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D5246-24 – Standard Test Method Technical Guide
🧪 Scope and Significance of D5246-24
ASTM D5246-24 covers the standard test method for the isolation and enumeration of the opportunistic pathogen Pseudomonas aeruginosa from water. Testing was performed on spiked samples using reagent grade water as the diluent. The intended matrices include surface waters, recreational waters, ground water, water supplies (especially rural nonchlorinated sources), waste water, and saline waters. The detection limit of this test method is one microorganism per 100 mL. As a causative agent of numerous infections in susceptible hosts, the membrane filtration procedure described in this standard provides a rapid and reliable means of detecting P. aeruginosa in water samples.
🔬 Key Terminology and Organism Profile
Accurate application of this standard requires a clear understanding of the specific organism characteristics and defined terminology. The following table summarizes the critical biological and procedural definitions provided in the standard.
| 🟦 Term / Feature | 📖 Definition or Specification |
|---|---|
| Pseudomonas aeruginosa | An aerobic, motile, gram-negative rod that produces fluorescent pigments and pyocyanin. |
| Biochemical Profile | Oxidase and caseinase positive; able to grow at 42 °C; may utilize acetamide. |
| Antibiotic Resistance | Relatively resistant to many antibiotics. |
| Refrigeration | Storage of samples at 2 °C to 8 °C. |
| Detection Limit | One microorganism per 100 mL. |
🛠️ Key Procedural Note: Strict temperature control is critical. The M-PA-C medium provides optimal selectivity when incubated at precisely 41.5 ± 0.5 °C for the full 72-hour duration, which effectively suppresses background flora while promoting P. aeruginosa growth.
⚙️ Test Method Procedure and Conditions
The standard procedure requires passing a water sample through a 0.45 µm (or equivalent) membrane filter. The filter is placed on M-PA-C selective medium and incubated under specific conditions. Following incubation, characteristic pink-brown to black colonies are enumerated. The table below outlines the precise parameters required for successful execution of the test.
| 📏 Parameter | 🎯 Specification |
|---|---|
| Membrane Filter | 0.45 µm or equivalent |
| Primary Isolation Medium | M-PA-C Agar |
| Confirmation Medium | Skim Milk Agar |
| Incubation Temperature | 41.5 ± 0.5 °C |
| Incubation Duration | 72 hours |
| Target Colony Appearance | Pink-brown to black |
| Validated Water Types | Reagent, Surface, Recreational, Ground, Rural, Waste, Saline |
Resulting colonies matching the target morphology are counted and reported as colony-forming units (CFU) per 100 mL. Verification on skim milk agar is recommended for definitive confirmation of P. aeruginosa.
⚠️ User Validation Requirement: Per Section 1.2 of the standard, while this test method was successfully tested with reagent water, it is the user’s responsibility to ensure the validity of the test method for their specific water matrix, including surface waters, saline waters, and chlorinated supplies.
❓ Frequently Asked Questions
🔍 What is the detection limit of ASTM D5246-24?
The standard specifies a detection limit of one Pseudomonas aeruginosa microorganism per 100 mL of sample.
💡 What media are specified for isolation and confirmation?
M-PA-C medium is specified for primary selective isolation. Colony verification is performed using skim milk agar.
⚡ What are the exact incubation conditions required?
The membrane filter on M-PA-C agar must be incubated at 41.5 ± 0.5 °C for a total duration of 72 hours.
📌 What types of water samples are covered by this standard?
The scope covers surface waters, recreational waters, groundwater, rural non-chlorinated water supplies, wastewater, and saline waters.