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Applied and Environmental Microbiology, February 2005, p. 1117-1121, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.1117-1121.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Rapid and Simple Quantification of Bacterial Cells by Using a Microfluidic Device

Chieko Sakamoto,^1, Nobuyasu Yamaguchi,^1, and Masao Nasu^*

Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan¹

Received 15 May 2004/ Accepted 14 September 2004

ABSTRACT

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.

Quantification of bacterial cells is fundamental to most microbiological studies, thus, rapid and simple techniques are required for this purpose. Culture methods are widely used for bacterial enumeration; however, for accurate enumeration of bacterial numbers, including injured or starved cells, it is important to use culture-independent techniques (16). Fluorescent staining is suitable for this purpose, and various fluorescent dyes have been used to detect bacterial cells by epifluorescence microscopy and flow cytometry (3, 10, 13, 21). Epifluorescence microscopic observation provides color and cell shape information; however, individual differences in results usually arise at visual counting (11, 17). Microscopic observation is also labor intensive. Flow cytometry offers rapid, sensitive, and reliable quantification of individual cells. However, a flow cytometer is relatively expensive, and maintenance is complicated for an unskilled operator. Simpler methods for the enumeration of bacterial cells are required.

In recent years, miniaturization of chemical and biological assays, particularly via microchip technology, has been investigated (19, 20, 22). Microchip-based analysis has many advantages: (i) chip-based analysis is small scale and is completed in a shorter time period than analysis using conventional devices, (ii) consumption of sample and reagent is low, (iii) risk of biohazard is low because a microchip is a closed system and most microchips are disposable, and (iv) sample preparation on a microchip is usually simple and analysis is performed automatically with high reproducibility. Microchip-based devices have been employed for many analyses, such as capillary electrophoresis (8), capillary gel electrophoresis (5, 24), PCR (12, 23), and flow cytometry. Flow cytometry on microfluidic chip-based devices has been used for cell sorting of bacteria (4, 6, 7) or on-chip staining (15). These applications concentrate on recovery of target cells or labeling efficiencies of bacteria rather than quantification of total cell numbers.

This study investigated a commercially available microfluidic device for eukaryotic cells for the enumeration of bacterial cells with emphasis on appropriate staining and analysis. The applicability of flow cytometry with the microfluidic device (on-chip flow cytometry) was then evaluated by comparison to conventional epifluorescence microscopic counts.

An Agilent 2100 Bioanalyzer (Agilent Technologies) was used for on-chip flow cytometry. This system is a commercially available microchip-based analysis system for simple flow cytometry primarily of eukaryotic cells, and among its advantages are that the apparatus is relatively inexpensive and does not require substantial user training or experience. This instrument is capable of two-color fluorescence detection. The blue LED has a maximum emission at 470 nm. The maximum emission of the red laser diode is at 630 nm. The detection windows are at 525 nm for the green and 680 nm for the far-red channel. The measured fluorescence values and event number are displayed as a histogram or dot plot on a personal computer.

Fluorescently stained bacterial cells and fluorescent beads were analyzed with the Cell Fluorescence LabChip kit (Agilent Technologies). This kit supplies microchips, priming solution, focusing dye, and cell buffer. First, fluorescently labeled bacteria or fluorescent beads were diluted in a ninefold volume of cell buffer. A microchip was primed with 10 µl of priming solution. Next, 10 µl of focusing dye for laser and LED alignment and 30 µl of cell buffer were added to certain wells on the chip. After addition of 10 µl of sample suspension to the sample wells, the chip was run on an Agilent 2100 Bioanalyzer. This microchip design has no cross-point between samples; therefore, it could analyze 6 samples in one chip. Cell numbers could be counted up to approximately 2,500 cells per sample in 4 min, and the total time of analysis was within 30 min for 6 samples (i.e., one full microchip). The instrumentation of this chip-based system and the design of the microfluidic chip have been described in detail by Preckel et al. (18).

For microscopic counting, the conventional total direct counting technique reported by Hobbie et al. was used (9). Fluorescently stained bacterial cells were filtered onto a black polycarbonate membrane filter (pore size, 0.2 µm; Advantec) and counted at a magnification of x1,000 with an E-400 epifluorescence microscope (Nikon). The bacterial number was adjusted to approximately 80 cells per field of view, and more than 2,000 cells per sample were counted.

