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Unique advantages of using low temperature scanning electron microscopy to observe bacteria

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Summary

Electron microscopy (EM) has greatly helped to elucidate our understanding of bacterial structure and function. However, several recent studies have cautioned investigators about artifacts that result from the use of conventional EM preparation procedures. To avoid these problems, the use of low temperature scanning electron microscopy (LTSEM) was evaluated for examining frozen, fully hydrated specimens. Spinach leaves (Spinacia oleracea L. cv. New Jersey), which were naturally infected or inoculated with bacteria, were used as the experimental material. 1 cm segments of the infected leaves were plunge frozen in liquid nitrogen, transferred to a cryochamber for sputter coating and then moved onto a cryostage in an SEM. After observation, some of the frozen, hydrated leaf segments were transferred onto agar medium to determine whether preparation for LTSEM was nondestructive to the bacteria. The other tissue segments were chemically fixed by freeze-substitution. The results indicated that after cryopreparation and observation in the LTSEM: (i) viable bacteria, which were recovered from the leaf sample, could be cultured on agar medium for subsequent study, and (ii) the frozen samples could be freeze substituted and embedded so that transmission electron microscopic (TEM) observations could be carried out on the same specimen. In conclusion, frozen, hydrated leaf tissue infected with bacteria can be observed using LTSEM and then can be either processed for TEM observation to obtain further structural details or recovered to culture the pathogenic bacteria for supplementary studies.

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Abbreviations

EPS:

extracellular polysaccharide

EM:

electron microscopy

LTSEM:

low temperature scanning electron microscopy

SEM:

scanning electron microscopy

TEM:

transmission electron microscopy

TSA:

tryptic soy agar

TSB:

tryptic soy broth

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Authors and Affiliations

  1. U.S. Department of Agriculture, BARC-East, Electron Microscopy Laboratory, ARS, Bldg. 177B, 20705, Beltsville, MD, USA

    Stéphane Roy & William P. Wergin

  2. U.S. Department of Agriculture, Horticultural Crops Quality Laboratory, Agricultural Research Service, Beltsville, Maryland

    Stéphane Roy, Isabelle Babic & Alley E. Watada

Authors
  1. Stéphane Roy
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  2. Isabelle Babic
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  3. Alley E. Watada
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  4. William P. Wergin
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Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology

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Roy, S., Babic, I., Watada, A.E. et al. Unique advantages of using low temperature scanning electron microscopy to observe bacteria. Protoplasma 195, 133–143 (1996). https://doi.org/10.1007/BF01279192

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  • DOI: https://doi.org/10.1007/BF01279192

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