Biological Consulting Services

of North Florida, Inc.

November 20, 2007

Luis Gomez

President

P.O. Box 221074

Hollywood FL 33022

(954) 922-5976

Dear Mr. Gomez,

Greetings.

We conducted the Poliovirus 1 challenge experiments on the BioActive™ disinfectant liquid sample that was produced onsite using the Aquamaster unit delivered to our laboratory. Challenge testing was conducted to evaluate the antiviral efficacy of the disinfectant. Various dilutions of the disinfectant were evaluated.  The BioActive™ disinfectant exhibited excellent antiviral efficacy in the challenge test devised. In the following pages, you will find a summery of the methodology used and the results of our analysis.

Should you have any further concerns please do not hesitate to contact me.

Best Regards,

George Lukasik, Ph.D.

Laboratory Director

Poliovirus 1 challenge Study 

Poliovirus 1 (Strain: chat, ATCC VR-1562)  was propagated on Buffalo Green Monkey Cells.  Cell free viral stocks are titrated and maintained at -80°C.  For Challenge experiments, an aliquot of the virus stocks was thawed rapidly and maintained at 4°C until use.

The viral challenge studies were conducted as per our standard laboratory protocol.  Briefly: 20 ml of Sterile Type 1 ASTM grade water (Rica Chemical Co., TX) and 20 ml of the various dilutions of Bioactive solutions were placed in sterile 50ml conical bottom polypropylene tubes (Fisher scientific, PA).  The Bioactive disinfectant was generated on site by the Aquamaster unit. The disinfectant was generated immediately prior to the challenge and dilutions of the disinfectant were prepared in ASTM type 1 deionized water.  To each of the water or disinfectant challenge tubes, approximately 1.0 x 10⁶ of the above Poliovirus was added.  The tubes were then inverted repeatedly at a moderated –slow speed for 60 seconds.  Following the exposure, a 0.1 ml aliquots of solution was removed and placed in 10 of Neutralizing Broth (Beckton Dickinson, MD) supplemented w/ 5% Fetal Bovine serum (Atlanta Biologicals, GA).  The viruses in the tubes were then enumerated on buffalo Green Monkey Cells as plaque forming units (pfu) by an agar overlay assay as per standard methods. Various volumes and dilutions were inoculated onto the cell monolayers in T-25 cell culture flasks (Corning NY).  The flasks incubated at 37°C for 72 hours and plaques were enumerated. All analyses were conducted in duplicates and experiments were repeated to verify results.

 The following table contains the results of the above-mentioned test.

Table 1. The reduction of Poliovirus 1 (CHAT, ATCC VR-1562) by the various dilutions of the Bioactive disinfectant generated on site by the Aquamaster unit.