Rapid on-site monitoring of Legionella pneumophila in cooling tower water using a portable microfluidic system

Abstract

Legionnaires' disease, predominantly caused by the bacterium Legionella pneumophila, has increased in prevalence worldwide. The most common mode of transmission of Legionella is inhalation of contaminated aerosols, such as those generated by cooling towers. Simple, rapid and accurate methods to enumerate L. pneumophila are required to prevent the spread of this organism. Here, we applied a microfluidic device for on-chip fluorescent staining and semi-automated counting of L. pneumophila in cooling tower water. We also constructed a portable system for rapid on-site monitoring and used it to enumerate target bacterial cells rapidly flowing in the microchannel. A fluorescently-labelled polyclonal antibody was used for the selective detection of L. pneumophila serogroup 1 in the samples. The counts of L. pneumophila in cooling tower water obtained using the system and fluorescence microscopy were similar. The detection limit of the system was 10⁴ cells/ml, but lower numbers of L. pneumophila cells (10¹ to 10³ cells/ml) could be detected following concentration of 0.5-3 L of the water sample by filtration. Our technique is rapid to perform (1.5 h), semi-automated (on-chip staining and counting), and portable for on-site measurement, and it may therefore be effective in the initial screening of Legionella contamination in freshwater.

Figure 1

Detail of the microfluidic device for on-chip staining and counting Long and short lengths are 50 mm and 25 mm, respectively. Width of the channel is 100 μm, with the exception of the “mixing part” (500 μm). Depth of the channel is 15 μm. Sample, fluorescent dye solution and sheath fluid were injected at each inlet. The process of on-chip staining and counting of bacterial cells is shown and includes: (i) the sample and fluorescent dye solution flowing separately and becoming mixed in the “mixing part” of the microchannel, (ii) alignment of the sample flow by the sheath fluid, and (iii) the flow of bacterial cells in the “detecting part” of the microchannel.

Figure 2

Portable microfluidic system for on-site bacterial monitoring.

Figure 3

Correlation between microfluidic counts and conventional fluorescence microscopic counts of L. pneumophila stained with a fluorescent antibody. Cultured L. pneumophila cells were spiked in cooling tower water. Error bars indicate the standard deviation (n = 5). Samples with 10¹ cells/ml of Legionella cells were counted following 1000-fold concentration by filtration. Samples with 10² and 10³ cells/ml of Legionella were 100-fold concentrated before counting, and samples with 10⁴ to 10⁶ cells/ml of Legionella were counted without concentration.

Figure 4

Monitoring of L. pneumophila in cooling tower water by fluorescence microscopy and the portable microfluidic system.