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AOAC Use-Dilution Test for Disinfectant Efficacy on Surfaces
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Summary
The AOAC Use-Dilution Test is a standardized method used to evaluate the bactericidal effectiveness of disinfectants on hard, non-porous surfaces. It uses carrier-based testing to replicate real-world conditions and provides pass/fail results. . It requires a specific number of contaminated carriers to show no microbial growth after treatment, and is widely recognized by the U.S Environmental Protection Agency for supporting disinfectant registration and label claims in the United States.
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Quick understanding of the test
AOAC Use Dilution Test for Disinfectants - Evaluating the antimicrobial efficacy of disinfectants intended to be used on inanimate surfaces
The AOAC Use Dilution Test is designed to evaluate the antimicrobial efficacy of disinfectants intended to be used on inanimate surfaces.
Application
- Salmonella enterica – AOAC 955.14
- Staphylococcus aureus – AOAC 955.15
- Trichophyton mentagrophytes – AOAC 955.17
- Pseudomonas aeruginosa – AOAC 964.02
- Stainless steel “penicylinders” (carriers) are inoculated with test organisms and dried.
- Contaminated carriers are exposed to disinfectant for a specified contact period.
- Carriers are transferred to a neutralizer/growth medium and incubated.
- Pre- and post-test carrier microbial concentrations are measured.
Benefits
- Ensures disinfectants meet regulatory and performance standards for killing microorganisms on inanimate surfaces.
- Essential for manufacturers to gain product approval from regulatory agencies like the EPA.
Turnaround Time
Passing criteria
- For Salmonella enterica: At least 59 out of 60 carriers must be disinfected.
- For Pseudomonas aeruginosa: At least 54 out of 60 carriers must be disinfected.
- For Staphylococcus aureus: At least 57 out of 60 carriers must be disinfected.
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What is AOAC Use-dilution test?
AOAC Use Dilution Test was first developed in 1955. It is a laboratory assay used to determine the efficacy of disinfectants on inanimate surfaces. This test method is specified by the US EPA as the required method for validating disinfectant claims.
AOAC Use Dilution Disinfectant test method uses “penicylinders” as carriers. These carriers are inoculated with test organisms and allowed to dry out. After this, inoculated carriers are suspended in a disinfectant solution and finally transferred to a broth growth medium to check the remaining test microorganisms.
Microorganisms used in AOAC Use-dilution test
Based on the type of the test organisms, AOAC Use Dilution Test methods can be selected and performed –
- AOAC 955.14 – Salmonella enterica
- AOAC 955.15 – Staphylococcus aureus
- AOAC 955.17 – Trichophyton mentagrophytes
- AOAC 964.02 – Pseudomonas aeruginosa
New Performance Standard Criteria
Performance criteria for Use dilution method is based on the maximum number of positive carriers out of 60 per test per microbe to achieve a passing outcome for a product. Previously, up to one positive carrier out of 60 tested was the performance standard for Staphylococcus aureus (AOAC 955.15) and Pseudomonas aeruginosa (AOAC 964.02). A reassessment of the method’s performance was conducted to reduce the error rate while maintaining a practical level of testing.
The following criteria must be met for a hospital disinfectant to be considered effective:
- Each microbe must be tested three times, with each test conducted using a separate batch of the product, totaling three batches. All three batches should contain the active ingredient(s) at the lower certified limit (LCL). Each test should be performed on a different day, and testing at a single laboratory is acceptable.
- Each test must use sixty carriers, ensuring no contamination in the subculture media.
- The performance standard for S. aureus allows a maximum of 0-3 positive carriers out of sixty, while for P. aeruginosa, the limit is 0-6 positive carriers out of sixty.
- For the product to be considered effective, it must pass all tests for both microbes.
AOAC Use-Dilution test method overview (955.14, 955.15, 955.17, and 964.02)
Inoculation of carriers
- 48-hour-old culture of test microorganisms is inoculated and dried on stainless steel mini cylinders (penicylinder) referred to as carriers.
- 60 carriers are required for testing.
- For product samples labeled as one-step cleaners/disinfectants the organic soil is added to the culture before performing the test.
- Three carriers are enumerated to determine pre-test carrier microbial concentrations before transferring to disinfectants.
Exposure to Disinfectant
- Each dry and contaminated test carrier is individually transferred into 10 mL of disinfectant and exposed for a specific contact period time.
Neutralization
- At the end of the contact period, carriers are immediately transferred to the neutralizer/growth medium. Three carriers are enumerated immediately to determine post-test carrier microbial concentrations. While other treated carriers are incubated for 48 hours.
Incubation & Growth Assessment
- After incubation, treated carriers are enumerated to estimate the number of viable cells.
Understanding AOAC Use dilution test results
In AOAC Use dilution test, results are calculated and compared to the following values.
- For Salmonella enterica – At least 59 of the 60 carriers must demonstrate complete disinfection
- For Pseudomonas aeruginosa – At least 54 of the 60 carriers must demonstrate complete disinfection
- For Staphylococcus aureus – At least 57 of the 60 carriers must demonstrate complete disinfection.
