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D5259-24 – Standard Test Method Technical Guide
🔬 Scope and Purpose of D5259−24
This standard, designated D5259−24, covers a membrane filter (MF) procedure for the detection and enumeration of enterococci bacteria in water. The targeted organisms include Enterococcus faecalis, E. faecium, E. avium, and their varieties. These bacteria are common in the feces of humans and other warm-blooded animals, making their presence in water a reliable indication of fecal pollution and the possible presence of enteric pathogens. The detection limit for this method is one colony forming unit (CFU) per volume filtered.
The test method has been validated for use with temperate fresh waters, marine ambient waters, and wastewaters. It is the responsibility of the user to determine its applicability for waters of untested types.
🧪 Membrane Filter Procedure and Media Specifications
The method provides a direct count of bacteria based on colony development on the surface of a membrane filter. A water sample is filtered through the membrane, which retains the bacterial cells. Following filtration, the membrane is placed on a selective medium (mE agar) and incubated. Colonies are then transferred to a confirmation medium (EIA agar). Target enterococci produce characteristic red to maroon colonies with a black or reddish-brown precipitate on the underside of the membrane.
| 🟦 Parameter | 📐 Specification |
|---|---|
| Detection Limit | 1 CFU / volume filtered |
| Primary Medium | mE Agar (selective) |
| Confirmation Medium | EIA Medium |
| Colony Appearance | Red to maroon with black or reddish-brown precipitate |
| Target Species | E. faecalis, E. faecium, E. avium |
💡 Validation for Untested Waters: This standard has been successfully used with temperate fresh and marine ambient waters, and wastewaters. However, it is the user’s responsibility to ensure the validity of this test method for waters of untested types.
📊 Referenced Standards and Quality Assurance
Compliance with D5259−24 requires the use of several ancillary standards for terminology, water quality specifications, and sampling practices. The following ASTM standards directly support the execution of this test method.
| 📑 Standard | 🎯 Title |
|---|---|
| D1129 | Terminology Relating to Water |
| D1193 | Specification for Reagent Water |
| D3370 | Practices for Sampling Water from Flowing Process Streams |
| D3870 | Practice for Establishing Performance Characteristics (Withdrawn) |
⚠️ Safety Precautions: This standard does not purport to address all safety concerns, if any, associated with its use. It is the responsibility of the user to establish appropriate safety, health, and environmental practices. Users must determine the applicability of regulatory limitations and refer to Section 9 of the standard for specific hazard statements.
❓ Frequently Asked Questions
🔍 What types of water can be tested with this method? The D5259−24 test method has been successfully used with temperate fresh waters, marine ambient waters, and wastewaters. It is the user’s responsibility to validate the procedure for waters of untested types.
💡 Which enterococci species are included under this definition? According to the standard, the enterococci group includes Enterococcus faecalis (E. faecalis), E. faecium, E. avium, and their variants.
⚡ What is the stated detection limit for this membrane filter procedure? The detection limit for this standard is one colony forming unit (CFU) per volume of water filtered.
📌 What does the presence of enterococci signify in a water sample? Enterococci in water are an indication of fecal pollution from humans or other warm-blooded animals, and can signal the possible presence of enteric pathogens.