly when chronic and non-healing, is inconclusive. These observations vary greatly because of (a) inconsistent in vitro test specifications, (b) use of different animal species models, (c) use of different organisms for determining the efficacy outcome. Therefore, overall results make direct comparisons less than ideal. While in vitro testing is required to select potential agents for clinical trials, these models will never totally mimic in vivo conditions. Thus, in light of published results on HOCl and data obtained in the present investigations, there is enough compelling evidence to show that Stabilized HOCl, which resembles the HOCl molecule made by neutrophils during oxidative burst (a natural defense process against invading microorganisms), could lend itself for safe and effective treatment modalities of infection. Because of HOCL’s broad-spectrum, fast-acting antimicrobial activity, the chances of developing resistance would be minimal and based on its safety profile the potential for collateral damage to infected tissues is also very low. Therefore, in vivo experiments in a chronic infected granulating wound model are planned to determine HOCL’s ability to persist in a hostile environment where the pH range may not be ideal and where inflammation may produce exudates that limit its use as a wound care agent.

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Defining the ideal disinfectant against microorganisms and the development to create this superior environmental friendly disinfectant.

September 15, 2013


An ideal disinfectant should posses high bactericidal activity, long shelf life, be ready for use without any preliminary activation and utilized after use without negative effect to the environment. Long storing of stable chemicals is available, but their utilization requires equivalent activation by another agent or energy. Therefore, combination of stability and easy utilization is impossible.

In order to exclude the preliminary activation stage before usage of a liquid chemical germicide, it should be noted that all variety of biocidal agents are belong to few classes of chemicals well-known for tens of years.

Appearance of new class of chemicals, which will meet the requirement, is unlikely. Modern tendency in developing new disinfectants is in search for activation means of known disinfectants, and not the creation of new ones. Addition of activators using an extra physical influence, i.e. creating conditions converting active ingredients into metastable state at the moment of disinfection, is one of main directions for the disinfection efficacy improvement.

Interaction disinfectants and microorganisms’ cell membrane.

Due to complexity and multi-functionality of microorganisms’ membrane specific interaction between membrane’s biopolymers and abovementioned chemicals is hardly studied at all.

Cytoplasmic membrane is extremely vitally important structure of any cells including microbes. Organic compounds are part of it and have many reactive groups that cause a high sensitiveness of membrane to damaging factors of different nature. It is known that high concentration of membrane-attacking agents destroy biopolymers of membrane, resulting in damaging lysis of microbe’s cell. The same chemicals in small doses affect membrane functions – change osmotic pressure, permeability, transport processes of molecules and ions through membrane, inhibit metabolic processes, bio-oxidation and cell divisions.

Cationic surfactants (quarternary ammonium compounds) are concentrated at membrane and bind with phosphatidic groups of its lipids; anionic surfactants such as alkaline detergents, alkyl- and arylsulfones, iodophors react with membrane lipids. Phenols and alcohols dissolve lipid’s fragments of membrane.

After disinfection treatment is completed, the moist surfaces get dry, so organic compounds are concentrated in a volume of porous material and turn into superfine and invisible to the eye film. Then it evaporates by sublimation with less intensity than under evaporation during wet treatment. Formed aerosol frequently has no smell that creates illusions of its harmlessness. One should take into account that in accordance with known physical laws each liter of the air in the room contains about some milliards of molecules of matter vaporized with natural course or due to sublimation even if its concentration could be hardly measured and does not exceed hundreds or thousands parts from maximum permissible concentration (MPC). During breathing as well as through the skin and mucous membrane such molecules penetrate to human organism (patients or medical staff) and each one of it keeps realizing its main function – suppression of vital function of cells, but this time in a human body. Stability of liquid chemical germicides creates their accumulation in organism followed by migration through digestive cycle.

Colonies of microorganisms form resistance to dry inefficient disinfectant and start using it as a nutrient medium. Processes as described above have recently become an object of attention; so it is in a stage of study now.

It is quite evident that the development of new liquid chemical germicides which allow bacteria to develop resistance in a short period of time, creates conditions for improvement of mutability mechanism of pathogens and initiates appearance of new isolates of microorganisms.

Today by efficacy of disinfectants is implied its spectrum of biocidal activity. Efficacy also relates to exposure time required for disinfection. However, taking a broad view on the subject we should say that disinfectant is effective only in the case it has a broad spectrum of biocidal activity and does not stimulate microorganism’s adaptation during a long-term use. In other words, effective disinfectant must be used for years with certainty that microorganisms could not form adaptation to it for principal reasons.

Mechanism of Antibacterial defence

Let us consider a mechanism of antibacterial defense created by nature and functions in internal environment of life organisms – from unicellular organisms up to human – over million of years and without any fail.

It is proved that leading role in bactericidal effect of neutrophils belongs to hypochlorous acid (HOCL) made by phagocytes. Under respiratory burst about 28% of oxygen used by neutrophils is spent for formation of HOCL. HOCL is generated from hydrogen peroxide and chloride-ions in neutrophils. Catalyst of this reaction is myeloperoxidase (MPO):

H2O2 + Cl [Cat (МPО)] HOCL + OH [9, 10].

Hypochlorous acid dissociates in aqueous media with formation of hypochlorite-anion and hydrogen-ion:

HOCL ClO + Н+.

Concentrations of HOCL and hypochlorite-anions ClO are almost equal at neutral pH. A decrease in pH shifts reaction balance towards to HOCL, and an increase of pH raises concentration of hypochlorite-anions.

A formation of H2O2 and HOCL in a short time (fractions of a second) in a little volume of aqueous media (parts of microliter, in a volume of active zone of phagocytosis) – inevitably must be followed by reactions of spontaneous decomposition and interaction of reaction products with formation of active particles similar to once formed by radiolysis or electrolysis of water.

