Gram staining

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Gram staining refers to a experimental method of placing bacteria into two sub-groups - Gram positive and Gram negative.

This method is used to differentiate between Gram Negative and Gram Positive bacteria. The method exploits the differing structures of the cell wall in both types of bacteria. In Gram positive bacteria, named like it is because it responds "positively" to staining, it is surrounded by a thick peptidoglycan layer, which offsets the need for it to have an additional outer membrane. This therefore sees a Gram positive bacterial cell wall, easily penetratable by the crystal violet stain used in Gram staining; it hence produces a purple stained bacteria as a result of the stain being retained by the thick peptidoglycan. In Gram negative bacteria, there is a reduced presence of peptidoglycan layers. It however has an additional outer membrane. This membrane stops the crystal violet stain from penetrating the cell wall and hence does not become purple in response to the crystal violet stain (also due to the lack of peptidoglycan, meaning the stain cannot be retained), but instead, once counterstained with safranin, turns pink/red colour, It's also useful to note there is a third group of bacteria, that do not respond to Gram staining. This group of bacteria can be categorised as Gram Neutral.




Main Purpose:

Identification and classification of infectious bacteria using a light microscope.


Gram-positive bacteria stains observed are purple/violet whereas Gram-negative stains observed are red/pink in colour following the completion of this method.


  1. Obtain the bacteria from a culture, transfer it into an inoculation loop and smear the bacterial sample on a glass microscope slide.
  2. Fix the bacteria to the slide so that it does not get washed away when chemicals are added to it. This is done by passing the glass slide over an open flame. The heat supplied fixes the bacteria.
  3. Add a few drops of a chemical called crystal violet onto the sample. The purple molecules of the crystal violet pass through the layers of the bacterial cell.
  4. Add iodine to the sample. The iodine molecules pass through the layers of the cell and bind with the crystal violet molecules. They clump together to form a larger crystal violet-iodine molecule.
  5. Wash the sample with alcohol which acts as a detaining agent.
  6. Apply Safranin (a red dye) to the sample. The Safranin molecules pass through the cell wall and bind to the lipids in the phospholipid bilayer.


After washing the bacterial sample with alcohol in Step 5, in Gram-positive the cell wall shrinks and the capsule gets dissolved and is washed away. In Gram-negative the same thing happens with the cell wall, however, not only does the capsule but the outer phospholipid bilayer also gets washed away. Due to the shrinking of the cell wall, the crystal violet-iodine molecules also get washed away. Thus, Gram-negative lose their colour and the bacteria would, therefore, look transparent underneath a light microscope after this step while the Gram-positive would appear purple/violet in colour because it has retained the crystal violet-iodine molecules[1]. After the addition of Safranin, Gram-positive appear purple and Gram-negative appear pink[2]. Even though they both have Safranin which would lead to a pink colour, the Gram-positive appear purple because of the purple crystal violet-iodine molecules still present in the cell are darker and overshadow the pink colour. Therefore, after this method, Gram-positive appear purple/violet and Gram-negative appear red/pink under a light microscope due to the differences in the physical and chemical properties of their cell walls.


A Gram positive bacteria should give a purple stain. This is because the thick layer of Peptidoglycan retains the purple crystal violet stain.

A Gram negative bacteria should give a pink stain. This is becaue it does not retain the crystal violet because the peptidoglycan layer is in the periplasm. So it is stained by the counterstain of Safranin.



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