Journal of Applied Bacteriology 1992, 73, 438-444
Characterization of bacteria by multiparameter flow cytometry R. Allman, A.C. Hannl, R. Mancheea and D. Lloyd Department of Pure and Applied Biology and 'Preclinical Electron Microscopy Unit, Department of Physiology, University of Wales College of Cardiff, Cardiff and 2Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire, UK 4084/01/92: accepted 6 July 1992
A n arc-lamp based flow cytometer was used to obtain high resolution measurements o f t h e light scattering characteristics a n d DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning a n d transmission electron microscopy of t h e bacterial samples demonstrate that t h e structural basis of t h e light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. T h e use of a third measurement parameter, DNA content, allowed much better discrimination of t h e organisms. Flow cytometry shows great promise as a method for t h e rapid discrimination and identification of bacterial populations. R . A L L M A N , A . C . H A N N , R . M A N C H E E AND D. L L O Y D . 1992.
INTRODUCTION
The need for new approaches to bacterial identification stems largely from the limitations of classical diagnostic microbiology. Clinical and environmental laboratories rely on a preliminary microscopical examination of the organism, followed by a series of biochemical tests. These often include the ability of the organism to grow under various nutritional and physical conditions, and its sensitivity to antibiotics and bacteriophages. Such an approach takes several days and often yields results which may be ambiguous, difficult to interpret, and results in only a presumptive identification. Therefore, a simple automated technique yielding unambiguous identification within minutes is highly desirable. Recent improvements in the sensitivity and specificity of flow cytometric instrumentation and techniques have made possible a wide range of microbiological investigations which in turn may offer the necessary techniques for the rapid characterization of bacterial populations. Previous attempts to demonstrate the use of flow cytometry for bacterial characterization have relied upon immunofluorescence-based methods (Phillips & Martin 1983, 1985, 1988; Barnett et al. 1984; Tyndall et al. 1985; Donnelly & Baigent 1986) or involved computerized analyses of measurements obtained with expensive duallaser instruments (Van Dilla et al. 1983; Sanders et al. 1990). Both of these approaches have major limitations. For routine use in the characterization of heterogeneous bac-
terial populations the techniques need to be simple and rapid, whilst the instrumentation needs to combine sensitivity with simplicity and low cost. T h e methods chosen must also be applicable to as wide a range as possible of different organisms. In this report we show how an arc-lamp based flow cytometer can be used to obtain high-resolution multiparameter measurements of bacteria. Simply and rapidly obtained dual parameter light scatter measurements (i.e. forward and wide-angle scatter) require no sample preparation or reagent additions and are, therefore, ideally suited to a rapid -diagnostic technique. We illustrate how such measurements can be used to differentiate a wide range of clinically important pathogenic bacteria. Electron microscopy of the samples has allowed us to draw some conclusions about how the different physical characteristics of the organisms in a population give rise to distinct light scattering properties, thus facilitating differentiation based on these measurements. Discrimination between different species in heterogeneous populations may be further enhanced by the measurement of a third parameter, i.e. DNA content of the cells. We have used these principles to characterize and quantify as many as eight different organisms in a mixed population.
MATERIALS AND METHODS Organisms and growth conditions
Correspondence t o : Dr R. Allman, Department of Pure and Applied Biology, 1inioersit.v of Wales College ofcardiff, PO Box 915, Cardiff CF1 STL, [IK.
