Biological Consulting Services

of North Florida, Inc.

July 12, 2007

Luis Gomez

President

P.O. Box 221074

Hollywood FL 33022

(954) 922-5976

Dear Mr. Gomez,

Greetings.

We conducted the microbiological challenge experiments on the Disinfectant Liquid sample that you delivered to our Laboratory on July 10, 2007. The testing was done according to the protocol we briefly discussed. 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

Staphylococcus aureus and E. coli  challenge 

 Staphylococcus aureus (ATCC 12600) and E. coli (ATCC 15597) stock cultures were obtained from American Type Culture Collection and are maintained at -80°C.  For Challenge experiments, overnight cultures from the frozen stocks were grown in 10 ml of (Tryptic Soy Broth (TSB, Beckton Dickinson, MD) at 36 ºC prior to the date of the experiments.  At the Day of Challenge, the broth cultures were centrifuged at 3K x G for 5 minutes and suspended in 10 ml of phosphate buffered saline (PBS, Fisher scientific, PA)

On July 11, 2007, Twenty ml of PBS and 20 ml of the solution delivered by Mr. Gomez was placed in 50ml conical bottom polypropylene tubes (Fisher scientific, PA).  To each of the tubes approximately 2.3 x 10⁶ of Staphylococcus aureus was added and the tubes were agitated rapidly. At 10, 30, 60, and 180 seconds following the addition of the bacteria 1 ml aliquots of PBS and disinfectant solution were removed and placed in 9 ml of PBS containing 0.02% sodium thiosulfate.   The microorganisms in each of the dilution tubes were enumerated by spread plating onto Plate Count Agar (PCA, Beckton Dickinson, MD) and incubation at 37°C for 24 hours. 

The same Experiment was repeated using E. coli.

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

Table 1. The reduction of E. coli (ATCC 15597) by the disinfectant delivered by Mr. Gomez at various time point following the introduction of the bacteria.

Table 2. The reduction of S. aureus (ATCC 12600) by the disinfectant delivered by Mr. Gomez at various time points following the introduction of the bacteria.