ASTM E2315 Standard Guide for Evaluating Antimicrobial Activity Using the Time-Killing Procedure

The standard "ASTM E2315 Standard Guide for Evaluation of Antimicrobial Activity Using Time-Killing Procedure," published by the American Society for Testing and Materials (ASTM), provides an example of a method that measures changes in aerobic microorganism populations over a specified sampling period when antimicrobial test materials are available. This standard provides various options for organism selection and growth, inoculum preparation, sampling times, and temperatures.

When the technique is applied as a specific test method, it is critical that the variables mentioned above be standardized. The antimicrobial activity of specific materials measured by this technique can vary significantly depending on the variables selected. It is important to understand the limitations of in vitro testing, particularly when comparing test results performed with different parameters. For example, test results from microorganisms requiring growth supplements or specific incubation conditions may not be directly comparable to those of organisms evaluated without the specified conditions. A knowledge of microbiological techniques is necessary for this procedure.

This procedure can be used to evaluate the in vitro reduction of the microbial population of test organisms after exposure to a test material.

Suspension time killingis a laboratory test that measures the antimicrobial performance of a liquid sample over a short period of time. A kill test measures the reduction in the number of known microorganisms in an inoculum over specified contact times. This method determines the effective kill rate of a finished product and is usually expressed as a percentage (for example, killing 99,9 percent of bacteria in 30 seconds).

The standard time-kill test can be performed on any liquid finished product, such as hand sanitizers, disinfectant cleaning products, antibacterial liquid soaps, mouthwashes, and more.

The contact time selected for suspension time-killing should be appropriate for the type of finished product being tested. For example, a hand sanitizer might be tested with a 30-second contact time, while a hard surface cleaner might be tested for a longer time, depending on the usage instructions.

The kill test requires a 250-g sample of your liquid product. The sample is inoculated with a mixed culture of four standard bacterial cultures. At specific intervals (e.g., 30 seconds, 1 minute, 2 minutes, 3 minutes, and 5 minutes), 1 g of the inoculated sample is removed and placed in a biostatic diluent to inactivate the biocides present in the sample. The diluent is then placed in a suitable medium and incubated, allowing the number of surviving microbes to be quantified using the plate count method.

ASTM E2315 is a critical tool for evaluating the effectiveness of antimicrobial agents. This standard includes a method for assessing the reduction in microbial populations upon exposure to in vitro antimicrobial test materials and changes in aerobic microorganism populations over a specified period. This standard is designed to evaluate the effectiveness of products such as disinfectants, hand sanitizers, biocides, and cleaning products. This standard emphasizes flexibility by providing a variety of options for organism selection, growth conditions, inoculum preparation, sampling times, and temperatures to suit different testing needs. However, it emphasizes the importance of standardizing these variables to ensure consistent and comparable results when the method is used as a specific test.

The main features of this standard are:

• Applicability: Suitable for testing liquid or solid antimicrobial agents that form a homogeneous solution in water.
• Microorganisms: Focuses on aerobic microorganisms, with options including bacteria, fungi, and yeasts such as Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Candida albicans.
• Variables: Allows for customization of test conditions (e.g., temperature, time intervals), but requires standardization for specific tests to ensure repeatability.
• Limitations: Results may vary depending on the variables selected, and comparisons between tests under different conditions (e.g., microorganisms requiring special growth supplements) may not be valid.

ASTM E2315 describes a time-kill procedure that quantitatively measures the reduction in microbial populations over time. The process includes the following steps:

• Preparation of microbial suspension: A standard microbial suspension is prepared using representative or standard strains appropriate for the intended use of the product.
• Exposure to test material: The antimicrobial test material (or its dilution) is mixed with the microbial suspension under controlled conditions such as a specific temperature (e.g. 25 degrees, alternatives such as 22 degrees, 30 degrees or 38 degrees depending on the application).
• Sampling and neutralization: Samples are taken at predetermined time intervals (e.g., 15 seconds, 30 seconds, 60 seconds, or longer) and neutralized to inhibit antimicrobial activity. Neutralization ensures accurate enumeration of surviving microorganisms.
• Quantification: Surviving microorganisms are counted using plate counting techniques and the reduction is calculated as log10 reduction or percentage reduction compared to the starting microbial population.

Because this standard does not specify pass/fail criteria, it allows users to define efficacy based on their specific needs. Each test concentration is typically performed in duplicate to ensure reliability.

The ASTM E2315 standard is widely used to evaluate the antimicrobial effectiveness of products and provides critical data for product development, validation, and regulatory compliance. Its importance lies in its ability to:

• Measuring antimicrobial effectiveness: Time-based kill testing measures how effectively an antimicrobial agent reduces microbial populations and provides insight into kill rates against bacteria, fungi, and other pathogens.
• Supporting product development: Manufacturers use this testing to optimize formulations and ensure products meet performance expectations before they are released to market.
• Ensuring regulatory compliance: Standardized protocol helps manufacturers demonstrate compliance with industry standards, increasing product reliability in global markets.
• Broad applicability: This method can be applied across all sectors, including healthcare (e.g. disinfectants), consumer products (e.g. hand sanitizers) and industrial applications (e.g. biocides).

This standard also acknowledges the limitations of in vitro testing, stating that results may not directly reflect real-world conditions and that comparisons between tests with different parameters must be made with caution.

ASTM E2315 emphasizes that users should determine appropriate safety and health practices because the procedure may involve hazardous substances, processes, or equipment. Users should also determine the applicability of regulatory requirements before conducting the tests.

As a result, the ASTM E2315 standard is a fundamental element in evaluating the effectiveness of antimicrobial products. Its flexible yet rigorous methodology supports product development and regulatory compliance by allowing manufacturers to evaluate microbial reduction under controlled conditions. By providing a standardized approach and ensuring reliable and reproducible results, this standard has become an indispensable tool for industries seeking to effectively combat microbial contamination.

Our organization, which has been serving for many years and meticulously following global developments in science and technology, employs a skilled team and extensive infrastructure to conduct testing, measurement, and analysis in a wide range of areas for businesses across all sectors. In this context, we also provide testing services in accordance with the ASTM E2315 Standard Guide for the Assessment of Antimicrobial Activity Using the Time Killing Procedure.