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 log10-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

OSL

Lab

absent (0) present (1)

1 2 3 4 1 2 3 4

7.0

6.5

6.0

5.5

5.0

4.5

Test LD

S.aureus

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.

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2011 UDM/OECD Technical Workshop

Test LD values for the experiments with P. aeruginosa

Multi-laboratory data set

OSL

Lab

absent (0) present (1)

1 2 3 4 1 2 3 4

7.0

6.5

6.0

5.5

5.0

4.5

Test LD

P. aeruginosa

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.

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2011 UDM/OECD Technical Workshop

Statistical Summary of Test LD Values

Multi-laboratory data set

Microbe OSL

Mean Test 

LD SEM Sr SR

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

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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.

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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 105to 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.

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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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