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Capillary Electrophoresis of Microbes Bull. Korean Chem. Soc. 2003, Vol. 24, No.

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Capillary Electrophoresis of Microbes
Byoung Geoun Moon, Yong-Ill Lee,† Seong Ho Kang,‡ and Yongseong Kim*

Division of Chemistry and Chemical Engineering, Kyungnam University, Masan 631-701, Korea

Department of Chemistry, Changwon University, Changwon 641-773, Korea

Department of Chemistry, Chunbuk National University, Jeonju 561-756, Korea
Received October 9, 2002

Direct analysis of microbes such as either gram-positive or gram-negative bacteria without cell lysis was
investigated using capillary electrophoresis. Bacteria cells were directly introduced into the microbore fused-
silica capillary, then separated under high electric field in less than 15 min. It was found that a proper dispersion
of bacteria cells was important for reproducible results. Migration behavior of bacteria at different storage
condition was investigated and many unexpected peaks were observed from bacteria stored at room
temperature due to the distortion of cells. This phenomenon was attributed to the change of size and shape of
the same bacterium and confirmed by the scanning electron microscopic images.
Key Words : Capillary electrophoresis, Bacteria, Microbes, Scanning electron microscopy

Introduction scence probes.9 Bacteria from infected urinary tract were
separated using a water-soluble polymer solution.16
Direct analysis of bacteria without pretreatment is important Certain bacteria have strong affinity to the cell surface of
in the area of diagnosis and profiling of some diseases, other bacteria due to strong hydrophilic and hydrophobic
evaluation of soil populations for agriculture, bioremedia- interactions. Therefore, chains or clusters of bacteria cells
tion, and quality control in fermentation processes.1 tend to be easily formed. In our study, we have investigated
Conventionally, bacteria cells have been lysed to investi- the formation of chains or clusters of bacteria and demon-
gate unique genetic materials or cell components.2,3 How- strated the successful separation and detection of intact
ever, many cell components such as lipoproteins, glyco- bacteria. Also, the activities of bacteria at different storage
proteins, lipids, lipopolysaccharides, and polynucleotides conditions for improved diagnosis were evaluated and
have similar structures in different bacteria, causing many confirmed by the analysis of scanning electron microscopic
unwanted peaks or negative identifications of bacteria.1,4 images.
Fluorescence assay and immunoassay have been introduced
and turned out to be efficient. However, the culture of Experimental Section
bacteria needs long time and the synthesis of selective
antibodies are required before analysis.5,6 Also, the sample Chemicals. Aerobacter aerogenes, Pseudomonas fluorescens
pretreatment process is time consuming and nonspecific and Micrococcus lysodeikticus were received as freeze-dried
absorption of antibody can cause negative results.7 Matrix- (Sigma-Aldrich Co.). Tris(hydroxymethyl)aminomethane
assisted laser desorption/ionization (MALDI) mass spectro- (TRIZMA base), boric acid, etheylenediaminetetra-acetate
metry has been employed for the direct analysis of bacteria (EDTA), poy(ethylene)oxide (PEO, Mw = 1,000,000 and
using specific biomarker ions in the bacteria cell.8 However, 400,000), phosphoric acid, potassium hydroxide were also
vast number of m/z peaks was sometimes observed due to purchased from Sigma-Aldrich.
many cell constituents, resulting in tough data analysis. Capillary electrophoresis. Buffer (4.5 mM Tris, 4.5 mM
Capillary electrophoresis (CE) would be a prominent boric acid, 0.1 mM EDTA, pH = 8.4) was diluted 8 : 1 by
analytical technique for direct analysis of bacteria since it is deionzed water. Poly(ethylene)oxide (Mw = 1,000,000 or
simple and efficient.9 Capillary electrophoresis has been 400,000) was dissolved in this buffer at the concentration of
widely applied to organic and inorganic ions as well as 0.5%. The polymer solution was stirred for 2 h, then ultra-
colloidal particles.10 Lately, capillary electrophoresis has sonicated for 1.5 h for complete dispersion. It was further
been used for the analysis of deoxyribonucleic acid (DNA), diluted to 0.0125% and ultrasonicated before the electro-
protein, virus, and bacteria.1,11,12 phoresis running.
In the analysis of intact microbes, capillary electrophoresis Bacteria samples were prepared by dissolving in the 8 : 1
showed the successful separation of a tabacco mosaic virus diluted buffer at the concentration of 1 mg/mL. They were
and Lactobacillus casei.13 The analysis of four types of precipitated by ultracentrifuge at 12,000 rpm and the super-
human rhinovirus with a surfactant was reported by capillary natant was decanted. They were redissolved in the 8 : 1
isoelectric focusing (CIEF).14 Yeast cells with different growth diluted buffer and completely dispersed by ultrasonication
phase showed different isoelectric points.15 The cell viability for 5 min. For the investigation of migration behavior of
was determined by capillary electrophoresis with the fluore- bacteria depending on age or growth condition, Areobacter

