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The Gram stain was developed by Christian Gram in 1884 and modified by Hucker in 1921.
The objective of Gram Stain
This test differentiates the bacteria into Gram-Positive and Gram-Negative Bacteria, which helps in the classification and differentiation of microorganisms. The Gram stain separates bacteria into two groups: (1) Gram-positive microorganisms that retain the primary dye (Crystal violet) and (2) Gram-negative microorganisms that take the color of the counterstain (usually Safranin O).
Principle of Gram Stain
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 walls of the two groups.
Gram-positive organisms contain a highly cross-linked layer of peptidoglycan that retains the primary dye, crystal violet (CV), following the application of the mordant, iodine (I). The iodine and crystal violet form a complex within the peptidoglycan. When decolorizer is applied to the cells, the CV-I complex remains within the cell, making it appear dark purple to blue.
The gram-negative organisms do not contain a thick cross-linked layer of peptidoglycan. The peptidoglycan is loosely distributed between the inner cell and the outer cell membranes. Following the application of the crystal violet and iodine, the CV-I complexes are not trapped 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 CV-I complex from the cells. The cells appear colorless. To make the colorless cells visible, a secondary stain, safranin, is applied, leaving the gram-negative cells pink.
Gram Stain Reagents
Primary stain: 2 g Crystal violet, 20 mL 95% ethyl alcohol, 0.8 g ammonium oxalate, and 100 mL distilled water.
Gram’s iodine: 2 g potassium iodide, 1 g iodine crystals, and 100 mL distilled water.
Decolorizer: 50 mL acetone and 50 mL ethanol.
Counterstain: 4.0 g Safranin, 200 mL 95% ethanol, and 800 mL distilled water
The procedure of Gram Stain
- Prepare and fix the specimen to the microscope slide before staining.
- Cover the smear with crystal violet, the primary stain, for 20 seconds.
- Gently rinse off the stain with water.
- Cover the smear with Gram’s iodine, the mordant, for 1 minute.
- Pour off the excess Gram’s iodine.
- Run the acid-alcohol decolorizer over the smear until the solution appears clear.
- Gently rinse with water.
- Cover the smear with safranin, the secondary or counterstain, for 20 seconds.
- Gently rinse the stain with water.
- Blot dry with bibulous paper.
Result Interpretation of Gram Stain
Gram-positive: Blue/Purple Color
Gram-Negative: Red/Pink Color
- Over-decolorization may result in the identification of false gram-negative results, whereas under-decolorization may result in the identification of false gram-positive results.
- Smears that are too thick or viscous may retain too much primary stain, making the identification of proper Gram stain reactions difficult. Gram-negative organisms may not decolorize properly.
- Cultures older than 16 to 18 hours will contain living and dead cells. Cells that are dead will be deteriorating and will not retain the stain properly.
- The stain may form a precipitate with aging. Filtering through gauze will remove excess crystals.
- Gram stains from patients on antibiotics or antimicrobial therapy may have altered Gram stain reactivity due to the successful treatment.
- Occasionally, pneumococci identified in the lower respiratory tract on a direct smear will not grow in culture. Some strains are obligate anaerobes.
- Toxin-producing organisms such as Clostridia, staphylococci, and streptococci may destroy white blood cells within a purulent specimen.
- Faintly staining Gram-negative organisms, such as Campylobacter and Brucella, may be visualized using an alternative counterstain (e.g., basic fuchsin).
Gram Positive: Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus, Clostridium, etc.
Gram Negative: E. coli, Salmonella Typhi, Shigella spp, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, Yersinia pestis, etc.