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Separating Microbes in the Manner of Molecules. 1. Capillary Electrokinetic Approaches Daniel W. Armstrong,* Georg Schulte,† Jeffrey M. Schneiderheinze, and David J. Westenberg‡
Department of Chemistry, University of MissourisRolla, Rolla, Missouri 65409
Selective, high-efficiency separations of intact bacteria may, in some cases, allow them to be identified and quantified in much the same way that molecules are done today. Two different capillary electrokinetic approaches were utilized. The first approach used a dissolved polymerbased CE separation that may be affected by size and shape considerations. Another approach uses capillary isoelectric focusing to separate bacteria by their surface charge or isoelectric point. Good peak shapes and extremely high efficiencies are observed (up to ∼1 600 000 theoretical plates/m). Careful sample preparation and separation runs are essential in order to obtain reproducible separations. Expansion of these types of rapid, efficient microbial separations could have profound effects on many branches of science and technology.
It has been recognized for decades that charged colloids and particulate matter will transport in direct current electric fields (i.e., electrophoresis).1-3 However, routine, high-efficiency separation and analysis of colloidal or larger particles by electrophoresis has not been as successful as it has for small molecules and macromolecules (particularly those of biological importance).4 With the advent of modern capillary electrophoresis (CE), the detection of single cells (such as red blood cells)5,6 and, in particular, the separation of the contents of lysed cells7-9 have been reported by several groups. The analysis of intact microbes (e.g., bacteria, viruses, etc.) is more limited. Most reports involve electropherograms of single organisms (one peak or multiple peaks per microbe).10-14 An early CE paper by Hjerte´n and coworkers showed that tobacco mosaic virus and Lactobacillus casei †
Current address: Bayer AG, Wuppertal, Germany. Department of Biology, University of MissourisRolla. (1) Tiselius, A. Nova Acta Regiae Soc. Sci. Ups., Ser. IV 1930, 7, 1-90. (2) Agarwal, K. N.; Hjerte´n, S. Acta Endrocrinol., Suppl., 1964, 93, 53-58. (3) Karger, B. L.; Snyder, L. R.; Horvath, C. An Introduction to Separation Science; John Wiley & Sons: New York, 1973. (4) See the special review issue: El Rassi, Z., Ed. Electrophoresis 1997, 18, 2123-2501. (5) Zhu, A.; Chen, Y. J. Chromatogr. 1989, 470, 251-260. (6) Rosenzweig, Z.; Yeung, E. S. Anal. Chem. 1994, 66, 1771-1776. (7) Hogan, B. L.; Yeung, E. S. Anal. Chem. 1992, 64, 2841-2845. (8) Cheung, N.-H.; Yeung, E. S. Anal. Chem. 1994, 66, 929-936. (9) Xue, Q.; Yeung, E. S. Anal. Chem. 1994, 66, 11755-1178. (10) Hjerte´n, S.; Elenbring, K.; Kilar, F.; Liao, J.-L.; Chen, A. T.; Seibert, C. J.; Zhu, M.-D. J. Chromatogr. 1987, 403, 47-61. (11) Grossman, P. D.; Soane, D. S. Anal. Chem. 1990, 62, 1592-1596. (12) Ebersole, R. C.; McCormick, R. M. Bio/Technology 1993, 11, 1278-1282. (13) Glynn, J. R., Jr.; Belongia, B. M.; Arnold, R. G.; Ogden, K. L.; Baygents, J. C. Appl. Environ. Microbiol. 1998, 64, 2572-2577. ‡
10.1021/ac990779z CCC: $18.00 Published on Web 11/13/1999
© 1999 American Chemical Society
would migrate through a capillary in a little over 3 min (under their experimental conditions).10 In a subsequent study, it was demonstrated that the orientation of tobacco mosaic virus affected its electrophoretic mobility.11 Recently the CE migration of the common cold virus and its contents were reported and discussed.14 Reports on the efficient analytical resolution of microbes from one another in a manner that might allow them to be effectively identified and quantified are not common. Ebersole and McCormick reported one of the few apparent separations of bacteria.12 The CE separations took ∼70 min and the efficiency and peak shapes were not good compared to the CE separations of molecules (n = 580-25 000 plates/m, by our calculations). A reasonable percentage of the microbes were thought to survive the separation process.12 In other approaches, bacteria were partially fractionated in conventional isoelectric focusing experiments using glass tubes containing aqueous solutions with appropriate pH and density gradients.15,16 The separations took from ∼6 to 24 h.15,16 Isoelectric focusing also has been used to determine the isoelectric point of bacteria17 and a virus.18 Free zone electrophoresis also was used early on in an attempt to purify certain microorganisms.19 Currently the characterization of microorganisms suffers from long analysis times and limited throughput.20 As yet, there are no broadly applicable, reproducible, highly efficient analytical approaches for the resolution