Antimicrobial Activity of ACS 200
Against Bartonella henselae
Test Solution: ACS 200 (Lot# 1606101)
May 24, 2016
1272 South 1380 West
Orem, UT 84058
Richard A. Robison, Ph.D.
Department of Microbiology and Molecular Biology
Brigham Young University
Provo, UT 84602
The purpose of this study was to determine the antimicrobial activity of ACS 200 on Bartonella. henselae (B34676) bacteria. This was accomplished by performing a standard kill-time suspension test using a 30 second contact time.
II. MATERIALS AND METHODS
A. Test Organism
The B. henselae test suspension was prepared by first streaking the organism on Tryptic Soy Agar (TSA) supplemented with 5% horse blood in 100mm x 15mm plastic petri dishes. These plates were incubated at 37 °C with 5% CO2 for 96 hours. The organism was then recovered by adding 2 ml of sterile physiological saline solution (PSS) and gently scraping the surface of the agar with a bent inoculating loop. The resulting suspension was the gently removed using a micropipette and transferred to a sterile 15 ml conical centrifuge tube. The bacterial suspension was then centrifuged and the pellet was re-suspended in 1 ml of sterile deionized water.
B. Disinfectant Solution
The sealed commercial product ACS 200 (Lot #: 1606101) was received on 3/23/2016 from Results RNA in a white spray bottle.
The neutralizer solution used was Dey-Engley Neutralizing Broth, prepared according to manufacturer’s directions.
D. Kill-Time Procedure
- A 9.9 ml of the test disinfectant solution was added to a sterile 50 ml polypropylene centrifuge tube. The tube was equilibrated in a 20 °C water bath. Thereafter, 0.1 ml of the henselae suspension was added to the test disinfectant at time zero. The tube was immediately vortex mixed.
- After the specified contact time (30 sec), 1.0 ml of organism/disinfectant suspension was added to 9.0 ml of neutralizer. The tube was mixed thoroughly.
- After 2 minutes, the neutralized suspension was serially diluted in sterile 9 ml PSS blanks.
- The number of viable organisms in selected dilution tubes was assayed by membrane filtration. One-ml aliquots were filtered and the apparatus was washed with about 100 ml of sterile PSS. The membranes were removed to horse blood TSA plates and incubated for 108 hours under the conditions mentioned in section A above.
- The number of colonies on each filter were then counted after 96 hours and 108 hours of incubation, and the 108 hour data was used to calculate log reduction and percent kill values.
- A titer of the test suspension was computed by performing membrane filtration assays on selected 1:10 dilutions in sterile PSS.
- A neutralizer control for the disinfectant was performed by adding 1 ml of disinfectant to 9 ml of neutralizer and inoculating this tube with 100 µl of 1:1×105 dilution of the test suspension. This tube was allowed to set for 20 minutes, and then diluted 1:10. One-ml aliquots from these tubes were assayed in duplicate by membrane filtration.
- Sterility controls for each solution were performed by filtering 1 ml samples in duplicate. Each bottle of PSS was checked for sterility by filtering 100 ml in duplicate.
B. henselae Titer
Dilution: 1:1×107 1:1×108
Number of Colonies: 230 18
Dilution: 1:1×100 1:1×101
Number of Colonies: 331 37
Expected Counts: 1:1×100 1:1×101
Percent Expected: 138%
ACS 200 (Received 3/23/2016)
Exposure Dilution of B. henselae/Disinfectant Suspension
Time 1:1×101 1:1×102
30 sec. 2 0
PSS 0, 0
TSA 0, 0
ACS200 0, 0
Neutralizer 0, 0
Results of the titer showed a B. henselae concentration of 2.47×109 colony forming units (CFUs) per ml in the original suspension. Inoculation of 9.9 ml of disinfectant with 0.1 ml of suspension produced an initial concentration of 2.47×107 B. henselae CFU per ml in the assay tube.
Results from these procedures allowed log reduction (LR) and percent kill (PK) values to be calculated using the formulas: (1) LR = -Log(S/S0); where S = concentration of viable organisms after the specified contact time; and S0 = concentration of viable organisms at time zero. (2) PK = (1 – (S/S0)) x 100. These values are shown below.
Product Contact Time Log Reduction Percent Kill
ACS 200 30 sec 6.39 99.99996
ACS 200 demonstrated a very rapid kill of the Bartonella henselae test organism. ACS 200 was able to effect a 6.39 log reduction (killing over 2.4 million organisms) in 30 seconds. Because the test solution produced almost complete kill, these numbers are based on very low counts. Shorter contact times may provide more accurate data.
Neutralization control data revealed that the neutralizer was able to adequately neutralize the test disinfectant. Observed counts were 138% of expected. Sterility controls showed all solutions and media to be sterile and free of extraneous contamination.
Dates: April 20-25, 2016
Richard A. Robison, Ph.D.
Brigham Young University
Provo, Utah 84062
Please Note: This report does not constitute endorsement by Richard A. Robison or Brigham Young University of the tested products in any way. The names ‘Richard A. Robison’ and/or ‘Brigham Young University’ may not be used in any type of promotional published material, either written or electronic, without express written permission from both parties.