Novel Esterase Substrate for the Quantification of Bacterial Viability

The determination of bacterial viability is important in the fields of microbiology, medicine, and food technology. Bacterial viability assays are commonly used in research for evaluating antimicrobial properties, for determining viability of environmental species and for assessing fermentative microbe efficacy in brewing applications ^[1,2]. Since a diverse range of microorganisms and pathogenic microbes grows on food sources and environmental reservoirs (e.g. water, air and soil), the accurate detection and quantification of bacterial viability is important for monitoring contamination. Traditionally, microbial viability assessment is determined utilizing plating and counting techniques, which involve careful sample dilution, plate-spreading, lengthy incubation times and manual counting ^[3]. Although these methods are inexpensive, interpretation of viable count data must be considered carefully because it is unable to account for viable but non-culturable bacteria. As such, fluorescence-based techniques for measuring bacterial viability have been developed. These techniques quantify and detect viable bacteria more efficiently and with greater sensitivity, and are amenable to be used in conjunction with assays for analysis of membrane integrity, respiratory activity or metabolic enzyme activity ^[4].

A frequently used fluorescence technique for monitoring metabolically active bacteria is based on measuring intracellular esterase activity using esterified fluorogenic substrates. Esterification of fluorogenic substrates is optimal for two reasons. First, it alters the charge of the substrate making it more neutral. This improves cell permeability allowing the substrate to passively cross intact cell membranes. Secondly, esterified substrates are non-fluorescent. The substrate does not become fluorescent until it has entered the cell and intracellular esterases cleave off the ester functional groups.

Carboxyfluorescein diacetate (CFDA), a derivative of fluorescein diacetate (FDA), is used in both flow cytometric and fluorescent microscope applications for the rapid detection and quantification of bacterial viability relative to esterase activity ^[5]. Although CFDA staining curtails many of the disadvantages of the traditional colony counting assays, pitfalls still remain. The poor cellular retention exhibited by CFDA contributes to a low signal-to-background ratio and a decrease in assay sensitivity. To address these limitations of CFDA, AAT Bioquest has developed MycoLight™ 520, a novel replacement with superior staining performance.

 

Brighter Staining of Viable Bacteria using MycoLight™ 520

---

In our comparison studies, Escherichia coli (E. coli) bacteria treated with MycoLight™ 520 and CFDA were evaluated using fluorescence microscopy. As illustrated in Figure 1D, MycoLight™ 520 exhibited improved intracellular retention and a significantly higher signal-to-background ratio compared to CFDA. A notable drawback of CFDA was the continuous dye leakage after 1 hour which generated high levels of background interference to the extent that individual cells were indistinguishable (Figure 1B). As for MycoLight™ 520, no significant change in the signal-to-background ratio was exhibited after 1 hour. Another benefit not illustrated by the fluorescence images, is that MycoLight™ 520 can be rapidly loaded into viable bacteria. MycoLight™ 520 only requires a 5 minute incubation time at 37 °C. Additionally, washing steps are not required, but an enhancer may be used to further improve the signal-to-background ratio of MycoLight™ 520. CFDA, however, requires a 30 minute incubation period and multiple washes after staining to reduce background interference.

MycoLight™ 520 Staining For Flow Cytometry

---

Furthermore, we investigated the efficacy of MycoLight™ 520 at quantifying live and dead bacteria utilizing flow cytometry. Accuracy was evaluated using different proportions of live and dead E. coli suspensions treated with MycoLight™ 520. Figure 2A illustrates the easily distinguishable staining patterns of live and dead bacterial populations treated with MycoLight™ 520. When measured in the FITC channel, peaks generated at higher intensities were representative of live bacteria while peaks generated at lower intensities illustrated the dead bacteria. The count of each sample was plotted against the percentage of live bacteria in order to generate a standard curve which can be used to accurately quantify the viability proportions of unknown sample (Figure 2B).

Conclusion

---

Our direct comparison of CFDA and MycoLight™ 520 demonstrates that MycoLight™ 520 is a more robust and sensitive tool for staining and differentiating live and dead bacteria. MycoLight™ 520 dye exhibited a more rapid cellular uptake, with an incubation time of 5 minutes at 37 °C. Furthermore, MycoLight™ 520's improved intracellular retention resulted in a significantly higher signal-to-background ratio, and allowed for staining to be detected for much longer periods of time. Based on these finding, MycoLight™ 520 is a superior alternative to CFDA in fluorescence microscope and flow cytometric applications for assessing bacterial viability.

Method

Sample Protocol for Microscopic Imaging or Flow Cytometer Assay

1. Bacteria should be cultured in appropriate medium to late-log phase to allow optimum detection.
2. For each sample, prepare bacteria in 0.5 mL of staining buffer at a density of 1X10⁶ " 1X10⁸ cells/mL.
3. Add MycoLight™ 520 into 0.5 mL of bacteria suspension, for microscope imaging, the addition of an enhancer is recommended.
4. Incubate bacteria with MycoLight™ 520 at 37 °C for 5 min.
5. Image bacteria using fluorescence microscope or analyze cells with a flow cytometer using the FITC filter set

Note: To exclude debris, it is recommended to set the threshold of the flow cytometer as the following: FSC >10,000, SSC>5,000.
Note: The efficiency of MycoLight™ 520 is highly strain dependent and the staining conditions should be optimized accordingly.

References

---

1. Oliver, Stephen P., Bhushan M. Jayarao, and Raul A. Almeida. "Foodborne pathogens in milk and the dairy farm environment: food safety and public health implications." Foodbourne Pathogens & Disease2, no. 2 (2005): 115-129.
2. Cui, Maojin, Zhuliang Yuan, Xiaohua Zhi, and Jianquan Shen. "Optimization of biohydrogen production from beer lees using anaerobic mixed bacteria." international journal of hydrogen energy34, no. 19 (2009): 7971-7978.
3. Miyanaga, Kazuhiko, Suguru Takano, Yuki Morono, Katsutoshi Hori, Hajime Unno, and Yasunori Tanji. "Optimization of distinction between viable and dead cells by fluorescent staining method and its application to bacterial consortia." Biochemical Engineering Journal37, no. 1 (2007): 56-61.
4. Kogure, Kazuhiro, Ushio Simidu, and Nobuo Taga. "A tentative direct microscopic method for counting living marine bacteria." Canadian Journal of Microbiology25, no. 3 (1979): 415-420.
5. Miyanaga, Kazuhiko, Suguru Takano, Yuki Morono, Katsutoshi Hori, Hajime Unno, and Yasunori Tanji. "Optimization of distinction between viable and dead cells by fluorescent staining method and its application to bacterial consortia." Biochemical Engineering Journal37, no. 1 (2007): 56-61.