Jafrul Hasan, Ph.D. Team Leader Office of Pesticide Programs Microbiology Lab U.S. Environmental Protection Agency Office of Pesticide Programs Microbiology Laboratory 

# The Quantitative Petri Plate Method 

# Standard Test Method for ASTM Work Item 32908: Standard Test Method 

## for Quantitative Petri Plate Method (QPM) for Testing Antimicrobial Towelette Products

## Overview of the Proposed QPM

2  

### Submitted the Proposed Method to/Presented at the ASTM 2011 Spring Meeting  

### Assigned Work Item #32908  

### Formed Work Group~24 members  

### Sent out Proposed Method to WG on May 18, 2011  

### Received Numerous Comments/Suggestions  

### Updated the Proposed Method/Developed a ‘Response Table’  

### Presented at the 2011 AOAC Annual Meeting in Sep, 2011  

### Distributed the Updated Method/Discussed Highlights 

### of the Updated Method at the ASTM Fall Meeting, 2011

# Work Group for WK 32908 (QPM)

3  

### John Mitchell  

### kathy Baxter  

### Mary Bruch  

### Lisa Hellickson  

### Terri Eastman  

### Sharon Hayden  

### Angela Hollingsworth  

### Rhonda Jones  

### Dan Klein  

### Dave Macinga  

### Linda Olson  

### Dave Rottjakob  

### Syed Sattar  

### Andrea Waggoner  

### Darla Goeres  

### Kelly Whitehead  

### Kyle Smith  

### Dena Clarke  

### Maria Curry  

### Jeff Van Komen  

### Diane Boesenberg  

### Samantha Broaders  

### Milady Brutofsky  

### Jafrul Hasan (Leader)

# Introduction

4 

###  

## The marketing and demand for antimicrobial towelettes used to treat hard non-porous inanimate surfaces is increasing .  

## No standard quantitative test method exists to 

## determine the efficacy of these products against Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica.  

## The method provides a standardized approach that is user-friendly, relevant, reproducible, and quantitative.

# Scope

5  

### To evaluate the performance of antimicrobial towelettes  

### Antimicrobial towelettes are diverse in size, matrix 

### composition and packaging .  

### Products also vary in label claims, such as disinfection and sanitizing claims . Antimicrobial towelettes are 

### designed to treat hard non-porous surfaces with or without soil load based on the label.  

### Formal training is necessary to conduct the tests since it may involve the use of hazardous materials, chemicals and infectious microorganisms .  

### Strict adherence to the protocol is necessary for the validity of the test results .

## Summary of Test Method

6  A quantitative test against S. aureus, P . aeruginosa and S. enterica.  Disinfectant and Sanitizing label claims are suitable.  Petri plates are inoculated with five spots ( 10 μL each) of the test organism.  A template detailing the pattern of inoculation is used.  Bacteria are exposed to the product by wiping the inoculated area using a corkscrew pattern for the appropriate contact time.  Bacterial survival is determined by neutralizing the test substance, scraping the Petri plate, and collecting the inoculum /neutralizer mixture.  Pooled mixture is diluted, filtered and plated.  The Mean log 10 density (LD) is calculated for treated carriers and compared to the Mean LD from the control carriers . This calculation is used to determine the efficacy of the product based on a Log Reduction (LR) value.

## Significance of Use

7  The method utilizes glass Petri dishes ( 150 mm 20 mm) as test and control carriers .  Provides a closed system for enumeration and easier manipulation of an antimicrobial towelette.  A template and a positive displacement pipette are used for inoculation.  One towelette per carrier, thus ensuring comparable treatment between carriers and eliminating the likelihood of cross contamination between carriers .  The circular motion of the product application ensures uniform coverage and contact of disinfectant to the inoculated surface.  Addition of neutralizer (20 mL, 2 times) to the carriers ensures thorough neutralization.  A procedure to conduct neutralization verification is provided.  The neutralization verification should be conducted for each class of test chemical.  The design of the test method minimizes any loss of viable organisms through wash off.  The use of organic soil load based on the product label claim is an option.  Adjustment (either dilute or concentrate) of the inoculum level to achieve desired control carrier counts and to accommodate different performance standards is optional.

# Inoculation Template

(to be used during inoculation and scraping)

8 Inoculation area
Inoculation spot

– 10 μL

# Wiping Schematic (1)

9 Start

END

# Wiping Schematic (2)

10 Start END

# Sterile Scraper

11

# Steps of QPM

12 A. Dried inoculum on plate13 

## B. Antimicrobial towelette folding- 1

## C. Antimicrobial towelette folding-2

14

## D. Wiping of plate

15

## E. Addition of neutralizer

16

## F. Scraping

17

## G. Collecting of mixture

18

# Calculations

19  

### Log 10 

### Reduction (LR)= LDC

### -LDT

 LDc = Mean log 10 Density of Control Carriers  LDT = Mean log 10 Density of Treated Carriers  

### Efficacy of Antimicrobial T

## owelette Products would be based on a Log Reduction (LR) value.

### Neutralization Verification Test

20 Treatments/ Controls Addition to Petri plates* Treatment Description Anticipated

Outcomes Test Chemical Neutralizer Test culture PBS Test Suspension Control (TSC) N/A N/A √ √ Inoculum titer for comparative purposes (A) A = 20 to 200

CFU/mL Neutralizer Effectiveness Control (NEC) √ √ √ N/A To measure if antimicrobial is effectively neutralized (B) A-B = within

0- 1 log difference Neutralizer Toxicity Control (NTC) N/A √ √ N/A To measure if neutralizer has any microbicidal activity (C) A-C = within

0- 1 log difference Carrier Sterility Control (CSC) N/A √ N/A N/A Sterility assessment of carrier and neutralizer No growth

# Next Steps

21  

### To be submitted for E35.15 Subcommittee balloting on Dec 8, 2011  

### Would notify all Workgroup members along with the 

### ‘Response Table’ and the updated proposed method  

### If Subcommittee Balloting appears favorable, the proposed method will be submitted for E35 Main 

### Committee balloting on January 24, 2012.  

### Plan to finalize as an ASTM Standard Method by May, 2012.

# Acknowledgements

22 

###  

## WK 32908 Work Group Members, ASTM E35.15  

## Luisa Samalot-Freire, EPA  

## Stephen Tomasino, EPA  

## Susan Lawrence, EPA