This procedure provides instruction for Gram staining of clinical smears, which allows the microscopist to visualize and categorize bacteria and yeast based on their reaction in the Gram stain, their size and morphology and their cellular arrangement. It also allows the Technologist to assess the quality of the clinical specimen for processing and provide presumptive diagnosis of infectious agents.
Procedure
- Flood the slide with crystal violet – leave on at least 30 seconds.
- Wash gently with tap water. A strong stream may cause the sample to wash away.
- Flood the slide with Gram’s iodine – leave on for 1 minute.
- Wash gently with tap water.
Flood slide with 95% ethyl alcohol until no more blue stain washes away.
Do not over-decolorize with the alcohol – decolorize just until the last of the excess crystal violet washes away. The timing of this step will vary depending on the nature of the specimen and the depth of the material on the slide. Over-decolorizing will result in Gram positive organisms appearing Gram negative. This step takes practice.
Wash gently with tap water.
- Flood slide with fuchsin or safranin – leave on for 1 minute.
- Wash gently with tap water.
- Tip slide and allow excess water to fall off slide.
- Clean off back of slide using gauze or bibulous paper.
- Allow slide to air dry.
Do not blot dry the smear – this may result in either the material lifting off the slide and adhering to the paper providing a poor smear for interpretation, or cross contamination from slides previously dried using the same book.
Interpretation
Organisms which retain the primary stain (Crystal violet) despite the application of the decolorizer will appear blue. These organisms are referred to as Gram positive.
Organisms which lose the primary stain (Crystal violet) after the application of the decolorizer and then are stained with the counterstain (fuchsin or safranin) will appear pink. These organisms are referred to as Gram negative.
Organisms which appear neither entirely Gram positive nor Gram negative are referred to as Gram variable.
Organisms appearing as rod forms are referred to as either Gram positive or Gram negative bacilli.
Organisms appearing as spheres (round) are referred to as either Gram positive or Gram negative cocci.