Gram Stain

Process
BY: BRADY PETERS

Gram Negative vs. Gram Positive

Bacteria

Gram positive bacteria differ from Gram negative bacteria in the

structure of their cell walls. The cell walls of Gram positive
bacteria are made up of 20x more peptidoglycan, a polymer
made of sugar and amino acids.

Step 1
Heat fix the slide so that bacterial cells are fixed to the slide.

For Gram positive and negative bacteria the same procedure is

followed.

Step 2
Flood the slide with crystal dye.

For a Gram positive bacteria, the individual crystal violet ions

penetrate the thick peptidoglycan layer of the cell as well as the
plasma membrane.

For a Gram negative bacteria, the individual crystal violet ions

penetrate the thin peptidoglycan layer of the cell as well as the
plasma membrane.

Step 3
Rinse the slides with water.

For Gram positive and negative bacteria the same procedure is

followed.

Step 4
Flood the slide with iodine.

For Gram positive and negative bacteria:

The iodine ions penetrate the cell wall and bind with the crystal
violet.

The iodine ions and crystal violet react, forming a crystal violetiodine complex that is a very large molecule and is insoluble in
water.

Step 5
Rinse the slides with water again.

For Gram positive and negative bacteria the same procedure is

followed

Step 6
Decolorize the slide with alcohol.

For Gram positive bacteria, the alcohol causes water to leave the
cell wall. Because of its larger size, the crystal violet-iodine
complex is blocked from moving easily through the cell wall and
thus is prevented from leaving the cell.

For Gram negative bacteria, the alcohol disrupts and dissolves

the outer membrane. Therefore the big crystal violet-iodine
complex is able to leave the cell.

Step 7
Rinse the slides with water.

For Gram positive bacteria, the crystal violet-iodine complex is

still stuck inside the cell. The cell is therefore stained purple.

For Gram negative bacteria, the crystal violet-iodine complex is

washed away, leaving colorless, unstained cells.

Step 8
Flood the slide with Safranin dye.

For Gram positive and negative Bacteria the same procedure is

followed

For Gram positive bacteria, safranin penetrates the cell, but since it is a
lighter color than the crystal violet-iodine complex, it is not visualized
over the purple color of the crystal violet-iodine.

For Gram positive bacteria, the cells appear purple.

For gram negative bacteria, safranin penetrates the cell and stains the
cell a reddish-pink color.
For gram negative cells, the cells appear reddish-pink.