and analysis of intact microbial mixtures. The time-honored way to identify bacteria is by isolation of pure cultures. This remains a dominant technique today but is far from universally applicable.16 In an effort to increase the speed, selectivity, and sensitivity of microbial assays, a number of different approaches are being investigated, including immunoassays, fluorescence assays, and polymerase chain reaction (PCR) detection.20,21 Field flow fractionation (FFF) has been used in microbial studies, and most recently, attempts have been made to use mass spectrometry (MS).20 However, MS approaches do not analyze the intact cells, (14) Okun, V. M.; Ronacher, B.; Blaas, D.; Kenndler, E. Anal. Chem. 1999, 71, 2028-2032. (15) Longton, R. W.; Cole, J. S., III; Quinn, P. F. Arch. Oral Biol. 1975, 20, 103-106. (16) Jaspers, E.; Overmann, J. Appl. Environ. Microbiol. 1997, 63, 3176-3181. (17) Harden, V. P.; Harris, J. O. J. Bacteriol. 1953, 65, 198-202. (18) Schnabel, U.; Groiss, F.; Blaas, D.; Kenndler, E. Anal. Chem. 1996, 68, 4300-4303. (19) Hjerte´n, S. In Cell Separation Methods; Bloemendal, H., Ed.; Elsevier: Amsterdam, 1977; pp 117-128. (20) Stinson, S. C. Chem. Eng. News 1999, 77 (March 29), 36-38. (21) Belgrader, P.; Benett, W.; Hadley, D.; Richards, J.; Stratton, P.; Mariella, R., Jr.; Milanovich, F. Science 1999, 284, 449-450.
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Table 1. Description and Properties of Six Bacteria and a Yeast bacteriuma
gram stain
diameter, µm
form
flagella
Escherichia coli K12 Pseudomonas putida Pseudomonas fluorescens Serratia rubidae Enterobacter aerogenes Micrococcus luteus Saccharomyces cerevisiae
negative negative negative negative negative positive
1.1-1.5 0.5-1 0.5-1 0.5-0.8 0.6-1 1-2 3-10
straight rods slightly curved rods slightly curved rods straight rods straight rods cocci, in irregular clusters speroidal or cylindrical cells
peritrichous flagella or nonmotile one or several flagella one or several flagella peritrichous flagella 4-6 peritrichous flagella nonmotile nonmotile
a
Note that the last microorganism in this column is not a bacterium, but rather is baker’s yeast.
but rather attempt to identify them indirectly by distinct patterns of their protein extracts, etc.22 Reproducibility can be a problem since cell growth conditions, age, and isolation/preparation procedures can significantly alter cellular composition.7-9,20 Clearly a highly selective and efficient separation approach (analogous to those that are routinely used for molecules) would be beneficial in studies involving microorganisms. In this first of a series of studies, we report on capillary electrokinetic approaches for the separation and identification of bacteria. EXPERIMENTAL SECTION Materials. The bacteria Escherichia coli K12, Pseudomonas putida, and Serratia rubidae were grown in-house. The starting cultures were transferred from solid agar to Nutrient Broth (Difco Laboratories, Franklin Lakes, NJ) and were grown for 24-36 h at 25 or 30 °C. Bacteria samples for isoelectric focusing were prepared using 0.5-1.0 mL of the above culture broth. The bacteria were pelleted for 3-5 min using a centrifuge (model 228, Fisher Scientific, Pittsburgh, PA) at 3400 rpm. The supernatant was removed, and the cells were washed with water to remove culture media constituents. This washing procedure was repeated twice. The bacteria were then resuspended in diluted Bio-Lyte Ampholyte (Bio-Rad, Hercules, CA), pH 3-10. Pseudomonas fluorescens type IV, Enterobacter aerogenes type III, Micrococcus luteus, and boric acid were purchased as freezedried samples from Sigma (St. Louis, MO). The viability of these bacteria was checked by microscopy and growth in culture. Saccharomyces cerevisiae was purchased from Wal-Mart. Poly(ethylene oxide) (PEO; Mn ) 600 000) and tris(hydroxymethyl)aminomethane (TRIS) were purchased from Aldrich (Milwaukee, WI). Disodium ethylenediaminetetraacetate (EDTA) was purchased from Fisher Scientific Co. (St. Louis, MO). Information on these microorganisms is compiled in Table 1. Methods. Isoelectric Focusing. Capillary isoelectric focusing was carried out using a Beckman PACE 5000 CE unit (Palo Alto, CA). Separations were performed using 50 m × 47 cm (40 cm to the detector) coated silica capillaries (Polymicro Technologies, Phoenix, AZ). The methylcellulose coating of the capillaries is prepared by a slightly modified procedure described by Hjerte´n.23 The pH gradient was generated with Bio-Lyte Ampholyte pH 3-10. The ampholyte was diluted with water to a final concentration of 0.5% (v/v). The anolyte and the catholyte were 20 mM phosphoric acid and sodium hydroxide, respectively. (22) Bundy, J.; Fenselau, C. Anal. Chem. 1999, 71, 1460-1463. (23) Hjerte´n, S.; Kubo, K. Electrophoresis 1993, 14, 390-395.
