Gram鈥檚 Stain Does Not Cross the Bacterial Cytoplasmic Membrane
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- 作者：Michael J. Wilhelm ; Joel B. Sheffield ; Mohammad Sharifian Gh. ; Yajing Wu ; Christian Spahr ; Grazia Gonella ; Bolei Xu ; Hai-Lung Dai
- 刊名：ACS Chemical Biology
- 出版年：2015
- 出版时间：July 17, 2015
- 年：2015
- 卷：10
- 期：7
- 页码：1711-1717
- 全文大小：343K
- ISSN：1554-8937
文摘
For well over a century, Hans Christian Gram鈥檚 famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram鈥檚 protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.