A model system consisting of eight different organisms was used. T h e organisms were as follows: Bacillus cereus
RAPID CHARACTERIZATION OF BACTERIA 439
NCTC 9947, Clostridium perfingens NCIMB 8875 (Type A), Legionella pneumophila (Kingston strain) supplied by Dr E.C. Hill, ECHA Microbiology Ltd, Unit M22, Cardiff Workshops, Lewis Road, Cardiff, Wales, UK, Listeria monocytogenes NCTC 11994 (Serotype 4B), Pseudomonas Juorescens CRA 424 (Biotype A), Salmonella typhimurium CRA 663, Staphylococcus aureus CRA 410 and Yersinia enterocolitica CRA 449. Bacillus cereus, List. monocytogenes, Salm. typhimurium, Staph. aureus and Y . enterocolitica were grown in Nutrient Broth (Oxoid); cultures (25 ml in 100 ml flasks) were incubated with shaking at 37°C. Pseudomonas juorescens was grown under similar conditions but at 25°C. Cultures of Cl. perfringens (200 ml) were grown at 37°C in Reinforced Clostridial Medium (RCM, Oxoid) in screw capped 250 ml bottles. Legionella pneumophila was grown on plates of Legionella Agar (Difco), incubated at 37°C. Bacillus cereus spores were produced in 1/10 normal strength nutrient broth containing 1% (w/v) NaCI. Cultures were incubated with shaking at 25°C for 4 d. Spores of Cl. perfringens were obtained in 1/10 normal strength RCM containing 1% (w/v) NaCI. Cultures were incubated in sealed bottles at 37°C for 4 d. Sampling regimes
Legionella pneumophila cells were washed off plates after 24 h growth with 5 ml of 10 mmol/l Tris HCI buffer, pH 7.6, immediately before fixation. All other organisms were sampled from exponential phase cultures after at least 10 generations, under conditions of ‘balanced growth’, established by repeated dilution of cultures into flasks containing fresh, prewarmed, growth medium. In all cases growth was measured as increase in A,,, . Fixation and staining and samples for flow cytometry
Samples (1 ml) of cell suspensions were washed in 1 ml of ice-cold 10 mmol/l Tris HCI, pH 7.6, and fixed in ice-cold 77% ethanol. Samples were stored at 4°C for 24 h before staining. Fixed bacteria were washed once in ice-cold 10 mmol/l Tris HCI, pH 7.6, and stained at 4°C with a combination of mithramycin (90 g/ml) and ethidium bromide (25 g/ml) in 10 mmol/l Tris HCI, pH 7.6, containing 10 mmol/l MgCI, , for 1 h. Flow cytometry
Forward light scatter, ‘wide-angle’ light scatter and DNA fluorescence were measured simultaneously with a Skatron Argus Flow Cytometer (Skatron, Tranby, Norway). Photomultiplier voltages were fixed for any given set of experiments, DNA fluorescence was collected using the Argus B1 filter-block. System performance was monitored
using 2.2 pm fluorescent latex spheres (Polysciences, Northampton, UK). Scanning electron microscopy
Cell suspension (1 ml) was fixed with 1 ml of 4% (v/v) glutaraldehyde/2% (v/v) paraformaldehyde in 0.1 mol/l phosphate buffer, pH 7.4, for 30 min at 4”C, followed by rinsing in 0.1 mol/l veronal acetate buffer, pH 7.4, for 30 rnin at 4°C and post-fixed in 1% OsO, in 0.1 mol/l veronal acetate for 1 h at 4°C. The cells were washed in 0.05 mol/l sodium maleate buffer, pH 6.0 for 30 rnin at 4°C and then immersed in 0.5% (w/v) uranyl acetate in 0.05 mol/l sodium maleate buffer, pH 7.4. Fixed cells were dehydrated in a graded series (50, 70, 80, 95, 100%) of ethanol and then transferred to a polycarbonate millipore filter (0-2 pm pore-size). The cells were critical pointdried for 3 rnin with CO, in a Samdri 780 critical point dryer. The cells were mounted on a specimen stub and coated with a thin layer of gold in a sputter coater (EM Scope Ltd UK). The specimens were examined in a Phillips EMWT/STEM electron microscope using a scanning electron detector at 80 kV accelerating voltage. Transmission electron microscopy
Cell suspension (1 ml) was fixed with 1 ml of 4% (w/v) glutaraldehyde/2% (w/v) paraformaldehyde in 0.1 mol/l cacodylate buffer, pH 7.4, at 4°C for 30 min. Cells were centrifuged in an MSE centrifuge at 1500 rev/min for 10 rnin and washed in four changes of 0.1 mol/l cacodylate buffer, p H 7.4. The pellet was washed with 0.05 mol/l sodium maleate buffer, p H 6.0, and then bloc-stained with 0.5% (w/v) uranyl acetate in 0.05 mol/l sodium maleate buffer, pH 6.0. Fixed cells were dehydrated in a graded series (50, 70, 80, 95, 100%) of ethanol and embedded in a 1 : 1 mixture of ethanol/araldite resin. The embedded cells were cured in a capsule for 48 h at 60°C. Sections (60 nm) were cut with a Reichart OM4 microtome. The sections were mounted on a copper grid coated with celloidin and carbon, and stained with 5% (w/v) aqueous uranyl acetate for 30 min at room temperature, followed by staining with Reynolds’ lead citrate (Reynolds 1963) for 15 rnin at room temperature. The sections were examined in a Phillips EM400T electron microscope at 80 kV accelerating voltage. RESULTS
Simultaneous measurements of forward light scatter, wideangle light scatter and DNA content were made on all samples.