Spontaneous decomposition of hydrogen peroxide in aqueous media is followed by formation of highly active biocides (in parenthesis appropriate reactions are presented):

HO2 – hydroperoxide-anion (H2O2 + OH HO2 + H2O);

О22 – peroxide-anion (OH + HO2 O22 + H2O);

О2 – superoxide-anion (O22 + H2O2 O2 + OH + OH );

НО2 – hydrogen peroxide radical (НO + H2O2 H2O + HO2);

HO2 – hydrogen super-oxide (O2 + H2O HO2 + OH).

At the same time it is possible the formation of extremely reactive singlet oxygen 1О2 : (ClO + H2O2 1О2 + H2O + Cl ). Participation of molecular oxygen ion-radical О2 in reactions of phagocytosis is determined experimentally. One of the described above could be the way of its formation.

Formation of free radicals СlO, Сl, НО is possible in aqueous media in presence of НСlО and СlO

HOCL + ClO ClO + Cl + НO.

By modern theory of catalytic processes, a formation of interim activated complex with myeloperoxidase as a catalyst seems also to be most possivle. A dissociation of this complex is followed by formation of О , and medium acidification:

HOCL + ClO [HOCL Cat (МПО) ClO ] 2Сl + 2O + Н+

Active hypochlorite radical СlO can participate in reactions of atomic oxygen (O ) and hydroxyl radical (НO ) formation:

СlO + СlO + ОН Сl + 2O + ОН.

Followed by formation of chlorine radicals:

OH + Cl Cl + OH.

Formed radicals and atomic oxygen take part in microbe’s destruction, oxidizing biopolymers, for example, by the following:

RH2 + OH RH + H2O;

RH2 + Cl RH + HCl;

RH2 + O RH + OH .

A metastable mixture of compounds formed during phagocytosis is a very effective mean for microbe’s destruction due to many spontaneous realized possibilities of changing (irreversible damage) of essential functions of microorganism’s biopolymers at a level of electron transmission. Metastable particles with different values of electrochemical potential possess universal spectrum of action, i.e. they are able to damage all large systematic groups of microorganisms (bacteria, mycobacteria, viruses, funguses, spores) and without damaging of human tissues and other multicellular system organisms.

That can be explained by texture and living activities of cells of that living organisms. Cells of multicellular organisms during their life process, for example, in oxygenase’s reactions of cytochrome P-450, during phagocytosis under microbe’s adhesion and cidal action produce a range of highly efficient oxidants. These cells have a strong chemical system of antioxidant protection with preventing a toxic effect of such compounds on vitally important cellular structures. Antioxidant properties of somatic cells are related to a presence of a strong three-layered lipoprotein’s shell that contains diene conjugates (–С=С–) possessing electron-donor properties and sulfhydric groups (SH). Microorganisms do not have strong mechanisms of antioxidant protection due to absence of mentioned chemical groups.

All somatic cells of living organisms are heterotrophs: their trophism depends on availability of nutritive materials in extracellular medium – glucose, amino acids, fatty acids. Though biological well-being of any somatic cell is up to place it keeps in a process of dispensing of trophic functions of all elements of multicellular system (cell is supported by cell).

Trophic functions of multicellular organisms cells are obeyed to interchangeability law. If a trophism of single cell is disturbed, then this disturbance can be corrected by neurotrophic regulation, functions of adjoining cells, reparative processes, nutritive function of blood and so on.

All microbe’s cells are autotrophs, so their nutrition depends on their own activity, in other words if enzymatic processes in microbe’s cell are depressed, it dies since there is no compensatory mechanism. Microbial cell gets all its trophic functions by enzymatic reactions only. An interaction between microbial cells in their habitat is not a compensatory one, that is to say susceptibility of microbe is in its autonomy.

Natural production of HOCL

Investigations carried out in recent decades indicate that all higher multi-cellular organisms including humans synthesize hypochlorous acid and highly-active meta-stable chlorine-oxygen and hydroperoxide compounds (a meta-stable oxidants’ mixture) in special cellular structures to combat microorganisms and foreign substances. Hypochlorous acid dissociates in aqueous medium forming hypochlorite-anion and hydrogen ion: НOСl OCl + Н+. When рН values are close to neutral, concentrations of НOСl and hypochlorite-anions OCl are approximately equal. Lower рН leads to shift of this reaction equilibrium towards higher concentration of НOСl; higher — towards higher concentration of hypochlorite-anions. Sodium hypochlorite demonstrates a considerably lower bactericidal ability than hypochlorous acid.

The highest bactericidal effect of oxygen chlorine compounds is observed with рН varying from 7.0 to 7.6, where concentrations of hypochlorite-ions and hypochlorous acid are comparable. This is due to the fact that the above compounds being conjugated acid and base (НOCl + Н2О + Н3О+ + OCl; OCl + Н2О + НOСl + ОН) form in the given range a meta-stable system capable of generating a number of compounds and particles possessing a much higher antimicrobial ability than hypochlorous acid: 1O2 — singlet molecular oxygen; СlO — hypochlorite-radical; Сl• — chlorine-radical (atomic chlorine); О — atomic oxygen; ОН — hydroxyl radical. Catalysts of reactions with chlorine-oxygen compounds are Н+ and ОНions present in water also in approximately equal quantity at рН value close to neutral one.

Chemical production of HOCL

A unique ability of hypochlorous acid to form meta-stable, universal in its scope of antimicrobial action oxidant mixture is widely employed in many disinfectant agents based on cyanuric acid salts (Aquatabs, Deochlor, Chlorsept, Presept, Javelion, Chlor-Clean, Sanival and others) making it possible to decrease active chlorine content in disinfectant working solutions at least 10-fold as compared to sodium hypochlorite solutions, antimicrobial activity of the former being higher. Let us take the mechanism of action of Johnson & Johnson’s Presept tablets as an example. The active ingredient is hypochlorous acid formed in the process of sodium dichloroisocyanurate interaction with water at a рН value of 6.2, maintained by adipic acid contained in the tablets.

However, the use of mentioned disinfectant agents based on cyanuric acid salts is unsafe for human and other warm-blooded organisms since it contain a chlorine organic compound, in particular, sodium dichloroisocyanurate, which, unlike inorganic chlorine-oxygen compounds, does not disappear leaving no traces during desiccation, but accumulates in the environment and human body.

