ISO 6461-2:1986

Overview

ISO 6461-2:1986 is an international standard developed by the International Organization for Standardization (ISO) that specifies a membrane filtration method for detecting and enumerating spores of sulfite-reducing anaerobes (clostridia) in water. These spores are key microbiological indicators due to their resistance to environmental stress and common water treatment procedures, making them valuable in assessing water quality and possible fecal contamination. The standard applies broadly to various types of water except when excessive particulate material would clog the membrane filter.

This method is designed to provide reliable and reproducible detection of clostridial spores, offering essential support for water quality monitoring and safety assurance.

Key Topics

• Scope of Application
  Suitable for examining all types of water, including drinking water, surface water, well water, and mineral waters. It excludes samples with high particulate content that could obstruct membrane filtration.

• Method Principle

  • Heat Selection: Samples undergo heat treatment at 75 ± 5°C for 15 minutes to eliminate vegetative bacteria, selecting for heat-resistant spores.
  • Membrane Filtration: The heat-treated water is filtered through a membrane with 0.2 µm pore size to retain spores.
  • Culturing: The membrane is placed on a selective sulfite-iron agar medium. This medium includes nutrient agar enhanced with sodium sulfite and iron sulfate, promoting growth and visualization of sulfite-reducing clostridia.
  • Incubation: Plates are incubated anaerobically at 37 ± 1°C for 20 ± 4 hours and again for 44 ± 4 hours.
  • Enumeration: Spores are identified by counting characteristic black colonies formed on the selective medium.

• Culture Media and Reagents

  • Nutrient agar enriched with sodium sulfite and iron(III) sulfate.
  • Alternative media such as tryptose-sulfite-agar may be used.
  • Preparation and quality control of media and reagents are critical for consistent results.

• Sampling and Reporting

  • Sample volumes vary depending on water contamination level, typically 100 ml for low contamination and less for highly polluted water.
  • Results are expressed as the number of spores per volume of water, typically referring to the 44-hour incubation count.
  • The test report must include method details, sample identification, and any deviations or incidents affecting results.

Applications

• Water Quality Monitoring
  Detection of spores of sulfite-reducing anaerobes is essential for monitoring microbial contamination, especially in drinking water, groundwater, surface water, and treated wastewater.

• Public Health and Safety
  Clostridial spores serve as indicators of fecal pollution and can help detect remote or intermittent pollution events due to their persistence in the environment.

• Treatment Efficiency Evaluation
  This method assists in evaluating the efficacy of water treatment processes, including chlorination and other disinfection methods, as clostridial spores often resist standard treatments.

• Environmental Studies
  Useful in microbiological studies of aquatic environments to track contamination sources and assess ecosystem health.

Related Standards

• ISO 8199: General guidance for microbiological examination of water and enumeration methods for microorganisms on culture media, supporting the interpretation of results from ISO 6461-2.

• ISO 5667 series: Guidelines for water sampling techniques (Part 2 and Part 3) ensuring representative sample collection and preservation for microbiological analysis.

• ISO 3696: Specifications for water used in laboratory testing, ensuring reagent and medium preparation does not interfere with microbial growth.

• ISO 7704: Evaluation and quality control of membrane filters used for microbiological analysis, crucial for reliable spore retention.

This standardized membrane filtration method facilitates accurate, reproducible detection and quantification of sulfite-reducing anaerobe spores in water, supporting comprehensive water quality control programs worldwide. It integrates rigorous sampling, heat treatment, selective culturing, and incubation techniques tailored for microbial water analysis professionals, environmental labs, and regulatory authorities focused on microbiological safety of water resources.