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ASTM E2315 Time-Kill Test for Antimicrobial Activity
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Summary
ASTM E2315 is a quantitative suspension test used to measure how rapidly a disinfectant or antiseptic kills microorganisms. It calculates microbial reduction in log values after a defined contact time under controlled laboratory conditions. The method is widely used by manufacturers to validate antimicrobial performance, compare formulations, and support regulatory and marketing claims in healthcare, personal care, and biocide industries.
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Quick understanding of the test
ASTM E2315: 2016 - Standard Test Method for Time-Kill Antimicrobial Activity
- Pseudomonas aeruginosa (ATCC 15442)
- Staphylococcus aureus (ATCC 6538)
- Enterococcus hirae (ATCC 10541)
- Escherichia coli (K12) (NCTC 10538)
- Salmonella typhimurium
- Lactobacillus brevis
- Enterobacter cloacae
- Microbial suspension is prepared and standardized.
- The test sample is mixed with the microbial suspension under defined conditions.
- At predetermined time intervals, a small amount of test mixture is collected and neutralized.
- Surviving microorganisms are quantified via plate counting.
- Microbial reduction over time is analyzed and recorded.
- This test quantitatively measures how effectively an antimicrobial agent reduces microbial populations over time, essential for gauging its kill rates against bacteria, fungi, and other pathogens.
- This test is crucial for developers of antimicrobial products, as it provides essential data that can influence formulation adjustments before market release
Turnaround Time
Percent reductions are calculated by comparing the initial microbial count with the number of surviving organisms after exposure.
Passing criteria
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What is ASTM E2315 Time-Kill Testing?
ASTM E2315 provides a standardized guide to evaluate the antimicrobial activity of test materials using a time-kill procedure. The method measures the reduction in a population of microorganisms after exposure to an antimicrobial substance for defined time intervals. It offers a flexible and reproducible approach to assess the performance of disinfectants, sanitizers, preservatives, and other antimicrobial agents under laboratory conditions.
ASTM E2315 is commonly used to support antimicrobial efficacy claims for disinfectants, antiseptics, and biocidal formulations. While not a surface carrier test, it provides foundational kill-rate data often required for product development validation and internal quality benchmarking.
Scope of ASTM E2315 Standard Guide
Scope of ASTM E2315 can be applied to liquid antimicrobial products evaluated in suspension against selected microorganisms. It is especially suitable for manufacturers looking for early-stage R&D screening , determining rapid kill claims (30 sec, 1 min), studying kill kinetics, comparing active ingredients and reformulation validation
Products Tested Under ASTM E2315
This test method can be used to determine the antimicrobial efficacy of various products
- Antibacterial liquid soaps
- Mouthwashes
- Disinfectants
- Hand sanitizers
- Surgical scrubs
- and other liquid antimicrobial formulations
ASTM E2315 Test Conditions & Contact Times
Test conditions | |
Bactericidal activity
Yeasticidal activity Fungicidal activity | Test organisms |
Pseudomonas aeruginosa (ATCC 15442) Staphylococcus aureus (ATCC 6538) Enterococcus hirae (ATCC 10541) Escherichia coli (K12) (NCTC 10538) Salmonella typhimurium Lactobacillus brevis Enterobacter cloacae Additional strains as per customer request Candida albicans (ATCC 10231) Saccharomyces cerevisiae Aspergillus brasiliensis (ATCC 16404) | |
Contact time | 15 seconds, 30 seconds, 60 seconds, or any time range from several minutes to hours. |
Temperature | 25 °C Additional temperatures are given depending on the intended use of the test product: Room temperature: 22 ± 2°C Human skin: 30 ± 2°C Warm water: 38 ± 2°C |
The test organisms chosen can be indicative of the microbial population found in the specific conditions in which the test product is used or may represent standardized strains.
ASTM E2315 Test Method Explained
Inoculum preparation
- For bacteria, 24-hour-old cultures cultivated in nutrient broth are used for the experiment. For fungi, spore preparation from a saline wash can be used.
Exposure Time & Contact Conditions
The microbial suspension is mixed with the antimicrobial product and exposed for defined time intervals.
Neutralization & Recovery
- After contact times(e.g., 30 seconds, 60 seconds), aliquots (typically 1 ml) are removed from the test mixture and neutralized to quench the antimicrobial activity of the test product.
- To determine the initial microbial concentration, a saline control is inoculated with the same test microbes.
Incubation
- The number of viable organisms from neutralized mixture are enumerated using the spread plate or pour-plate technique.
- The plates are then incubated for 24-48 hours at a specified temperature.
Enumeration & Log Reduction Calculation
- The microbial population is enumerated and expressed in terms of CFU/mL.
- Log₁₀ reduction or percent reduction are calculated by comparing the initial microbial count with the number of surviving organisms after exposure.
Log Reduction Interpretation (3-log, 5-log etc.)
Log reduction represents the magnitude of microbial kill:
- 3-log = 99.9% reduction
- 5-log = 99.999% reduction
A 3-log reduction means the microbial count has been reduced by 1,000 times. A 5-log reduction means a 100,000-fold reduction.
Required log reduction depends on product claims, intended application or as per client’s specifications.
