PROCESS INVOLVED IN GRAM STAINING By Amarjeet Gram

PROCESS INVOLVED IN GRAM STAINING • By –Amarjeet

Gram staining is a common technique used to differentiate two large groups of bacteria based on Their different cell wall constitutenta. The gram Stain procedure distinguished between gram positive and gram negative

• Principle of gram staining 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 wall 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 ctystal voilet -iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria appears blue or purple in colour

• In case of negative bacteria, cell wall also takes up the cv-iodine complex but due to the thin layer of peptidog-lycan and thick outer layer which is formed of lipids , cviodine 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

Gram staining procedure 1. Flood air dried , heat – fixed smear of cells for 1 minute with crystal violet staining reagent 2. wash slide in gentle and indirect stream of tap water for 2 second 3. Flood slide with the mordant : gram‘s iodine wait 1 minute 4. Wash slide in a gentle and indirect stream of tap water for 2 sec

5. Flood slide with decolorizing agent (acetone-alcoholdecolorizer). Wait 10 -15 sec or add drop to slide untill decolorizing agent running from the slide runs clear. 6. Flood slide with a counterstain , saffranin. Wait 30 sec to 1 mint 7. Wash slide in a gentile and indirect stream of tap water untill no color

Appear in the effluent and then blot dry with absorbent paper 8. Observe the results of the staining procedure under oil immersion (100 X)using a bright feild microscope Results: . Gram- negative bactria will stain pink/red. Gram – positive bacteria will stain blue/ purple

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