Gram Staining: Principle, Procedure and Results MSc. Sarah Ahmed

Gram staining Hans Christian Gram in 1884 It is a differential stain which distinguishes between gram positive and gram negative bacteria depending on the difference in the cell wall structure.

CRYSTAL VIOLET Primary stain Violet colored, stains all micro-organism GRAM IODINE Mordant Forms Crystal violet iodine complexes DECOLORIZER Acetone + Methanol Removes Crystal violet iodine complex from thin peptidoglycan layers Dissolves outer layer of Gram negative org GRAM SAFRANINE Counter stain Red colored Stains thin walled Gram neg organism

Organizing the Staining Bottles Dr.T.V.Rao MD

Principle: Gram positive bacteria when treated with a basic dye such as crystal violet , then with iodine (will fix the stain to the cell) and then subjected to a subsequent treatment with a decolourizing agent such as alcohol/acetone mixture remain stained with the basic dye and the decolourizer doesn,t remove the dye , however, gram negative bacteria when treated similarly will be readily decolourized with the alcohol acetone mixture and become invisible , thus to render the unstained gram negative bacteria visible, a counterstain is applied.

Q- Why gram positive bacteria resist decolourization by alcohol/acetone mixture but the gram negative ones can not? Because the cell wall of gram negative bacteria has high lipid content and low peptidoglycan content so when treated with acetone/alcohol mixture dissolves their lipids creating pores in the cell wall through which crystal violet dye will diffuse, and gram negative bacteria become unstained. On the other hand, gram positive bacteria has high peptidoglycan content and low lipid content so when treated with the decolourizer , it will dehydrate the cell wall causing it to shrink and the pores to close and thus the diffusion of the dye will be blocked

Gram Staining Procedure/Protocol: 1-Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results. 2-Wash slide in a gentle and indirect stream of tap water for 2 seconds. 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 seconds. 5-Flood slide with decolorizing agent (Acetone-alcohol decolorizer). Wait 10-15 seconds or add drop by drop to slide until decolorizing agent running from the slide runs clear . 6-Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute. 7-Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper. 8-Observe the results of the staining procedure under oil immersion (100x) using a Bright field microscope. Results: Gram-negative bacteria will stain pink/red and Gram-positive bacteria will stain blue/purple.

Gram stained Escherichia coli Type of stain: differential stain Name of stain: gram stain Gram reaction: gram negative Colour: pink Size: small Shape: rod shaped Arrangement: single Name of m.o: Escherichia coli Gram stained Escherichia coli Type of stain: differential stain Name of stain: gram stain Gram reaction: positive gram Colour: violet Size: intermediate Shape: rod-shaped Arrangement: chains Name of m.o: Bacillus sp. Gram stained bacillus sp

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