Physical Address
304 North Cardinal St.
Dorchester Center, MA 02124
D5916-96 – Standard Test Method Technical Guide
🔬 Scope and Significance of D5916-96
This test method is designed for the detection and enumeration of Clostridium perfringens spores and vegetative cells from marine water, sediment, wastewater, ambient water, and drinking water. C. perfringens is defined biochemically as an obligate anaerobic, Gram-positive, spore-forming, nonmotile bacillus (0.9–1.3 by 3.0–9.0 µm) that ferments sucrose and lactose with stormy gas production, does not ferment cellobiose, and produces acid phosphatase. Due to its highly resistant endospores—single, oval, subterminal, and less than 1.0 µm in diameter—the organism serves as both an indicator of present fecal contamination and a conservative tracer of past pollution events. These spores are notably resistant to heat, drying, and chemical disinfectants that would kill vegetative forms, preserving the organism for long periods.
⚠️ Safety Responsibility: C. perfringens produces toxins capable of causing gas gangrene and gastroenteritis. This standard does not purport to address all safety concerns. The user must establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
⚙️ Membrane Filtration Procedure and Incubation
Appropriate volumes of water are passed through membrane filters with retention characteristics per ASTM D3863. The membrane is placed on mCP agar, a selective medium modified by Armon and Payment from the formulation of Bisson and Cabelli. Incubation is conducted under strict anaerobic conditions at a precise temperature of 44.5°C for 24 hours. The selectivity of the mCP medium and the elevated incubation temperature suppress competing bacteria while promoting the recovery of C. perfringens.
💡 Methodological Note: Because of the high selectivity of the mCP medium, presumptive counts are normally reported for routine monitoring. The standard recommends that verification steps be conducted only when specific investigative or regulatory requirements dictate.
📊 Enumeration and Confirmation Characteristics
Following incubation, colonies that appear yellow to straw-colored on the mCP agar are presumptively identified as C. perfringens. When exposed to ammonium hydroxide fumes, these colonies shift to a distinct dark pink or magenta color. Counts are reported as colony forming units (CFU) per 100 mL. Confirmed identification is based on the specific metabolic and morphological traits defined in the standard.
| 🟦 Characteristic | 📏 Specification |
|---|---|
| Colony Morphology (mCP) | Yellow to straw-colored; turns dark pink/magenta with NH₄OH |
| Cellular Morphology | Gram-positive, nonmotile bacillus (0.9–1.3 x 3.0–9.0 µm) |
| Spore Morphology | Single oval subterminal spore (< 1.0 µm diameter) |
| Lactose Fermentation | Positive (stormy gas production) |
| Cellobiose Fermentation | Negative |
| Acid Phosphatase | Positive |
| 📐 Test Parameter | ⚡ Critical Requirement |
|---|---|
| Filtration Medium | Membrane Filter (per ASTM D3863) |
| Culture Medium | mCP Agar (Armon & Payment modification) |
| Incubation Time | 24 hours |
| Incubation Temp. | 44.5°C |
| Atmosphere | Strict Anaerobic |
❓ Frequently Asked Questions
🔍 What sample matrices are validated for this method?
The standard validates the method for marine water, sediment, wastewater, ambient water, and drinking water. It is the user’s responsibility to ensure the validity of the test method for waters of untested matrices.
💡 How are presumptive C. perfringens colonies verified?
Verification requires demonstrating the specific traits defined in Section 3.2.1: Gram-positive and nonmotile; stormy gas production from lactose; negative cellobiose fermentation; and positive acid phosphatase production. Spore staining confirms the characteristic subterminal endospores.
⚡ Why is the strict anaerobic incubation at 44.5°C critical?
The thermophilic temperature of 44.5°C is highly selective, favoring the growth of C. perfringens while suppressing many competing mesophilic bacteria, including other Clostridium species. The anaerobic atmosphere is required due to the organism’s obligate anaerobic metabolism.
📌 What makes C. perfringens a “conservative tracer”?
The organism produces highly refractile endospores that are resistant to heat, drying, and chemical disinfectants that would kill vegetative bacteria. This resistance preserves the organism for long periods, making it a persistent indicator of past or remote fecal contamination events in environmental waters and sediments.