Mechanism of Gram Staining

1. Put bacteria on plate (if from another plate then dilute agar first);
2. Warm the plate to fix bacteria;
3. Add gram stain; 20-30 seconds; wash it;
4. Add iodine (mordant)
5. Add drops of  alcohol, to wash out the gram negative bacteria’s color – 20 – 40 seconds;
6. Put safranin (counter stain) 20-40 seconds.

Watch DrBeen video explaining these points

https://members.drbeen.com/library/gram-staining/SkUhjG0unx-?topic=Microbiology&subTopic=General

What Happens?

• Gram positive (+ive) bacteria turns purple
• Gram negative(-ive) bacteria turns red

Go to TC 2:57 in the video for illustration of how it happens:

• Covering the Gram +ive plasma membrane (phospholipid bi-layer), there are up to 60 peptidoglycan (PG) layers.
• The gram -ive plasma layer is covered by up to three PG layers and an outer membrane containing lipopolysaccharides.
• Beta-Lactamase is the most important factor in the periplasmic space, enzymes that can break down penicillins. 
  
  

What happens to the gram stain?

• The small molecules of dye get trapped in the PG layer of gram +ive bacteria and in both the outer membrane and the PG layers of the gram -ive bacteria.
• At this stage both gram +ive and gram -ive look purple under the microscope.
• When iodine (+) is added, it attaches to the molecules of dye (-) and creates a bigger molecule, which gets trapped in the PG layers.  Color does not change.
• When alcohol is added it dissolves the lipid portion and dehydrates the cells.
• In the gram +ive cells the water is pulled out by the alcohol and the 60 layers are compressed, trapping the molecules of dye, keeping the purple color.
• In the gram -ive cells, the alcohol dissolves the more abundant lipids, and the three PG layers are too weak to trap the dye molecules so they escape, making the cells colorless.
  NB: Using too much alcohol on the gram +ive cells will eventually break down the PG layers allowing the dye to escape so you have to be careful.
• The final step is to add safranin, a counterstain, that will turn the colorless gram -ive cells red, but will not be absorbed by the compressed gram +ive cells which stay purple.

And that is how gram staining works!