The most efficient antimicrobial agents among all generally known liquid sterilizing and disinfectant means, which demonstrate very low toxicity or no toxicity at all for warm-blooded animals, are electrochemically activated solutions, in particular Neutral electrolyzed Water.

Electrochemical activated HOCL

Maximum use of fundamental difference between living organisms of micro- and macro-biological life is an ideological basis of electrochemical activated biocidal liquids.

As physicochemical process electrochemical activation is an electrophysical and electrochemical influence on water that contains ions and molecules of dissolved substances in it. It takes place under conditions of minimal heat release in the area of dimensional charge at the electrode surface (anode or cathode) of electrochemical system at non-equilibrium charge transfer through the interface “electrode – electrolyte” by electrons.

As a result of electrochemical activation water converts into a metastable (activated) condition showing increased reactivity in different physical-chemical processes during some tens hours. Electrochemical activation allows directly change a composition of dissolved gases, acid-base and redox characteristics of water within the bigger scale then under the equivalent chemical regulation. Chemical reagents (oxidants or reducing agents) in metastable condition can be generated from water and dissolved substances. It is used in processes of water purification and disinfection as well as for water or diluted electrolyte solution transformation into ecologically friendly biocidal (disinfecting/sterilizing solution), cleaning, extractive and other functionally useful liquids.

An Electrolytic Flow Cell is used for electrochemical transformation of water and dissolved substances. A distinctive feature of a Flow Cell is in combination of properties of ideal displacement reactor and ideal mixing reactor in one element as well as high technical and economic characteristics at processing of fresh water and low-mineralized liquids.

Very seldom electrochemically activated solutions (Electrolyzed Water or Super-Oxidized Water) is identified with hypochlorous acid. This is due to inadequate awareness and natural tendency to simplify comprehension by classifying electrochemically activated solutions to well-known hypochlorite ones on the basis of their formal resemblance.

Neutral Electrolyzed Water, unlike 0.5-5.0% hypochlorite solutions possessing only disinfectant ability, is a sterilizing solution at oxidant concentration 0.005 to 0.05%.(5-500ppm)

Benefits of Electrochemical activated HOCL

Active ingredients of Electrochemical activated HOCL (Neutral Electrolyzed Water) are chlorine-oxygen compounds НOСl (hypochlorous acid) and OCl (hypochlorite-ion).

The combination of active these active chlorine-oxygen substances avoid that microorganisms adapt or become resistant to Neutral Electrolyzed Water, while low total concentration of chlorine-oxygen compounds guarantee absolute safety for man and the environment in the process of its long-term application.

In other words, a mixture of metastable chlorine-oxygen compounds eliminates microbes’ ability for adaptation to bactericidal effect of Neutral Electrolyzed Water. Thus, only a small concentration of chlorine-oxygen compounds guarantees for absolute safety for man and environment under long-term use of Electrolyzed Water.

Neutral Electrolyzed Water is considered non-toxic due to low content of active substances HOCL and OCL, therefore there is no need to remove it from treated surfaces after treatment.

Total content of active chlorine-oxygen compounds in Neutral Electrolyzed Water oxidant content varies from 50 to 500ppm which is many times lower than in most working solutions of disinfectants routinely used today. Neutral Electrolyzed Water causes no coagulation of protein protecting microorganisms and thanks to its loose structure easily penetrates into micro-channels of living and nonliving matter.

Environmentally friendly electrochemically-activated Neutral Electrolyzed Water has “life time” that is necessary for procedure of disinfection. After its use it spontaneously degrades without formation of toxic xenobiotics and does not require any neutralization before discharging to sewerage.

A chemical potential of molecules and ions in Neutral Electrolyzed Water is much higher than in hypochlorite solutions. A low mineralization of Neutral Electrolyzed Water and its hydration ability helps penetration through cell membrane, creates conditions for intensive osmotic and electro-osmotic oxidant’s transfer into intracellular media. The osmotic transfer of oxidants through shells and membranes of microbe’s cells is more intensive than through membranes of somatic cells due to inherent difference in osmotic gradient of these types of cells. Electrically charged cluster structures formed by dissolved gas molecules in water and electron-active components of medium promote high-speed electro-osmotic carry of oxidants into bacterial cell, because this clusters produce strong local electric fields with high heterogeneity in zones of contact with biopolymers.

Neutral Electrolyzed Water kills microorganisms of bacterial, viral and fungous etiology (Staphylococcus aureus, Pseudumonas aeruginosa, Escherichia coli, hepatitis B virus, poliomyelitis virus, HIV, adenovirus, pathogens of tuberculosis, salmonellosis, dermatomycosis and others). By its efficacy Electrolyzed Water greatly exceeds chloramines, sodium hypochlorite and overwhelming majority of other disinfectants and sterilizing agents.

A sum of active chlorine-oxygen compounds in Neutral Electrolyzed Water (total oxidant content) is within 50 to 500 mg/l, that is many times less than in most solutions of currently used disinfectants. Neutral Electrolyzed Water does not cause coagulation of protein that protects microorganisms and, due to its loosened structure, easily penetrate into pinholes of living and lifeless matter.

Neutral Electrolyzed Water is produced from dilute solution of sodium chloride in drinking water. Total mineralization of initial solution for Neutral Electrolyzed Water is within 0,5 to 5,0 g/l.


To sum up, it can be concluded that the most effective disinfecting liquid in terms of their functional properties and simultaneously very low-toxicity is Neutral Electrolyzed Water (meta-stable low-mineralized chlorine-oxygen antimicrobial solutions), which have no alternative as long as life on Earth is represented by various forms of protein bodies existing in electrolyte of aqueous solutions of mainly sodium and chlorine ions.