and this phenomenon can be attributed to a decreased this work.9 scanning electron microscopy. 1 Byoung Geoun Moon et al. PACE 5000. while were performed between wash steps and the supernatant was Pseudomonas fluorescens has flagella. sample was obtained with high separation efficiency. Initially. aerogenes flagella aerobes soil. Vol. then When the bacteria samples were well dispersed with ultra- 0. However. feasible since they produce many unwanted peaks in the Electrophoresis run was performed by using Beckman electropherogram. substances from soil. Each bacteria sample with the great suppression of chain or cluster formation was injected by 0. many bacteria can secret proteins. irregular cluster nonmotile 30 oC strict skin of man and other animal. Pre-electrophoresis sonication for 5 min before injection. deionized different migration times as shown in Figure 1a and 1c. The images of that the formation of chains or clusters are favorable under bacteria stored at 0 oC and at 23 oC were compared by the normal buffer condition. Micrococcus lysodeikticus is non-motile. The capillary was preconditioned by of chain or cluster resulting in several strong peaks with washing with 0. Most of other small biomolecules in the buffer. and while Micrococcus lysodeikticus is non-motile. The SEM image was taken by Hitachi S-4200 at coccus lysodeikticus is close to sphere. deikticus and Pseudomonas fluorescens at 8 : 1 diluted runn- Each sample was then washed twice with 60%. ing is either positive or negative.5 N phosphoric acid for 2 min. enzymes. We believe that a lower since the mobility of each bacterium under electric field is buffer concentration changed the local environment of bacteria determined by those factors. Aerobacter Table 1. Therefore.) with 75 micrometer i. No. Korean Chem.000 rpm in Table 1. Ultracentrifuge with 12. and Gram stain. lysodeikticus or regular packets aerobes soil. The shape of Micro- decanted. Pseudo.5-1 motile rods ú 4-6 peritrichous 37 oC facultative faces of man other animal. tion. sewage Micrococcus positive 1-2 spheres. the surface bacteria are less than 2 micrometer in diameter. Therefore. than in diluted urine. cell surface. resulting in bacteria is unique (rod.82 Bull. The resolution at 10 fluorescens is rod-like. As compared to Figure 1b and 1d. 24. and living ration but different sample buffer concentration was reported16 environment. Total length of the capillary was 27 cm with the effec. dust.and extracellular components for the detection of Escherichia coli in urinary tract. and the cell surface of other electropherograms. Also. while Pseudomonas 10 kV and magnified by 20000 times.5-1 slightly curved rods one or several 25-30 oC strict soil and water fluorescens flagella aerobes Aerobacter negative 0.5 psi for 10 s and run at 10 kV. A fused silica capillary (Polymicrotechnology Figure 1 shows the electrophoretic separation of Micro- Co. As shown in Table 1. Micrococcus lysodeik- run before bacteria sample injection was underwent for 5 ticus and Pseudomonas fluorescens showed a strong peak min for the stabilization of baseline. interference of microbe analysis or migration time shifts.0125% PEO running buffer for 2 min. A UV (Figure 1b and 1d). Soc.000). Size and shape are so critical in electrophoresis concentration of running buffer. coccus lysodeikticus and Pseudomonas fluorescens. 80%. ing buffer with 0. water . the mobility shift at the same running buffer concent- amphoteric and sensitive to pH. In general. Baseline resolution for the mixed kV was around 2.0 nm and a cold-cathode field emission Micrococcus lysodeikticus and Pseudomonas fluorescens was employed for the generation of the second electron. sphere spheroidal). kinds of bacteria. bacteria are Even. 70%. was employed for the separa. intra. proper dispersion. The classes and properties of bacteria Optimum Gram Diameter Aerobe or Bacterium Form Flagella growth Existence in nature stain (µm) anaerobe Temperature Pseudomonas negative 0. The formation of chains or caused by the age or changes in growth conditions. ionic strength. required before every injection of bacteria samples. The shape of properties of mixed bacteria may be altered. in those bacteria cells are so diverse that the separation of Escherichia coli in concentrated urine had slower mobility bacteria under electric field has to be carefully controlled. We found that ultrasonication was absorption detection at 214 nm was employed. aerogenes was stored at 0 oC or at room temperature for 15 clusters makes the electrophoretic separation of bacteria less days. water.d.000. Without tion. water for 3 min. meaning Scanning electron microscopy (SEM). 2003. and the use of internal standard was Certain bacteria have strong affinity to organic or inorganic suggested for the identification of bacteria or comparing surface. bacteria cells were Figure 2 shows the separation of mixed Micrococcus lyso- fixed by glutaraldehyde 20% solution and stored for 24 h. chains or clusters of bacteria In order to investigate variations of shape or size of microbes cells tend to be easily formed. different migration Results and Discussion times were observed for Micrococcus lysodeikticus and Pseudomonas fluorescens in Figure 2 compared to in Figure Table 1 shows the characteristics of bacteria employed in 1. 1 N potassium hydroxide for 2 min.0125% PEO (Mw = 1. bacteria tend to form many different sizes tive length of 20 cm. As shown 90% and 95% of methanol. generating mobility shifts and improved resolu- monas fluorescens and Aerobacter aerogenes have flagella.

