Power of Scanning Electron Microscopy and Energy Dispersive X-Ray Analysis in Rapid Microbial Detection and Identification at the Single Cell Level

Abstract

The demand for rapid, consistent and easy-to-use techniques for detecting and identifying pathogens in various areas, such as clinical diagnosis, the pharmaceutical industry, environmental science and food inspection, is very important. In this study, the reference strains of six food-borne pathogens, namely, Escherichia coli 0157: H7 ATCC 43890, Cronobacter sakazakii ATCC 29004, Salmonella Typhimurium ATCC 43971, Staphylococcus aureus KCCM 40050, Bacillus subtilis ATCC 14579, and Listeria monocytogenes ATCC 19115, were chosen for scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis. In our study, the time-consuming sample preparation step for the microbial analysis under SEM was avoided, which makes this detection process notably rapid. Samples were loaded onto a 0.01-µm-thick silver (Ag) foil surface to avoid any charging effect. Two different excitation voltages, 10 kV and 5 kV, were used to determine the elemental information. Information obtained from SEM-EDX can distinguish individual single cells and detect viable and nonviable microorganisms. This work demonstrates that the combination of morphological and elemental information obtained from SEM-EDX analysis with the help of principal component analysis (PCA) enables the rapid identification of single microbial cells without following time-consuming microbiological cultivation methods. Rapidly detecting and identifying biological threat microorganisms without traditional culture or chemical-based methods are highly important. The widely used identification techniques are nucleic acid-based, biosensor-based and immunologically based techniques. Real-time PCR multiplex PCR, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), and oligonucleotide DNA microarray are examples of some common nucleic acid-based identification techniques 1-4. These techniques have higher sensitivity, specificity, and reliability and can detect multiple pathogens in an automated manner with several constraints, such as sensitivity to PCR inhibitors and complicated primer design, and the methods cannot differentiate viable and nonviable cells 5,6. All of these nucleic acid-based techniques are slow processes that require 4-72 h to detect microbes 1-6. Electrochemical, optical and mass-based biosensors are commonly used to detect microbes. These automated, label-free, real-time detection processes can handle a large number of samples. Biosensor-based processes have several drawbacks, such as long incubation time, numerous washing steps, low specificity, interference with the food matrix and unsuitability for lesser cells 7-10. The lateral flow immunoassay and enzyme-linked immunosorbent assay (ELISA) are two immunological-based detection techniques with several advantages and disadvantages; most importantly, these techniques are also slow processes that require 3-10 h 11,12. These limitations increase the overall cost of the detection process due to costly logistics trails and restrict autonomous operation 13,14. ...

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