Biological Consulting Services

of North Florida, Inc.

September 04, 2007

Luis Gomez

President

P.O. Box 221074

Dear Mr. Gomez,

Greetings.

We conducted the microbiological challenge experiments on the disinfectant liquid sample that you delivered to our Laboratory. As per our conversation, testing was conducted to satisfy US EPA request of analysis according to AOAC Method 961.02 (AOAC Official methods of Analysis; 2005). The disinfectant liquid that you provided exhibited excellent antibacterial and antifungal efficacy by passing the above-mentioned test using various microorganisms.  In the following pages, you will find a summary 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

AOAC Official Method 961.02 Germicidal Spray Products as Disinfectants (2005)

Staphylococcus aureus (ATCC 6538), E. coli O157:H7 (ATCC 43895) Pseudomonas aeruginosa (ATCC 15442), Listeria monocytogenes (ATCC 4428), Salmonella choleraesuis (ATCC 10708), Tricophyton mentagrophytes (ATCC 9533) and Stachybotrys chartarum (ATCC 9182) stock cultures were obtained from American Type Culture Collection and were maintained as per the described methodology and in AOAC 961.02 and the referenced related AOAC protocols.  For Challenge experiments, cultures were grown at the temperature and incubation times specified in the specified method. E. coli O157:H7 and Listeria monocytogenes were grown as per method 991.47.   Fungal spores of Tricophyton mentagrophytes and Stachybotrys chartarum were grown and harvested as outlined in Method 955.17.  All media used for microbial growth were manufactured by Beckton Dickinson (Sparks, MD) and were purchased from ThermoFisher Scientific (Waltham, MA).  All media used was new and was purchased specifically for the project.  Positive and negative controls were performed as outlined in the Method. 

On August 06, 2007, Mr. Luis Gomez Delivered nine 1-gallon bottles labeled

“Bio Active™”.  On the same day, one botStaphylococcus aureus,  E. coli O157:H7, Pseudomonas aeruginosa, Listeria monocytogenes, and Salmonella choleraesuis.  Ten-microliters of the bacterial suspension was placed and spread onto sterile 25x25 mm glass slides (Fisher Scientific, PA). Ten slides for each bacterial species were used for challenge; additionally, one un-inoculated slide was used as a negative control and one inoculated slide was used as a positive growth control.  The inoculum was allowed to dry at 37°C for 40 minutes. The slides were the sprayed for 10 seconds with the provided solutions; the glass slides were completely covered with solution. The slides were allowed to incubate at room temperature for 10 minutes.  After drying, the slides were removed, excess liquid was shaken off, and they were placed into 20 ml of appropriate growth media and allowed to incubate as per method requirements.   The tubes were examined for microbial growth at 48 and 72 hour intervals.  Sub cultures were also removed and examined for growth as described in the method.  Tubes demonstrating no growth following the incubation period were inoculated  with 10-100 cfu of the respective microorganisms and were observed for growth after 24 hours; this was done to ensure the absence of residual antimicrobial residual effect.

On August 27, 2007, Mr. Luis Gomez Delivered one additional 1-gallon bottle labeled “Bio Active™”.  On the same day, the Tricophyton mentagrophytes and Stachybotrys chartarum were harvested earlier that morning as described in AOAC method 955.17.  The above mentioned challenge was repeated using the spore inoculate as per AOAC 961.02

All data is summarized in the following Tables..

Table 1. Inactivation of bacterial and fungal species by Bioactive™ Spray disinfectant provided by Enviro-Tech Engineering.  Test Conducted as per AOAC Official Method 961.02; Germicidal Spray Products as Disinfectants (2005)