Antimicrobial testing is important to prove the effectiveness of disinfectants, sanitizers, and other antimicrobial agents before they are released as safe products in the market. The AOAC International maintains such standards and the industries follow AOAC test standards for regulatory requirements, development of new products, and quality assurance. This paper details the AOAC test standards and requirements for antimicrobial testing.

Introduction to AOAC International

AOAC International is an independent global association and not-for-profit organization that develops and verifies analytical methods. This association develops methods that generally set the standard for other regulatory bodies, overseas and domestically, and in significant use by industrial professionals and at the academic level. The AOAC standards guarantee users that the antimicrobial products will work against a wide variety of microbes—bacteria, viruses, fungi, and protozoa.

Importance of AOAC Test Standards

AOAC test standards are very important for a number of reasons:

• Regulatory Compliance:  Certain standards have to be followed in order to secure approval from the relevant authorities to market the products. The regulatory bodies include the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA).
• Consumer Safety: Ensuring that the antimicrobial products are effective gives confidence to the public and consumers to use such products.
• Product Development: Improved testing standards enable manufacturers to develop better, more efficient, and superior antimicrobial products.
• Market Credibility: As a matter of fact, consumers will also establish trust in the products with tested formulations which have been validated as per AOAC standards.  This increases the credibility of the products and their performance in the markets.

Key AOAC Test Standards for Antimicrobial Testing

1. AOAC Use-Dilution Test (AOAC 955.14, AOAC 955.15, and AOAC 964.02)

One of the commonly applied methods for assessing the effectiveness of disinfectants is the AOAC Use-Dilution Test. It measures the potential of the disinfectant to kill specific test organisms which are inoculated on and absorbed into the surface.

• Procedure: Inoculation of the test organisms on the stainless-steel cylinders, drying, and subsequent exposures to the disinfectant followed by transfer of the carriers into the neutralizing solution. After neutralization, the bacterial inoculum from these treated carriers is cultured for the detection of the residual surviving organisms.
• Test Organisms:
  • AOAC 955.14 – Salmonella enterica
  • AOAC 955.15 – Staphylococcus aureus
  • AOAC 964.02 – Pseudomonas aeruginosa
• Evaluation: The disinfectant should, over a given ‘contact time’, reduce the number of organisms to a level stated. This criterion differs based on the test organisms. In case of Salmonella enterica, 59 out of 60 carrier tests must show complete disinfection, for the disinfectant to be considered effective. Similarly for Staphylococcus aureus, 57 out of 60 carriers and for Pseudomonas aeruginosa, 54 out of 60 carrier tests must demonstrate complete disinfection.

2. AOAC Germicidal Spray Products Test (AOAC 961.02)

The test’s objective is to determine the effectiveness of spray disinfectants on hard, non-porous treated materials.

• Procedure: The test surfaces (generally 60 non-porous surfaces) are inoculated with test organisms followed by treatment with the spray disinfectant. The surfaces are then rinsed with a predetermined rinsing solution after exposure for a specified contact time to nullify the antimicrobial activity of the disinfectant. The live bacterial inoculum from the surface after neutralization is enumerated by broth culture or plate count method. 
• Test Organisms: The most frequently used organisms in the test are Staphylococcus aureus and Pseudomonas aeruginosa.
• Measurement: The disinfectant is considered effective if 59 out of 60 surfaces are completely sterilized by the disinfectant spray. .

3. AOAC Fungicidal Activity of Disinfectants (AOAC 955.17)

This standard tests the effectiveness of fungicidal products.

• Procedure: Inoculate different concentrations of disinfectants with fungal spores, allow them to incubate for a defined contact time, and then transfer an inoculum from each of the concentrations in liquid growth media to allow the surviving spore to proliferate.
• Test Organisms: The common fungal organisms used are T. mentagrophytes and Candida albicans.
• Evaluation: Maximum effective dilution of the disinfectant which kills all the spores is determined.  

4. AOAC Sporicidal Activity Test (AOAC 966.04)

The test evaluates the ability of a disinfectant to kill highly resistant bacterial spores.

• Procedure: A known population of spores is inoculated to the spore carriers prior to exposure to the test chemical. After the desired time of contact, the spore carriers are transferred to a neutralizing medium for culturing.
• Test Organisms: The most widely used test organisms are Bacillus subtilis and Clostridium sporogenes.
• Assessment: The disinfectant should provide a definite extent of reduction in the viability of spores to indicate effectiveness.

Some of the other test methods include:

1. AOAC 965.13: Used for establishing the efficiency of the disinfectant in the product used for the disinfection of swimming pools and for maintaining their microbial quality.
2. AOAC 955.16: The procedure is used for the evaluation of antimicrobial efficacy of a test product by the chlorine equivalency method, relating to a referenced or otherwise known concentration of free chlorine. It is specifically developed for food processing environments.

Requirements for AOAC Test Standards

1. Test Validation and Verification

• Validation: Importance of the above procedures in the validity of the AOAC methods with respect to accuracy, precision, and reproducibility. The testing should be carried out over a wide variety of conditions and conducted by a number of operators, such that the results are reproducible and accurate.
• Verification: After the method is validated, it will be verified in the laboratory where it is intended to be used. The method will be executed with known controls to ensure it gives the expected right results.

2. Proper Handling and Preparation of Test Organisms

• Culturing: Test organisms have to be prepared for the experiments using specific protocols and conditions to guarantee reproducibility and reliability.
• Storage: Proper storage conditions have to be upheld for the viability and representativeness of the organisms.
• Inoculation: The technique to inoculate carriers or surfaces with test organisms has to be carried out very carefully to ensure uniform contamination.

3. Standardized Testing Conditions

• Contact Time: The contact time of the disinfectant shall be noted and followed in accordance with the prescribed process.
• Temperature and Humidity: The specified range of environmental conditions shall be assured with respect to temperature and humidity, within which the integrity of the obtained results is maintained.
• Neutralization: After the action of the disinfectant has been completed, a neutralizing solution is applied to provide a full stop to the action of the disinfectant. It allows the correct enumeration of survivors.

4. Quality Control and Proficiency Testing

• Quality Control: Routine quality control practices like the use of control organisms and standard reference materials should be adopted in order to maintain the reliability of test results.
• Proficiency Testing: Participation in Proficiency Testing Programs is undertaken to ensure that laboratories are competent in displaying their capability to deliver results that are accurate and of reliable quality.

At Microbe Investigations Switzerland, we specialize in conducting a wide range of AOAC tests to ensure your products meet the highest standards of safety and efficacy. Our team of expert microbiologists is well-versed with the specific requirements of various AOAC test standards, providing you with accurate and reliable results that support regulatory compliance and product integrity. From antimicrobial efficacy to contamination control, MIS offers comprehensive testing solutions tailored to your needs.

To learn more about our AOAC testing services or to discuss how we can help ensure your products meet these rigorous standards, please contact our specialists today.