By Ronald L. Barnes and D. Kevin Caskey

In addition to testing bacteria, dye tests were also run. This consisted of mixing indigo blue with the agarose solution. Indigo blue is commonly used to measure the amount of ozone present in water. For the purposes of this experiment, the indigo blue was used as an indicator of how well AOP reacted with the artificial biofilms. Indigo blue is actually bleached by ozone, so quantitative results could be obtained. Leaving bacteria out of these tests, the same run times were repeated as before. Both visual observations and actual instrument analysis were used to measure these results. The average reduction of dye was measured at 95 percent for water and 65 percent for air.

Test methods
All testing was performed in the lab of Dr. Gopi K. Podila, Department of Biological Sciences, University of Alabama in Huntsville. The P. aeruginosa came from a TSB culture concentrated at 500,000 cells/mL. The bacteria were diluted 100x into a two-percent agarose solution that was cooled and maintained at 40°C (104°F) for a final concentration of 5,000 cells/mL. One milliliter of this mixture was placed onto a 1.5 x 1 glass slide and allowed to solidify. After the various treatments, the films were placed in phosphate buffered saline (PBS), homogenized and one mL was spread onto tryptic soy agar (TSA) plates. After overnight incubation at 37°C (98.6°F), the bacteria were enumerated using standard plate counts. As an added control, the original agarose and bacteria mixture was diluted and counted in the same manner before solidification to ensure the actual biofilm was concentrated at 5,000 cells/mL. The dyed slides were prepared by adding one gram of indigo blue into 100 mL of the two-percent agarose mixture. After thoroughly mixing, the solution was kept at 50°C (122°F) to prevent solidification while the slides were being prepared. A spectrophotometer was used to quantify the amount of dye that was bleached.

For both the bacterial and dye tests, negative controls were used. Plain distilled water was used for the aqueous test while compressed air was used for the gas tests. In both cases, the bacterial counts were very close to 5,000 cells/mL. Additionally, it was found that neither water pressure nor air pressure was able to substantially move the films off the slides.

To achieve uniform thickness of the slides, the fine adjustment knob of a light microscope, coordinate graph paper and a paper clip were utilized. The graph paper was marked with increments to count the number of times the knob was turned.

The paper clip was attached to the fine adjustment knob to help gauge half turns, quarter turns, etc. A complete turn equals 200 micrometers. A slide was dipped once into a two-percent agarose solution with no bacteria. The slides and agarose were maintained at 40°C in a water bath. The uniformity of temperatures was done in order to ensure an even spreading of the agarose, thus eliminating one variable that could potentially skew the data. A mark was placed on the slide prior to dipping, followed by another on top of the film after the agarose had solidified on the slide. Both marks cannot be in focus simultaneously; therefore the bottom mark was brought into focus followed by the top mark. The number of turns to do this gives the thickness. The initial thickness was determined to be 500 micrometers, which was within range of the initial target of 450 micrometers to 700 micrometers. It was found that dipping the slides twice in the mixture at 40°C consistently produced the same thickness.

These tests are an indicator of how the use of ozone in aqueous and gaseous forms can work in relation to the cleanliness of jetted tub plumbing. The test data suggests that gaseous ozone has an impact on mature biofilms. When combined with aqueous AOP, it appears there is a ‘one-two punch’ on biofouling. The conditions under which these tests were performed would not exist in a new jetted tub. Therefore, it is a reasonable assumption that using this type of system with the whirlpool system of a new jetted tub can be of benefit in terms of keeping biofouling under control.

About the study
This study was jointly funded by Pentair Water Spa and Bath, a distributor of the Prozone Unit. For additional information, contact Rodney Tripp at Rodney.tripp@

About the product
Prozone’s Advance Oxidation Hybrid Generator forces ozone to react via a free radical pathway with activation by UV radiation. The model PZ6-CT, tested and discussed here, is manufactured by Prozone Water Products. For more information visit the company’s website, or contact the sales department at (800) 632-6462.

About the authors
Ronald L. Barnes is founder and CEO of Prozone Water Products Inc. (established in 1977), Ecozone (founded in 1984) and Vistek Corp. (started in 1984). He has a Bachelor’s Degree in physics and a Master’s Degree in physics and electronic engineering. A successful consultant, (International Ozone Association, NASA, ASHRAE, The National Pool and Spa Institute, GE, Boeing and Lockheed), Barnes is a member of the International Ozone Association and several related organizations. He holds 10 patents for ozone water treatment, ozone generators, plasma generators, optical storage scanners, electrophoresis, laser Doppler radars and an ozone contact lens cleaner. He can be contacted at (256) 539-4570 or [email protected]

D. Kevin Caskey is a Project Manager and microbiologist for Prozone Water Products. He holds Bachelor and Master’s Degrees in microbiology. Prior to rejoining Prozone, where he was originally a Project Manger, he worked at Vanderbilt University in Nashville as a Lab Manager while conducting his own research projects. He can be reached at (256) 539-4570 or [email protected]

About the companies
Pentair, a $3.2 billion corporation with global headquarters in Golden Valley, Minn., is dedicated to bringing safe, clean water to the world. The Spa and Bath division is a supplier of a full range of products to spa and whirlpool bath OEM manufacturers looking for quality products and a reliable supplier. For product information please call (800) 467-0827. Prozone Water Products, Intl., headquartered in Huntsville, Ala., manufactures ozone generators and advanced hybrid ozone generators. Since the creation of a prototype in 1977, the systems have evolved into commercial pool, spa and whirlpool bath purification systems. Call (800) 632-6462 for more information.


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