19.2: Introduction

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The Gram Stain is a differential stain because it separates bacteria into two groups based on differences in their cell wall structure. The protocol involves more steps than a simple stain, but is still performed on air-dried, heat-fixed smear preps. The smear prep is critical. If the smear is too thick the bacteria will not stain evenly, nor will they decolorize evenly. This can be a major source of error in evaluating the Gram reaction of a culture.

Bacteria are differentiated into two groups called Gram Positive bacteria and Gram Negative bacteria. The stain complexes with the peptidoglycan (PG) layer of bacterial cell walls and is not effective, nor utilized diagnostically on non-bacterial cells. Bacteria without cell walls, or those with unusual cell wall structures do not Gram Stain well. The cell walls of Gram Positive bacteria have a very thick, highly cross-linked PG layer, while Gram Negative bacteria have a thin PG layer.

During the procedure the first, or primary, stain is Crystal Violet. Crystal Violet will attach to the PG layer staining the cells purple.
This action is enhanced with the use of a mordant, Iodine, in the second step. A mordant is a substance that helps fix a stain to its target, in this case the PG in the cell wall. At this point in the procedure all cells, both Gram + and Gram -, will appear purple due to the Crystal Violet/Iodine complex.
The next step is the most critical in the Gram Stain procedure because it is the step responsible for differentiating the two groups. If not done carefully, incorrect or ambiguous results will occur. A decolorizer, acetone/alcohol solution, is dripped over the slide to remove Crystal Violet from Gram – cells. Gram + cells will retain the Crystal Violet/Iodine complex due to the many layers of the PG. Gram – cells will lose the Crystal Violet stain and would appear unstained once more. This allows them to be stained a different color with another stain.
The secondary, or, counterstain used is Safranin and colors the cells pink. Some safranin will also attach to the Gram + cells, but the dark purple color from the Crystal Violet obscures any additional pink staining.

Contributors and Attributions

Kelly C. Burke (College of the Canyons)