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Gram Stain Troubleshooting

The Gram-stain is a delicate procedure that requires attention to detail. Improper smear preparation and staining techniques often yield false results.

Smear Preparation

  • Bacterial smear: If the smear is applied in too thick of a layer, Gram-negative bacteria appear darker and could be mistaken for Gram-positive.
    Try to always apply only a single layer of the organism you want to study.

  • Fixation: If bacteria are over-heated during fixation, the cell wall is destroyed and the bacteria are not capable of retaining Crystal Violet.
    All bacteria will, as a result, appear Gram-negative.

  • Expired culture smears: If culture smears are too old, the bacterial cell wall might have already broken down, rendering them incapable of retaining the Crystal Violet stain.
    All bacteria would, therefore, appear Gram-negative.

Staining

Decolorizing frequently yields false results in the Gram-stain.

  • Under-decolorization: If alcohol (decolorizer) is washed away before it had any effect on the cell wall, the Crystal Violet molecules will not escape the Gram-negative cell wall.
    As a result, all bacteria appear Gram-positive (purple) after counterstaining with Safranin.


  • Over-decolorization: Leaving alcohol (decolorizer) on the slide for too long disrupts the cell wall of both Gram-positive and Gram-negative bacteria.
    The Gram-positive bacteria can not retain Crystal Violet, and therefore, all bacteria appear Gram-negative (pink) after counterstaining with Safranin.
    First image presents the outcome of under-decolorization. Two bean shaped structures, representing bacteria cells, are coloured in purple. A blue arrow goes out of each of them towards two cell wall cross sections, one for gram positive, and one for gram negative bacteria. In both structures, even though they differ from each other, purple molecules of crystal violet are present, together with orange spheres representing iodine. Second image presents the outcome of over-decolorization. Two bean shaped structures, representing bacteria cells, are decolourized. A blue arrow goes out of each of them towards two inner cell membrane cross sections, one for gram positive, and one for gram negative bacteria. In both structures, the purple molecules of crystal violet and orange molecules of iodine are floating above the membrane, not trapped inside.

Figure 1. Under-decolorization. Over-decolorization.