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

Muhammad Saiful Islam Khan,Yun-Ji Kim,Se-Wook Oh

doi:10.1038/s41598-020-59448-8

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.

Highlights

In recent studies, the application of electron microscopy (EM) is rare in the area of microbial detection, whereas EM played some crucial role in detecting the cause of infectious diseases in earlier studies[27]
Energy dispersive X-ray (EDX) microanalysis is an elemental analysis technique related to EM based on representative X-ray generation that determines the types of elements present in the target analyte
The morphological information obtained from scanning electron microscopy (SEM) and the chemical composition determined by energy dispersive X-ray (EDX) techniques for the same individual cell has been elucidated, and principal component analysis (PCA) analysis was performed to distinguish these findings

Summary

Introduction

The application of electron microscopy (EM) is rare in the area of microbial detection, whereas EM played some crucial role in detecting the cause of infectious diseases in earlier studies[27]. This study sought to demonstrate the power of the SEM-EDX analysis method for the identification and clear distinction among individual cells of six commonly encountered pathogenic microbes. The morphological information obtained from SEM and the chemical composition determined by EDX techniques for the same individual cell has been elucidated, and PCA analysis was performed to distinguish these findings.

Results

Conclusion