Flow cytometer detects bacteria in environmental waters

The rapid and accurate detection of bacteria in aquatic environments has typically been challenging due to the complexity of the natural water microbial populations. Traditional agar plate methods for analysing aquatic bacteria are subjective and laborious, and rely on being able to adequately culture bacteria.

The good news is, flow cytometers with a high sensitivity of detection provide tools for detecting and analysing microbes independent of their cultivability, as the size, number, nucleic acid content, activity and classification of bacteria can be derived from scattered light and fluorescence signals using flow cytometry. The Agilent NovoCyte flow cytometer can detect very small particles (down to 0.1 µm) with high sensitivity, and so can be applied to various microbial studies by combining multiparameter analytic capability and convenient fluidic maintenance.

In testing the flow cytometer’s capabilities, water samples from multiple environment waters were stained with either SYBR Green I or SYTOX Green I; both are fluorescent dyes which bind to nucleic acids. The instrument was cleaned prior to running samples, filtered buffers were used, and an unstained sample was run to distinguish background noise from fluorescent signal and appropriate scatter and fluorescent thresholding was undertaken.

Using the NovoCyte flow cytometer, the bacteria in natural water could be differentiated into two groups: bacteria of low nucleic acid content (LNA) and of high nucleic acid content (HNA) (Figure 1). It is broadly accepted that HNA is active bacteria, whereas LNA is inactive, dead or a dormant population.

Figure 1: Detection of bacteria in natural waters.

Total bacterial cell counts in multiple environmental waters was also quantified using the NovoCyte flow cytometer (see Table 1). Results demonstrated that natural waters contained the highest bacterial counts of the samples tested and bottled water the lowest. Furthermore, bacterial counts in still water were 10 to 100x higher than that of spring and mountain stream sources.

Table 1: Total cell counts of bacteria in various water samples.

The detection of bacteria in various water samples is essential to maintaining sanitary and healthy drinking conditions, and the NovoCyte flow cytometer can easily and efficiently detect and quantify bacteria in water from several sources. With detection sensitivity coupled to the automatic cell counts measured for each sample, the NovoCyte is an efficient instrument for this application.

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