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Before each separation the capillary was washed for 2 min with water and ampholyte. Samples were introduced into the capillary by a 0.5 psi pressure injection for 90 s followed by a second injection of ampholyte for 129 s. After focusing for 5 min (voltage +20 kV), samples were mobilized with a low-pressure (0.5 psi) rinse while the 20-kV voltage was maintained. Detection of analytes was carried out at 280 nm, using an on-line UV detector. All separations were carried out at 23 °C under thermostated conditions. Typically, the current initially increased sharply to ∼4 µA and then decreased to 1 µA, over a 5-min period. The blind side of the capillary, the part behind the detection window and the end of the capillary, was not blocked with a basic compound like N,N,N′,N′-tetramethylenediamine (TEMED) or sodium hydroxide because bacteria have an isoelectric point at lower pH values. There is no risk that certain bacteria are focused in this blind side of the capillary. Capillary Electrophoresis of Bacteria in Dissolved Polymer Solutions. A stock buffer solution containing 4.5 mM TRIS, 4.5 mM boric acid, and 0.1 mM EDTA was prepared by dissolving appropriate amounts of each reagent in deionized water yielding a buffer of pH 8.4. This buffer solution was then diluted 8:1 with deionized water. The pH of this diluted buffer was 8.4; 0.2 g of PEO was then added to 40 mL of the diluted stock buffer to give a final concentration of 0.5%. This heterogeneous polymer solution was dispersed by placing it in an ultrasound bath (Fischer model FS-28, 720 W at 43 kHz) for 4 h at ∼55 °C. The mixture was removed from the bath and left overnight to dissolve completely. If the mixture is not dispersed in an ultrasound bath it takes much longer to dissolve. However, it should be noted that extensive sonication may cause some degradation of the dissolved polymer.24 For example, the relative viscosities of aqueous solutions (0.5% at 22 °C) of sonicated and stirred polymer are 4.36 and 4.89 cP, respectively. The µEOF of the same sonicated vs stirred polymer solutions were 6.38 × 10-4 and 5.77 × 10-4 cm2/V‚s, respectively. All buffers and polymer solutions were prepared fresh daily. The bacteria samples were prepared by dispersing appropriate amounts of the bacteria cells in the diluted stock buffer to a concentration of 1 mg/mL. The bacteria cells were immersed in the buffer solution for 45 min, yielding a turbid solution of cells. The cells were then centrifuged, the supernatant was decanted, and fresh diluted stock buffer was added. The sample tubes containing solutions of S. cerevisiae (yeast) and M. luteus were then placed in an ultrasound bath (model FS-28; Fisher Scientific Co.) at room temperature for 3 min. This served to disperse these cells which otherwise tend to aggregate over time. Microscopic examination of the dispersed cells indicated that they were intact, individual
entities. The cells were then injected separately or mixed and separated. This entire process was repeated for each injection. All polymer-based capillary electrophoresis experiments were performed on a Beckman P/ACE 2100 coupled to a computer equipped with Gold data acquisition software. Fused-silica capillary with an 100-µm i.d. was purchased from Polymicro Technologies, Inc. The running buffer was prepared by diluting the polymer solution with the diluted stock buffer to give a final polymer concentration of 0.0125%. The choice of this polymer concentration was determined empirically as indicated in the Results and Discussion. The column used for the separations was 27 cm in length (20 cm to window). The column was washed for 1.5 min with 0.5 N phosphoric acid, 0.5 min with water, 1.5 min with 1 N KOH, and 0.5 min with water, followed by 1 min with the running buffer. The bacteria samples were then pressure injected for 8-10 s. The separation was performed at a voltage of 10 kV and a temperature of 23 °C with thermostated control. On-line detection of the bacteria was accomplished at 214 nm. The RSDs (n ) 4) for the electrophoretic mobilities of individual microorganisms using this CE approach were as follows: S. cerevisiae 1.8%, M. luteus 1.5%, E. aerogenes 1.9%, and P. fluorescens 2.0%. The retention order of the microbes did not vary in either method with multiple injections (provided the conditions were