Australasian Biotechnology (backfiles) AusBiotech ISSN: 1036-7128 Vol. 6, Num. 3, 1996

Rapid Microbial Analysis using Flow Cytometry.

Dan Deere PhD, Graham Vesey PhD and Duncan Veal PhD -
Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences, Macquarie University NSW 2109, Australia.

Code Number: AU96004
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This article will describe how flow cytometry permits rapid, quantitative, relatively low cost microbial analysis that is readily automated. The application of flow cytometry to microbiological analysis has, until recently, been limited by the sensitivity, reliability and cost of instrumentation combined with a lack of specific fluorescent tags for target microorganisms. Continuing advances in both instrumentation and biological reagents have now reached the level of advancement required to present flow cytometry as a valuable tool for research and routine microbial analysis.

Why Flow Cytometry?

Most routine microbial analyses still use culture-based methodologies, the foundations of which were developed more than a century ago. Such techniques are generally slow, taking from one day to several weeks to give results, and are extremely difficult to automate. More recently molecular techniques, based on the polymerase chain reaction (PCR), have become available that can be complete within a few hours. With clean samples these techniques can provide a very sensitive method of detection. However, many samples contain inhibitors of the PCR reaction, such as humic acids or metal ions, and these can severely limit detection sensitivity. Laborious clean-up procedures are required, and even then some samples may not be clean enough to permit PCR. Further limitations of PCR based methods come from the difficultly in quantification and the problems of carry-over contamination from previous reactions.

Instrumentation

Flow cytometers are used to quantitatively measure the optical characteristics of particles, such as cells, as they are presented, in single file, into a focused light beam (Fig. 1). Cells can be analysed at rates of up to 40,000 per second as they are carried within a fast flowing fluid stream termed the sheath flow. In the first flow cytometer, the light source used was the headlamp from a Ford motorcar. This flow cytometer was developed by the US military for the detection of microorganisms used in biological warfare. In modern flow cytometers the light source used is either a high pressure mercury vapour lamp or an assortment of different lasers.

As particles pass through the light beam three parameters are measured: forward angle light scatter (FALS), side angle light scatter (SALS) and fluorescence (FL). FALS provides information on the size of a particle while SALS correlates with cell refractibility and is thought to provide information on surface properties and internal structure. Natural fluorescence, (autofluorescence), is emitted by cellular components such as flavin nucleotides, pyridine and photosynthetic pigments. However, most examples of the use of flow cytometry for microbial analysis make use of fluorescent dye labels rather than autofluorescence. The cheapest and simplest flow cytometers are analysis only instruments. After analysis by the flow cytometer, samples are discarded as waste. Their operating simplicity and reproducibility make them ideal for use in quality control procedures.

Approximately double the cost buys a sorting flow cytometer. These use a variety of mechanisms to physically collect droplets containing single particles that have particular characteristics determined by the operator (Fig. 2). The large range of user changeable options on sorting instruments makes them considerably more difficult to operate than analysis only models.

Fluorescent labels

A range of instruments are available that can detect cellular microorganisms and measure their light scatter and fluorescence properties. Particles that are sufficiently different in measurable properties can be discriminated and, using cell sorting instruments, physically separated from one another. For example, it is possible to differentiate groups of microorganisms such as algae, protozoa, yeasts or bacteria simply by measuring the light scatter and autofluorescence of the individual cells. However, even with the best resolution realisable using conventional flow cytometry, most microorganisms within any particular phylum would be impossible to tell apart without artificially introducing some difference. For example, the majority of bacterial species, incredibly diverse although they are, appear very similar in terms of their light scatter and autofluorescent properties. To overcome this limitation, labelling techniques are used that discriminate groups of microorganisms by making them appear different when analysed by the flow cytometer. It is the fluorescent properties of particular target microorganisms that are modified for this purpose. Therefore, any discussion of the advances in, and utility of, flow cytometry would be incomplete without a discussion of the parallel advances in fluorescent labelling technology.

The macromolecular constitution of microorganisms can be elucidated in some detail using flow cytometry to quantitatively measure the fluorescence of cells stained with dyes that have specificity for protein, RNA or DNA. For example, stains specific for DNA are widely used to estimate ploidy.

