Monitoring Bacterial Growth using a Capacitively-Coupled Contactless Conductivity Detector (C4D)

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Figure 1

Abstract

An automatic electrical bacterial growth sensor (EBGS) was developed, using capacitively-coupled contactless conductivity detection (C4D) with multiple channels. Eight disposable tubes containing culture samples of E. coli were monitored simultaneously using non-invasive C4D detection. High resolution growth curves were recorded by plotting the conductivity against incubation time. The results of the growth rate of E. coli were more accurate than those obtained with optical density and contact conductivity methods. The method could also be used for other tasks, such as the investigation of toxic/stress effects from chemicals and antimicrobial susceptibility testing. All of these performances required neither auxiliary devices nor additional chemicals and biomaterials. Taken together, this strategy has advantages of simplicity, accuracy, reproducibility, affordability, versatility and miniaturization, reducing the financial and labor costs.


Figure 2

Introduction

Insight into the nature of bacterial growth is of fundamental significance in many fields, such as medicine research, clinical diagnosis, food safety, fermentation industry and environmental monitoring. This includes the speed of proliferation, stress and/or toxic effect of coexistent chemicals, and physiological activities. Growth-based measurement is still ‘gold standard’ method for antimicrobial susceptibility testing and is employed to study the life revolution at gene level.

Various techniques for charactering the bacterial growth have been established. The plate counting and PCR (Polymerase Chain Reaction) methods are accurate and reproducible, but labor-intensive. Online monitoring methods have been developed, for example optical-based patterns, including turbidity, imaging, fluorescence, diffraction and reflectance. Electrochemical techniques such as impedimetric sensors are a very promising choice for monitoring bacterial growth, however, electrode deterioration and nonspecific binding are unavoidable since that the working electrodes must be in galvanic contact with the medium. This results in erratic measurements that decrease the accuracy.

This application note shows how C4D (capacitively-coupled contactless conductivity detection) can be used to solve problems faced by traditional electrical and electrochemical methods for online monitoring bacterial growth, as well as to realize automatic high-throughput measurement while equalling plate counting method and PCR method in precision.


Equipment Required

  • ER818 Octal Contactless Conductivity System, which includes:
    • ER815 Contactless Conductivity Detector
    • ET128 Octal headstage, Figure 1 shows a photograph of the headstage of the eight- channel C4D, with eight bacterial culture tubes inserted in.
  • A computer with Chart software or your own software to record the data (for example, Tera Term, LabVIEW, C#, WinWedge or HyperACCESS)


Experiment

The growth of bacteria at desired temperature transforms uncharged or weakly charged substrates, e.g. yeast, peptone, and sugar into highly charged end products, such as amino acids, aldehydes, ketones, acids, and other metabolites, causing an alteration in ionic concentration, increasing the conductivity of the medium, which is online monitored with C4D, allowing to character the process of bacterial growth. This is shown in Figure 2. The C4D electrodes, applying and recording the sine wave, are shown in pink.


Figure 3

Results

Characterize Growth of Escherichia Coli with EBGS

Figure 3 shows a typical curve of E. coli growth in LB medium; E. coli in LB medium is the blue line while pure LB medium is the red line (control). This is generated by plotting normalized apparent conductivity value (NACV) against incubation time over a period around 45000 s (12.5 h). It is a classical S-shaped cure similar to those obtained with OD method.

Figure 4
Figure 5

Quantitative Determination of Viable Escherichia Coli

Figure 4 shows the plots of NACV of different initial inoculum (CFU Colony-Forming Unit) of Escherichia coli as a function of incubation times obtained with the EBGS. The linear relationship between the logarithmic values of initial inoculum of Escherichia coli and detectable times is showed in Figure 5. This suggests that the proposed C4D method can also be used to rapidly quantify target bacteria based on the growth kinetics, just like employing optical, PCR, calorimetry and other electrochemical methods, though the conductivity responses cannot indicate the cell concentration directly.

Figure 6

Study of Stress Effect of Salinity on Escherichia Coli Growth

The bacterial growth is often affected by many environmental factors, e.g. salinity, temperature and pH. To elucidate the capacity of the EBGS for these kinds of investigation, salinity gradient was used to probe Escherichia coli growth. Figure 6 shows typical growth curves of E. coli growth in LB medium, in which the concentration of NaCl is 1.0, 2.0, 3.0 and 4.0 g/L, respectively. Apparently, the higher concentration of NaCl, the lower maximum growth rate and maximum outputs can be obtained, due to its inhibition effect on bacterial growth. The result is in agreement with those obtained with conventional optical method. Of note, there is an interesting phenomenon on the curves – the salinity plays almost little effect on the lag phases.


Reference

Online monitoring of bacterial growth with electrical sensor. Xuzhi Zhang, Xiaoyu Jiang, Qianqian Yang, Xiaochun Wang, Yan Zhang, Jun Zhao, Keming Qu, and Chuan Zhao. Anal. Chem., 2018, 90 (10), pp 6006–6011 DOI: 10.1021/acs.analchem.8b01214