Primary stain: The blue-violet appearance of Gram-positive cells is caused by crystal violet, the primary (first) stain.
Iodine mordant: Crystal violet binds to the iodine mordant causing this large molecule to become trapped in the layers of peptidoglycan of Gram+ cells.
Decolorizer: This step does not remove the crystal-violet stain trapped within the Gram-positive cell wall, but does remove the stain from the thin layer of peptidoglycan of Gram-negative bacteria.
Application of Primary Stain: 1. Heat fixed bacterial smear, Gram-positive control on left, Gram-negative control on right, and unknown bacteria in center; 2 & 3. Flood the slide with primary stain, crystal violet stain; 4. Rinse after 1 min.
Application of Mordant: 1 & 2. Next apply the iodine mordant to the slide; 3. Leave on for 1 minute; 4. Rinse.
Application of Decolorizer: 1. Decolorizer, Acetone Alcohol, used in Gram-stain; 2. Hold slide at angle and run a stream of decolorizer over slide for 10 to 15 seconds, until negative control loses color; 3. Rinse.
Application of Counterstain: 1. Safranin secondary stain; 2 & 3. Apply safranin counterstain to slide and leave on for 1 minute; 4. Rinse; 5. Gram-stained slide with positive control on left, negative control on right and unknown bacteria in center. More Gram stain photos.
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