### 2011 UDM/OECD Technical Workshop Improvements to the AOAC Germicidal Spray Products as Disinfectants Test – A Carrier Enumeration Procedure and Establishment of a Log Density Range for



### Test Microbes on Inoculated Carriers.

Jafrul Hasan U.S . Environmental Protection Agency Office of Pesticide Programs Microbiology Laboratory

### 2011 UDM/OECD Technical Workshop



# Background

• The AOAC Germicidal Spray Products as Disinfectants (GSPT) test method 961.02 measures the efficacy of spray products on hard inanimate surfaces.

– The method lacks procedures for assessing microbial load on inoculated glass slide carriers (carrier counts). 

### • Without a standard procedure to measure carrier counts, the associated efficacy data may be less reliable and repeatable . 

### • An enumeration procedure currently used by the EPA and a proposal to establish a carrier count range based on historical data collected by 4 laboratories over an 11 year period is 

### provided.

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### 2011 UDM/OECD Technical Workshop 

# Carrier Enumeration Methodology

4. Serially dilute in phosphate buffered dilution water. 5. Plate and enumerate .

S. aureus colonies on trypticase soy agar (TSA) after 1 -2 days at

36 1 C 1. Randomly extract 6 carriers for enumeration .

Inoculated glass carriers (25 25 mm) 3. Vortex carriers

– 1 min. for P. aeruginosa and 2 minutes for S. aureus . 2. Place each carrier separately into a tube of 20 mL letheen broth.

Carriers in 20 mL letheen broth

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### 2011 UDM/OECD Technical Workshop 

# Multi-laboratory data set
• Multi-laboratory carrier count data set from 4 labs 

## performing the GSPT combined and analyzed • Data set contained a total of 116 independent 

## efficacy tests with 6 control carriers per test for a total of 696 carriers analyzed over an 11 year period (1999-2010) 

## • Data were log 10

## -transformed to generate mean Test Log Density ( Test LD ) values

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### 2011 UDM/OECD Technical Workshop



# Distribution of Tests Multi-laboratory data set P. aeruginosa S. aureus Organic Soil Load Organic Soil Load Lab Absent Present Total Absent Present Total 1 2 1 3 5 1 6 2 6 6 12 6 6 12 3 3 5 8 4 5 9 4 20 12 32 20 14 34 Total: 31 24 55 35 26 61

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### 2011 UDM/OECD Technical Workshop 

## Test LD values for the experiments with S. aureus

Multi-laboratory data set Figure 1

Proposed range

Test LD values for tests with S. aureus . Each point is the Test LD for one test (total tests 61 ) . Each solid line denotes the mean of Carrier LD values for the specified OSL level. The dashed line is at 5 . 0 , the minimum acceptable value for each Test LD . The red arrows stretching from 5.0 to 6.5 indicate the proposed range for acceptable carrier counts. 6

### 2011 UDM/OECD Technical Workshop 

### Test LD values for the experiments with P. aeruginosa

Multi-laboratory data set Figure 2

Proposed range

Test LD values for tests with P . aeruginosa . Each point is the Test LD for one test (total tests 55 ) . Each solid line denotes the mean of Carrier LD values for the specified OSL level. The dashed line is at 5 . 0 , the minimum acceptable value for each Test LD . The red arrows stretching from 5.0 to 6.5 indicate the proposed range for acceptable carrier counts. 7

### 2011 UDM/OECD Technical Workshop 

# Statistical Summary of Test LD Values

Multi-laboratory data set Microbe OSL Mean Test LD SEM Sr S R P.a. Absent 6.09 0.10 0.231 0.291 P.a. Present 5.97 0.15 0.492 0.534 S.a. Absent 6.23 0.05 0.227 0.234 S.a. Present 6.29 0.06 0.208 0.222 Sr = repeatability standard deviation (within-lab variability) of the Test LD SR = reproducibility standard deviation (total variability among labs) of the Test LD SEM = standard error of the mean

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### 2011 UDM/OECD Technical Workshop



# Discussion Multi-laboratory data set
• During the collection of the 116 independent efficacy tests, 

### each laboratory used the same carrier count procedure (previously described) . • No significant differences between mean log densities for cultures with and without OSL . • Mean log densities for S. aureus and P. aeruginosa were very similar • Repeatability and reproducibility standard deviations were 

### acceptably small • Recommend procedural changes to method 961 .02 :

– Addition of a carrier count procedure

– Carrier count range of 5 . 0 to 6 . 5 9

### 2011 UDM/OECD Technical Workshop 

# Evaluation of microbial persistence on



# glass slide carriers

• An additional aspect of the procedure includes the 

## holding time of the inoculated carriers . • 3 of the 4 laboratories conducted an additional study to evaluate the stability of the test organisms on the carriers over a 6 hour period .

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### 2011 UDM/OECD Technical Workshop 

## Mean Test LD Values for S. aureus and P. aeruginosa

Persistence data set Figure 3 – Mean Test LD of S. aureus OSL absent present Figure 4 – Mean Test LD of P. aeruginosa OSL absent present

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### 2011 UDM/OECD Technical Workshop



# Discussion Persistence data set • Decrease in mean log density (LD) per hour  0 . 02 logs/hour for S. aureus  0 . 25 logs/hour for P. aeruginosa 

## • Strong decreasing linear trend in the LDs over time for 

## P. aeruginosa ; • LDs for S. aureus remained stable • Similar variability for the LDs with and without an organic soil load (OSL) for S. aureus 

## • Statistically significant decrease between carriers for LDs with and without an OSL for P. aeruginosa • Based on the data presented in Figures 3 -4 , dried inoculum on carriers persists at the proposed minimum 

## mean carrier counts of 5 . 0 for up to 2 hours . 12

### 2011 UDM/OECD Technical Workshop



# Conclusions Based on the analysis of this historical data set, the following procedural changes to method 961.02 are proposed for S. aureus and P. aeruginosa : 1. Inclusion of the carrier enumeration procedure described above 2. Acceptable range of mean carrier counts (LD/carrier):

- 5.0 to 6.5 (1.0 105 to 3.2 106 CFU/carrier) 

### 3. Use-period of 2 hours (after drying) for inoculated carriers 4. Procedural changes for S. enterica may be considered at a later date

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### 2011 UDM/OECD Technical Workshop 

## Proposed procedural revisions to the GSPT Operating Technique: The mean LD across carriers is the mean LD for the test. The mean LD must be at least 5.0 (corresponding to a geometric mean density of 1.0 × 105) and not above 6.5 (corresponding to a geometric mean density of 3.2 × 106); a mean LD below 5.0 and above 6.5 invalidates the test. Note: If the GSPT is strictly followed, mean LD values of at least 5.0 are expected; values lower than 5.0 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: 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. 14

### 2011 UDM/OECD Technical Workshop



# Acknowledgements
• Martin A. Hamilton and Gordon C. Hamilton, Big Sky Statistical Analysts LLC, and Al Parker, Montana State University’s Center for Biofilm Engineering 

## • Kiran Verma, Quality Assurance Officer for the EPA Microbiology Laboratory • Michigan Department of Agriculture, East Lansing, MI; 

## • Ohio Department of Agriculture, Reynoldsburg, OH; • North Carolina Department of Agriculture, Raleigh, NC.

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