Wednesday, May 6, 2009

Biological Organism Reduction With Hydrogen Peroxide

Hydrogen Peroxide is a strong oxidizer used for high-level disinfection and sterilization. It produces reactive hydroxyl free radicals and ions that can attack membrane lipids, DNA and other essential cell components.

3 to 6% solution is generally used for an exposure time of 10 to 600 minutes depending on the microorganism targeted. In general, vegetative bacteria are most susceptible and bacterial spores the most resistant. Concentrations of 3 to 6% have been used for the disinfection of ventilators, soft contact lenses, and tonometer biprisms. Still, liquid disinfection with hydrogen peroxide is not widely used in the medical industry.

Vaporized hydrogen peroxide, however, is now being used for low-temperature sterilization by way of a plasma sterilizer. Two commercially available plasma sterilizers, the Sterad 1001 and the Plazlyte2, both require a vacuum and high concentrations of hydrogen peroxide to operate. The low pressure plasma in the vacuum environment induces free radicals and ions which enhance the bactericidal process. Used for sterilizing surgical instruments, they require extensive containment enclosures.

The three common states of matter are solids, liquids, and gases. Plasma, the fourth state of matter, however, comprises 99% of the visible universe. Sir William Crookes, an English physicist, first identified plasma in 1879. The word “plasma” was not applied to ionized gas until 1929, by an American chemist, Dr. Irving Langmuir.

Plasma consists of free-moving electrons and ions that can be formed when energy is applied to molecules, e.g.:

H2 + energy ----> 2 H+ + 2 e-

Many different forms of energy can be used: thermal, electrical, or light, for example. With insufficient sustaining power, plasmas recombine into neutral gases, liquids, or solids. Examples of common plasmas include the sun and stars, lightning, fluorescent or neon light, lasers, and vacuum tube discharge.

Binary Ionization Technology

Binary Ionization Technology™ (BIT™, Photo 1), developed by Intecon (US Patent #6,343,425), is the process of passing a cleaning and disinfecting mist through plasma (ionized gas), which results in a more effective and drastically shorter disinfection/cleaning process. The chief advantage of BIT over previous H2O2/plasma systems is that BIT works at standard atmospheric conditions and does not require a vacuum; it can be used in the open air without special enclosures for containment.

A weak solution of hydrogen peroxide was chosen as the first system to evaluate since it is a chemical that is commonly used in cleaning and disinfecting. Hydrogen peroxide is relatively inexpensive, leaves no residue, and is effective in disinfecting open wounds. The reactivity of hydrogen peroxide is easily seen in the foaming that occurs when it is applied to an open wound. The foaming occurs because the hydrogen peroxide dissociates into water and oxygen in the presence of enzymes found in open wounds. However, hydrogen peroxide is known to be relatively slow in disinfecting. At ambient temperatures and pressure, 20 minutes of contact is recommended to disinfect a wound.

The BIT reaction functions in a way similar to vacuum based H2O2/plasma systems; the plasma efficiently transfers energy and activates the hydrogen peroxide. The activated species then kill microbes and recombine into water and oxygen.

The difference between BIT and vacuum based H2O2/plasma systems is that BIT uses a fluidized bed of liquid particles instead of a vacuum generated vapor, thus creating a significantly larger reservoir of H2O2 molecules to react. BIT also utilizes “atmospheric cold plasma” technology that allows the creation of suitable plasma without a vacuum.

The plasma activated species of hydrogen peroxide (ions, hydroperoxy, hydroxyl or other free radicals, excited hydrogen peroxide molecules, etc.) is extremely effective at killing microbes. The initial studies, which demonstrate this, were performed with Serratia marcescensý a Gram negative bacillus. This organism was selected for two reasons: It is the organism specified in the ASTM “Standard Method for Evaluation of Health Care Personnel Handwash Formulation.” It is also a stringent organism to test in a system using hydrogen peroxide as it is an organism with high cellular catalase activity.

The first experiment showed that while hydrogen peroxide without the plasma has no detectable effect in 60 sec., the hydrogen peroxide through the plasma field gave near total kill in the same amount of time. The next experiment showed that the level of kill exceeds a 6-log reduction, the level typically used to indicate sterilization potential, in as little as 15 sec. The final experiment with Serratia marcescens again shows extremely high efficacy of activated hydrogen peroxide, this time at very low concentrations (0.3%, in 15 sec.).

More recent studies have been performed using B. subtilis, an accepted Anthrax surrogate, and B. stearothermophilus, which is very resistant to hydrogen peroxide. As shown in the table, BIT using 3% hydrogen peroxide through a 10.5 KV plasma arc for 15 sec. was effective at killing both spores.

The Center for Biological Defense at the University of Southern Florida tested the effectiveness of BIT against anthrax surrogates. Weaponized B. atrophaeus spores and B. atrophaeus vegetative cells were exposed to four BIT pulses, each 15 sec. long. BIT achieved an 8-log reduction on the spores and a 9-log reduction on the vegetative cells.

Intecon has also experimented with additives such as iso- and n-propyl alcohol, chelates, organic acids, and balanced ionized air. These further the effectiveness of metal and particle removal and may play a key role if reduction in endotoxins is an objective. The potential mechanisms of these additives may include decreasing surface tension, neutralizing static attractive forces or enhancing free-radical formation.

Reduction of Spores in a Simulated A/C Duct

The bacterial spore strips used were of very high concentrations, i.e. 106 spores per strip. Two types were used: B. stearothermophilus and B. subtilis var. niger. The B. stearothermophilus is known to be resistant to hydrogen peroxide and represents a significant challenge for H2O2-based sterilization. The B. subtilis var. niger is a large Gram positive rod that is closely related to the spore that causes anthrax (B. anthracis) and is commonly used to test sterilization effectiveness against anthrax spores.


>When the BIT system was off and the fan was on, all strips clearly indicated no observable decrease in growth.

> When the hydrogen peroxide was injected without the BIT ionization being turned on, all strips clearly indicated no observable decrease in growth.

> When the BIT system was turned on, there was no growth of spores; there was complete kill.

>BIT effectively killed all spores at all four locations— i.e., 1, 2, 4, and 6 ft.


BIT was demonstrated to be totally efficacious in a simulated A/C duct. Given the above results and the previous work Intecon has done with Serratia marcescens, BIT has proven sterilization (kill) capability against bacteria and spores. Additionally, viruses are much less robust than spores and should be easily killed by the BIT system.

The inclusion of a BIT system in existing heating/air conditioning systems can ensure that airborne bio-agents are not passed on to other rooms and are killed each time the air is exchanged through the system (usually several times per hour).


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