440 R . ALLMAN ET A L .
Figure 1 shows dual parameter contour plots of forward scatter versus wide-angle scatter for mid-exponential phase cells in steady state growth, together with the two Grampositive spore types. With Salm. typhimurium as a ‘marker’ organism, it can be seen that some of the organisms can be distinguished purely on the basis of their differing light scatter properties. It is also apparent however that some of the organisms (Salm. typhimurium, Y. enterocolitica, L.
pneumophila, Ps. Jluorescens, Staph. aureus) have very similar (overlapping) scattering characteristics. We have used scanning and electron microscopy to obtain measurements of size and shape as well as characterization of surface and internal structures to determine which of these factors are contributing to the light scatter profiles. Figure 2 shows the scanning electron micrographs of the organisms. I t can be seen by comparing Figs 1 and 2
Fig. 1 Dual parameter contour plots of forward scatter us wide angle scatter of the 10 cell types. In each case Salmonella typhimurium was used as a ‘reference’ organism. Bacillus cereus showed a much larger scattering signal than the other organisms and this is shown compared to Closrridium perfringens. The photomultiplier gain in this contour plot (Fig. Id) was reduced by a factor of 2 to bring the larger cells on scale. Photomultiplier settings were constant for all other samples
RAPID CHARACTERIZATION OF BACTERIA 441
Fig. 2 Scanning electron micrographs of the organisms (magnification = 1 1 000 x ). (a) Bacillus cereus, (b) Clostridium perfringens, (c) Legionella pneumophila, (d) Listeria monocytogenes, (e) Pseudomonas Juorescens, (f)Salmonella typhimurium, (9) Staphylococcus aureus, (h) Yersinia enterocolitica, (i) Bacillus cereus spores, ( j) Clostridium perfringens spores
442 R . ALLMAN ET A L .
that the generally held assumption that the intensity of forward light scatter is correlated with cell-size does not necessarily hold for mixed populations. Thus, List. monocytogenes which is larger than L. pneumophila gives a relatively small forward scatter signal. That spores of B. cereus and CI. perfringens give a forward scatter signal which is out
of proportion to their size, may be explained on the basis of a high value for their refractive index. Figure 3 shows the structural characteristics of the organisms as revealed by transmission electron microscopy. It is also apparent from this set of photographs that drawing conclusions about the structural basis of the wide-angle scatter signal is difficult.
Flg. 3 Ultrathin sections of the organisms (magnification = 25 OOO x ). (a) Bacillus
cereus, (b) Clostridium perfringens, (c) Legionella pneumophila, (d) Listeria rnonocytogenes, (e) PseudomonasPuorescens, (r) Salmonella typhimurium, (9) Staphylococcus aureus, (h) Yersinia enterocolitica, (i) Bacillus cereus spores, ( j) Clostridium perfiingens spores
RAPID CHARACTERIZATION OF BACTERIA
It is clear from these figures that organisms which have differing morphologies, internal structures or surface structures give rise to differing wide-angle scattering intensities. However, this series again shows up an anomaly. Salmonella typhimurium and L. pneumophila are of similar size and shape, but their internal surface structures are markedly different. On this basis we would expect them to give rise to distinctive wide-angle scattering signals. In this case, this is clearly not so and we must conclude that the overall ‘effective refractive indices’ of the organisms are similar.
443
Figure 4 shows how the organisms, including those with overlapping light scatter characteristics can be further resolved on the basis of analysis of the intensity of fluorescence emission from ethidium bromide/mithramycinstained DNA. It was not possible to distinguish the two spore populations (Bacillus and Clostridium) under these conditions. I t was evident that both spore samples were composed of subpopulations containing differing quantities of DNA.
