Consistent with previous studies , we also found that gram-positive bacteria were less likely to decolorize when fixed to slides with methanol rather than heat. In our experience, S. aureus cells that were fixed with methanol consistently were stained a dark purple color. Identically stained cells that were heat-fixed varied in color, ranging from dark purple to pink.
The decolorization step is the most critical of the Gram-stain procedure. Students tend either to under-decolorize, leading to gram-negative cells falsely appearing gram-positive (purple), or, more commonly, to over-decolorize, in which case gram-positive bacteria falsely appear gram-negative (pink). Using methanol as a fixative agent would help to eliminate this problem. Gram-positive bacteria fixed with methanol would likely still appear purple in color even if excessive amounts of decolorizing agent
were used.