Webinar - Best Practices for Environmental Monitoring

Legionella Detection and Identification Methods

Key Points

  • Gram -ve non-spore forming rod
  • Require L-cysteine
  • Motile by means of polar flagella
  • Aerobic
  • Optimum growth temperature 36°C
  • Intracellular pathogen of macrophages

Legionella1 Legionella pneumophila infects the lungs and causes a sometimes fatal form of pneumonia. It is the causative organism of Legionaires Disease and Pontiac Fever. Legionnaires' disease can have symptoms like many other forms of pneumonia: a high fever, chills and a cough. Some people may also suffer from muscle aches and headaches. These symptoms usually begin 2 to 14 days after being exposed to the bacteria.

Classically Legionella spp. are found in cooling towers, air conditioners, spa equipment, fountains, humidifiers and showers, hot and cold water systems, oil/water emulsions used for lubricating lathes, misting devices, decorative fountains and water features, dentistry tools, TMV's (thermostatic mixing valves). The mode of transmission is through inhalation of airborne droplets.Legionella detection and identification methods fall into 2 categories: those methods that are aimed at the clinical diagnosis of disease and those aimed at monitoring and identifying risk in water systems. These methods are listed separately.

Prevention of Legionnaires' disease depends on good maintenance of possible sources, including regular cleaning and disinfection and the application of other physical (temperature - keeping hot water at 60°C and cold below 20°C) or chemical measures (biocide).

There have been reports in several journals of viable but non-culturable [VBNC] Legionella. In some instances it would appear that these are associated with free-living amoebae - a reported consequence of this is significantly increased resistance to biocides. Particular care must be taken when choosing the optimal test method for evaluating such samples.

There are many contract testing companies that specialise in Legionella management and testing of water.

Detection Techniques

For clinical samples, culture of the causative organism is regarded as essential for definitive proof of diagnosis (some patients do not produce antibodies) and allows for subgrouping and molecular typing (epidemiological data).

Typically a charcoal yeast extract (CYE) agar or buffered charcoal yeast extract (BCYE) agar is used. Incubation is at 35-37°C for 3 to 10 days in a moist atmosphere.

Legionella is typically isolated from water by filtration followed by heat or acid treatment then plating onto BCYE with glycine vancomycin polymyxin cyclohexamide (GVPC) agar. An alternative media, GVPN replaces the potentially hazardous cyclohexamide with natamycin. Or MWY (BCYE with glycine, polymxin B, anisomycin and vancomycin) contains dyes that will colour the target colonies aiding in their identification. Incubation is for up to 10 days at 36°C.

As an alternative to growth on agar there are a variety of technologies which may provide rapid results e.g.: antibody/antigen interactions using direct or indirect fluorescent immunoassay methods; molecular methods such as PCR/nucleic acid. Sometimes combinations of these techniques are used to further enhance the speed to result. In some situations these may be the method of choice if VBNC legionella are suspected in the sample.

PCR can eliminate the need for identification in the event of a positive result and may also provide quantitative information.


Legionella spp. have an absolute growth requirement for L-cysteine and without this amino acid they are unable to grow, colonies with typical morphology on selective media are subcultured onto BCYE and BCYE without L-cysteine. Those isolates that grow on BCYE but fail to grow on this media may be presumptively identified as Legionella spp. Quicker identification can be carried out using latex agglutination, direct immunofluoresence or molecular techniques.

Immunological methods such as latex agglutination allows rapid elimination of negative samples, positives can then be checked out with more targeted tests.

Molecular methods using PCR or nucleic acid techniques are routinely used as confirmatory tests.


There are 8000 - 18,000 cases of Legionaires disease requiring hospitalization annually in the USA - many more infections are not reported or are undiagnosed. In the UK 542 cases were provisionally reported for 2006 [figures do not include Pontiac fever].

AFNOR is currently developing the AFNOR Validation Mark for water. The examination of the first certification, for a Legionella PCR Kit, commenced at the beginning of 2007.


Related News:

Why Bio-Rad? Learn More Here!

Bio-Rad Resource Page for Industrial Diagnostic Safety SolutionsVisit Bio-Rad's new Resource Page: you will find useful information on food, beverage, industrial water and veterinary safety diagnostics. Watch our webinars and training videos and explore our digital library.

iQ-Check® Free DNA Removal Solution for Legionella

Bio-Rad has introduced a new easy and fast solution to the problem of detecting free DNA in environmental water testing which can lead to overestimation of number of Legionella present in the sample.

Legionella Analysis in 2 Hours Including VBNC Detection with rqmicro

Detect Legionella in less than 2 hours including VBNCQuantify bacterial pathogens, e.g. Legionella, within just two hours with the rqmicro lab-on-a-chip technology. The automated and highly specific method is based on the combination of immunomagnetic separation, microfluidics and flow cytometry.

New Simple, Accurate Culture Testing Method for L. pneumophila in Water

The Legiolert test is based on a bacterial enzyme detection technology that signals the presence of L. pneumophila through utilisation of a substrate present in the Legiolert reagent.

Healthcare Facilities Begin Work to Prevent Legionella Growth in Water Systems

EMLab P&K offers laboratory and planning services to Medicare certified healthcare facilities who are building and instituting water management plans in response to the Centers for Medicare and Medicaid Services’ recent memorandum.

Choose test method guide:

Select by Organism:
Select by Test Method:
Microbiology Insider - Validation and Verification
Creating anaerobic and microaerophilic incubation

© 2001 - 2018 Rapid Test Methods Ltd • Please join us on Twitter, Facebook and LinkedIn • RSS Feed