II. Indications
- Evaluation of suspected Bacterial Infection (e.g. acute Urethritis, Pelvic Inflammatory Disease, Pneumonia)
III. Specimens
- Infected body fluids
- Aspirated abscesses
- Exudates
IV. Technique
- Thin smear of material spread on slide
- Allow slide to air dry
- Fix material onto the slide
- Place side on warmer at 70 degrees Celsius or
- 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)
- Perform until no violet color washes off slide
- 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
- Allow excess water to run off slide
- Allow slide to air dry or blot with bibulous paper
- Examine under high power (100x) oil immersion
V. Interpretation
- Gram Positive (Stains dark blue to purple)
- Bacterial cells that absorb and retain crystal violet (blue stain)
- Gram Positive Bacteria have a thick peptidoglycan cell wall layer that retains the blue stain
- Gram Positive Cocci (e.g. Staphylococcal and Streptococcal organisms)
- Bacterial cells that absorb and retain crystal violet (blue stain)
- Gram Negative (Stains pink to red)
- Bacterial cells that fail to retain crystal violet (blue stain), will instead absorb safranin (red stain)
- Gram Negative organisms have a relatively thin peptidoglycan cell wall layer
- Unlike Gram Positive Bacteria, the crystal violet (blue dye) is washed off with acetone-Alcohol rinse
- Gram Negative Rods (e.g. Enterobacteriaceae)
- Gram Negative Coccobacilli
- Bacterial cells that fail to retain crystal violet (blue stain), will instead absorb safranin (red stain)