AOAC Use-dilution test vs AOAC 960.09 vs AOAC 961.02
Parameter | AOAC Use dilution test ( (AOAC 955.14, 955.15, 955.17, 964.02) | AOAC 960.09 | AOAC 961.02 |
Purpose | To evaluate the antimicrobial efficacy of disinfectants intended for use on inanimate, hard, non-porous surfaces | To evaluate the sanitizing effectiveness of disinfectants intended for pre-cleaned food contact surfaces | To evaluate the germicidal disinfectant efficacy of ready-to-use spray products on hard, non-porous surfaces |
Test strains | Salmonella enterica, Staphylococcus aureus, Trichophyton mentagrophytes, Pseudomonas aeruginosa | Staphylococcus aureus and Escherichia coli | Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica |
Test type | Carrier based test | Suspension test | Carrier-based test |
Passing criteria | Qualitative pass/fail based on number of carriers showing no growth | Quantitative reduction criterion : ≥ 5 log (99.999%) reduction | Qualitative pass/fail assessment based on presence or absence of microbial growth on treated carriers |
Why products fail AOAC Use-dilution test
Products fail AOAC use dilution test due to several reasons, including:
- Insufficient antimicrobial strength
- Poor surface wetting ability
- Inadequate contact time
- Ineffective neutralization
Regulatory relevance of AOAC Use-dilution test
AOAC Use dilution test is recognized by United States Environmental Protection Agency as a validated method for disinfectant claims in the United States. It is widely used to support hospital disinfectant claims, ensuring products meet efficacy requirements against specific microorganisms on hard, non-porous surfaces. This test plays a critical role in regulatory compliance, and is often required for registering hard surface disinfectants, making it essential for market access and brand credibility.
When should you choose AOAC Use-dilution test?
Choose this test if your product:
- Is intended for hard surface disinfection
- Requires EPA registration support
- Needs real-world efficacy validation
- Is a hospital or industrial disinfectant
Why choose MIS
Microbe Investigations Switzerland (MIS) has world-class equipment and facilities based on the latest technology to perform AOAC Use-dilution tests.
For more information on the AOAC disinfectant testing method, reach out to our experts here. We would be happy to provide you with a quote and solve other related queries.
We are constantly betrothed in providing excellent customer service and support, ensuring that our testing analysis meets your needs and expectations.
Frequently Asked Questions
DR. Martinoz Scholtz
AOAC International (formerly known as the Association of Official Analytical Chemists) is a globally recognized, independent, not-for-profit association that develops and validates analytical methods used by regulatory agencies and industry to ensure the safety and integrity of foods, beverages, dietary supplements and other products.
The dilution method evaluates the ability of disinfectants to kill specific microorganisms at various concentrations. In this method, the test substance (disinfectant) is diluted in a series of decreasing concentrations and exposed to a microbial suspension. The highest dilution that kills the microorganisms is considered the effective concentration.
AOAC Use Dilution test for disinfectants is applicable to liquid-based disinfectants used on hard surfaces.
The AOAC Use Dilution test for disinfectants takes 4-5 days to complete.
We test for AOAC Use Dilution using the following microbial strains: Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538P), Enterococcus hirae (ATCC 10541), Klebsiella pneumoniae (ATCC 4352), MRSA (ATCC 33591), Salmonella enterica (ATCC 10708), Candida albicans (MTCC 3017). Additional strains can be added on request.
To pass the AOAC Use-Dilution Method test, a disinfectant must achieve a 99.9% reduction of test organism under specified conditions. Additionally, the product must demonstrate consistent performance over multiple replicates and meet the established regulatory or industry standards for the particular type of microorganism being tested, such as bacteria or fungi.
AOAC Use dilution test is used to evaluate the efficacy of liquid disinfectants on hard, non-porous surfaces by checking whether the bacteria survive after treatment.
No, the AOAC Use-Dilution Method is specifically designed for testing on hard, non-porous surfaces. It may not be suitable for porous surfaces or materials that can absorb the disinfectant which could affect the test results.
Common challenges in microbial analysis include controlling variables, preparing test surfaces, managing microbial variability, and effectively neutralizing disinfectants to prevent further microbial kill during analysis.
The frequency of disinfectant product testing depends on regulatory requirements, product changes, and quality assurance protocols. It should typically be conducted during product development and before the market release. Products should also be tested periodically as a part of ongoing quality control efforts during manufacturing processes.
The AOAC Use Dilution Method is crucial for standardizing disinfectant efficacy testing approaches, ensuring safety of the product, supporting compliance, and building trust in disinfectant products, as it helps manufacturers meet regulatory and industry standards.
AOAC Use-Dilution is a carrier-based, qualitative test that evaluates whether a disinfectant can kill bacteria on contaminated surfaces, with results interpreted as growth or no growth. Whereas, suspension tests are quantitative and measure how much the microbial count is reduced in a liquid system. Carrier tests better replicate real-world conditions compared to suspension tests.
A disinfectant passes if a specified number of carriers show complete disinfection (e.g., ≥59/60 for Salmonella, ≥57/60 for Staphylococcus, ≥54/60 for Pseudomonas)
Yes, It is an EPA-recognized method required to substantiate disinfectant claims for product registration in the U.S.
AOAC Use Dilution test for disinfectants is applicable to liquid-based disinfectants used on hard surfaces.
Yes, AOAC Use dilution testing can be customized based on product type and specific client requirements. At MIS, we offer bespoke testing services, tailoring study design to match your product needs.
AOAC Use-dilution test typically takes 3-4 weeks to complete
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