September 8, 2013


There is an epidemic of healthcare acquired infections within hospitals, out-patient surgical centers, nursing homes and medical clinics. The number of hospital acquired infections alone is staggering. About 1 in every 15 patients get an infection while hospitalized and up to 98,000 Americans die from these infections each year. That makes infections the most common complication in hospital care and one of the nation’s top 10 causes of death. In California, an estimated 200,000 patients develop hospital infections each year, resulting in 12,000 deaths.

The problem is much larger than official statisitcs because the numbers fail to account for millions of patients treated in outpatient surgery centers, community clinics, nursing homes and other care facilities.

About six and one half percent of patients admitted to US hospitals—nearly 5,500 daily, or two million annually—get sick from a hospital-acquired infection. This adds 19 days of hospitalization and $43,000 in costs totaling more than $45 billion a year to U.S. medical bills

Under the new Affordable Healthcare Law consumers will be able to learn hospital infection rates. Hospital Infection rate information will be posted on a Department Health and Human Services website called Hospital Compare. This new reporting requirements applies to hospitals that participate in Medicare and Medicaid programs which are virtually every hospital in the country. Beginning in October 2012, Medicare payments to hospitals will be tied to how well they protect patients from these infections. Hospitals with infection rates exceeding national averages will lose 1 percent of their Medicare funding, starting in 2015. This is a huge dollar amount considering the federal government spent $563 billion last year on 49 million recipients and Medicare spending is expected to grow to $970 billion by 2021.

II        MARKET

Hospital Cleaning is the removal of all dust, oil, and organic materials such as blood, secretions, excretions and microorganisms. Cleaning reduces or eliminates the populations of potential pathogenic organisms. It is accomplished with water, detergents and mechanical action. Hospital Disinfection is the inactivation of disease producing organisms. Disinfection does not destroy high levels of bacterial spores. Disinfectants are used on inanimate objects. Disinfection usually involves chemicals, heat or ultraviolet light. Levels of chemical disinfection vary with the type of product used.

 There are three types of cleaning and disinfection markets within hospitals and healthcare facilities. These are critical, semi-critical and non-critical.

A.        Critical Applications

Medical devices and items that represent a high risk for infection if they are contaminated with any microorganism. Objects that enter sterile tissue or the vascular system must be sterile because any microbial contamination could transmit disease. Critical cleaning and disinfection includes surgical instruments, cardiac and urinary catheters, implants, and ultrasound probes used in sterile body cavities. These items are to be sterilized with steam if possible. Heat-sensitive objects can be treated with EtO, hydrogen peroxide gas plasma; or if other methods are unsuitable, by liquid chemical sterilants.

B.         Semi-Critical Application

Devices to include vaginal-rectal ultrasound probes, endoscopes, laryngoscope blades, cystoscopes, esophageal manometry probes, anorectal manometry catheters, respiratory/anesthesia equipment, all GI scopes, transesophageal echocardiogram probes and rhinoscopes. Medical devices and equipment that contact mucous membranes or non-intact skin minimally require high-level disinfection.

C.        Non-Critical Applications

Devices are those that come in contact with intact skin but not mucous membranes.  Intact skin acts as an effective barrier to most microorganisms; therefore, the sterility of items coming in contact with intact skin is “not critical.”  Non-critical items are divided into non-critical patient care items and non-critical environmental surfaces.  Non-critical patient-care items are bedpans, blood pressure cuffs, crutches and computers.


 A segment within the non-critical environmental surfaces market is Terminal Room Cleaning. Terminal Room Cleaning means a thorough cleaning of a patient room after being discharged. The concept is to eliminate the residual bacteria left in a “contaminated room” whether it is a hospital room, OR room, ER room, nursing home room or any room in which another patient can potentially come into contact. The potential market of Terminal Room Cleaning is huge. For example, there are 35,000,000 patient “discharges” per year in more than 7000 hospitals and 15,000 outpatient surgery centers.

Transmission of many healthcare acquired infections are related to contamination of patient surfaces, in-room equipment, high touch surfaces with patient rooms.

Patients shed microorganisms into their environment by coughing, sneezing or having diarrhea. Bacteria and viruses can survive for weeks or months on dry surfaces in a patient environment. When another patient, doctor, nurse or visitor, touches that surface the microorganisms are transmitted throughout the hospital. The following are example of “at-risk” patient environments.

  1. Acute Care, the patient environment is the area inside the curtain, including all items and equipment used in his/her care, as well as the bathroom that the patient uses.
  2. Intensive Care Units (ICUs), the patient environment is the room or bed space and items and equipment inside the room or bed space.
  3. Nursery/Neonatal setting, the patient environment is the bassinet and equipment outside the bassinet that is used for the infant.
  4. Ambulatory Care, the patient environment is the immediate vicinity of the examination or treatment table or chair and waiting areas.
  5. Long-term care, the resident environment includes their individual environment (e.g., bed space, bathroom) and personal mobility devices (e.g., wheelchair, walker).

Terminal Room cleaning is performed by the Environmental Services Staff. The cleaning includes emptying trash and removing any loose items, changing bed linen, wiping the mattress with a disinfectant, washing walls with detergent, cleaning bathroom sink and toilet with a disinfectant, wiping all bed rails, tables, light switches, door handles, telephone, call buttons, privacy curtain and other “high touch” items with a disinfectant then mop the floor with a detergent cleaner and disinfectant. Once the Environmental Staff completes the terminal room cleaning, the Environmental Service Supervisor inspects the room. The Supervisor will look for any visible dirt, blood, secretions, etc. They will also use a bio-luminescence meter to measure bacterial contamination. If the Environmental Service Supervisor rejects a room, the entire room is re-clean and disinfected.


In the US the average time from patient discharge to another patient occupying the same room is 27 minutes. The work required (as noted above) by the Environmental Service Staff to terminally clean the discharged patient room in the 27-minute timeframe is almost impossible. This creates extreme pressure and stress on the Environmental Service Staff resulting in poor cleaning and very high job turnover. Other factors contributing to poor cleaning and high turnover is the use of toxic and corrosive detergents and disinfectants. To improve cleaning performance, stronger and more toxic chemicals are required. However, these chemicals slow down cleaning time. The Staff must be more careful in handling these chemicals, adding a rinse step and allow time for the room to dry and “air out”.