they can grow even under unculturable conditions. when it was stored at room temperature (slightly active) for 15 days. 24. a strong peak with several minor peaks was observed (Figure 3a). When Aerobacter aerogenes was stored at 0 oC (complete- ly dormant). Other condition: fused silica capillary. and 0.d.5 mM boric acid.4) was composed of 4.. Soc. + 10 kV running voltage.0 mg/mL.5 psi. As suggested by Amstrong.0125% poly(ethylene)oxide (Mw = 1. 75 micrometer i. 4. bacteria at different growth stages may be advantageous in obtaining information regarding the behavior of microbes under high electric field. Figure 2. Electropherograms of mixed Micrococcus lysodeikticus This gives the idea that the identification and separation of and Pseudomonas fluorescens. Running buffer was further diluted 8 : 1 from that used and storage condition. the analysis of were taken for Aerobacter aerogenes as shown in Figure 4a . It was known that the size of bacteria became smaller under those condi- tions probably due to the secretion of cellular components.5 mM Tris. however. scanning electron microscopic images it difficult to identify microbes. Vol. 20 cm effective length.000).05X TBE buffer at the concentration of 1. bacteria were dispersed in an ultrasonic bath for 5 min. For figure 1b and 1d. Since bacteria are living organism.16 the use in figure 1. 1c and 1d). Korean Chem. For figure 1a and 1c. room temperature. of internal standards would produce the reproducible results for both qualitative and quantitative analysis of microbes. 1 83 Figure 1.1. for 10 s.17 The change of size and shape and the formation of chains or clusters would cause the mobility shifts and unwanted peaks. However. pressure injection at 0. aerogenes was stored at two different temperature conditions To confirm the changes of size and shape at different and compared as shown in Figure 3.000. Electropherograms of Micrococcus lysodeikticus (Fig.1 mm EDTA. Bacteria samples were dissolved in 8:1 diluted 0.5 mg/mL. Each bacterium concentration was microbes should be taken in consideration of growth stage 0. 1a and 1b) and Pseudonomas fluorescens (Fig. Running buffer (ph = 8. bacteria were injected into the column without the treatment by ultrasound.Capillary Electrophoresis of Microbes Bull. 2003. 0. The variation may make storage conditions. No. many noticeable peaks were obtained (Figure 3b) indicating the formation of chains or clusters. Other conditions are the same as in figure 1. 27 cm total length.