Fig. 4 Dual parameter (forward scatter vs DNA content) contour plots of the organisms. The use of DNA content as one of the measurement parameters provides much improved resolution of the organisms
444 R . A L L M A N ET A L
DISCUSSION
REFERENCES
Light scattering by organisms is a complex function of their size, shape and refractive indices (Salzman el a l . 1979). T h e amount of light scattered in the forward direction (low angle) shows good correlation with size or protein content for any given bacterial species. Many studies have shown that changes in morphology of bacteria result in altered angular dependency of the light scatter signal (Scrienc et al. 1984; Dubelaar et al. 1987; Wittrup et a l . 1988; Allman et al. 1990). In our study we have tried to use flow cytometric analyses of forward and wide-angle scatter as the basis of a method for characterizing bacterial populations. It is clear that we can indeed discriminate between a range of organisms in a mixed population purely on their light scattering characteristics. However, electron microscopy of the same samples of cells has shown that attempting to draw anything other than empirical deductions about the physical basis of these scattering profiles is not possible. Thus from the results presented we may conclude that forward scatter is a good measure of cell size within a homogeneous population (Allman et al. 1990). This is not so for different species, however, where factors such as refractive index have a substantial influence, i.e. two differing cell types of similar size do not necessarily give similar forward scatter profiles. We may also deduce that wide-angle scattering is dependent upon some structural characteristics other than cell size, although defining exactly which features are contributing to this scatter signal is not possible. Overall refractive index is obviously important in this case too. Salmonella typhimurium and Y . enterocolitica which apparently are very different structurally give very similar scattering profiles and thus may have similar overall refractive indices. Irrespective of the bases of the observed differences in scattering profiles of different organisms, they are a very useful first step for the flow cytometric characterization of bacterial populations. The fact that no sample preparation (other than fixation) is required makes such measurements even more attractive. We have also demonstrated that a third measurement parameter, that of fluorescently-stained DNA gives greatly enhanced discrimination between mixed bacterial populations. Genome size, gross organizational characteristics and replication properties are unlikely to be similar in different species, thus providing measurable differences to be exploited for identification by fluorescence intensity measurements.
A L L M A N R., , HA", A . C . , P H I L L I P S ,A.P., M A R T I N , K . L . & L L O Y D ,D . (1990) Growth of Azotobacter vinelandti with correlation of coulter cell size, flow cytometric parameters, and ultrastructure. Cytometry 11, 822-83 1. B A R N E T T J, . M . , C U C H E N S M , . A . & B U C H A N A NW ,. (1984) Automated immunofluorescent speciation of oral bacteria using flow cytometry. Journal of Dental Research 63, 104& 1042. D O N N E L L YC, . W . & B A I G E N TG, . J . (1986) Method for flow cytometric detection of Listeria monocytogenes in milk. Applied and Environmental Microbiology 52, 689-695. D U B E L A A RG.B.J., , V I S S E R ,J . W . M . & D O N Z E , M . (1987) Anomalous behaviour of forward and perpendicular scattering of cyanobacteria owing to intracellular gas vacuoles. Cytometry 8, 405412. P H I L L I P S ,A.P. & M A R T I N ,K . L . (1983) Immunofluorescence analysis of B a d u s spores and vegetative cells by flow cytometry. Cytometry 4, 123-131. P H I L L I P SA, . P . & M A R T I N K , . L . (1985) Dual parameter scatter-flow immunofluorescence analysis of Bacillus spores. Cytometry 6, 126-129. P H I L L I P SA, . P . & M A R T I NK , . L . (1988) Limitations of flow cytometry for the specific detection of bacteria in mixed populations. Journal of Immunological Methods 106, 109-1 17. R E Y N O L D SE, . S . (1963) The use of lead citrate at high pH as an electron opaque stain for electron microscopy. Journal of Cell Biology 17, 208. S A L Z M A NG, . C . , W I L D E R ,M . E . & J E T T , J . H . (1979) Light scattering with stream-in-air flow systems. Journal of Histochemistry and Cytochemistry. 27, 264. S A N D E R SC, . A . , Y A J K O ,D . M . , H Y U N ,W . , L A N G L O I S , R . G . , N A S S O S P.S., , F U L W Y L EM R .,J . & H A D L E Y , W. K . (1990) Determination of guanine plus cytosine content of bacterial DNA by dual laser flow cytometry. Journal of General Microbiology 136, 359-365. S C R I E N CF, . , A R N O L D , B. & B A I L E YJ, . E . (1984) Characterisation of intracellular accumulation of poly-B-hydroxybutyrate (PHB) in individual cells of Alcaligenes eutrophus H16 by flow cytometry. Biotechnology 6 Bioengineering 26, 982-987. T Y N D A L LR, . L . , H A N D ,R.E., M A N N ,R . C . , E V A N SC ,. & J E R N I G A N , R.(1985) Application of flow cytometry to detection and characterisation of Legionella spp. Applied and Environmental Microbiology 49, 852-857. V A N D I L L A , M.A., L A N G L O I S ,R . G . , P I N K E L , D . , Y A J K O ,D . & H A D L E YW , . K . (1983) Bacterial characterisation by flow cytometry. Science 220, 620-622. W I T T R U P ,K . D . , M A N N ,M.B., F E N T O N D , .M., TSAI, L.B. & B A I L E Y J, . E . (1988) Single cell light scatter as a probe of refractile body formation in Escherichia coli. Biotechnology 6, 423426.
