Advertisement

Influence of the Novel Rapid Preparation Method on Sample Imaging Evaluation for Pathogenic Microorganisms Scanning Electron Microscopy

  • Mingzhu Song
  • Xirui Wang
  • Wei Yao
  • Yang Su
Conference paper
Part of the Advances in Intelligent Systems and Computing book series (AISC, volume 856)

Abstract

This study provides a novel rapid and effective sample processing method for scanning electron microscopy observation of bacterium and fungi. We investigated the relationships between image quality and instrument operating parameters. The procedure consists of seven stages, including fixing, cleaning, dehydration, replacement, drying, spraying and observation. The incorporation of tertiary butyl alcohol (TBA), which played important roles in both dehydration and cleaning, was optimized using six distinct tertiary butyl alcohol concentrations (30%, 50%, 70%, 85%, 95% and 100%, v/v). Electrically conductive adhesive transfer tape was chosen as the carrier on which samples were mounted for SEM observation. Saved the time of preparation and cut down the use of expensive toxic reagents. The methodology established in this study helps to improve the efficiency for identifying the ultra microstructures of pathogenic microorganisms. The method describes the complete method development and validation process and is well suitable for pathogenic microorganisms scanning electron microscopy sample imaging evaluation.

Keywords

Biofilm Pathogenic microorganisms Sample preparation SEM 

Notes

Acknowledgments

We are grateful to Canquan Mao Professor from Laboratory of Molecular Evolution and Applied Biology, School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, P. R. China for helping us with language editing and research platform. This work was financially supported by Sichuan Provincial Department of Education 2017 Natural Science Key Project (No. 17ZA0095); Sichuan Provincial Key Laboratory of Medicinal and Edible Plant Resources Development Fund Project (No. 10Y201705).

Conflict of Interest.

Mingzhu Song and Xirui Wang contributed equally to this work, as co-author of the first author. All authors have no conflicts of interest.

References

  1. 1.
    Rajesh, P.S., Ravishankar Rai, V.: Quorum quenching activity in cell-free lysate of endophytic bacteria isolated from Pterocarpus santalinus Linn., and its effect on quorum sensing regulated biofilm in Pseudomonas aeruginosa PAO1. Microbiol. Res. 169, 561–569 (2014)CrossRefGoogle Scholar
  2. 2.
    Ali Ahmed, A.B., Taha, R.M.: Chapter 31—The perspectives of the application of biofilm in the prevention of chronic infections. In: Se-Kwon, K. (ed.) Advances in Food and Nutrition Research, pp. 403–416. Academic Press, Cambridge (2011)Google Scholar
  3. 3.
    Tollkamp-Schierjott, C.: The influence of different SEM parameters on CD measurement results. Microelectron. Eng. 6, 673–678 (1987)CrossRefGoogle Scholar
  4. 4.
    Song, M.Z., Wang, X.R., Mao, C.Q., Yao, W.: The discovery of a potential antimicrobial agent: the novel compound natural medicinal plant fermentation extracts against Candida albicans. IOP Conf. Ser. Mater. Sci. Eng. 301, 012026 (2018)CrossRefGoogle Scholar
  5. 5.
    Prakash, D., Nawani, N.N.: A rapid and improved technique for scanning electron microscopy of actinomycetes. J. Microbiol. Methods 99, 54–57 (2014)CrossRefGoogle Scholar
  6. 6.
    Jiang, W., Pan, H., Wang, F., Jiang, M., Deng, X., Li, J.: A rapid sample processing method to observe diatoms via scanning electron microscopy. J. Appl. Phycol. 27, 243–248 (2014)CrossRefGoogle Scholar
  7. 7.
    Cheng, C.F., Wu, K.M., Chen, Y.T., Hung, S.L.: Bacterial adhesion to antibiotic-loaded guided tissue regeneration membranes - a scanning electron microscopy study. J. Formos. Med. Assoc. Taiwan yi zhi 114, 35–45 (2015)CrossRefGoogle Scholar
  8. 8.
    Yamada, H., Chikamatsu, K., Aono, A., Mitarai, S.: Pre-fixation of virulent Mycobacterium tuberculosis with glutaraldehyde preserves exquisite ultrastructure on transmission electron microscopy through cryofixation and freeze-substitution with osmium-acetone at ultralow temperature. J. Microbiol. Methods 96, 50–55 (2014)CrossRefGoogle Scholar
  9. 9.
    Munnecke, A., Servais, T.: Scanning electron microscopy of polished, slightly etched rock surfaces: A method to observe palynomorphsin situ. Palynology 20, 163–176 (1996)CrossRefGoogle Scholar
  10. 10.
    Faganello, J., Arruda, W., Schrank, A., Vainstein, M.H.: An alternative method to prepare samples of the pathogenic yeast Cryptococcus neoformans for scanning electron microscopy analysis. J. Microbiol. Methods 64, 416–419 (2006)CrossRefGoogle Scholar
  11. 11.
    Sarmiento, P.L., Ciarmela, M.L., Sanchez Thevenet, P., Minvielle, M.C., Basualdo, J.A.: Comparison of preparation techniques of mixed samples (fungi-helminth eggs) for scanning electron microscopy by critical point drying. Parasitol. Res. 99, 455–458 (2006)CrossRefGoogle Scholar
  12. 12.
    Priester, J.H., Horst, A.M., Van de Werfhorst, L.C., Saleta, J.L., Mertes, L.A., Holden, P.A.: Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy. J. Microbiol. Methods 68, 577–587 (2007)CrossRefGoogle Scholar
  13. 13.
    Jiang, W.K., Pan, H.Q., Wang, F.X., Jiang, M.L., Deng, X.Y., Li, J.B.: A rapid sample processing method to observe diatoms via scanning electron microscopy. J. Appl. Phycol. 27, 243–248 (2015)CrossRefGoogle Scholar
  14. 14.
    Thai, T.Q., Nguyen, H.B., Saitoh, S., Wu, B., Saitoh, Y., Shimo, S., Elewa, Y.H., Ichii, O., Kon, Y., Takaki, T., Joh, K., Ohno, N.: Rapid specimen preparation to improve the throughput of electron microscopic volume imaging for three-dimensional analyses of subcellular ultrastructures with serial block-face scanning electron microscopy. Med. Mol. Morphol. 49, 154–162 (2016)CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.School of MedicineChengdu UniversityChengduPeople’s Republic of China
  2. 2.School of Materials Science and EngineeringSouthwest Jiaotong UniversityChengduPeople’s Republic of China
  3. 3.School of Tourism, Economics and ManagementChengdu UniversityChengduPeople’s Republic of China
  4. 4.Sichuan Academy of Medical Sciences and Sichuan Provincial People’s HospitalChengduPeople’s Republic of China

Personalised recommendations