Objective: Develop a validation system for Bio Shield's photo-catalytic surface coatings that meet the following base criteria:

There are three spore formers utilized in many cases, depending on the type of decontamination or sterilizing technique being employed. They are; bacillus stearothermophilus, (http://en.wikipedia.org/wiki/Bacillus_stearothermophilus) bacillus subtilis, and bacillus pumilus. Besides demonstrating an unusual degree of resistance they are also harmless to humans, so they can be used in any environment without danger to products, personnel or employees.

The object of this validation protocol is to inoculate a series of stainless steel coupons (discs) with a known quantity of b. subtilis and expose the inoculated coupon to the photo-catalytic reaction taking place in the purification chamber. Following exposure, the disc would be placed in sterile growth media and incubated to measure the presence of any remaining cells which would still be viable. A summary of steps follows:

ˇ Initially we would have a predetermined number (~100) stainless steel discs manufactured under sterile conditions. (see attached photographs)

ˇ The discs would be shipped to NRL to be coated with NuTiO titanium dioxide (TiO2). Following the coating process, each disc would be placed in a separate sealable plastic envelope and shipped to a fully accredited laboratory.

ˇ The laboratory will be responsible for culturing and hand applying the b. subtilis spores at a log3 concentration (1000 organisms) per disc.

ˇ Prior to testing, a number of "control" discs (inoculated but untreated in the photo-catalytic chamber) would be placed in sealable vials containing 10 ml of tryptic soy broth (growth media) and incubated at 37 degrees centigrade. The incubator will have been calibrated and monitored by a display thermometer inside the chamber.

(Note): When the discs are placed in the tryptic soy broth and subjected to the heat of the incubator, the bacteria begin to subdivide. As they multiply, the bacteria release an acid which turns the clear growth media cloudy. We would time the number of hours it takes for the untreated inoculated discs to cloud the liquid. For example, we feel this might take place in an average of ~24 hours.

ˇ Prior to exposing the discs in the photo-catalytic chamber, the air purification unit would be turned on and allowed to warm up for ~30-60 minutes, to assure it is operating at peak efficiency.

ˇ For each test, the warmed up test unit would be turned off. One or more discs would then be removed from the airtight container and placed in the photo-catalytic chamber, attached to the surface of the TiO2 coating within the chamber, such that the inoculated/TiO2 coated side of the disc is facing the UV light. The unit would then be turned back on and the disc exposure timed for 15 seconds. After 15 seconds, the test unit would be turned off and the disc (s) would be removed and placed in new sealable plastic bags and stored in a separate airtight container.

ˇ The same procedure as described in the above paragraph would be repeated in exactly the same manner, except that the exposure times for each subsequent test would be increased (i.e.30 s, 60 s, 5 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours, 24 hours or other times as agreed ). Each set of discs would be marked appropriately to identify the time of exposure.

ˇ Following the exposure step, each individual disc would be placed in its own sealable vial of 10 ml tryptic soy broth and the vials would be placed in the heated incubator along with a "control" (inoculated but untreated disc) in its own vial. The vials would then be visually monitored (using a light box) and the elapsed time measured until each vial turns cloudy. If the "control" vial clouds in "x" hours and any of the treated vials take longer to cloud then it is safe to assume that the unit is killing some of the b. subtilis. Therefore lesser resistant organisms that might be present in the air can be safely assumed to have succumbed to the chamber's photo-catalytic effect. If any of the treated vials remain clear after ~7 days of incubation all the b. subtilis can be assumed to have been killed and "sterilization" has been achieved for that disc.