Using stronger and more toxic cleaning and disinfecting chemicals does not always provide the level of disinfecting required by hospital guidelines. The over prescribed use of antibiotics have created “super-bugs”. These “super-bugs” can develop a resistance to disinfectants. There are sixteen hospital identified “super-bugs”. A few of these are MRSA (methicillan resistant staphylococcus aureaus), C. diff (clostridium difficle), VRE (vancomycin resistant enterococci) and acinetobactor baumannii.

To reduce the human factor in terminal room cleaning and eliminate the chemical resistance of “super-bugs” new technologies have been developed and are currently marketed. One new technology is called VHP (vaporize hydrogen peroxide). VHP meets and exceeds hospital guidelines for environmental surface disinfection.  The guideline for hospital cleaning was developed by HICPAC (Hospital Infection Control Procedures Advisory Committee). This committee is Infection Control doctors and researchers within the medical community specializing in Non-Critical Environmental surface disinfection. The level of surface disinfection for terminal room cleaning is called 6-log reduction. 6-log cleanliness is basically a sterile surface. VHP provides 6-log surface cleanliness but requires 4 hours to clean, disinfect and “air out” the room. In addition the Vaporized Hydrogen Peroxide equipment cost more than $200,000 and requires a company representative located full-time at the hospital to operate the equipment.

Another new technology for terminal room cleaning is UV-C light. UV light has been used for surface disinfection for many years. Used properly UV-C can provide a 6-log level of disinfection. However, UV-C is difficult to use because the light must be directed at an exact angle to the surface, the light requires a long contact time and the light must be checked regularly to insure the proper wavelength. A properly cleaned and disinfected room using UV-C equipment takes more than 90 minutes.

These technologies and others meet the HICPAC cleaning guidelines for terminal room cleaning but they do not come close to the time requirements for most hospitals. Electrolyzed Water is the only new technology that can provide 6-log disinfection within the 27-minute time requirement. In addition electrolyzed water is non-toxic, requires no chemical storage, mixing, dries faster and does not require Staff to wear protective clothing. Electrolyzed water can eliminate the pressure and stress of the Environmental Service Staff reducing turnover. It has no odor or chemical residue that can cause patient sensitivities.

After years of working in hospitals with Environmental Service and Infection Control Professionals, the most important cleaning solution proved to be electrolyzed alkaline water. Alkaline water’s cleaning performance is due to its alkalinity and very negative ORP (oxidation-reduction potential). The more negative the solution the greater cleaning power and faster drying properties. Electrolyzed Alkaline Water’s negative ORP has a very short shelf life. It is usually less than 1 hour in an open container exposed to air.  The key for electrolyzed water technology’s acceptance in hospitals is making the Environmental Service Staff job easier, safer and less pressure. As a result, electrolyzed water must be a direct replacement to detergents and work in their cleaning process. For example, the Environmental Service Staff at the start of their shift fill an open container with a detergent solution and add 8 to 10 micro-fiber mop heads. One mop head per room is used to mop walls and floors. As the staff changes mop heads and agitates the solution, the ORP of ordinary electrolyzed alkaline water is quickly lost. However, a patent-pending product enhancement (enhanced alkaline water) preserves the negative ORP and actually continues the electrolysis process maintaining the alkaline water above pH11. The product will keep the alkaline water’s pH and ORP for at least 1 day. Enhanced alkaline water can be used into the mop head containers, spray bottles or other applicators. The product will maintain negative ORP with the addition of dyes, surfactants or other cleaning aids.

Once surfaces are cleaned with alkaline water, the surface has a negative charge. At this point, the electrolyzed acidic water disinfectant can be applied with an electrostatic spray device. This device will put a 5 to 10 mil coating on every surface within the room. Electrostatic sprayers can reach every side of a surface even if the sprayer is not pointed directly at the surface. Electrostatic spraying of a patient room takes less than 3 minutes. This technique enables the Environmental Surface Staff to take more time cleaning with the alkaline water and still finish under the 27-minute time requirement.

Electrolyzed Water technology and application equipment can reduce a hospital’s overall chemical costs, cut Environmental Service labor requirements and reduce the hospital liability insurance premiums. This is proven technology that has been used in Japanese hospitals for more than 20 years. In Japan electrolyzed water technology has reduce healthcare acquired infections to less than 2%.

For more information, please contact

How bleach kills germs

August 27, 2013

Bleach has been killing germs for more than 200 years but it was only since 2008 that U.S. scientists figured out how the cleaner does its dirty work.

It seems that hypochlorous acid, the active ingredient in bleach, attacks proteins in bacteria, causing them to clump up much like an egg that has been boiled, a team at the University of Michigan reported in the journal Cell on Thursday.

The discovery, which may better explain how humans fight off infections, came quite by accident.

“As so often happens in science, we did not set out to address this question,” Ursula Jakob, who led the team, said in a statement.

The researchers had been studying a bacterial protein called heat shock protein 33, which is a kind of molecular chaperon that becomes active when cells are in distress, for example from the high temperature of a fever.

In this case, the source of the distress was hypochlorous acid or hypochlorite.

Jakob’s team figured out that bleach and high temperatures have very similar effects on proteins.

When they exposed the bacteria to bleach, the heat shock protein became active in an attempt to protect other proteins in the bacteria from losing their chemical structure, forming clumps that would eventually die off.

“Many of the proteins that hypochlorite attacks are essential for bacterial growth, so inactivating those proteins likely kills the bacteria,” Marianne Ilbert, a postdoctoral fellow in Jakob’s lab, said in a statement.

The researchers said the human immune system produces hypochlorous acid in response to infection but the substance does not kill only the bacterial invaders. It kills human cells too, which may explain how tissue is destroyed in chronic inflammation.

“Hypochlorous acid is an important part of host defense,” Jakob said. “It’s not just something we use on our countertops.”