Characterization of bacteria by multiparameter flow cytometry R. Allman, A.C. Hannl, R. Mancheea and D. Lloyd Department of Pure and Applied Biology and 'Preclinical Electron Microscopy Unit, Department of Physiology, University of Wales College of Cardiff, Cardiff and 2Chemical Defence Establishment, Porton Down, Salisbury, Wiltshire, UK 4084/01/92: accepted 6 July 1992
A n arc-lamp based flow cytometer was used to obtain high resolution measurements o f t h e light scattering characteristics a n d DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning a n d transmission electron microscopy of t h e bacterial samples demonstrate that t h e structural basis of t h e light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. T h e use of a third measurement parameter, DNA content, allowed much better discrimination of t h e organisms. Flow cytometry shows great promise as a method for t h e rapid discrimination and identification of bacterial populations. R . A L L M A N , A . C . H A N N , R . M A N C H E E AND D. L L O Y D . 1992.
INTRODUCTION
The need for new approaches to bacterial identification stems largely from the limitations of classical diagnostic microbiology. Clinical and environmental laboratories rely on a preliminary microscopical examination of the organism, followed by a series of biochemical tests. These often include the ability of the organism to grow under various nutritional and physical conditions, and its sensitivity to antibiotics and bacteriophages. Such an approach takes several days and often yields results which may be ambiguous, difficult to interpret, and results in only a presumptive identification. Therefore, a simple automated technique yielding unambiguous identification within minutes is highly desirable. Recent improvements in the sensitivity and specificity of flow cytometric instrumentation and techniques have made possible a wide range of microbiological investigations which in turn may offer the necessary techniques for the rapid characterization of bacterial populations. Previous attempts to demonstrate the use of flow cytometry for bacterial characterization have relied upon immunofluorescence-based methods (Phillips & Martin 1983, 1985, 1988; Barnett et al. 1984; Tyndall et al. 1985; Donnelly & Baigent 1986) or involved computerized analyses of measurements obtained with expensive duallaser instruments (Van Dilla et al. 1983; Sanders et al. 1990). Both of these approaches have major limitations. For routine use in the characterization of heterogeneous bac-
terial populations the techniques need to be simple and rapid, whilst the instrumentation needs to combine sensitivity with simplicity and low cost. T h e methods chosen must also be applicable to as wide a range as possible of different organisms. In this report we show how an arc-lamp based flow cytometer can be used to obtain high-resolution multiparameter measurements of bacteria. Simply and rapidly obtained dual parameter light scatter measurements (i.e. forward and wide-angle scatter) require no sample preparation or reagent additions and are, therefore, ideally suited to a rapid -diagnostic technique. We illustrate how such measurements can be used to differentiate a wide range of clinically important pathogenic bacteria. Electron microscopy of the samples has allowed us to draw some conclusions about how the different physical characteristics of the organisms in a population give rise to distinct light scattering properties, thus facilitating differentiation based on these measurements. Discrimination between different species in heterogeneous populations may be further enhanced by the measurement of a third parameter, i.e. DNA content of the cells. We have used these principles to characterize and quantify as many as eight different organisms in a mixed population.
MATERIALS AND METHODS Organisms and growth conditions
Correspondence t o : Dr R. Allman, Department of Pure and Applied Biology, 1inioersit.v of Wales College ofcardiff, PO Box 915, Cardiff CF1 STL, [IK.