For specifically labelling microorganisms, targeted antibodies, lectins and nucleic acid probes can be linked to a range of fluorochromes that can be detected using flow cytometry. Antibodies can be used to detect particular target species or to look for cell lines expressing particular antigens. For example, a fluorescent antibody is routinely used by water supply companies to label the parasitic protozoa Cryptosporidium and Giardia for monitoring their presence in water supplies (Vesey et al. 1994a). Cell sorting is used to physically separate the labelled protozoa onto slides for microscopic confirmation. It is possible to detect one oocyst in 10 litres of water using this method. The cell sorting and microscopic confirmation step is required because some non-target particles have, by chance, the same fluorescence and scatter properties as the labelled protozoa. Lectins bind specifically to certain microbial compounds such as cell surface polysaccharides and glycoproteins. However, they have a relatively low specificity when used for microbial detection and have not been widely used for flow cytometry.

Where antibodies of the desired specificity are not available, nucleic acid probes can be targeted to complementary nucleic acid sequences within target cells. The naturally amplified target to which nucleic acid probes are bound is the RNA of the ribosomes (rRNA) (Amman et al. 1995). Portions of the rRNA sequence have evolved at different rates and regions have been identified that are highly conserved (throughout a phylogenetic kingdom) through to highly variable (up to strain specific). This has permitted the design of probes that label cells according to their phylogeny, described by Delong et al. (1989) as phylogenetic stains. This technique has potential applications for detection of specific microorgansisms. For example, Cryptosporidium oocysts can be labelled by hybridisation with a fluorescent oligonucleotide that has specificity for a portion of the rRNA sequence of the parasite (Vesey 1996). Wallner et al (1995) used oligonucleotide probes with a range of specificities to perform a systematic analysis of the microbial community of activated sludge. Interestingly, this direct flow cytometric approach revealed that the different subdivisions of microorganisms were present in proportions markedly different to those found after examination using culture based techniques.

Methods for assessing the structural constitution, phylogeny and antigenic properties of populations of microorganisms still don't give the whole picture. Various cell functions can also be assessed using an ever increasing range of dyes. Many of these functions can be considered vital for cell survival and maintenance and the term "viability dyes" is often used to refer to molecules that stain cells in relation to these functions. For example, healthy bacterial cells possess a membrane potential (negative inside) such that they will take up the positively charged dye rhodamine 123 (Diaper et al. 1992) whilst excluding the negatively charged oxonols (Deere et al. 1995). The use of viability dyes and flow cytometry has several advantages over conventional culture techniques for measuring viability. Not only are the flow methods more rapid, but non-culturable and injured organisms are also detected and analysed. Since the flow cytometric method is quantitative, the result is more informative than simply live/dead and can be used to indicate the magnitude of the measured property.

Reporter gene expression can be detected using substrates that are cleaved to fluorescent products, these products being retained within the cell. The same principles can be applied to detect endogenous enzymatic activity. An even more direct approach makes use of the Green Fluorescent Protein reporter gene, the product of which, when expressed within cells, makes them fluoresce. Their are many other cellular properties that can be measured using an ever increasing variety of fluorescent dyes, these include dyes that stain bacterial cells in relation to Gram staining properties, or give information on intracellular pH or calcium ion concentration.

Multiple labelling

Flow cytometers measure particle fluorescence at as many as four wavelength ranges simultaneously. This means that cells can be marked with multiple fluorescent labels, of different colours, at the same time. For example, it is possible to use the green fluorescence of FITC to visualise protein, the red fluorescence of propidium iodide to label nucleic acid and simultaneously quantify both. Multiple labelling can also be used to greatly increase the specificity of detection protocols. There is always a chance that non-target cells may pick up sufficient label, or have sufficient autofluorescence at the wavelength of the label, to give false positive signals. Where two differently coloured target specific labels are used, the chances of non-target particles having the same fluorescence properties as the target becomes much more remote. For detection of a single species in complex environmental samples, dual colour labelling has been shown to greatly enhance specificity. For example, experiments in which the spores of the slime mould Dictyostelium were seeded into water demonstrated that detection could reliably be achieved, without sorting and confirmatory microscopy, using two differently labelled antibodies to give dual colour labelling (Vesey 1996). The enhanced specificity of this method over a one colour label permits detection using an analysis only cytometer, no sorting being required.

