Unveiling Legionella: A Comprehensive Exploration of ISO 11731:2017 Procedure for Accurate Enumeration

January 24, 2024 by
Unveiling Legionella: A Comprehensive Exploration of ISO 11731:2017 Procedure for Accurate Enumeration
Kadesha Dawkins


Legionella, the elusive bacterium inhabiting surface waters, takes center stage in ISO 11731:2017, a pivotal standard governing health and hygiene criteria in legionellosis prevention. Defined by RD 865/2003, this procedure unravels the mysteries of Legionella, delving into its morphology, characteristics, and the intricate methodology required for precise enumeration.

Legionella Characteristics

A bacillus with dimensions ranging from 0.3 to 0.9 μm wide and 1.5 to 5 μm in length, Legionella exhibits weak Gram staining (gram-negative) and motility through polar or subpolar flagella. As a strict aerobic microorganism, it thrives in surface waters like lakes and rivers, becoming part of the natural bacterial flora.

Legionella Prevention and Control

 Addressed by RD 865/2003, Legionella prevention is achieved through routine maintenance of piping systems, microbial status monitoring, and proper disinfecting processes. Legionella analysis becomes the linchpin, assessing risks and determining the effectiveness of preventive and corrective measures.

Legionella Counting Procedure

  • Pre-Treatment and Selective Isolation

    • Sample division into ACID, HOT, and UNTREATED portions.
    • Streaking onto Legionella BCYE Agar and another medium for diversified analyses.
  • Membrane Filtration + Elution: Concentration/Dilution

    • Generation of three subsamples—Unconcentrated, concentrated, and diluted.
    • Utilization of membrane filtration, elution, and a meticulous process of concentration and dilution.
  • Sample with a High Concentration of Accompanying Microorganisms

    • HOT followed by ACID treatment.
    • Dilutions and selective isolation for precise enumeration amid microbial complexity.
  • Sample with an Extremely High Concentration of Accompanying Microorganisms

    • Plates' incubation at 36 ± 2ºC for 7/10 days.
    • Growth observations at intervals, followed by confirmatory subcultures to distinguish Legionella from microbial companions.
  • Common Procedure Regardless of Sample Type

    • Unified selective isolation on Legionella BCYE Agar and BCYE + AB Agar for baseline comparison across diverse sample compositions.


ISO 11731:2017 emerges as a meticulous guide through Legionella enumeration, considering varied sample scenarios. From pre-treatment nuances to confirmatory measures, this standardized procedure not only upholds health and hygiene criteria but also serves as a beacon for preventive and corrective measures against legionellosis. In the realm of microbiology, it stands as a key to understanding, assessing risks, and ensuring the safety of water distribution networks.

Legionella BCYE Agar 1311

AB Agar 6022

Legionella GVPC Agar 6025

Legionella MWY Agar 6067

REST Client (Huachao Mao)