This post has been posted in 2008. Mentioned Journal article available upon request.


June 22, 2011

JUNE 8, 2011, 2:06 P.M. ET
Associated Press

NEW YORK — It sounds like a late-night infomercial: Kill germs and clean surfaces with nothing more than water and a few volts of electricity! Pay pennies a gallon! Strong enough to kill germs but gentle on your skin! The use of electricity and water to clean and disinfect has been embraced by some food and hospitality businesses looking to save money and go green by swapping out conventional products.
At busy Whole Foods on Manhattan’s Union Square, workers keep battery-operated spray bottles designed to keep surfaces clean with water packing an electrical charge. Also available are electrolyzed oxidizing water products, or EO water, which are cleaning systems that use salt and electricity to create solutions for cleaning kitchens, prison floors and hotel rooms.  No, these are not miracle elixirs.

While users of the two different types of systems say they save money, start-up costs are far higher than simply buying a bottle of bleach. They’re not suitable for every cleaning job, and different zapped water treatments can lose potency over time. Critics say some of the claims for electrolyzed water in particular — it’s touted as everything from a health drink to a skin treatment — are overblown. Still, studies have shown water exposed to a charge works as a cleaner.
“We use it everywhere,” said Mary Ann Flynn, appearance manager for the Culinary Institute of America in Hyde Park, N.Y. The school uses EO water. “They fill mop buckets with it. They fill bottles so that the students and the chefs use it in the kitchen.”
The electrolyzed water systems vary, but a common type creates separate streams of disinfectant and cleaner by
running a charge through water exposed to salt. The disinfectant stream mainly contains hypochlorous acid, a form of chlorine. Viking Pure, one of several makers active in the United States, claims its sanitizing solution is effective against a long list of pathogens ranging from listeria to swine flu virus. A big selling point of the machines it sells is that users make the cleaner on the spot so they don’t have to transport chemicals. Viking Pure’s president, Walter Warning, said the “acid water” is so gentle you can spray it on your skin. The same salt-and-electricity process also creates a separate stream of sodium hydroxide, a common ingredient in cleaners. This “alkaline” stream can be used as a general purpose cleaner and degreaser.

Deborah Stone, housekeeping manager for Carolina Designs rental agency at North Carolina’s Outer Banks, swears by it and said some of the biggest problems are convincing workers they can clean without suds. “It’s very difficult for the cleaners to comprehend that because there is no smell and because there are no bubbles, they don’t get the sense that they’re actually cleaning,” Stone said. “You still have those die-hard people that want the suds and the pretty smell.”
Academic researchers have found that electrolyzed systems can be effective cleaners and disinfectants when the process is done correctly.

Professor Ali Demirci of the Department of Agricultural and Biological Engineering at Pennsylvania State University has researched the use of EO water to decontaminate various food products and clean dairy equipment. He has found it works well for both cleaning equipment surfaces and killing bacteria. Professor Yen-Con Hung of the Department of Food Science & Technology at the University of Georgia has studied electrolyzed water for years and said it can be more effective than bleach in many cases. Researchers note that EO water performs best on smooth surfaces. Bassam Annous, a research microbiologist for the federal Agricultural Research Service, has found it does not work well ridding lettuce and apples of E. coli because the water-based solution cannot penetrate the minute crevices where the bacteria can lurk. “This is not a silver bullet,” Hung said. “EO water is not perfect.”

Bob Brown, who is in charge of food safety support for Whole Foods, said that a number of stores in the
mid-Atlantic and Midwest are starting to use the sprayers for cleaning glass and other surfaces, like conveyor
belts. “It’s better for the environment if you’re not using chemicals,” Brown said. “So it’s a green technology that’s
available.” How green? That’s hard to quantify precisely.

In the case of the electric spray bottles, there are no chemicals. Both the spray bottles and the EO water require
electricity, though not much. Activeion’s spray bottle runs on a rechargeable 12-volt battery. Bob Schildgen, aka Mr. Green, the environmental advice columnist at Sierra Club, said comparing a chemical-based cleaner to an electricity-based one is apples to oranges.  “It’s extraordinarily difficult to compare such different processes and come to a firm conclusion on it,” he said.

Copyright 2011 Associated Press

Chlorine – A Great Disinfectant!

December 16, 2010

There are distinct differences between a Sodium Hypochlorite solution, a Calcium Hypochlorite solution and an onsite generated Hypochlorous Acid solution.

Sodium Hypochlorite Solution (NAOCL)

Sodium Hypochlorite solution often called bleach usually containing LYE is manufactured at a factory, stored, shipped to distribution centers, stored again and then sold.

Calcium Hypochlorite Solution (CAOCL)

Dry Calcium Hypochlorite tablets produce a “FRESH” Hypochlorite solution when mixed with water. In tests done, a solution produced with the proper Calcium Hypochlorite tablet, can maintain “Free Available Chlorine” or Hypochlorous Acid the
active disinfectant in this Calcium Hypochlorite solution, for ONLY about 4 hrs, then it starts rapidly degrading.

Hypochlorous Acid Solution (HOCL)

Until now, HOCl has simply been thought of as a transient byproduct in the ubiquitous chlorine chemical family. However, HOCl generated by ECA technology carries with it fewer negative hydroxides than the previous HOCl formed via disassociation from sodium hypochlorite. Because of this, ECA-generated HOCl behaves uniquely and must be considered separately from chlorine. HOCl as a stand-alone chemical, separate from chlorine, has not been available in the market until now. This breakthrough results in a need for a paradigm shift in biocidal approaches. HOCl is an “old”, well appreciated chemical but is now “new” availabie as onsite generated solution.

1. Free available Chlorine content

For a chlorine solution to be a good disinfectant it must meet the Chlorine Demand. The chlorine demand is the amount of Free Available Chlorine (FAC) often called Hypochlorous Acid (HOCl), needed to disinfect or oxidize organic matter before a FAC residual is reached. If the chlorine demand is not met then complete disinfection has not been obtained. One of the best signs that the Chlorine Demand has not been met is the strong chlorine odor.

