2011 UDM/OECD Technical Workshop

Establishment of Maximum Log Density 

Value for Test Microbes on Inoculated 

Carriers

Stephen F. Tomasino, Ph.D.

U.S. Environmental Protection Agency

Biological and Economic Analysis Division

Office of Pesticide Programs

Microbiology Laboratory
2011 UDM/OECD Technical Workshop

Background

• EPA has worked closely with AOAC 

INTERNATIONAL (AOAC) and user-community to 

enhance the Use-Dilution Method (UDMs) 

– Comprehensive editorial revisions to each of the three 

UDMs were published in 2006 

• Procedural revisions to methods 955.15 and 954.02

adopted as First Action in 2009 

– Enumeration procedure to estimate the viable test microbe population 

dried on inoculated penicylinders (carrier counts)

– Establishment of a minimum level of viable cells of 1.0 × 106

CFU/carrier (log10 density of 6.0) to qualify efficacy results

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2011 UDM/OECD Technical Workshop

Background

• Minimum carrier count requirement based on multilaboratory data collected over an 8 year period (1999 –

2006) 

• 242 independent tests conducted by 4 laboratories; all 

mean log10 densities (LD) at least 6.0 and did not 

exceed 7.5

• For all microbes and OSL treatments, mean LD (±

SEM) was 6.7 (± 0.07) per carrier (5.39 × 106

CFU/carrier)

• Mean LD for six carriers per test showed acceptable 

repeatability (0.29) and reproducibility (0.32) SDs

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2011 UDM/OECD Technical Workshop

Present Investigation

• In addition to the previous data, utilizes 1999 – 2006 

multi-laboratory data to support the establishment of a 

maximum carrier count as an official modification to 

methods 955.15 and 964.02. 

• Data from 270 independent tests conducted from 2006 

– 2010 added to the original data set 

– Total of 512 tests (3,072 total carriers for carrier counts) 

– S. aureus and P. aeruginosa have been prioritized

• Establishing a maximum carrier count, in conjunction 

with the published minimum carrier count, will further 

standardize the UDM and provide increased confidence 

in product efficacy results within and between 

laboratories. 

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2011 UDM/OECD Technical Workshop

Data Collection

• 12 year (1999 – 2010) collection period

• Data collected under the Antimicrobial Testing 

Program by EPA and 3 state laboratories concurrently 

with performing the efficacy evaluations

• All testing conducted in accordance with GLPs

• Stock culture initiation/maintenance, test culture 

preparation, and the carrier inoculation steps detailed 

in the UDMs closely followed. 

– No dilution of the test cultures was performed 

– Each laboratory followed specified protocol for 

determining carrier counts

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2011 UDM/OECD Technical Workshop

Data Collection

• Carrier count data associated with 512 individual efficacy 

tests against P.a. and S.a. with and without a 5% (v/v) 

organic soil load (OSL) added to the inoculum (depending 

on product label claim)

• Horse serum was used as the OSL in the majority of the 

tests

• Nutrient broth used as the test culture medium

• 6 carriers/test day for a total of 3,072 total carriers

• All data (raw data and spreadsheets) peer-reviewed by EPA 

laboratory staff and verified by the Quality Assurance Unit 

prior to the statistical analysis

• All data were deemed valid and were included in the 

statistical analysis

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2011 UDM/OECD Technical Workshop

Summary for the mean log densities (1999 – 2010)

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Microbe OSL Laboratory No. of Tests Mean

Pseudomonas

aeruginosa

absent 1 9 6.802

absent 2 34 6.924

absent 3 20 6.988

absent 4 16 7.188

present 1 36 6.694

present 2 62 6.515

present 3 46 6.901

present 4 41 6.794

Staphylococcus

aureus

absent 1 9 6.896

absent 2 30 6.803

absent 3 24 7.018

absent 4 15 6.894

present 1 30 6.690

present 2 59 6.586

present 3 39 7.016

present 4 42 6.723
2011 UDM/OECD Technical Workshop

Summary for the mean log densities, averaged across 6 

carriers per test, for each microbe and OSL combination

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Microbe OSL No. of Tests No. of Labs Mean Sr SR

Pseudomonas

aeruginosa

absent 79 4 6.973 0.256 0.296

present 185 4 6.730 0.261 0.306

Staphylococcus

aureus

absent 78 4 6.902 0.229 0.246

present 170 4 6.755 0.258 0.315

Microbe & OSL 

combined:

512 4 6.801 0.279 0.311
2011 UDM/OECD Technical Workshop

Distribution of Mean Test LDs

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Microbe

OSL

Laboratory

P. aeruginosa S. aureus

Absent Present Absent Present

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

8.0

7.5

7.0

6.5

6.0

Test LD

Figure 1. Each point is the mean log density for the 6 carriers in one of the 512 Use-dilution method tests. The points 

aligned vertically are tests in the same laboratory for the specified combination of OSL and microbe. The horizontal 

dashed green line represents the current 6.0 log minimum. The horizontal dashed black line represents the mean log 

density across all carriers (6.8). The horizontal dashed blue line represents log densities in the 90th percentile (7.16), 

and the horizontal dashed red line represents log densities in the 95th percentile (7.25).
2011 UDM/OECD Technical Workshop

Discussion

• Of 512 mean LD values, the smallest was 6.0 and the highest was 7.8 

• With the exception of two tests, the mean LD values did not exceed 7.5 

• Across both microbes and OSL treatments, the mean LD/carrier was 6.8; 

comparable to mean LD reported for initial 8-year data set

• Mean LD across 6 carriers/test showed good repeatability (0.28) and 

reproducibility (0.31)

• Across microbes, presence of OSL reduced mean LD by 0.20 logs; 

difference is of no practical importance 

• The 90th and 95th percentile of the overall LD values were 7.16 and 7.25, 

respectively. 

