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Journal of Electron Microscopy Advance Access published online on September 19, 2009
Journal of Electron Microscopy, doi:10.1093/jmicro/dfp045
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Examination of electron stains as a substitute for uranyl acetate for the ultrathin sections of bacterial cells

Kaoru Yamaguchi*, Ken-ichiro Suzuki and Kenji Tanaka

Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan

* To whom correspondence should be addressed. E-mail: yamaguchi-kaoru{at}nite.go.jp

Electron staining reagents were examined to find a possible substitute for uranyl acetate (UA) in electron microscopy of bacterial ultrathin sections. Four kinds of stains, platinum blue (Pt-blue), oolong tea extract (OTE), potassium permanganate (KMnO4) and phosphotungstic acid (PTA), were examined in comparison with UA either with or without post-staining with lead citrate (Pb). Electron microscopy was performed on sections from Spurr-embedded cells of a Gram-positive bacterium, Bacillus cereus NBRC 13597, and a Gram-negative bacterium, Escherichia coli NBRC 3301. Both Pt-blue and OTE showed staining similar to each other and to that of double staining with UA and Pb in B. cereus, while in E. coli the cytoplasmic membrane appeared less dense when compared with UA and Pb. KMnO4 stained excessively to some extent, but showed images of the best contrast in the cytoplasmic membrane comparable with UA and Pb among the four reagents. PTA could stain the peptidoglycan layer but gave images of low quality for both bacteria. This study demonstrated that none of the reagents examined showed staining results of the same quality or better than the conventional method with UA and Pb. However, stains of Pt-blue, OTE and KMnO4 could possibly be an alternative candidate for the UA according to the structure in question.

Keywords     electron staining, bacteria, freeze substitution, platinum blue, oolong tea extract, potassium permanganate

Received      2 March 2009, accepted 13 August 2009


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