identical). However, the electrophoretic mobilities were altered by changing the polymer concentration (in the CE technique) or slightly altering the pH gradient in the capillary isoelectric focusing (CIEF) techniques. This will be discussed in the subsequent section. Prominent, intermittent spikes that would indicate precipitate formation were not observed under the conditions outlined above. RESULTS AND DISCUSSION Devising an effective, efficient separation of intact microorganisms is an attractive concept. However, it can be fraught with practical and conceptual difficulties, particularly with regard to reproducibility, efficiency, universality, and lability of the analytes. Bacteria, in particular, are easily damaged, lysed, or altered. Figure 1 shows the CE separation of a lysed cell and the corresponding intact P. fluorescens. The multitude of peaks from the lysed organism represent various proteins, protein conjugates, polynucleotides, cell fragments, some small molecules, etc. Because of the ease with which cells are disrupted, attempts have been made to use their constituents (i.e., proteins, etc.) and the peak patterns of lysed cells or cell extracts to identify them.20,22 Instead, the focus of this work is on production of distinct, resolved peaks from intact cells. The preparation and separation of bacteria must be done under carefully controlled and relatively mild conditions so that (A) the cells remain intact, (B) the separation is reproducible, and (C) high efficiencies are achieved. Seemingly small changes, which would have little affect on the separation of molecules, can negate the viable separation of microbial cells. Table 2 lists many of the factors that can affect a high-efficiency analytical separation of microbes. A complete evaluation of all of these factors is beyond the scope of this initial report. They will undoubtedly be the subject of many future studies, several of which are currently underway.26 Obviously, the ionic strength and pH of the prepara(24) Smith, W. B.; Temple, H. W. J. Phys. Chem. 1968, 72, 4613-4619.
Figure 1. Capillary electropherograms of (A) lysed P. fluorescens and (B) intact P. fluorescens, tr ) 6.6 min. The separation conditions for both were identical (0.0125% of dissolved PEO in pH 8.4 running buffer; see Experimental Section). The former bacteria represented by electropherogram A were lysed by warming them with 0.15% sodium dodecyl sulfate prior to injection. Table 2. Some Characteristics of Microbes and Microbial Solutions That Must Be Recognized and Properly Controlled in Order To Obtain Reproducible Separations Comparable to those of Molecules 1. 2. 3. 4.
5. 6. 7. 8. 9. 10.
microbes are generally amphoteric there are few, if any, pure microbe standards microbes can be very sensitive to the environment (pH, ionic strength, O2, etc.) and lyse under adverse conditions many microbes secrete a variety of substances; these secretions can cause the appearance of spurious or unwanted peaks, lysis of other cells, altered mobilities, or elution times and have other undesired effects some microbes aggregate to form chains, clusters, etc. some microbes attach to other microbes some microbes readily attach to surfaces solutions of microbes can change with time (e.g., multiply, lyse, etc.) even the smaller microbes can be orders of magnitude larger than most molecules on a per particle basis, microbial solutions are usually more dilute than solutions of most molecules; this can result in sampling problems
tion solution and the running buffer are very important. Surfactants should be avoided, as they tend to contribute to bacterial lysis. The nature of the buffer also can have significant effects on the (25) Cota-Robles, E. H.; Stein, S. M. Bacterial Cell Breakage or Lysis. In Practical Handbook of Microbiology; O’Leary, W. M., Ed.; CRC Press: Boca Raton, FL, 1989; pp 563-574. (26) Armstrong, D. W.; Schneiderheinze, J. M.; Schulte, G., 1999. Unpublished results.
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Figure 3. Capillary electropherogram showing the separation of three bacteria and baker’s yeast (S. cerevisiae) where small amounts of 600 000 MW PEO are added to the running buffer. Note the relatively short migration times and the high efficiency. The arrow denotes the migration time of the EOF marker mesityl oxide. See the Methods section for other experimental conditions.
