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 Bacillus subtilis challenge experiments using the BioActive™ disinfectant liquid sample that was produced onsite using the Aquamaster unit that was delivered to our laboratory. Challenge testing was conducted to evaluate the sporocidal efficacy of the disinfectant. Various dilutions of the disinfectant were evaluated.  The BioActive™ disinfectant exhibited excellent sporocidal 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

Bacillus subtilis challenge Study 

Bacillus subtilis (ATCC 19659) was propagated on Plate Count Agar and Tryptic Soy Broth (Beckton Dickinson, MD).  The original ATCC strain is maintained at -80°C.  A purified spore suspension of B. subtilis was produced as per ASTM E2111-00 (Standard Quantitative Carrier Test Method To Evaluate the Bactericidal, Fungicidal, Mycobactericidal and Sporicidal Potencies of Liquid Chemical Germicides).  Spores were generated 48 hours prior to challenge.

The spore 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 I deionized water.  To each of the water or disinfectant challenge tubes, approximately 1.0 x 10⁶ of the above purified spore suspension was added.  The tubes were then inverted repeatedly at a moderated –slow speed for 60 seconds.  Following the exposure, a 100 µl aliquots of solution was removed and placed in 10 of Neutralizing Broth (Beckton Dickinson, MD).  The spores in the dilution tubes were then enumerated by spread plating onto Plate Count Agar (Beckton Dickinson, MD) as per laboratory standard method.  The plates were then incubated at 37°C for 48 hours and the colonies 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 Bacillus subtilis spores (ATCC 19659) by the various dilutions of the Bioactive disinfectant generated on site by the Aquamaster unit.