In general flow cytometry, the bacterial number is often calculated from a ratio of event number derived from bacterial cell comparison to a known number of internal standard particles. On-chip flow cytometry has high reproducibility; therefore, we expected that on-chip flow cytometry was able to calculate bacterial number from a standard curve determined by fluorescent beads. Quantification with a standard curve eliminates addition of standard particles to each sample, and this leads to simple preparation of samples.

A standard curve for quantification of bacterial cells was constructed using fluorescent beads which were 0.5, 0.7, 1.0, and 1.7 µm in diameter (Fluoresbrite YG Microspheres; Polysciences). A standard curve for quantification of large cells, such as eukaryotic cells, was also constructed using fluorescent beads which were 2.8, 6.0, and 11 µm in diameter (Fluoresbrite YG Microspheres; Polysciences) as well as a bead that was 5 µm in diameter (blue beads; Agilent Technologies). Fluorescence beads were diluted by sterilized deionized water just before the experiments were performed and were analyzed by on-chip flow cytometry (final concentration, 4 x 10⁵ to 5 x 10⁶ particles ml^–1).

Correlation between the number of on-chip flow cytometric events and the number of inoculated beads is shown in Fig. 1. All dots were distributed linearly and were positively correlated (Fig. 1A, r² = 0.96; B, r² = 0.90). Also, both standard curves for quantification of bacteria (Fig. 1A) and large cells (Fig. 1B) were nearly identical. From the regression line shown in Fig. 1A, the following expression could be calculated for the enumeration of bacterial number: bacterial number (cells ml^–1) = (event number) x (2.8 x 10³) – (3.1 x 10⁵), where event number was the event number of on-chip flow cytometry within a range of 190 to 1,900.

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FIG. 1. Standard curves with fluorescent beads analyzed by on-chip flow cytometry. Shown are analyses of beads with a diameter of 1.7 µm (), 1.0 µm (x), 0.7 µm (+), and 0.5 µm (–) (A), as well as beads with a diameter of 11 µm (), 6.0 µm (), 5 µm (), and 2.8 µm () (B). The line indicates the linear regression.

As confirmation of its expression accuracy, total direct counts (TDC) of Escherichia coli O157:H7 ATCC 43888 was determined with the expression. E. coli O157:H7 was cultured in Luria-Bertani (LB) liquid medium (10 g of Bacto-tryptone [Difco, Detroit, Mich.], 5 g of yeast extract, 10 g of NaCl in 1 liter of distilled water) at 30°C to stationary phase. Bacterial cells were harvested by centrifugation (5,000 x g, 5 min), washed twice with phosphate-buffered saline (PBS) (130 mM NaCl, 10 mM Na₂HPO₄, 10 mM NaH₂PO₄ [pH 7.2]), and suspended in 70% (wt/vol) ethanol at 4°C for bacterial fixation.

Fixed E. coli O157:H7 were stained with SYBR Green II (excitation, 497 nm; emission, 520 nm; Molecular Probes, Eugene, Oreg.) or double stained with SYBR Green II and TO-PRO 3 (excitation, 642 nm; emission, 661 nm; Molecular Probes). SYBR Green II, which was provided by the manufacturer as a 10,000x solution in dimethyl sulfoxide (DMSO), was added to the bacterial suspension (final dilution, 10x). The final dilution of TO-PRO 3 was 10 µM (a 1 mM solution in DMSO was provided by the manufacturer). These bacterial cells were stained for 5 min at room temperature (approximately 25°C) under dark conditions. After being stained, samples were analyzed immediately by on-chip flow cytometry or were observed by epifluorescence microscopy (bacterial numbers, 4 x 10⁵ to 6 x 10⁶ cells ml^–1).

At microscopic observation, bacterial cells stained with SYBR Green II were counted under blue excitation (Nikon B-2A cube with excitation filter EX 450-490, dichroic mirror DM 505, and absorption filter BA 520). Bacterial cells stained with TO-PRO 3 were observed under red excitation (Nikon CY5 HYQ cube with excitation filter EX 590-650, dichroic mirror DM 660, and absorption filter BA 663-735).

