Gram Staining

Gram staining is a common technique used to differentiate two large groups (gram-positive, gram-negative) of bacteria based on their different cell wall constituents. Gram staining technique is a common staining method used in microbiology, it is used in classifying and identification of microorganisms. With gram staining, bacteria are divided into two groups as ”Gram-Positive" and "Gram-Negative".
 
Gram-Positive Bacteria
 
After the staining process, bacteria that take purple and blue colour on microscope are called gram-positive bacteria. The reason for these colours is that the cell walls of gram-positive bacteria retain the mixture of crystal violet/iodine. Gram-Positive bacteria have thicker cell walls than Gram-Negative bacteria.
 
Gram-Negative Bacteria
 
Gram-negative bacteria lose the crystal violet stain in Gram's method of staining. During Gram staining, the outer membrane of Gram-negative bacteria deteriorates from the alcohol added to the sample and is not able to retain the blue colour. The counterstain is added to provide contrast by staining the decolourized Gram-negative bacteria. Gram-negative bacteria are observed as red and pink coloured cells because of the counterstain. Gram negative bacteria are often pathogenic. Gram-negative bacteria can cause many types of infections and are spread to humans in a variety of ways.
 
Gram Staining Procedure;

The crystal violet dye is added to the preparation prepared for Gram staining and left for 1 minute and washed with iodine-lugol solution to remove the crystal violet.
Iodine-lugol solution is added to the preparation and left for 1 - 2 minutes. Then the iodine-lugol solution is removed by washing with distilled water.

A solution of 96% ethyl alcohol or ether-acetone is allowed to drop on the preparation for 15 to 30 seconds, washed with distilled water, and with the addition of safranine as an opposite dye, and held for 40 to 50 seconds.
The preparation is allowed to dry in the air by washing with distilled water, immersion oil is added to the preparation and examined with a 100 ile lens.Purple colored bacteria are evaluated as Gram positive and pink-red colored bacteria as Gram negative.All solutions required for Gram staining are also marketed as ready commercial kits.