The most crucial staining method is Gram staining.o Not all bacteria can be seen with Gram stain; when exposed to a red dye like safranin, they turn colorless and stain pink.o The reason for this discrepancy is that gram-positive bacteria can hold onto the crystal violet-iodine complex when a lipid solvent--typically acetone-alcohol--is present.o Acetone-alcohol treatment causes gram-negative bacteria to lose their purple dye due to their thin peptidoglycan and outer lipid-containing membrane.