Gram staining is a widely used microbiological technique to differentiate bacterial species
into two main groups Gram-positive and Gram-negative, based on the structural differences
in their cell walls.
The technique, developed by Hans Christian Gram in 1884, involves a series of steps that
lead to the visualization of these differences under a microscope.
๐๐ซ๐ข๐ง๐๐ข๐ฉ๐ฅ๐ ๐จ๐ ๐๐ซ๐๐ฆ ๐๐ญ๐๐ข๐ง๐ข๐ง๐
The principle of Gram staining is based on the differences in the thickness and composition
of the peptidoglycan layer in bacterial cell walls
๐๐ซ๐๐ฆ – ๐๐จ๐ฌ๐ข๐ญ๐ข๐ฏ๐ ๐๐๐๐ญ๐๐ซ๐ข๐
These bacteria have a thick peptidoglycan layer in their cell walls, which retains the primary
stain (crystal violet) after a series of treatments, appearing purple under the microscope.
๐๐ซ๐๐ฆ – ๐๐๐ ๐๐ญ๐ข๐ฏ๐ ๐๐๐๐ญ๐๐ซ๐ข๐
These bacteria have a thin peptidoglycan layer and an outer membrane that does not retain
the crystal violet stain after treatment with a decolorizer (alcohol or acetone). Instead, they
take up the counterstain (safranin) and appear red or pink.
๐๐๐ฒ๐ฝ๐ ๐ถ๐ป ๐๐ฟ๐ฎ๐บ ๐ฆ๐๐ฎ๐ถ๐ป๐ถ๐ป๐ด ๐ฝ๐ฟ๐ถ๐บ๐ฎ๐ฟ๐ ๐ฆ๐๐ฎ๐ถ๐ป๐ถ๐ป๐ด (๐๐ฟ๐๐๐๐ฎ๐น ๐ฉ๐ถ๐ผ๐น๐ฒ๐)
A purple dye, crystal violet, is applied to the bacterial smear. This dye penetrates and stains
all the cells.
๐๐ผ๐ฟ๐ฑ๐ฎ๐ป๐ (๐๐ผ๐ฑ๐ถ๐ป๐ฒ ๐ง๐ฟ๐ฒ๐ฎ๐๐บ๐ฒ๐ป๐)
Iodine is added as a mordant, which forms a complex with crystal violet, making the dye
molecules larger and more difficult to wash out of the cell.
๐๐ฒ๐ฐ๐ผ๐น๐ผ๐ฟ๐ถ๐๐ฎ๐๐ถ๐ผ๐ป (๐๐น๐ฐ๐ผ๐ต๐ผ๐น ๐ผ๐ฟ ๐๐ฐ๐ฒ๐๐ผ๐ป๐ฒ ๐ง๐ฟ๐ฒ๐ฎ๐๐บ๐ฒ๐ป๐)
The smear is treated with alcohol or acetone, which dehydrates the thick peptidoglycan layer
in Gram-positive cells, trapping the crystal violet-iodine complex inside.
In Gram-negative cells, the alcohol disrupts the outer membrane and the thin peptidoglycan
layer, allowing the crystal violet-iodine complex to be washed out.
๐๐ผ๐๐ป๐๐ฒ๐ฟ๐๐๐ฎ๐ถ๐ป๐ถ๐ป๐ด (๐ฆ๐ฎ๐ณ๐ฟ๐ฎ๐ป๐ถ๐ป)
A red counterstain, safranin, is applied.
Gram-negative cells, which have lost the crystal violet, take up the safranin and appear red or
pink.
Gram-positive cells retain the purple color of crystal violet, so the safranin does not alter their
appearance.
๐๐๐ฌ๐ฎ๐ฅ๐ญ๐ฌ
Gram-Positive Bacteria: Appear purple.
Gram-Negative Bacteria: Appear red or pink