Strengths of Time-kill testing
The strengths of the time kill test method include its ability to:
- Provide quantitative data on antimicrobial efficacy.
- Assess a wide range of microorganisms and products.
- Offer reproducible and standardized results.
- Support compliance with regulatory requirements.
Limitations of Time-Kill Testing
- ASTM E2315 is in vitro time-kill study, so results may not fully represent real-world environment or product use.
- Microorganisms that require special nutrients or incubation conditions may produce results that can’t be directly compared to organisms grown without those conditions.
- Assesses antimicrobial activity in liquid suspensions, not on solid surfaces, fabrics, or complex materials.
ASTM E2315 vs Other Standards
Feature | ASTM E2315 (Time-Kill Test) | ASTM E1054 (Neutralizer Evaluation) | EN 1276 |
Purpose | Measures antimicrobial activity by quantifying reduction of a microbial population over defined contact times in a suspension test. | Evaluates the effectiveness of neutralizers/inactivators to stop antimicrobial action during efficacy tests. | Phase 2 step 1 suspension test for assessing the bactericidal activity of chemical disinfectants and antiseptics. |
Mandatory Test Organisms | Pseudomonas aeruginosa (ATCC 15442), Staphylococcus aureus (ATCC 6538), Enterococcus hirae (ATCC 10541), Escherichia coli (K12) (NCTC 10538) , Salmonella typhimurium, Lactobacillus brevis, Enterobacter cloacae | Neutralizer tests use the same organisms as the efficacy tests to confirm neutralizer performance. | Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus hirae |
Scope of Products | Liquid antimicrobial products: disinfectants, hand sanitizers, antimicrobial soaps, mouthwashes, surgical scrubs, and similar formulations. | Neutralizers and inactivators used across antimicrobial efficacy assays | Disinfectants and antiseptics intended for food, industrial, domestic and institutional areas |
Results Interpretation | No fixed pass/fail in the standard; performance interpreted based on claimed log reduction (e.g., 3-log, 5-log)reduction | Results are interpreted by comparing microbial recovery in neutralizer controls versus growth controls. | ≥5-log reduction for bactericidal claim under specified conditions |
Regulatory Relevance of ASTM E2315
ASTM E2315 data is often submitted as supporting evidence for EPA disinfectant validation in the United States, specifically to assess kill kinetics and contact-time performance during product development, but it does not replace EPA-mandated efficacy methods. In FDA OTC antiseptic products, this test method is applicable as a non-clinical, in vitro approach for measuring antimicrobial performance, but is not alone a requirement for compliance.
In the area of hospital disinfectant efficacy claims, ASTM E2315 is a quantitative method generating log reduction data that can be used to support rapid or time-dependent antimicrobial performance. In general, this test method is frequently used for label claim substantiation, providing defensible, repeatable log reduction data consistent with the use and contact time claims, but acceptance is subject to the regulatory testing requirements.
Why Choose Our Lab for ASTM E2315 Testing
At Microbe Investigations Switzerland, we assist our customers in identifying the antimicrobial efficacy of their products. Our highly qualified microbiologists follow systematic testing procedures in accordance with ASTM E2315 guidelines. Equipped with top-notch instruments and advanced facilities, our labs ensure a meticulous antimicrobial evaluation process by significantly reducing the likelihood of errors.
Request ASTM E2315 Testing
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Frequently Asked Questions
DR. Martinoz Scholtz
ASTM E2315 assesses the antimicrobial Activity Using a Time-Kill Procedure. It measures how rapidly and to what extent a test antimicrobial product kills a known inoculum of microorganisms within specific contact times.
Log reduction is calculated by comparing viable counts at the beginning and post exposure:
log reduction = log₁₀(initial count) − log₁₀(final count). A higher log reduction equals greater kill.
A 3-log reduction means a 99.9 % kill of organisms, and a 5-log reduction means a 99.999 % kill.
ASTM E1054 is used to validate neutralizers (inactivators) used in antimicrobial tests to ensure that product’s antimicrobial action at the exact sampling point. . ASTM E2315 is the efficacy test, while E1054 ensures neutralization is effective so results are accurate.
ASTM E2315 is often used as a pre-registration or supporting study to screen formulations and generate preliminary efficacy data. However, final regulatory approval for surface disinfectants typically relies on carrier-based efficacy methods rather than suspension time-kill testing alone.
The latest published version is ASTM E2315-23, released April 1, 2023
ASTM E2315 itself does not set pass/fail criteria; acceptance thresholds (e.g., 3-log, 5-log) depend on your claim requirements or regulatory guidelines.
Neutralization must be validated (often per ASTM E1054) to show that the neutralizer stops the antimicrobial activity without harming organisms, ensuring surviving counts reflect true kill at contact times.
ASTM E1052 is specifically a virucidal suspension test measuring virus inactivation in liquid; ASTM E covers bacterial/fungal kill kinetics in suspension. They target different organism types and endpoints.
E2315 is a suspension test (liquid phase), while useful for antimicrobial activity, it is not a dedicated surface contact test. Surface efficacy usually requires a surface carrier or quantitative surface test method.
Log reduction quantifies how many orders of magnitude of viable microorganisms are killed. A higher log reduction means more effective kill.
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