# Procedural Revision to the Use - dilution Method: Establishment of Maximum Log Density Value for Test Microbes on Inoculated Carriers Stephen F. Tomasino , Ph.D. U.S. Environmental Protection Agency Office of Pesticide Programs Microbiology Laboratory (Presented at the 2011 AOAC INTERNATIONAL Meeting)2 

# DISCLAIMER This information is distributed solely for the purpose of pre - dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency Determination or Policy . The information provided should not be cited or referenced.

# Background • EPA worked closely with AOAC INTERNATIONAL (AOAC) and usercommunity to enhance the Use - Dilution Method (UDMs)

– Comprehensive editorial revisions to each of the three UDMs 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)

– E stablishment of a minimum level of viable cells of 1.0 × 10 6 

### CFU/carrier (log 10 

### density of 6.0) to qualify efficacy results 

## • Minimum carrier count requirement based on multi - laboratory data collected over an 8 year period (1999
– 2006 )

– 242 independent tests conducted by 4 laboratories; all mean log 10 

### densities (LD) at least 6.0 (1.0 × 10 6 

### CFU/carrier) and did not exceed 7.5 (3.2 × 10 7 

### CFU/carrier)

– For all microbes and OSL treatments, mean LD (± SEM) was 6.7 (± 0.07) per carrier (5.39 × 10 6 

### CFU/carrier)

– Mean LD for six carriers per test showed acceptable repeatability (0.29) and reproducibility (0.32) SDs 3

# Present Investigation • Utilizes the 1999
– 2006 multi - laboratory data, along with an additional four years’ worth of 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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# 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 

### • 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 

### • 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 5

## Summary for the mean log densities, averaged across 6 carriers per test, for each combination of microbe, OSL and laboratory (1999
– 2010)

6 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 Table 1.

## Summary for the mean log densities, averaged across 6 carriers per test, for each microbe and OSL combination

7 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 Table 2.

# Distribution of Mean Test LDs

8 M i c ro b e O S L La bo rat o r y S . a u re u s P . ae ru g i n os a P res e n t A bs e n t P res e n t A bs e n t 4 3 2 1 4 3 2 1 4 3 2 1 4 3 2 1 8 . 0 7 . 5 7 . 0 6 . 5 6 . 0TestLD

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 95 th percentile (7.25).

# Discussion
• Of 512 mean LD values, the smallest was 6.0 (1.0 × 10 6 

### CFU/carrier) and the highest was 7.8 (5.7 × 10 7 

### CFU/carrier) • With the exception of two tests, the mean LD values did not exceed 7.5 • Across the 16 combinations of laboratories, microbes and OSL treatments, the mean LD ranged from 6.5 to 7.2 • Across both microbes and OSL treatments, the mean LD/carrier was 6.8 (6.32 × 10 6 

### CFU/carrier); 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 95 th 

### 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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# Discussion (cont.) • Additional data support continued use of current minimum mean carrier count requirement (6.0 logs per carrier) • To mitigate potential variability in the efficacy results that may be caused by a wide range in carrier counts, 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 )

– Provides procedures to standardize inoculum and specifies an acceptable range for carrier counts for each microorganism (0.5 - 2.0 × 10 6 

### CFU/carrier for S. enterica , and 1 - 5 × 10 6 

### CFU/carrier for S. aureus and P. aeruginosa). 

## • 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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# Proposed Revisions
• Calculate the log 10 density (LD) for each carrier by taking the log 10 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 (corresponding to a geometric mean density of 1.0 × 10 6) and not above 7.0 (corresponding to a geometric mean density of 1.0 × 10 7 ); 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 (e.g., carrier drying and culture incubation conditions), 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. 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 (nutrient broth or synthetic broth). 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; OD readings of 0.2 to 0.4 are typical for harvested test cultures for both S. aureus and P. aeruginosa . Sterile broth medium should always be used to blank the spectrophotometer. • Retesting guidance: For tests where the product passes and the mean carrier counts are above 7.0 logs, no retesting is necessary. For test where the product fails and the mean carrier counts are below 6.0 logs, no retesting is necessary.
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