Gram stain mechanism
1. Crystal violet enters the cytoplasm and stains cells purple.
2. Iodine (the mordant) forms large crystals with the dye that are
too large to escape through the cell wall.
3. Decolorizing with 95% alcohol dehydrates the peptidoglycan of
gram-positive cells TO
MAKE IT MORE IMPERMEABLE TO THE CRYSTAL VIOLET-IODINE.
The effect on gram-negative cells is quite different; ALCOHOL
DISSOLVES THE OUTER MEMBRANE of gram-negative cells and even
LEAVES SMALL HOLES IN THE
THIN PEPTIDOGLYCAN LAYER through which crystal violet-iodine
diffuses.
4. Safranin stains the colorless gram-negative bacteria pink.
Questions:
What happens when we use too much alcohol in step 3
(over-decolorizing)? Can it
also make holes in the cell wall, consequently release the dye
complex, and allow the cells to stain pink with safranin in the
last step?
Ref:
1. Tortora, G. J., B. R. Funke, and C.L.Case. (2005) Microbiology:
An Introduction.Pearson Education, Inc., p.85 (Ch.4).
Note: The Gram reaction is named after the Danish physician, Christian Gram, who developed this staining technique in 1884.
