The Gram stain, developed in 1884 by Danish microbiologist Hans Christian Gram, is a common staining procedure used in the lab to differentiate bacteria based on the physiological properties of their cell walls.
Bacterial specimens are heat-fixed to the glass slide and a crystal violet dye and iodine are applied. The iodine and dye form purple insoluble complexes within the cell. The specimen is then treated with an alcohol solvent which will extract the dye complex through a permeable cell wall. Gram-positive bacterial cell walls are not permeable to this complex and therefore retain the purple dye. Gram-negative bacterial cell walls allow the dye complex to be extracted in this step and therefore appear colorless until they are counterstained with safranin. Gram-negative bacteria appear pink due to the counterstain.
It is possible to receive a "false Gram-negative" if the gram-positive bacteria are old, dead, or damaged and the cell wall is not intact. There is no equivalent "false Gram-positive," but a false Gram-positive can occur if the decolorization step is accidentally omitted.
The major difference between Gram-positive and Gram-negative bacteria is cell wall structure. The Gram-positive bacterial cell wall has two major components, peptidoglycan and teichoic acid. There are also additional polysaccharides and proteins that vary according to species. The outer cell wall is thick and chiefly comprised of murein, a peptidoglycan only found in prokaryotes. The inner surface of this cell wall touches the cell membrane, a lipid bilayer. Gram-positive cell walls have low lipid content, no endotoxin, no periplasmic space, and no porin channel.
Gram-negative bacteria have three layers in their cell wall, not counting periplasmic space. Like Gram-positive bacteria, they are surrounded by a cell membrane, but the peptidoglycan layer is thin and does not directly touch the cell membrane. Additionally, the peptidoglycan layer does not contain teichoic acid, although it does contain a murein lipoprotein which binds this layer to the unique outer cell membrane. This outer cell membrane is a phospholipid bilayer that contains lipopolysaccharide (LPS). LPS is the component of the cell wall which contains Lipid A, or endotoxin. Also unique in the Gram-negative outer membrane are porin proteins which allow passage of nutrients.
Gram staining is still clinically important as the etiologic agents of many bacterial infections are readily seen on Gram-stained smears of pus or fluids. Medical personnel may use this information to guide management of infection before cultures are available.
This "animated" (with appropriate and/or inappropriate sounds) PowerPoint presentation can be utilized in the classroom to provide:
1. An introduction (brief history and structural basis) to the Gram Stain.
2. Stepwise instructions for the techniques.
3. "Cartoons" of how the cells appear at each step.
4. Views of typical Gram morphologies.
Please note: This tutorial works best in Microsoft Internet Explorer 5.0 or higher. Upon clicking on the link to the tutorial above, you will see frames containing the outline and the slide show. You may use the outline on the left-hand side to scroll through the slide show, but you will not hear the sound effects. In IE (but not Netscape) in the lower right-hand corner of your screen there is a "Full slide show" icon. If you click on this, you will be able to view the full slide show with sound effects. Use the space bar to advance the slides, use the ESC key to exit the slide show at any time.
Legend written by:
Kristen Catlin
American Society for Microbiology
Washington D.C. 20036
kcatlin@asmusa.org | 