If a chlorine solution is does not contain enough HOCl to satisfy the chlorine demand of the surface or product to be disinfected, chloramines will form as chlorine and nitrogen-based materials combine. Examples of nitrogen-based materials are proteins and blood. Chloramines are responsible for the obnoxious odor sometimes associated with chlorine disinfection. The obnoxious, pungent, eye-stinging smell of chloramines, mistakenly identified as free chlorine, indicates that the chlorine/water mix is not effective. There is not enough HOCl to satisfy the chlorine demand

2. Chlorine Efficacy determined by pH

Chlorine in water splits into two forms, Hypochlorous Acid (HOCl) and Hypochlorite Ion (OCl-). At the high pH the chlorine provided by bleach contains a maxiimum of Hypochlorite Ion. The chlorine produced by onsite Electrolyses in an Aquaox System contains a maximum concentration of Hypochlorous Acid (HOCl).

How much of each is present in a chlorine solution is totally dependent upon the pH of the solution. As pH rises, less Hypochlorous Acid and more Hypochlorite Ion is in the solution. As the pH rises, less germ killing power is available. According to a University of Illinois study, HOCl is 120 times more effective as a sanitizer than the -OCl ion. The ideal pH of a disinfecting chlorine solution is a pH of 6-7.

Most FRESH Calcium Hypochlorite solutions have a pH of between 7 and 8.  ALL (fresh or old) Sodium Hypochlorite solutions, (“bleach”) have a pH of 10.25+ producing NO HOCl at all! These solutions produce only the OCl- ion, a very poor disinfectant which is from 80 to 120 times less effective as a disinfectant than HOCl, providing that there is any chlorine left in the stock solution.

3. Contact time

The amount of time that chlorine is present during treatment is called the contact time. Contact times are calculated to determine the amount of time that a disinfectant must be present in the system to achieve a specific kill of microorganisms, for a given disinfectant concentration. A long contact time  means that disinfection alone will not be sufficient treatment and additional methods will be necessary to eliminate the microorganisms.  The contact time is directly related to the chemicals’ efficiency of eliminating bacteria and viruses from the water. HOCl requires by far the shortest contact time to achieve a 99% kill of E. coli (Reynolds, 1996).

4. Shelf-life and added lye

Finally, just as champagne or carbonated water “go flat” on sitting as the bubbly carbon dioxide gas escapes into the air, chlorine escapes from a Hypochlorite solution thus weakening its germ killing value. In order to slow this escape, bleach manufacturers add Sodium Hydroxide (lye) to their product causing the pH to rise dramatically. Lye burns animal and plant tissues; it saponifies (converts) fats in poultry and meat products. Hypochlorous Acid dispensed from Aquaox Systems contains NO LYE!

According to all the technical literature, depending on storage conditions; ALL Hypochlorite solutions will lose half of their potency in less than thirty days. Light, temperature and age are the biggest factors.  The biggest misconception is that liquid household bleach (Sodium Hypochlorite) does not loose potency until you make a Sodium Hypochlorite solution; “liquid household bleach” is already a Sodium Hypochlorite solution, that starts degrading soon after manufacture, so a “bleach” bottle bought at a retail store or chemical supply house is, NOT a FRESH Hypochlorite solution. It is a Hypochlorite solution with an unknown chlorine content, so when we make a solution all we are doing is diluting an already weak Hypochlorite solution even more. All literature recommends that if you are using “chlorine bleach”, daily tests should be conducted by a laboratory to assure its potency.

Why Use onsite produced Hypochlorous Acid solutions instead of Calcium or Sodium Hypochlorite solutions?

1. Onsite electrolyses of a brine solution in Aquaox Systems produce a maximum of Hypochlorous Acid whereas pH can be accurately set and controlled anywhere between 3-7.

2. At an pH of ~5 the Hypochlorous Acid solution consist almost solely of Free Available Chlorine and maximum disinfection is achieved.

3. Hypochlorous Acid requires the shortest contact time to eradicate a microorganism.

4. As Hypochlorous Acid is produced onsite, there is no need of mixing and dilution of Hypochlorite solutions with unknown chlorine content. Shelf life is no issue, as Hypochlorous Acid solutions are produced on demand. Therefore no addition of Lye is required, as shelf life became more or less irrelevant.


George Clifford White, Handbook of Chlorination and Alternative Disinfectants. Third Edition, Van Nostrand Reinhold, New York, 1999.

George R. Dychdala. Chlorine and Chlorine Compounds. In: Block SS, ed. Disinfection, Sterilization, and Preservation, 5th ed. Philadelphia Lippincott Williams & Wilkins, 2001.


January 27, 2010


Chlorine is one of the most commonly used disinfectants for water disinfection. Chlorine can be applied for the deactivation of most microorganisms and it is relatively cheap. Chlorine is commercially available as gaseous Chlorine (CL2) and as Sodium Hypochlorite liquid or powder (NaOCL).

Both gaseous Chlorine (CL2) and Sodium Hypochlorite (NaOCL) have very limited disinfecting properties. It is the formation of chlorine by-products such as Hypochlorous Acid (HOCL), Hypochlorite Ion (OCL-), Hydrochloric Acid (HCL) and Oxygen (O) that inhibit disinfecting properties.

Gaseous Chlorine

Gaseous Chlorine (CL2) is commercially available and mostly used in disinfecting mains water.
When gaseous Chlorine (CL2) added to water (H2O) the following hydrolysis reaction takes place:

Cl2 + H2O = H+ + Cl- + HOCl

Sodium Hypochlorite

Sodium Hypochlorite is produced adding gaseous Chlorine (CL2) to caustic soda (NaOH). When this is done, Sodium Hypochlorite (NaOCL), water (H2O) and salt (NaCl) are produced according to the following reaction:

Cl2 + 2NaOH + → NaOCl + NaCl + H2O

Chlorine reacts with sodium hydroxide to Sodium Hypochlorite (NaOCl). Sodium Hypochlorite is known as Bleach. Bleach (NaOCL) cannot be combined with acids. When NaOCL comes in contact with acids the hypochlorite becomes instable, causing poisonous gaseous Chlorine (CL2) to escape.