TDC of E. coli O157:H7 calculated with the determined expression are shown in Fig. 2. These samples did not have internal standard particles added. E. coli O157:H7 double stained with SYBR Green II and TO-PRO 3 showed a stronger red signal than cells single stained with SYBR Green II alone. There was a strong correlation between the on-chip flow cytometric count and the epifluorescence microscopic count within a range of 4 x 10⁵ to 5 x 10⁶ cells ml^–1 (Fig. 2A, r² = 0.95; B, r² = 0.96). Therefore, it was possible to say that TDC determined by on-chip flow cytometry with a standard curve and by epifluorescence microscopy were almost identical. Enumeration with a standard curve did not require the addition of internal standard particles; therefore, we achieved simple and easy sample preparation for the determination of TDC by on-chip flow cytometry.

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FIG. 2. Relationship between on-chip flow cytometric count (on-chip FCM) and epifluorescence microscopic count (EFM) of E. coli O157:H7. Bacterial cells were stained with SYBR Green II (A) or double stained with SYBR Green II and TO-PRO 3 (B). On-chip flow cytometric counts were determined by the standard curve (Fig. 1A). The line represents the linear regression.

For the specific detection of E. coli O157:H7 in a mixed bacterial population, fluorescently labeled antibody staining was applied. Polyclonal anti-E. coli direct antibody (O.E.M. Concepts, Toms River, N.J.) was labeled with Alexa Fluor 647 (excitation, 650 nm; emission, 668 nm) by use of a commercial kit (Alexa Fluor 647 Monoclonal Antibody Labeling kit; Molecular Probes). E. coli O157:H7 and Pseudomonas putida ATCC 12633 were cultured in LB liquid medium and fixed as described above. Pure culture of E. coli O157:H7, pure culture of P. putida, and a mixture of the two were stained for 30 min at room temperature by adding a one-third volume of bovine serum albumin (12% [wt/vol] in PBS) and 2 µg of fluorescent antibody. These bacterial samples stained with Alexa Fluor 647-labeled antibody were counterstained with SYBR Green II (final dilution, 10x) and analyzed by on-chip flow cytometry (final concentration of bacterial cells, approximately 2.5 x 10⁶ cells ml^–1). At microscopic observation, bacterial cells stained with Alexa Fluor 647-labeled antibody were observed under red excitation (Nikon CY5 HYQ cube).

In three samples, it was confirmed by fluorescent micrographs that the Alexa Fluor 647-labeled antibody reacted to E. coli O157:H7 specifically (data not shown). The same fluorescently labeled samples were analyzed by on-chip flow cytometry to count E. coli O157:H7. E. coli O157:H7 demonstrated strong signals derived from Alexa Fluor 647 (red fluorescence) and SYBR Green II (green fluorescence) (Fig. 3A), while P. putida showed a weaker red signal (Fig. 3B). Weak fluorescence appeared in the negative control (Fig. 3D, sterilized deionized water) and was considered background, which was then removed from all samples under 0.9 on the green fluorescence channel. After elimination of the background, the borderline between E. coli O157:H7 and P. putida was determined to be 8 on the red fluorescence channel (Fig. 3C). On-chip flow cytometric events are plotted based on two colors of fluorescence intensity. Separation of each population is not due to bacterial size but rather to the fluorescence intensity of Alexa Fluor 647. The total E. coli population plus the P. putida population (Fig. 3C) was defined as total bacteria. Therefore, a mixture of E. coli O157:H7 and P. putida was separated into two populations by fluorescent antibody staining, and both the number of E. coli O157:H7 and TDC could be determined simultaneously by on-chip flow cytometry.

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FIG. 3. On-chip flow cytometric analysis of E. coli O157:H7 (A), P. putida (B), a mixture of E. coli O157:H7 and P. putida (C), and sterilized deionized water (D) double stained by Alexa Fluor 647-labeled anti-E. coli direct antibody and SYBR Green II. The box labeled E. coli shows the area of E. coli O157:H7, and the P. putida box shows the area of P. putida.

