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Applied and Environmental Microbiology, July 2005, p. 4086-4096, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.4086-4096.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water

Pilar Delgado-Viscogliosi,1* Tristan Simonart,1 Virginie Parent,1 Grégory Marchand,1 Marie Dobbelaere,1 Eric Pierlot,1 Véronique Pierzo,1 Florence Menard-Szczebara,2 Elisabeth Gaudard-Ferveur,2 Karine Delabre,2 and Jean Marie Delattre1

Département Eaux et Environnement, Institut Pasteur de Lille, F-59019 Lille Cedex,1 Laboratoire Central, Anjou Recherche, Veolia Water, F-94417 Saint-Maurice, France2

Received 10 September 2004/ Accepted 8 February 2005

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.

* Corresponding author. Mailing address: Département Eaux et Environnement, Institut Pasteur de Lille, F-59019 Lille Cedex, France. Phone: 33 3 20 87 71 20. Fax: 33 3 20 87 73 83. E-mail: pilar.viscogliosi{at}pasteur-lille.fr.

Applied and Environmental Microbiology, July 2005, p. 4086-4096, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.4086-4096.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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