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 log10 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 plate
13

B. Antimicrobial towelette folding-1
C. Antimicrobial towelette folding-2

14
D. Wiping of plate

15
E. Addition of neutralizer 

16
F. Scraping

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G. Collecting of mixture

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Calculations

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 Log10 Reduction (LR)= LDC-LDT

 LDc = Mean log10 Density of Control Carriers

 LDT = Mean log10 Density of Treated Carriers 

 Efficacy of Antimicrobial Towelette Products would be 

based on a Log Reduction (LR) value.
Neutralization Verification Test

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Treatments/

Controls

Addition to Petri plates* Treatment 

Description

Anticipated 

Outcomes Test 

Chemical

NeutralizerTest 

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

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 WK 32908 Work Group Members, ASTM 

E35.15

 Luisa Samalot-Freire, EPA

 Stephen Tomasino, EPA

 Susan Lawrence, EPA