. Soc. 3rd ed. Johnson. 4. 211. Tomas. using capillary electrophoresis without cell lysis. Vol. Also. S. Figure 4a and 4b correspond to figure 3a and 3b. C (Fig. and 4b. bacterium sample was dispersed by ultrasonication for 5 min before injection. respectively. figure 4b (Aerobacter capillary electrophoresis would be useful since it shows the aerogenes stored at room temperature for 15 days) shows great potential as a fast and simple diagnosis tool. W. This change would cause toward the separation of bacteria with cell fixation and/or many spurious peaks as shown in figure 3b since the using a lab-on-a-chip technique is under progress. 25. E. Schulte. The European wise. Schneiderheinze. Elisha. A. Lastovica. We believe that direct analysis of pathogenic microbes by respectively. Anal. O. Electropherograms of Aerobacter aerogenes stored at 0 o aerogenes. W. turned out to be important for the analysis of bacteria. Duche. 3b). Namane. the Korea Science & Engineering Foundation.. SEM was performed using Hitachi S-4200 at 10 kV Formation of more chains or clusters was observed with Aerobacter and magnified 20.. 359. the storage condition was Lett. We found D. Lett. Compared to figure 4a. ent environment. Conclusions References Microbes with different size and shape were separated 1. 3b.. . capillary inlet was critical for reproducible results... 3b). 2002.. 1992. J. 71. SEM images clearly suggest that the growth condition Acknowledgment. K. Figure 4. Westenberg. This work was supported by grant No. 2003. D. 2002. unwanted peaks due to the formation of chains for Listeria Genome Consortium.. Armstrong. On. 1997. Anal. For figure 3a and experimental section. 3a) or stored at room temperature for 15 days (Fig. F. The scanning electron microscopic images of Aerobacter Figure 3. M.000 times. Chem. 198. F. electrophoretic mobility is the function of size and shape. Other. clusters were observed. G.. No. 24. American temperature showed different migration behavior and it Public Health Association: Washington. see the aerogenes stored at room temperature (Fig. J.. 3. that a proper dispersion of microbes before injection to the FEMS Microbiol. J. Alexander. Tremoulet. 6.. Ransom. Matsheka.. FEMS Microbiol. 5. D. For the preparation of bacteria. could be attributed to the change of size and shape at differ. DC... 2. Study distorted shapes with uneven sizes. 5465.. C.84 Bull. B. B. Labadie. Compendium of Methods for Electropherograms of bacteria stored at 0 oC or at room the Microbiological Examination of Food. Sharar. 210. L. should be carefully considered to separate and identify R05-2001-000-00238-0 from the Basic Research Program of bacteria.. M. 17. This change was confirmed by analysis of the scanning electron microscopic images. G. F. J. Splittsoesser. L. Vanderzant. 1 Byoung Geoun Moon et al. Korean Chem. I. B.. C. G. L. J. Figure 4a and 4b correspond to figure 3a and 3b.. Chem. 1999. Rose. A. P. A. W. Martinie. M.

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