Double staining with crystal violet and methylene blue is appropriate for colonic endocytoscopy: an in vivo prospective pilot study

Dig Endosc. 2014 May;26(3):403-8. doi: 10.1111/den.12164. Epub 2013 Sep 10.


Background and aim: Endocytoscopy (EC) at ultra-high magnification enables in vivo visualization of cellular atypia of gastrointestinal mucosae. Clear images are essential for precise diagnosis by EC. The aim of the present study was to evaluate the optimal staining method for EC in the colon.

Methods: Thirty prospectively enrolled patients were allocated 1:1:1 to three distinct staining methods: 0.05% crystal violet (CV) alone, 1% methylene blue (MB) alone, or CV+MB (CM double). Normal rectal mucosae were stained with each dye and videos of EC images were recorded. Visibility of nuclei and gland formation after staining were evaluated as 'recognizable' or 'not recognizable'. Time for each parameter to become 'recognizable' was measured, and the average times for the three staining regimens were compared.

Results: MB alone and CM double staining resulted in 'recognizable' (102 ± 27 vs 89 ± 22 s, P=0.263) nuclei within comparable periods of time, whereas CV alone was unable to identify nuclei. Gland formation became 'recognizable' sooner after CM double staining than after MB alone (61 ± 16 vs 108 ± 24 s, P<0.001).

Conclusions: Double staining with CV and MB, which rapidly provided recognizable images of both nuclei and gland formation, is an appropriate staining regimen for colonic EC.

Keywords: crystal violet; endocytoscopy; methylene blue; staining regimen; ultra-high magnification.

Publication types

  • Comparative Study

MeSH terms

  • Aged
  • Analysis of Variance
  • Colon / pathology*
  • Colonoscopy / methods*
  • Cytodiagnosis / methods
  • Female
  • Gentian Violet / pharmacology*
  • Humans
  • Image Enhancement / methods
  • Intestinal Mucosa / pathology
  • Japan
  • Male
  • Methylene Blue / pharmacology*
  • Middle Aged
  • Pilot Projects
  • Prospective Studies
  • Sensitivity and Specificity
  • Staining and Labeling / methods*


  • Gentian Violet
  • Methylene Blue