PRECISION AND ACCURACY OF RECOVERY OF LEGIONELLA-PNEUMOPHILA FROM SEEDED TAP WATER BY FILTRATION AND CENTRIFUGATION

Determination of the concentration of Legionella pneumophila in enviro nmental water sites may be useful for the prediction of the risk of a particular site's causing Legionnaires' disease as well as for experim ental studies of environmental growth or remediation. The precision an d accuracy of recovery of two different L. pneumophila strains from se eded tap water samples were studied, with either filtration or centrif ugation used to concentrate the bacteria. L. pneumophila grown on BCYE alpha agar or in Acanthamoeba castellanii was used to seed sterile ta p water. Water samples were then either filtered (0.2-mu m pore size) or centrifuged. An average of 53% (95% confidence interval [CI], 47 to 58%; n = 45) of the seeded L. pneumophila organisms were recovered by filtration with flat polycarbonate membranes. This recovery was signi ficantly higher (P < 0.01) than that obtained by filtration with cast membranes (mean, 13%; 95% CI, 11 td 38%; n = 4) or by centrifugation a t 3,800 x g for 30 min (mean, 14%; 95% CI, 2 to 25%; n = 9) or at 8,15 0 x g for 15 min (mean, 32%; 95% CI, 28 to 36%; n 19). Recovery of L. pneumophila was not significantly different whether the bacteria were grown on plates or in amoebae, Use of a selective medium did not decre ase the recovery efficiency, but preplating acid treatment of specimen s caused an approximate to 30% bacterial loss. The lower limit of dete ction of L. pneumophila from 50 mi of seeded tap water was highly vari able and was in the range of 1 to 10 CFU/ml when filtration was perfor med with single-layer membranes and the membranes were either plated d irectly or first minced and suspended in water before plating. Filtrat ion with single-layer membranes is the superior method for the recover y of L. pneumophila from seeded tap water. All concentration methods h ave highly variable recovery rates, making accurate quantification of low concentrations of L. pneumophila difficult. Even the most efficien t recovery method can be very inaccurate.