Figure 2. Decrease in EOF velocity with increasing concentrations of PEO in running buffer. The EOF marker was mesityl oxide, the voltage was 10 kV, and the length of the 100-µm-i.d. capillary was 27 cm (20 cm to the detector).
separation of colloidal-sized bacteria. Proper pretreatment of specific microbial samples and choice of operating conditions (see Experimental Section and Figure 1) help negate the factors listed in Table 2. Two different approaches were found to be effective for the high-efficiency resolution and identification of intact bacteria and yeast. The first involves capillary electrophoresis using high-molecular-weight, water-soluble polymers. The second approach, involves CIEF and makes use of differences in the surface charge of the bacteria-cell wall. Dissolved Polymer CE. Without the polymer additive, the velocity of the electroosmotic flow (EOF) is rapid, which minimizes the electrophoretic contribution of the microbes which then tend to elute near EOF. Addition of PEO (average Mn ) 600 000) caused the expected decrease in the EOF velocity (Figure 2) but more importantly produced a differential migration and separation of several bacteria and yeast (Figure 3). Under the conditions of this separation, the microbes are negatively charged and migrate in a direction opposite to that of the EOF. The electrophoretic mobility of all four microbes was affected by the concentration of the added PEO. Higher concentrations of PEO produced longer migration times and different electrophoretic mobilities for all microbes (Figure 4). Since these curves are not parallel to one another, different elution orders can be obtained at different PEO concentrations (Figure 4). Importantly, the peak shapes are good and the efficiencies are high (n = 850 000 plates/m for M. luteus). The earliest eluting peak (i.e., closest to the EOF) was that for S. cerevisiae (yeast) which also happened to be the largest microbe in the study (e.g., 3-10 µm in diameter, Table 1). The other three bacteria are approximately the same size (∼1 µm). However, the M. luteus is spherical while P. fluorescens and E. aerogenes are rod-shaped (Table 1). It appears that the polymeric matrix within the running buffer may induce some size and shape selectivity; 5468 Analytical Chemistry, Vol. 71, No. 24, December 15, 1999
Figure 4. Concentration effect of 600 000 MW PEO (dissolved in the running buffer) on the electrophoretic mobility of four microorganisms.
however, further studies are needed to discern the exact mechanism and all factors contributing to selectivity. These are currently underway. Capillary Isoelectric Focusing. Under specific conditions some bacteria can be resolved from one another by taking advantage of their difference in surface charge (or more exactly their isoelectric point). To achieve reproducible results for the isoelectric focusing of bacteria, both the capillary and microbial pretreatment procedures (see Experimental Section) must be carried out exactly. The nature and concentration of the ampholyte also exerted a strong influence on the separation. Figure 5 shows the separation of three rod-shaped bacteria using CIEF. The E. coli is a little over 1 µm in diameter while P. putida and S. rubidae are a little less than 1 µm in diameter (Table 1). Again, good peak
taken to prevent aggregation; see Experimental Section). The analysis and control of these effects are beyond the scope of the current work and will be covered in subsequent reports.26
Figure 5. Electropherogram showing the CIEF separation of three bacteria of similar size. The separation conditions are outlined in the Experimental Section. Note the high efficiencies and relatively short analysis times.
shapes are observed and the efficiencies are excellent (n = 1 600 000/m for E. coli). In both the polymer CE and CIEF approaches described in this work, comparable peak shapes, efficiencies, and elution times were obtained for these microbes whether they were run individually (Figure 1B) or as mixtures (Figures 3 and 5). Under different experimental conditions, the presence of certain microbes can affect the integrity25,26 or elution26 of others. Also some microbes can aggregate to form clusters (e.g., M. luteus), which can affect the number and location of its peak(s) (unless precautions are
CONCLUSIONS Two different capillary electrokinetic methods can be used to achieve high-efficiency separation of a variety of bacteria and baker’s yeast. Polymer-containing CE running buffers may separate microbes via size and shape, among other factors. Capillary isoelectric focusing can separate similar size bacteria by surface charge. Meticulous sample preparation and exact separation conditions are needed to ensure reproducibility. Effective, broadly applicable analytical methods for the separation and quantitation of microbes (analogous to what is now done for molecules) could revolutionize aspects of microbiology involving the diagnosis and profiling of some diseases, evaluating soil populations for agriculture, bioremediation, quality control in fermentation processes, environmental studies, and many other areas of study. It has not escaped our notice that the coupling an effective microbial separation with MS, FT-IR, etc., would be a highly desirable direction for this work and would greatly simplify the use and interpretation of most spectroscopic analyses of microbes. Finally, the mechanism of microbial separations, particularly in semidilute polymers solutions, has not yet been addressed adequately. This will undoubtedly occur as interest in microbial and colloidal separations, in general, increases.
Received for review July 15, 1999. Accepted September 30, 1999. AC990779Z
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