Gram Stain

Thin smear of material spread on slide
1. Allow slide to air dry
Fix material onto the slide
1. Place side on warmer at 70 degrees Celsius or
2. Pass slide through Bunsen Burner flame 3-4 times
Overlay the slide with crystal violet (blue dye) for 1 minute
Wash slide thoroughly with water
Overlay slide with Gram Iodine and wait for 1 minute
Wash slide thoroughly with water
Flood slide surface with decolorizer (acetone-Alcohol)
1. Perform until no violet color washes off slide
2. May require up to 10 seconds
Wash slide completely with water
Overlay smear with safranin counterstain (red dye) and wait for 1 minute
Wash slide thoroughly with water
1. Allow excess water to run off slide
Allow slide to air dry or blot with bibulous paper
Examine under high power (100x) oil immersion

Gram Positive (Stains dark blue to purple)
1. Bacterial cells that absorb and retain crystal violet (blue stain)
  1. Gram Positive Bacteria have a thick peptidoglycan cell wall layer that retains the blue stain
2. Gram Positive Cocci (e.g. Staphylococcal and Streptococcal organisms)
Gram Negative (Stains pink to red)
1. Bacterial cells that fail to retain crystal violet (blue stain), will instead absorb safranin (red stain)
  1. Gram Negative organisms have a relatively thin peptidoglycan cell wall layer
  2. Unlike Gram Positive Bacteria, the crystal violet (blue dye) is washed off with acetone-Alcohol rinse
2. Gram Negative Rods (e.g. Enterobacteriaceae)
3. Gram Negative Coccobacilli