Gram Staining : Principle, Procedure and Interpretation

                     Gram Staining

Gram staining is the common, important and most used differential staining technique in Microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884. This test differentiate the bacteria into Gram Positive and Gram Negative bacteria, which helps in the classification and differentiation of microorganisms.

Principle:-

The two major groups of bacteria can be divided into Gram Positive and Gram Negative. The Gram stain technique is based on the differential structure of the cellular membranes and cell wall of the two groups.

Gram positive organisms contain a thick layer of peptidoglycan that retains the primary dye, crystal violet, following the application of the mordant, iodine. The iodine and crystal violet form a complex within the peptidoglycan. when decolorizer applied to the cells, making it appear dark purple to blue.

The Gram Negative organisms do not contain a thick layer peptidoglycan. The peptidoglycan loosely distributed between the inner cell and the outer cell membranes. Following the application of the crystal and iodine, the crystal violet complexes are not traped within the peptidoglycan. Application of the acid alcohol decolorizer dehydrates the outer cellular membrane, leaving holes in the membrane and effectively washing or removing the crystal violet complex from the cells. The  cells appear colorless. To make the colorless cells visible, a secondary stain, safranin is applied, leaving the gram negative cell pink.

Reagents Used :- 

° Crystal Violet (Principle stain)

° Iodine (Mordant)

° Decolorizer (95% alcohol)

° Saffarnin (Counterstain)

Procedure :-

• Take a clean, grease free slide.
• Prepare the smear of suspension on the clean slide with a loopful of sample.
• Air dry and heat fix.
• Crystal violet was poured and kept for about 30 second to 1 minute and rinse with water.
• Flood the gram's iodine for 1 minute and wash with water.
• Then, wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.
• Add safranin for about 1 minute and wash with water.
• Air dry, Blot dry and observe under Microscope.         

     

Interpretation :- 

Gram positive :- Blue/ Violet

Gram negative :- Red/Pink