A model system consisting of eight different organisms was used. T h e organisms were as follows: Bacillus cereus
RAPID CHARACTERIZATION OF BACTERIA 439
NCTC 9947, Clostridium perfingens NCIMB 8875 (Type A), Legionella pneumophila (Kingston strain) supplied by Dr E.C. Hill, ECHA Microbiology Ltd, Unit M22, Cardiff Workshops, Lewis Road, Cardiff, Wales, UK, Listeria monocytogenes NCTC 11994 (Serotype 4B), Pseudomonas Juorescens CRA 424 (Biotype A), Salmonella typhimurium CRA 663, Staphylococcus aureus CRA 410 and Yersinia enterocolitica CRA 449. Bacillus cereus, List. monocytogenes, Salm. typhimurium, Staph. aureus and Y . enterocolitica were grown in Nutrient Broth (Oxoid); cultures (25 ml in 100 ml flasks) were incubated with shaking at 37°C. Pseudomonas juorescens was grown under similar conditions but at 25°C. Cultures of Cl. perfringens (200 ml) were grown at 37°C in Reinforced Clostridial Medium (RCM, Oxoid) in screw capped 250 ml bottles. Legionella pneumophila was grown on plates of Legionella Agar (Difco), incubated at 37°C. Bacillus cereus spores were produced in 1/10 normal strength nutrient broth containing 1% (w/v) NaCI. Cultures were incubated with shaking at 25°C for 4 d. Spores of Cl. perfringens were obtained in 1/10 normal strength RCM containing 1% (w/v) NaCI. Cultures were incubated in sealed bottles at 37°C for 4 d. Sampling regimes
Legionella pneumophila cells were washed off plates after 24 h growth with 5 ml of 10 mmol/l Tris HCI buffer, pH 7.6, immediately before fixation. All other organisms were sampled from exponential phase cultures after at least 10 generations, under conditions of ‘balanced growth’, established by repeated dilution of cultures into flasks containing fresh, prewarmed, growth medium. In all cases growth was measured as increase in A,,, . Fixation and staining and samples for flow cytometry
Samples (1 ml) of cell suspensions were washed in 1 ml of ice-cold 10 mmol/l Tris HCI, pH 7.6, and fixed in ice-cold 77% ethanol. Samples were stored at 4°C for 24 h before staining. Fixed bacteria were washed once in ice-cold 10 mmol/l Tris HCI, pH 7.6, and stained at 4°C with a combination of mithramycin (90 g/ml) and ethidium bromide (25 g/ml) in 10 mmol/l Tris HCI, pH 7.6, containing 10 mmol/l MgCI, , for 1 h. Flow cytometry
Forward light scatter, ‘wide-angle’ light scatter and DNA fluorescence were measured simultaneously with a Skatron Argus Flow Cytometer (Skatron, Tranby, Norway). Photomultiplier voltages were fixed for any given set of experiments, DNA fluorescence was collected using the Argus B1 filter-block. System performance was monitored
using 2.2 pm fluorescent latex spheres (Polysciences, Northampton, UK). Scanning electron microscopy
Cell suspension (1 ml) was fixed with 1 ml of 4% (v/v) glutaraldehyde/2% (v/v) paraformaldehyde in 0.1 mol/l phosphate buffer, pH 7.4, for 30 min at 4”C, followed by rinsing in 0.1 mol/l veronal acetate buffer, pH 7.4, for 30 rnin at 4°C and post-fixed in 1% OsO, in 0.1 mol/l veronal acetate for 1 h at 4°C. The cells were washed in 0.05 mol/l sodium maleate buffer, pH 6.0 for 30 rnin at 4°C and then immersed in 0.5% (w/v) uranyl acetate in 0.05 mol/l sodium maleate buffer, pH 7.4. Fixed cells were dehydrated in a graded series (50, 70, 80, 95, 100%) of ethanol and then transferred to a polycarbonate millipore filter (0-2 pm pore-size). The cells were critical pointdried for 3 rnin with CO, in a Samdri 780 critical point dryer. The cells were mounted on a specimen stub and coated with a thin layer of gold in a sputter coater (EM Scope Ltd UK). The specimens were examined in a Phillips EMWT/STEM electron microscope using a scanning electron detector at 80 kV accelerating voltage. Transmission electron microscopy
Cell suspension (1 ml) was fixed with 1 ml of 4% (w/v) glutaraldehyde/2% (w/v) paraformaldehyde in 0.1 mol/l cacodylate buffer, pH 7.4, at 4°C for 30 min. Cells were centrifuged in an MSE centrifuge at 1500 rev/min for 10 rnin and washed in four changes of 0.1 mol/l cacodylate buffer, p H 7.4. The pellet was washed with 0.05 mol/l sodium maleate buffer, p H 6.0, and then bloc-stained with 0.5% (w/v) uranyl acetate in 0.05 mol/l sodium maleate buffer, pH 6.0. Fixed cells were dehydrated in a graded series (50, 70, 80, 95, 100%) of ethanol and embedded in a 1 : 1 mixture of ethanol/araldite resin. The embedded cells were cured in a capsule for 48 h at 60°C. Sections (60 nm) were cut with a Reichart OM4 microtome. The sections were mounted on a copper grid coated with celloidin and carbon, and stained with 5% (w/v) aqueous uranyl acetate for 30 min at room temperature, followed by staining with Reynolds’ lead citrate (Reynolds 1963) for 15 rnin at room temperature. The sections were examined in a Phillips EM400T electron microscope at 80 kV accelerating voltage. RESULTS
Simultaneous measurements of forward light scatter, wideangle light scatter and DNA content were made on all samples.
