Trypan blue dye enters viable cells incubated with the pore-forming toxin HlyII of Bacillus cereus

PLoS One. 2011;6(9):e22876. doi: 10.1371/journal.pone.0022876. Epub 2011 Sep 6.


Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability.

MeSH terms

  • Adenosine Triphosphate / biosynthesis
  • Animals
  • Bacillus cereus / metabolism*
  • Bacterial Proteins / pharmacology*
  • Cell Membrane Permeability / drug effects
  • Cell Survival / drug effects
  • Coloring Agents / metabolism*
  • Hemolysin Proteins / pharmacology*
  • Macrophages / cytology
  • Macrophages / drug effects
  • Macrophages / metabolism*
  • Mice
  • Trypan Blue / metabolism*


  • ApxII toxin, bacteria
  • Bacterial Proteins
  • Coloring Agents
  • Hemolysin Proteins
  • Adenosine Triphosphate
  • Trypan Blue