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)
VI. References
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Related Studies
| Definition (NCI) | This is a method (named after the Danish bacteriologist, Hans Christian Gram) for staining and identifying bacteria. It is based on the differential retention of a crystal violet-iodine complex within the cell membrane. Gram-positive organisms will appear blue-black or purple while Gram-negative organisms will appear red. |
| Definition (NCI_CDISC) | A microscopy staining method for staining and identifying bacteria, based on the differential retention of a crystal violet-iodine complex within the cell membrane. Gram-positive organisms will appear blue-black or purple while Gram-negative organisms will appear red. |
| Concepts | Laboratory Procedure (T059) |
| SnomedCT | 62777006 |
| LNC | LP6301-8 |
| English | Bacterial stain, routine, gram's stain, gram stain, GRAM STAIN, Gram stain, Gram stain method (procedure), Gram stain method, Gram stain (procedure), Gram Staining Method |
| Italian | Colorazione di Gram |
| Japanese | グラム染色, ｸﾞﾗﾑｾﾝｼｮｸ |
| Spanish | tinción de Gram, tinción bacteriana, de rutina, Tinción de Gram, tinción de Gram (procedimiento), método de tinción de Gram (procedimiento), método de tinción de Gram |
| Czech | Gramovo barvení |
| Hungarian | Gram festés |
| Portuguese | Coloração de Gram |
| Dutch | Gramkleuring |
| French | Coloration de Gram |
| German | Gram-Faerbung |
