ACS 200 Borrelia Burgdorferi Kill Time Study

KILL TIME STUDIES
Antimicrobial Activity of Advanced Cellular Silver (ACS) 200
Using Borrelia burgdorferi

Test Solution: ACS 200 Submitted April 29, 2009
September 15, 2009
PREPARED FOR:
Results RNA, LLC

I. PURPOSE

The purpose of this study was to estimate the antimicrobial activity of ACS 200 test formulation onBorrelia burgdorferi. Since there is no current protocol for disinfectant testing with Borrelia burgdorfer, and since this organism does not form colonies on agar, loss of motility observed through dark field microscopy was used to determine the extent of kill.

II. MATERIALS AND METHODS

A. Test organism.
The test suspension was prepared by growing Borrelia burgdorferi (ATCC 35210; Type strain) in 10ml of BSK H Medium Complete (Sigma Aldrich) for approximately six days at 37 °C without shaking.
B. Kill Time Procedure.
1. A 0.9 ml aliquot of the test solution was added to a 2 ml cryovial. Then, 100 µl of the B. burgdorferi culture was added at time zero.
2. The solution was vortexed and 30 µl of the suspension was placed on a slide with a cover slip.
3. The slide was immediately placed on the microscope for observation under dark field microscopy.
4. A control slide using PSS was prepared in an identical manner.
5. The test and control slides were observed for loss of motility every 2 minutes. Motility counts from representative fields were recorded until there was no more movement observed in the test solution.
D. Controls.
1. A baseline count was established by counting several fields of motile organisms on a control slide and estimating an average number of motile organisms per field.

III. RESULTS

Borrelia burgdorferi baseline count:
Mean number of motile organisms: 130/field
The estimated volume of 1 field is approximately 3.4 X 10⁶ ml. Therefore, the starting concentration of the B. burgdorferi culture was approximately 3.8 X 10⁷ organisms/ml.

ACS 200:
(Received 04/29/09)
Time	No. motile organisms
1.5 min	17
4 min	1
6 min	1
8 min	0
PSS:
(Control)
Time	No. motile organisms
1 min	11
4 min	10
6 min	3
8 min	10

Since a 1:10 dilution of the culture was performed in both the test and PSS control suspensions, the number of live organisms observed after 8 minutes in the PSS control was about 77% of that expected.

IV. CONCLUSION

From these preliminary studies, it appeared that most of the B. burgdorferi motility ceased after about 4 minutes of exposure to the ACS 200 solution. No motility was observed after 8 minutes of exposure.
Date of test: September 15, 2009