ISO 11731:2017: Isolation and Enumeration of Legionella with Membrane Filtration

Legionnaires’ Disease is a type of pneumonia that is caused by Legionella bacteria, which commonly grows in warm, stagnant water. Infection is acquired by inhalation of contaminated water from man-made water sources [1]. Due to the public health risks associated with Legionella, routine monitoring of water systems is an important component of water quality management programs. ISO 11731: 2017 provides standardized methods in the isolation and enumeration of Legionella in water [2].

Overview of ISO 11731:2017

Clause 8 of ISO 11731:2017 describes the analytical workflow used for Legionella testing, including:

  • Sample preparation (8.1)
  • Concentration of microorganisms from water samples (8.2)
  • Sample pretreatment (8.3)
  • Culture methods (8.4)
  • Confirmation of Legionella colonies (8.5)

Among these sections, Clause 8.2 is particularly important because it outlines the membrane filtration techniques commonly used to concentrate microorganisms prior to culturing.

Membrane Filtration Approaches

Membrane filtration procedures for the isolation and enumeration of Legionella utilize equipment commonly found in microbiology laboratories. Laboratories processing smaller sample volumes often use a single filtration assembly such as Rocker Scientific VF3 Glass Filtration Set or a Rocker Scientific VF6 Glass Filtration Set.

 

For higher throughput workflows, glass microanalysis filters paired with stainless steel vacuum manifolds are often preferred. Multi-place, autoclavable manifolds allow laboratories to process multiple water samples simultaneously, improving efficiency while maintaining consistent filtration conditions. Depending on the model, manifolds can concentrate up to 12 samples in a single filtration cycle.

Direct Filtration Method (8.2.2)

The direct filtration method described in section 8.2.2 utilizes Mixed Cellulose Ester (MCE) membranes to capture microorganisms from water samples. Following filtration, the membrane filter is carefully transferred to a Petri Dish containing the growth media. Common membrane specifications used for Legionella testing include MCE or cellulose nitrate membranes with pore sizes of 0.2 µm or 0.45 µm and a diameter of 47 mm. Examples include:

Concentration and Recovery Method (8.2.3)

The concentration and recovery method described in section 8.2.3 incorporates an additional recovery step prior to culturing. Water samples are filtered through a Polyethersulfone Membrane or a Hydrophilic Polycarbonate Membrane Filter this concentrates the cells on the membrane to avoid loss of residual cells and maximum cell collection. After filtration, the membrane is removed from the filter holder and placed in a sterile container with a screw cap, with optional sterile glass beads. 5 to 10 ml of sterile diluent is then added, and the sample is shaken vigorously. An ultrasonic water bath or a vortex mixer may also be used to improve recovery efficiency. Following recovery, the suspension may be plated directly or subjected to membrane filtration before transfer to culture media using the direct filtration workflow described in section 8.2.2.

Selection of filtration equipment should be based on laboratory throughput requirements and sample volume. The choice between direct filtration and concentration/recovery methods depends largely on the water source and expected microbial load. Clean water, like those present in municipal water systems distributed into the household is expected to have fewer cells so a concentration step ( 8.2.3) is preferred. In contrast, environmental samples from stagnant water systems, cooling towers, ponds, or rainwater collection systems may contain higher microbial loads, making direct filtration an effective and efficient approach.

Whether utilizing a single filtration assembly or a high-throughput vacuum manifold, proper membrane selection and sample handling are critical for achieving accurate and reproducible Legionella testing results in accordance with ISO 11731:2017.

References:
([1] About Legionnaires' Disease | Legionella | CDC

[2] ISO 11731:2017 - Water quality — Enumeration of Legionella