440 R . ALLMAN ET A L .
Figure 1 shows dual parameter contour plots of forward scatter versus wide-angle scatter for mid-exponential phase cells in steady state growth, together with the two Grampositive spore types. With Salm. typhimurium as a ‘marker’ organism, it can be seen that some of the organisms can be distinguished purely on the basis of their differing light scatter properties. It is also apparent however that some of the organisms (Salm. typhimurium, Y. enterocolitica, L.
pneumophila, Ps. Jluorescens, Staph. aureus) have very similar (overlapping) scattering characteristics. We have used scanning and electron microscopy to obtain measurements of size and shape as well as characterization of surface and internal structures to determine which of these factors are contributing to the light scatter profiles. Figure 2 shows the scanning electron micrographs of the organisms. I t can be seen by comparing Figs 1 and 2
Fig. 1 Dual parameter contour plots of forward scatter us wide angle scatter of the 10 cell types. In each case Salmonella typhimurium was used as a ‘reference’ organism. Bacillus cereus showed a much larger scattering signal than the other organisms and this is shown compared to Closrridium perfringens. The photomultiplier gain in this contour plot (Fig. Id) was reduced by a factor of 2 to bring the larger cells on scale. Photomultiplier settings were constant for all other samples
RAPID CHARACTERIZATION OF BACTERIA 441
Fig. 2 Scanning electron micrographs of the organisms (magnification = 1 1 000 x ). (a) Bacillus cereus, (b) Clostridium perfringens, (c) Legionella pneumophila, (d) Listeria monocytogenes, (e) Pseudomonas Juorescens, (f)Salmonella typhimurium, (9) Staphylococcus aureus, (h) Yersinia enterocolitica, (i) Bacillus cereus spores, ( j) Clostridium perfringens spores
442 R . ALLMAN ET A L .
that the generally held assumption that the intensity of forward light scatter is correlated with cell-size does not necessarily hold for mixed populations. Thus, List. monocytogenes which is larger than L. pneumophila gives a relatively small forward scatter signal. That spores of B. cereus and CI. perfringens give a forward scatter signal which is out
of proportion to their size, may be explained on the basis of a high value for their refractive index. Figure 3 shows the structural characteristics of the organisms as revealed by transmission electron microscopy. It is also apparent from this set of photographs that drawing conclusions about the structural basis of the wide-angle scatter signal is difficult.
Flg. 3 Ultrathin sections of the organisms (magnification = 25 OOO x ). (a) Bacillus
cereus, (b) Clostridium perfringens, (c) Legionella pneumophila, (d) Listeria rnonocytogenes, (e) PseudomonasPuorescens, (r) Salmonella typhimurium, (9) Staphylococcus aureus, (h) Yersinia enterocolitica, (i) Bacillus cereus spores, ( j) Clostridium perfiingens spores
RAPID CHARACTERIZATION OF BACTERIA
It is clear from these figures that organisms which have differing morphologies, internal structures or surface structures give rise to differing wide-angle scattering intensities. However, this series again shows up an anomaly. Salmonella typhimurium and L. pneumophila are of similar size and shape, but their internal surface structures are markedly different. On this basis we would expect them to give rise to distinctive wide-angle scattering signals. In this case, this is clearly not so and we must conclude that the overall ‘effective refractive indices’ of the organisms are similar.
443
Figure 4 shows how the organisms, including those with overlapping light scatter characteristics can be further resolved on the basis of analysis of the intensity of fluorescence emission from ethidium bromide/mithramycinstained DNA. It was not possible to distinguish the two spore populations (Bacillus and Clostridium) under these conditions. I t was evident that both spore samples were composed of subpopulations containing differing quantities of DNA.
