Gram Staining

Gram Staining:

Gram staining is a common staining method or technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process.

When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in colour. In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. Then when again stained with safranin, they take the stain and appears red in color.
Reagents Used in Gram Staining:
  • Crystal Violet, the primary stain
  • Iodine, the mordant
  • A decolorizer made of acetone and alcohol (95%) 
  • Safranin, the counterstain
 Procedure of Gram Staining:
1.Take a clean, grease free slide.
2.Prepare the smear of suspension on the clean slide with a loopful of sample.Air dry and heat fix:
The first consideration is the correct preparation of the smear. Make a thin film of the material on a clean glass slide, using a sterile loop or swab for viscous specimens. Air dry, then heat fix the slide by passing it several times through a flame (the slide should not become too hot to touch). Failure to follow these directions may cause staining artifacts and disrupt the normal morphology of bacteria and cells.

Gram staining

3.Crystal Violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.
4.    Flood the gram’s iodine for 1 minute and wash with water.
5.  Then ,wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.
6. Add safranin  for about 1 minute and wash with water.
7.   Air dry, Blot dry and Observe under Microscope.

As shown below, organisms that retain the violet-iodine complexes after washing in ethanol stain purple and are termed Gram-positive, those that lose this complex stain red from the safranin counter stain are termed Gramnegative

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3.   Henry George Liddell. Robert Scott. A Greek-English Lexicon (revised and augmented edition, Oxford, 1940) s.v. γράμμα Archived 2015-07-17 at the Wayback Machine, citing the 10th-century work Geoponica and a 4th-century papyrus edited in L. Mitteis, Griechische Urkunden der Papyrussammlung zu Leipzig, vol. i (1906), 62 ii 27.
4.   "System of Measurement Units - Engineering and Technology History Wiki". Archived from the original on 29 April 2018. Retrieved 29 April 2018.
5.    "Circulating Coin Designs". Japan Mint. Archived from the original on 18 September 20

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