Kendra Liu, Narine Arabyan, Nguyet Kong, Marie-Pierre Forquin Gomez, and Bart C. Weimer

Department of Population Health and Reproduction

School of Veterinary Medicine, University of California – Davis, CA, USA

ACKNOWLEDGMENTSI am grateful to Dr. Weimer for his guidance and helpful

discussions. I also acknowledge and appreciate all the help and

support received from everyone in the Weimer Lab.

CONCLUSIONAlternative method to quantify bacterial and eukaryotic cells.

Numbers determined by this new method were similar to

those obtained from traditional methods of plating the cells for

bacteria or hemocytometer for eukaryotic cells.

The physiological state of the cells were differentiated into

live and dead cells with the dyes SYTO62 and SYTOX,

respectively.

INTRODUCTION

Quantification and detection of the physiological state of the

cells is fundamental to microbiological studies. Traditional

methods of bacterial enumeration and detection of

physiological state via microscopic observations are labor and

time intensive (Ikeda et al. 2009). Using traditional culturing

methods, compromised or dead cells would not be able to be

enumerated. Therefore, a new, fast, and accurate method of

cell enumeration and detection of physiological state of the

cell is necessary to develop. This new method needs to be

culture independent. One suitable way is the use of

fluorescent dyes to stain the cells and flow cytometry. This

instrument uses two-color fluorescence detection. The red

laser diode has an excitation range of 620-645nm with max

emission of 630nm and detection range of 674-696nm

(Sakamoto et al. 2005). The blue LED has an excitation range

of 458-482nm with max emission of 470nm and detection

range of 510-540nm (Sakamoto et al. 2005).

Microchip based analysis has many advantages over the

conventional methods (Müller & Nebe‐von‐Caron 2010). This

provides a rapid, sensitive, and reliable quantification of

individual cells. The analysis is small scale and is completed

in a shorter time period. The assay takes only thirty minutes to

run six samples and it counts 2500 cells in four minutes

(Sakamoto et al. 2005). The consumption of the samples and

reagents are low. Lastly, preparation of samples is simple and

the analysis is performed with high reproducibility.

The aim of this investigation was to develop an assay that will

quantify and detect the physiological state of the cells: Madin-

Darby Canine Kidney (MDCK) epithelial cell line and

Salmonella enterica Typhimurium ST14028 using Agilent

2100 Bioanalyzer System on-chip flow cytometry.

ABSTRACT

This study describes the use of cell fluorescence assays

on the Agilent 2100 Bioanalyzer System. The Agilent

2100 Bioanalyzer System is the first commercially

available instrument capable of measuring cell

fluorescence for cell sorting. This instrument uses two-

color fluorescence detection—the red laser diode and

blue LED. The cell assay makes use of microfluidic chip-

based flow cytometry to quantify and detect the

physiological state of the cells. Salmonella enterica spp.

enterica serovar Typhimurium ST14028 and Madin-Darby

Canine Kidney (MDCK) epithelial cells were quantified

using the Agilent 2100 Bioanalyzer System . The

physiological states of the cells were detected using

SYTO62 and SYTOX dyes for live and dead cells,

respectively. Numbers determined by this method were

similar to those obtained by CFU counts from plates and

microscopic count using a hemocytometer, respectively.

With low cell and reagent consumption and running up to

six samples at a time, data analysis using the software on

the Agilent 2100 Bioanalyzer is quick and simple.

.

EXPERIMENTAL APPROACH RESULTS

Figure 1. Detection of live bacterial cells

with SYTO62 red fluorescent dye. 10µM is

the optimum concentration of SYTO62 to

detect live bacterial cells.

Figure 3. Optimization of SYTOX green

fluorescent dye. 5µM is the optimum

concentration of SYTOX to detect dead

bacterial cells.

Grow cells to mid-

exponential phase

Development of an assay to quantify and detect the physiological state of the cells

CONTACT INFORMATION

Bart C. Weimer, Ph.D. (bcweimer@ucdavis.edu)

Narine Arabyan (narabyan@ucdavis.edu)

Kendra Liu (kkliu@ucdavis.edu)

UC Davis (VM:PHR) VetMed3B – Room 4016

1089 Veterinary Medicine Dr. Davis, CA 95616

(530) 752-6426

http://weimermicrolab.wix.com/thelab

REFERENCES

Ikeda, M., Yamaguchi, Nobuyasu & Nasu, Masao, 2009. Rapid On-chip flow Cytometric

Detection of Listeria monocytogenes in Milk. Journal of Health Science, 55(5), pp.851-856.

Johnson, S., Nguyen, V. & Coder, D., 2001 Assessment of Cell Viability. Current Protocols in

Cytometry.

Müller, S. & Nebe-von-Caron, G., 2010. Functional single-cell analyses: flow cytometry and cell

sorting of microbial populations and communities. FEMS Microbiology Reviews, 34(4), pp.554-

587.

Sakamoto, C., Yamaguchi, N. & Nasu, M., 2005. Rapid and Simple Quantification of Bacterial

Cells by Using a Microfluidic Device. Applied and Environmental Microbiology, 71(2), pp.1117-

1121.

Figure 4. Quantification of dead

bacterial cells.

Figure 7. Differentiation of live and dead

MDCK cells when new and old/overgrown

MDCK cells were used with both 1 µM

SYTOX and 2 µM SYTO62.

Figure 8. Quantification of MDCK cells.

Bacterial cellsSalmonella enterica

Typhimurium ST14028

Grow cells for 14 hours

Eukaryotic cellsMadin-Darby Canine

Kidney epithelial cells

Incubate with SYTO62 / SYTOX for 1 hour at room

temperature in the dark

Count cells using

hemocytometer

Grow cells for 24 hours

30 minutes for

6 samples

Dilute cells to 106 cell/mL

Live Cells: SYTO62 Dead Cells: SYTOX

Load onto the cell chip

Calculate number of cells from event number

Red laser diode Blue LED

Detection of Live Bacterial Cells With SYTO62

Detection of Dead Bacterial Cells With SYTOX

Figure 2. Quantification of live cells from

event numbers.

Detection of Live and Dead MDCK Cells With

Both SYTO62 and SYTOX

Detection of Live and Dead Bacterial Cells With

Both SYTO62 and SYTOX

Figure 5. Detection of Live and Dead cells

with 10 µM SYTO62 and 5 µM SYTOX.

Figure 6. Quantification of Live and

Dead cells with 10 µM SYTO62 and 5

µM SYTOX.

FUTURE DIRECTIONS

Identify the different populations of cells during

Salmonella infection