Fig. 4 Dual parameter (forward scatter vs DNA content) contour plots of the organisms. The use of DNA content as one of the measurement parameters provides much improved resolution of the organisms
444 R . A L L M A N ET A L
DISCUSSION
REFERENCES
Light scattering by organisms is a complex function of their size, shape and refractive indices (Salzman el a l . 1979). T h e amount of light scattered in the forward direction (low angle) shows good correlation with size or protein content for any given bacterial species. Many studies have shown that changes in morphology of bacteria result in altered angular dependency of the light scatter signal (Scrienc et al. 1984; Dubelaar et al. 1987; Wittrup et a l . 1988; Allman et al. 1990). In our study we have tried to use flow cytometric analyses of forward and wide-angle scatter as the basis of a method for characterizing bacterial populations. It is clear that we can indeed discriminate between a range of organisms in a mixed population purely on their light scattering characteristics. However, electron microscopy of the same samples of cells has shown that attempting to draw anything other than empirical deductions about the physical basis of these scattering profiles is not possible. Thus from the results presented we may conclude that forward scatter is a good measure of cell size within a homogeneous population (Allman et al. 1990). This is not so for different species, however, where factors such as refractive index have a substantial influence, i.e. two differing cell types of similar size do not necessarily give similar forward scatter profiles. We may also deduce that wide-angle scattering is dependent upon some structural characteristics other than cell size, although defining exactly which features are contributing to this scatter signal is not possible. Overall refractive index is obviously important in this case too. Salmonella typhimurium and Y . enterocolitica which apparently are very different structurally give very similar scattering profiles and thus may have similar overall refractive indices. Irrespective of the bases of the observed differences in scattering profiles of different organisms, they are a very useful first step for the flow cytometric characterization of bacterial populations. The fact that no sample preparation (other than fixation) is required makes such measurements even more attractive. We have also demonstrated that a third measurement parameter, that of fluorescently-stained DNA gives greatly enhanced discrimination between mixed bacterial populations. Genome size, gross organizational characteristics and replication properties are unlikely to be similar in different species, thus providing measurable differences to be exploited for identification by fluorescence intensity measurements.
A L L M A N R., , HA", A . C . , P H I L L I P S ,A.P., M A R T I N , K . L . & L L O Y D ,D . (1990) Growth of Azotobacter vinelandti with correlation of coulter cell size, flow cytometric parameters, and ultrastructure. Cytometry 11, 822-83 1. B A R N E T T J, . M . , C U C H E N S M , . A . & B U C H A N A NW ,. (1984) Automated immunofluorescent speciation of oral bacteria using flow cytometry. Journal of Dental Research 63, 104& 1042. D O N N E L L YC, . W . & B A I G E N TG, . J . (1986) Method for flow cytometric detection of Listeria monocytogenes in milk. Applied and Environmental Microbiology 52, 689-695. D U B E L A A RG.B.J., , V I S S E R ,J . W . M . & D O N Z E , M . (1987) Anomalous behaviour of forward and perpendicular scattering of cyanobacteria owing to intracellular gas vacuoles. Cytometry 8, 405412. P H I L L I P S ,A.P. & M A R T I N ,K . L . (1983) Immunofluorescence analysis of B a d u s spores and vegetative cells by flow cytometry. Cytometry 4, 123-131. P H I L L I P SA, . P . & M A R T I N K , . L . (1985) Dual parameter scatter-flow immunofluorescence analysis of Bacillus spores. Cytometry 6, 126-129. P H I L L I P SA, . P . & M A R T I NK , . L . (1988) Limitations of flow cytometry for the specific detection of bacteria in mixed populations. Journal of Immunological Methods 106, 109-1 17. R E Y N O L D SE, . S . (1963) The use of lead citrate at high pH as an electron opaque stain for electron microscopy. Journal of Cell Biology 17, 208. S A L Z M A NG, . C . , W I L D E R ,M . E . & J E T T , J . H . (1979) Light scattering with stream-in-air flow systems. Journal of Histochemistry and Cytochemistry. 27, 264. S A N D E R SC, . A . , Y A J K O ,D . M . , H Y U N ,W . , L A N G L O I S , R . G . , N A S S O S P.S., , F U L W Y L EM R .,J . & H A D L E Y , W. K . (1990) Determination of guanine plus cytosine content of bacterial DNA by dual laser flow cytometry. Journal of General Microbiology 136, 359-365. S C R I E N CF, . , A R N O L D , B. & B A I L E YJ, . E . (1984) Characterisation of intracellular accumulation of poly-B-hydroxybutyrate (PHB) in individual cells of Alcaligenes eutrophus H16 by flow cytometry. Biotechnology 6 Bioengineering 26, 982-987. T Y N D A L LR, . L . , H A N D ,R.E., M A N N ,R . C . , E V A N SC ,. & J E R N I G A N , R.(1985) Application of flow cytometry to detection and characterisation of Legionella spp. Applied and Environmental Microbiology 49, 852-857. V A N D I L L A , M.A., L A N G L O I S ,R . G . , P I N K E L , D . , Y A J K O ,D . & H A D L E YW , . K . (1983) Bacterial characterisation by flow cytometry. Science 220, 620-622. W I T T R U P ,K . D . , M A N N ,M.B., F E N T O N D , .M., TSAI, L.B. & B A I L E Y J, . E . (1988) Single cell light scatter as a probe of refractile body formation in Escherichia coli. Biotechnology 6, 423426.
