Biological Consulting Services

of North Florida, Inc.

May 20, 2009

Luis Gomez

President

P.O. Box 221074

Hollywood FL 33022

Dear Mr. Gomez,

We have completed the antiviral efficacy study on using the Bioactive solution produced by the Aquamaster.    The testing was done according to the protocol we briefly discussed and have used previously in disinfectant studies.  The protocol used is comparable to ASTM E 1053-97 (Standard Test Method for Efficacy of Virucidal Agents Intended for Inanimate Surfaces) and AOAC Official Method 961.02 (Germicidal Spray Products as Disinfectants).  Influenza A (H1N1) virus was used to determine antiviral efficacy of disinfection. 

According to the observed results the Bioactive solution exhibited significant antiviral properties.  It completely killed the exposed viral inoculum on the treated surfaces. 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

Stock Virus and Cell Culture Preparation

Influenza A (H1N1; ATCC VR-1469) virus was propagated and enumerated as Most  Probable Numbers (MPN ) using MDCK cell monolayers (ATCC CCL-34) as the host.   Cells were grown in 6 well cell culture plates. For enumeration, aliquots of a sample containing the virus are inoculated on freshly prepared monolayers of MDCK monolayers.  The cells are then incubated in dMEM (MediaTech, USA) media containing trypsin at 35°C and 5% CO₂ for 3-8 days.  Cells are monitored routinely microscopically for signs of degeneration.  Cells in wells demonstrating signs of infectivity (Cytopathic effects; CPE) are recorded as positive (+) and ones that do not demonstrate any CPE are recorded as negative (-).  The most probable number of infectious virus in a sample is then calculated using MPNCALC software (version 0.0.0.23). For Challenge experiments, virus stocks (typically 2 x 10⁶ pfu/ml) are thawed at the day of experiment.  They are then diluted 1/10 in Class 1 ASTM reagent water supplemented with 1% Bovine Serum Albumin (BSA). 

Challenge Study; May 07, 2009

The protocol used is comparable to AOAC Official Method 961.02 (Germicidal Spray Products as Disinfectants) and also ASTM E 1053-97 (Standard Test Method for Efficacy of Virucidal Agents Intended for Inanimate Surfaces).  Briefly, fifty microliters of the above virus dilution was evenly spread on the surface of fifteen 25 x 25 mm glass slides (Fisher Scientific, PA).   The slides were allowed to air dry at 25° C.  Following, ten of the inoculated glass slides were spayed with freshly produced Bioactive solution.  The slides were saturated with the bioactive solution. A total contact time of 5 minutes was allowed. The glass slides were then placed in a sterile 50 ml centrifuge tube (Fisher scientific, PA) containing 10 ml sterile Neutralizing Buffer (Beckton Dickinson, Sparks, MD). Glass slides containing viral inoculums and not sprayed with bioactive were used as positive controls.  The recovered viable viral mpn from the positive control slides were used to calculate challenge concentration and percent reduction. Ten fold dilutions of the viral suspensions were performed in PBS.  The number of viable (infectious) Influenza A in each of the tubes was enumerated by MPN procedure described above Table 1 below presents the results of the above-mentioned test.

 Table 1. The efficacy of Bioactive sanitizer on the inactivation of Influenza A (H1N1; ATCC VR-1469) during a five minute contact time.  

*Data represents an average of three trials for each test point. Most Probable Number (MPN) of Influenza A virus was enumerated using EPA ICR comparable Methodology (EPA 600/R-95/178, 1998).  For enumeration, aliquots were inoculated on freshly prepared monolayers of MDCK (CCL-34) cells and CPE was checked during a 7 day incubation period Cells were incubated at 35°C in a 5% CO₂  atmosphere.