• ~10% of mean carrier counts across the study period were above 7 logs 

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2011 UDM/OECD Technical Workshop

Discussion (cont.)

• Additional data support continued use of current 

minimum mean carrier count requirement (6.0 logs per 

carrier) 

• A maximum carrier count of 7.0 logs is proposed as a 

procedural change for methods 955.15 and 964.02. 

– Thus, the mean carrier counts must be between 6-7 logs for 

a valid test. 

• Proposal to establish lower and upper limits is in line 

with the AOAC Hard Surface Carrier Test (HSCT) 

Methods (991.47, 991.48 and 991.49) 

– Specifies an acceptable range for carrier counts for each 

microorganism (0.5-2.0 × 106 CFU/carrier for S. enterica, 

and 1-5 × 106 CFU/carrier for S. aureus and P. aeruginosa).

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2011 UDM/OECD Technical Workshop

Discussion (cont.)

• To reduce the potential of acquiring carrier 

counts above upper limit, additional revisions 

to the method are recommended to allow for 

dilution (using sterile broth) of the final 48-54 

hr test culture. 

• To adjust the microbial titer, harvested 

inoculum may be diluted in sterile broth prior 

to the addition of organic burden (if required). 

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2011 UDM/OECD Technical Workshop

Proposed Revisions

• Calculate the log10 density (LD) for each carrier by taking the 

log10 of the density (per carrier).

• The mean LD across carriers is the mean LD for the test.

• The mean LD must be at least 6.0 and not above 7.0; a mean 

LD below 6.0 and above 7.0 invalidates the test. 

• Note: If the Use-dilution method is strictly followed, mean LD 

values of at least 6.0 are expected; values lower than 6.0 may 

be indicative of a dilution error, poor media quality, 

interference by environmental parameters , contamination, or 

lack of adherence to the method.

• The prescribed mean minimum and maximum carrier counts 

also account for the addition of 5% OSL to the inoculum.

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2011 UDM/OECD Technical Workshop

Proposed Revisions

• Note: For the purpose of achieving mean carrier counts 

within a range of 6.0 – 7.0, dilution of the final test culture 

may be performed using the culture medium (sterile) used 

to generate the final test culture. 

• Dilution of the final test culture (e.g., one part culture plus 

one part sterile broth) should be made prior to the addition 

of the OSL to the inoculum.

• In addition, the use of a spectrophotometer to measure 

optical density (OD @ 650 nm) is recommended to provide 

a tool for assessing the need to dilute the final test culture

• Sterile broth medium should always be used to blank the 

spectrophotometer. 

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2011 UDM/OECD Technical Workshop

Data Summary for Staphylococcus aureus

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Date Dilution Carrier Count Titer OD

07/18/2011

undiluted 5.7×106 2.2×108 0.243

1:2 3.9×106 8.5×107 0.109

1:5 2.0×106 3.4×107 0.043

1:10 1.0×106 2.2×107 0.014

07/20/2011

undiluted 9.2×106 1.1×108 0.176

1:2 3.3×106 4.1×107 0.076

1:5 9.4×105 1.9×107 0.050

1:10 1.6×106 1.2×107 0.018

07/25/2011

undiluted 9.3×106 1.4×108 0.245

1:2 5.9×106 9.7×107 0.118

1:5 1.8×106 2.3×107 0.033
2011 UDM/OECD Technical Workshop

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4

4.5

5

5.5

6

6.5

7

7.5

8

Staphylococcus aureus Pseudomonas aeruginosa* Salmonella enterica**

Mean Log10 Carrier Counts

Test Microbe

Mean Carrier Counts Associated with Undiluted and Diluted 

Test Cultures

Undiluted

1:2

1:5

1:10

*1:10 dilution not performed

** 1:10 dilution not performed, 2 trials 

performed instead of 3
2011 UDM/OECD Technical Workshop

Acknowledgements

• Cord Hamilton, Big Sky Statistical Analysts, 

Bozeman, MT 59715

• Michigan Department of Agriculture, Laboratory 

Division, William C. Geagley Laboratory, East 

Lansing, MI 

• Ohio Department of Agriculture, Consumer 

Analytical Laboratory, Reynoldsburg, OH 

• North Carolina Department of Agriculture, 

Constable Laboratory, Raleigh, NC 

• Denis Franks and Nicholas Solomotis, MLB –

culture dilution data

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