BIOTECHNOLOGY
Flow cytometer detects common bacteria in water
Paul Mortensen
Australian scientists have developed a laser-based method that swiftly detects outbreaks of microorganisms such as cryptosporidium and giardia in swimming pools and water supplies. The technique provides results overnight instead of the more-typical seven-day waiting period.
Monitoring water and food for microbial pathogens typically requires isolating and growing infectious agents in the laboratory. However, some agents of infection are difficult or impossible to grow, including giardia and cryptosporidium, which are common agents of waterborne diarrheas. To detect these agents in food or water, they must be specifically tagged with fluorescent stains so that they can be identified under a microscope.
These techniques are slow, relatively insensitive, and very labor-intensive. Furthermore, most staining methods are incapable of determining the species of the organism or if it is alive. Such problems have severely limited the effectiveness of air- and water-quality monitoring, making it difficult to quickly and sensitively detect a "needle in an environmental haystack" to safeguard public health. Scientists in the Australian Environmental Flow Cytometry Group at Macquarie University (Sydney) are using modified laser-based flow cytometers to quantitively measure the optical characteristics of cells as they pass--single-file--through a laminar flow into a focused beam of laser light (see figure).
Ruling out the dissimilar
The majority of bacterial species appear very similar in terms of light-scattering and autofluorescent properties. Unlike previous tests, this new, faster technology is able to test and rule out background particles that often obscure dangerous bacteria.
The researchers studied the autofluorescence of water samples to determine the optimal excitation source and optimal fluorescent labels for minimizing background autofluorescence--thereby enhancing detection of the organisms. They identified two major peaks as optimum--at 640 to 700 nm and 390 to 510 nm. A water-cooled argon-ion laser was used for excitation, producing 50 mW of 351-nm light to fluoresce the target cells.
Analyzing environmental samples with a flow cytometer is applying the instrument to something other than what it was designed for. During the analysis of an environmental water sample, a flow cytometer must analyze in excess of 5 billion particles per second. And this rate is only an average over the entire sample; instantaneous rates of analysis may exceed this figure.
This analysis rate is so high because, in the case of detecting pathogenic organisms in water, researchers are looking for very low numbers (up to 1 in 1012). Thus, to guarantee full recovery of targets from a sample, they must ensure that each particle passing through the analysis region of the cytometer is not a target particle; if it is, then it is sorted.
Mark Gauci, laser engineer, provided the breakthrough. His background in laser physics helped accelerate the findings of microbiologists. Chief researcher Duncan Veal noted that a very basic example was the problem with instrumentation. "We wanted the flow cytom eter to see or recognize only the particles we were interested in. Traditionally, we would have twiddled the knobs until we achieved better separation. Mark [Gauci] made a mathematical model of the problem and within weeks had resolved it." That model recorded the emission spectra of particles as a function of time and, via the Fourier transform, converted them to a spatial spectrum.
The target cells were labeled with fluorochromes (rare-earth chelates), which, when excited by the ultraviolet light, have a much longer fluorescence-emission time than average particles in water samples. By detecting the light emitted from particles some time after they were excited, the researchers could get an accurate idea of what the fluorochrome was bound to.
The opportunity for early warning means that water companies can head off public-health problems. This work has sparked international interest and attracted annual industry and government partnerships of several million dollars.