Gram Stain - A Simple Yet Useful Tool

Ten Important points on Gram stain:

1. Differential stain, which categorizes bacteria broadly into Gram positives and Gram-negatives
2. Gram reaction is due to the differences in the cell wall structure between Gram-positive and negative bacteria.
3. Four steps in Gram staining are primary stain(methyl violet), mordent(Grams iodine), Decolourizer(acetone/alcohol) and counterstain (dilute carbol fuschin).
4. Decolourization is the crucial step of Gram staining.
5. Gram-positive bacteria have more acidic protoplasm and a thick peptidoglycan layer. It retains the primary stain, resists decolourization and appears violet.
6. Gram-negative bacteria have a thin peptidoglycan layer, and the cell wall outer layers are dissolved by lipid solvents. It undergoes decolourization and appears pink.
7. Gram stain helps identify organisms directly from the specimen and aids the clinician in selecting treatment. It also helps in biochemical reactions and antibiotic sensitivity testing. 
8. Gram-negatives can appear as Gram negatives when over decolourization is done, ancient cultures, repeated subcultures, autolytic enzymes, and antibiotic treatment.
9. Gram-negatives can appear as Gram positives due to under decolourization.
10. Certain bacteria cannot be viewed by Gram staining like mycoplasma (lack cell wall), mycobacteria (acid-fast), rickettsia and chlamydia (intracellular).