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"Gram Stain"[MeSH Terms]
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CDC Gram stain procedure crystal violet iodine decolorizer safranin
These notes describe the Gram staining procedure, used to differentiate bacteria into Gram-positive and Gram-negative types.
Transcription and interpretation
Materials required
- Specimen
- Instruments:
- Glass slide
- Cotton
- Platinum loop
- Bunsen burner
- Light microscope
Solutions required
- Primary stain: 5% solution of crystal violet in distilled water.
- Iodine solution: iodine 1 g + potassium iodide 2 g in about 100 mL distilled water.
- Iodine acts as a mordant, fixing the crystal-violet dye complex.
- Counterstain: likely carbol fuchsin, prepared as a 1-in-10 solution in neutral red.
Visible procedure
A. Preparation and fixation of the smear/film
- Take a clean glass slide.
- Take a specimen from the “petri dish” using a wire/platinum loop.
- Put a drop of distilled water on the slide and mix/spread the specimen to form a thin smear.
- The rest of the steps are cut off in the image, but normally the smear is air-dried, heat-fixed, then stained.
Standard sequence
- Crystal violet
- Gram’s iodine
- Decolorization with alcohol/acetone
- Counterstain with safranin or dilute carbol fuchsin
- Examine under the oil-immersion objective of a light microscope.
Result: Gram-positive bacteria retain crystal violet and appear purple. Gram-negative bacteria become decolorized and take up the counterstain, appearing pink/red. This matches the standard CDC Gram-stain sequence described in the CDC Gram-stain procedure.
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