KILL-TIME STUDIES
Antimicrobial Activity of ACS 200
Against Bartonella henselae

Test Solution: ACS 200 (Lot# 1606101)
May 24, 2016

PREPARED FOR:

Results RNA
1272 South 1380 West
Orem, UT 84058
BY:
Richard A. Robison, Ph.D.
Department of Microbiology and Molecular Biology
Brigham Young University
Provo, UT 84602

I. PURPOSE

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.

C. Neutralizer

The neutralizer solution used was Dey-Engley Neutralizing Broth, prepared according to manufacturer’s directions.

D. Kill-Time Procedure

  1. 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.
  2. 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.
  3. After 2 minutes, the neutralized suspension was serially diluted in sterile 9 ml PSS blanks.
  4. 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.
  5. 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.

E. Controls

  1. A titer of the test suspension was computed by performing membrane filtration assays on selected 1:10 dilutions in sterile PSS.
  2. 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.
  3. 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.

III. RESULTS

B. henselae Titer

Dilution:                                  1:1×107            1:1×108

Number of Colonies:                230                   18

264                   28

Neutralization Control

Dilution:                                  1:1×100            1:1×101

Number of Colonies:                331                   37

347                   56

Expected Counts:                  1:1×100            1:1×101

245                   25

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

0                      0

Sterility Controls:

Material           Counts

PSS                     0, 0

TSA                     0, 0

ACS200              0, 0

Neutralizer         0, 0

IV. DISCUSSION

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

Performed by:

                                                          

Marcus Jackson

Research Associate

 

Supervised by:

                                                          

Richard A. Robison, Ph.D.

Professor

4007B LSB

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.