Hypochlorous Acid and Hypochlorite Ion formation

Hypochlorous Acid (HOCL) and Hypochlorite Ion (OCL-) are the by-products of Sodium Hypochlorite (NaOCL) in water (H2O). NaOCL reacts with water (H2O) to Hypochlorous Acid (HOCl) and Hypochlorite Ions (OCl-).

NaOCl + H2O → HOCl + NaOH-

Hypochlorous Acid formation

Hypochlorous Acid (HOCL) is the by-product of gaseous Chlorine (CL2) in Water. Gaseous Chlorine (CL2) reacts with water to Hypochlorous Acid (HOCL).

Cl2 + H2O -> HOCl + H+ + Cl-

Oxygen formation

Depending on the pH value, Hypochlorous Acid (HOCL) expires to Hypochlorite Ions (OCL-).
Cl2 + 2H2O -> HOCl + H3O + Cl-
HOCl + H2O -> H3O+ + OCl-

This falls apart to Chlorine and Oxygen atoms:

OCl- -> Cl- + O

The efficacy of disinfection is determined by the pH.

Disinfection will take place optimally when the pH is between 5 and 7, as then a maximum of HOCL is present.
HOCL reacts faster than OCl- ; HOCL is 80-100% more effective than OCL-. HOCL does not evaporate and does not cause severe corrosion like CL2. CL2 exposed in air can be very explosive and evaporation should be avoided. For this reason, the ideal pH is between 6 and 7, as no CL2 is present.

The level of HOCL will decrease when the pH value is higher than 5. The level of HOCL will decrease when the pH value is lower than 5. With a pH value of 6.5 the level of HOCL is more than 90%, whereas the concentration of OCL- is less than 10%.

Free Available Chlorine

Free Available Chlorine (FAC) is chlorine that is present in the form of Hypochlorous Acid, hypochlorite ions or as dissolved elemental chlorine. FAC includes all chlorine species that are not combined with ammonia (or other nitrogenous compounds) to form chloramines. It is ‘free’ in the sense that it has not yet reacted with anything, and ‘available’ in the sense that it can and will react if needed.

A pH value of 6 to 7 is the most effective and the safest pH-range, due to absence of chlorine gas. Therefore when Free Available Chlorine is mentioned, it is assumed that Free Available Chlorine solely consists of HOCL and OCL-

Free Available Chlorine compounds with regard to pH .Hypochlorous Acid (red) and Hypochlorite Ion (green)

Superiority of Hypochlorous Acid compared to Hypochlorite Ion

Hypochlorous Acid (HOCl, which is electrically neutral) and Hypochlorite Ions (OCl-, electrically negative) will form Free Available Chlorine (FAC) when bound together. This results in disinfection. Both substances have very distinctive behavior.

The cell wall of pathogenic microorganisms is negatively charged by nature. As such, the cell wall only penetrated by the neutral Hypochlorous Acid (HOCL), not by negatively charged Hypochlorite Ion (OCL-).
HOCL can penetrate slime layers, cell walls and protective layers of microorganisms and effectively kills pathogens as a result. The microorganisms will either die or suffer from reproductive failures.

The pH neutral Hypochlorous Acid (HOCL) can penetrate cell walls of pathogenic microorganisms whereas the negatively charged Hypochlorite Ion (OCL-) cannot penetrate cell walls.

Besides the neutrality of HOCL, it is a much more reactive and is a much stronger disinfectant than OCL-, as HOCL is split into hydrochloric acid (HCl) and atom air Oxygen (O). Oxygen is a very powerful disinfectant.

Neutral Electrolyzed water (HOCL) guarantees optimal disinfecting

The disinfecting properties of Chlorine in water are based on the formation and oxidizing power of Oxygen and HOCL. These conditions occur when the pH is between 6 and 7.

Neutral Electrolyzed Water (NEW) produced onsite from a AQUAOX System has a pH of 6.5. At this pH more than 90% of the free available chlorine is HOCL, less than 10% OCL- and no CL2 are formed. The strength of Free Available Chlorine (FAC) in NEW is pre-set to 300+ppm. To make a solution with 300+ppm FAC from commercially available bleach (NaOCL), it is diluted in water (H2O).

The problem with diluting bleach in water is twofold:

1) The volume to dilute bleach is very small. Small differences in the volume of bleach added to water causes significant differences in terms of pH and Free Available Chlorine (FAC).
2) The fact that water has naturally different pH levels, causes that addition of the same volume of bleach still result in a different pH. Although at each dilution 300+ppm FAC can be measured, the pH of the mixture and consequently the amount of active compounds HOCL and OCL- may vary considerably.

Therefore, disinfecting properties using bleach vary whereas the disinfecting properties of NEW are kept stable. As a result NEW may exceed the disinfecting properties of bleach by 300 times.


When producing HOCL by acidifying NaOCL, relatively high prices and possibility of side reactions limit the use of weak organic acids; use of cheaper inorganic acids provokes gaseous chlorine discharge and a raise of toxicity level. Because of it, the method above is only used for water treatment, where residual chlorine concentration values do not exceed 0.5-5mg/l.

Dilution of gaseous chlorine in water to produce HOCL according to equation demands special safety measures and is only used for disinfecting large volumes of water, where active chlorine concentration is below 10-15mg/l. Nowadays all the companies that manufacture gaseous chlorine stopped gaseous chlorine production and started NaOCL manufacture exclusively because of safety considerations.

Neutral Electrolyzed Water onsite produced by AQUAOX Systems is a unique method of non-reagent synthesis of HOCL. We would like to point out once more that the unique quality of the AQUAOX System is the possibility of directed pH regulation in the 6.0-7.0 ranges, while working with solutions of any mineralization, whereas electrolyses of sodium chloride solutions have identical biocidal activity if pH and FAC concentration are equal.

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