The Gram stain is used to differentiate members of the domain Bacteria based on the comparative biochemistry of their cell envelopes. Some members of the domain Bacteria do not stain using the standard method (e.g., Legionella spp.) or using any alternative staining method (e.g., Treponema pallidum) whereas some members of the domain Archaea, particularly halophiles, can be stained using the Gram method.

The Gram stain reaction exploits the differential permeability of microbes with a comparatively thick, murein sacculus retaining the crystal violet–Gram’s iodine complex after extraction with the Gram’s decolorizer compared with microbes possessing a thinner, murein sacculus that will not retain the crystal violet–Gram’s iodine complex after extraction with decolorizer.

The opening slide of this animation depicts two microscope fields. The left depicts purple, rod-shaped, gram-positive microbes and the right depicts red, rod-shaped, gram-negative microbes. By selecting one of the two fields the user then may proceed through a series of interactive slides that animate the interaction of bacterial cell envelope molecules with the sequence of Gram stain reagents leading to the final staining results.

Gram positive sequence
The first slide depicts the gram-positive cell envelope. Successive slides depict the addition of the Gram’s stain reagents in stepwise fashion. The salient results of the addition of the Gram’s stain reagents on gram-positive cell envelope molecules are depicted. Molecules of crystal violet combine with iodine molecules to form a complex within the microbial cell. The addition of decolorizer effectively dehydrates the peptidoglycan layer preventing the extraction of the crystal violet–iodine complex from the interior of the cell. The final step consists of the addition of safranin counterstain. The color from the retained crystal violet-iodine complex obscures the safranin counterstain to give the cells a dark blue, or purple, appearance via high resolution light microscopy.

Gram negative sequence
The first slide depicts the gram-negative cell envelope. Successive slides depict the addition of the Gram’s stain reagents in stepwise fashion. The salient results of the addition of the Gram’s stain reagents on gram-negative cell envelope molecules are depicted. Molecules of crystal violet combine with iodine molecules to form a complex within the microbial cell. The addition of Gram decolorizer effectively extracts the outer membrane and dehydrates the peptidoglycan layer. It is presumed that the inner membrane is extracted by the Gram decolorizer, but remains associated with the murein sacculus (1). However, the thinner dehydrated peptidoglycan layer cannot prevent the extraction of the crystal violet–iodine complex from the interior of the cell through the peptidoglycan layer. The final step consists of the addition of safranin counterstain. As the crystal violet-iodine complex has been extracted from the cells, the safranin counterstain gives the cells a pink, or red, appearance via high resolution light microscopy.

Educational significance of this animation
The Gram stain technique elucidates important information about the comparative structure of the cell envelope of most of the domain Bacteria and some members of the domain Archaea (particularly halophiles). This animation depicts the salient molecular interactions between the constituent molecules of both gram-positive and gram-negative microbial envelopes (independently) during the stepwise addition of the Gram stain reagents resulting in the final, differential staining results.

This animation was created using Macromedia Flash version MX2004.

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