Application of flow cytometry to studies of pathogenic free-living amoebae

Abstract

Species of small, free-living amoebae of the genera Naegleria and Acanthamoeba can cause fatal amoebic meningoencephalitis. Previous investigations have shown that pathogenic amoebae are associated with thermally altered water. Flow cytometric techniques for identifying species of pathogenic and nonpathogenic amoebae from such water have been developed, using immunofluorescence and fluorescein-bound concanavalin A. Flow cytometry is accomplished with a cytofluorograph, in which cells are dispersed in a suspended carrier liquid and passed in front of a focused argon ion laser beam. Cells are then distinguished by the degree of scattered light (size) or fluorescence. Flow cytometry techniques have proven efficient for environmental samples, as indicated by the identification of pathogenic Naegleriafowleri and nonpathogenic Naegleri gruberi and Acanthamoeba castellanii isolated from the Savannah River Plant in South Carolina. Cytofluorographic analysis of environmental samples has several advantages over the current methods of isolation and classification of free-living amoebae. With this system, it is possible to rapidly identify species and quantitate mixtures of pathogenic amoebae in environmental samples. Cytofluorographic analysis of amoebic isolates reduces the time presently required to screen environmental sites for pathogenic amoebae. The cytofluorograph permits detection and species identification of nonthermophilic Naegleria spp. and Acanthamoeba spp. that could not easily be isolated for species identification by conventional methods. Other advantages of flow cytometry over fluorescent microscopy include a high degree of statistical precision due to the large numbers measured, high immunofluorescent titers, and elimination of subjectivity and fluorescence fading.

High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining

“…To use a wider range of protozoa, and to screen the largest number of samples, we modified our isolation procedures slightly and implemented a new high-throughput automated method to detect the presence of giant viruses infecting protozoa in liquid media by flow-cytometry. Flow cytometry-based methods have been previously described for their applications in studies involving pathogenic free-living amoeba ( Muldrow et al, 1982 ; Avery et al, 1995 ) but have not focused on isolation-related studies. We associated enrichment methods with flow cytometry.…”

Application of flow cytometry to detection and characterization of Legionella spp

“…These various procedures for detecting and analyzing Legionella populations are laborious, time consuming, and costly. We have previously described the application of flow cytometry (FCM) in detecting the presence of the amoebic pathogen Naegleria fowleri in thermally altered water (10). Ingram et al (8) have described the cytofluorographic profiles of laboratory cultures of Legionella pneumophila.…”

Biology of Naegleria spp.

“…Environmental samples, particularly those from heated waters of >40°C, often contain thermophilic nonpathogenic N. lovaniensis growing in competition with pathogenic N. fowleri (205). Flow cytometric techniques for rapidly identifying and quantitating species of pathogenic and rionpathogenic amoebae in environmental samples have been reported (152). Rapid identification of environmental isolates of Naegleria spp.…”