The future of flow cytometry in Microbiology

The possibilities offered by flow cytometry are constantly being expanded through development of an ever increasing range of biological labelling reagents combined with improvements in the instruments themselves. In the past, flow cytometers have been developed with the analysis of mammalian cells in mind. The types of samples analysed by the microbiologist are often very different to these. For example, microbial cells are typically smaller than mammalian cells and the samples containing the microorganisms are often very much more complex than body tissues and blood. Currently, developments in cytometry hardware are underway which are specifically designed for aiding the analysis of more complex samples. These developments include the use of array detectors to image particles (Vesey et al., 1994b) and to enable spectral fingerprinting to characterise the fluorescence of micro-organisms and fluorochromes (Gauci et al., 1996). For field sampling, the use of modern solid state lasers may even enable the development of small, robust, portable models. Future developments in instruments and biological labelling techniques are set to position flow cytometry as a major tool in microbiological research and surveillance, both complementing and replacing many existing methods.

Further reading

General flow cytometry

Shapiro, H.M. (1995) "Practical Flow Cytometry, Third Edition" (Wiley-Liss: New York). 452 pages

Darzynkiewicz, Z., Robinson, P. and Crissman, H. A. (1994). "Methods in Cell Biology vol 42: Flow Cytometry, Second Edition, parts A & B" (Academic Press: Orlando, Florida).

Flow cytometry in microbiology

Lloyd, D. (Editor). (1993) "Flow cytometry in Microbiology" (Springer-Verlag: London)

Porter, J., Deere, D., Pickup, R. and Edwards, C. (1996) Flow cytometry: New insights into environmental bacteriology. Cytometry, 23, 91-96

McFeters, G. A., Feipeng, P. Y., Pyle, B. H. and Stewart, P. S. (1995). Physiological assessment of bacteria using fluorochromes. J. Microbiol. Meth., 21, 1-13

Pore, R. S. (1994) Antibiotic susceptibility testing by flow cytometry. J Antimicrob. Chemotherapy, 34, 613-627.

Cited References

Amann, R. I., Ludwig, W., Schleifer, K. H. (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev., 59, 143-169.

Deere, D., Porter, J., Edwards, C. and Pickup, R. (1995) Evaluation of the suitability of bis-(1,3-dibutylbarbituric acid)trimethine oxonol, (diBA-C4(3)-), for the flow cytometric assessment of bacterial viability. FEMS Microbiol. Lett., 130, 165-170.

DeLong, E. F., Wickham, G. S. and Pace, N. R. (1989) Phylogenetic stains: Ribosomal RNA-based probes for the identification of single cells. Science, 243, 1360-1363.

Diaper, J. P., Tither, K. and Edwards, C. (1992) Rapid assessment of bacterial viability by flow cytometry. Appl. Microbiol. Biotechnol., 38, 268-272.

Gauci, M., Vesey, G., Narai, J., Veal, D., Williams, K. and Piper, J. (1996) Single particle spectra in flow cytometry. Cytometry accepted

Vesey, G., Hutton, P., Champion, A., Ashbolt, N., Williams, K. L., Warton, A. and Veal, D. (1994a) Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water. Cytometry, 16, 1-6.

Vesey, G., Narai, J., Ashbolt, N., Williams, K., & Veal, D. A. (1994b) Detection of specific microorganisms in environmental samples using flow cytometry. IN "Flow Cytometry, Second Edition, Methods in Cell Biology, vol 42" edited by Z. Darzynkiewicz P. Robinson and H. A. Crissman (Academic Press, Orlando, Florida). pp 488 - 521.

Vesey, G (1996) PhD Thesis, Macquarie University, Sydney.

Wallner, G., Erhart, R. and Amann, R. (1995) Flow cytometric analysis of activated sludge with rRNA-targeted probes. Appl. Environ. Microbiol., 61, 1859-1866.

Figure Legends

Figure 1. How a Flow Cytometer "Sees" Particles.

Figure 2. Flow Cytometric Sorting.

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