Both the numbers of E. coli O157:H7 and TDC were determined by on-chip flow cytometry and epifluorescence microscopy to evaluate the accuracy of on-chip flow cytometric enumeration (Table 1). The ratio of E. coli O157:H7 to TDC analyzed by on-chip flow cytometry was 97% in the sample of E. coli O157:H7. In other words, almost all E. coli O157:H7 cells were detected by on-chip flow cytometry. Moreover, no cell was counted as E. coli O157:H7 in the P. putida sample. In the mixed sample of E. coli O157:H7 and P. putida, the ratio of antibody-labeled bacteria to TDC was 56% for on-chip flow cytometry and 50% for epifluorescence microscopy; once again, nearly identical. In conclusion, antibody-labeled bacteria could be detected and enumerated specifically from mixed bacterial samples by on-chip flow cytometry. Fluorescent antibody staining is an established method for detecting and identifying species of bacteria that have been cultured and whose antibody preparations are available. E. coli as well as other bacteria can be enumerated through the use of suitable fluorescent antibodies by this on-chip flow cytometry method.

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TABLE 1. Number of E. coli O157:H7 and total bacterial cells determined by on-chip flow cytometry and epifluorescence microscopya

Following the study with cultured cells, on-chip flow cytometry was applied to quantification of bacteria in river water samples. River water samples were collected at Kitahashi (Neyagawa River) in a commercial area and Hirano (Hirano River) in an industrial site of Osaka, Japan. These samples were obtained during April 2004. After sampling, bacteria in the river water samples were fixed by heat treatment at 95°C for 10 min.

For clear discrimination between bacteria in river water and background, river water samples were double stained with SYBR Green II and TO-PRO 3 as described above. TDC in river water samples were determined by both on-chip flow cytometry and epifluorescence microscopy with double-staining techniques (Table 2). The ratio of on-chip flow cytometric count to microscopic count was about 80% in Kitahashi and 120% in Hirano. These differences could be regarded as within a standard limit of error or individual differences in TDC of environmental samples. Bacteria in river water can be enumerated reliably by on-chip flow cytometry by using a double-staining technique.

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TABLE 2. Bacterial numbers in river water samples determined by on chip flow cytometry and epifluorescence microscopya

This study focused on a simple and rapid technique for quantification of bacteria by using on-chip flow cytometry. The time required for enumeration of bacterial cells was approximately 90 min per 6 samples for epifluorescence microscopy, while on-chip flow cytometry took only 30 min. Although cell numbers counted by both methods were almost identical (2,000 cells per sample on average), the time required for on-chip analysis was about threefold less. Furthermore, we demonstrated that total bacteria and antibody-labeled bacteria could be counted simultaneously in a bacterial mixture with double-staining by fluorescent antibody and nucleic acid stain. The time required for on-chip flow cytometric count was not increased (30 min) by the double-staining method. Additional operations were not required during on-chip flow cytometric analysis, because the data were acquired automatically. After completion of the analysis, no complex maintenance of the flow cytometer was required.

In addition to speed, ease of sample preparation is also important for bacterial quantification. We investigated an on-chip flow cytometric quantification method without addition of internal standard beads to samples by formulating a standard curve. Thus, sample preparation became even simpler than that for general flow cytometry, which often requires the addition of internal standard beads.

A large number of analytical microchip-based devices have been developed since the concept termed Micro-Total Analysis Systems (µ-TAS) was introduced (14). Several microchip-based devices are useful for concentrating and mixing samples (1, 2). An ideal selection of fluorescent antibodies and dyes in combination with the optimal microchip-based devices for pre- and posttreatment analyses is expected to make on-chip flow cytometry a routine method for the quantification of bacterial cells.

ACKNOWLEDGMENTS

This research was partially supported by a grant-in-aid for exploratory research from the Ministry of Education, Science, Sports, and Culture (grant no. 16651013).

 
* Corresponding author. Mailing address: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8170. Fax: 81-6-6879-8174. E-mail: nasu{at}phs.osaka-u.ac.jp.

FOOTNOTES

C.S. and N.Y. contributed equally to this work.

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Applied and Environmental Microbiology, February 2005, p. 1117-1121, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.1117-1121.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sakamoto, C. Articles by Nasu, M. Search for Related Content PubMed PubMed Citation Articles by Sakamoto, C. Articles by Nasu, M. Agricola Articles by Sakamoto, C. Articles by Nasu, M.