The Gram Stain

Gram staining is the first step and common technique used to differentiate Gram-positive bacteria and Gram-negative bacteria. Bacterial cell walls contain the constituent peptidoglycan.


In the photo above you’ll see Staphylococcus aureus gram positive stained purple and Escherichia coli gram-negative stained pink.

Bacterial cell walls lack membranes around their organelles. A major component of a prokaryotic cell wall is the structure of peptidoglycan. The peptidoglycan gives the cell shape and surrounds and surrounds the cytoplasmic membrane. Peptidoglycan consists of a polymer of disaccharides (glycan) which is cross-linked by short chains of amino acid monomers. The backbone of the peptidoglycan molecule consists of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) that are connected via peptide bridges. The NAM molecule varies slightly between bacterial species. The peptidoglycan molecules are transported across the cytoplasmic membrane by bactoprenol; a carrier molecule. The peptidoglycan provides receptor sites for viruses and antibiotics.

In Gram-Negative bacteria the cell wall is composed of a single layer of peptidoglycan surrounded by a membrane structure called an outer membrane. The outer membrane contains a unique lipopolysaccharide (LPS) component. The LPS is an endotoxin that generates a strong immune response when come into contact with. The gram-negative bacteria do not retain the crystal violet which stains the peptidoglycan, but are able to retain he safranin which is a counterstain added after the crystal violet. The safranin is responsible for the red or pink color that gram-negative bacteria appear under the microscope.

Gram-positive bacteria are stained dark blue or purple due to the thick layer of peptidoglycan which retains the crystal violet. Gram-positive bacteria lack the LPS that gram-negative have, but contain a group of molecules called teichoic acids. Teichoic acids give the bacteria an overall negative charge due to the presence of phosphodiester bonds between the monomers. The primary function is unknown, but they are believed to serve as a means of adherence for the bacteria.

The process of gram staining is quite simple;

First make a slide of cell sample that needs to be stained. Heat fix the slide by carefully passing it through a Bunsen burner a few times. Then add the primary stain crystal violet and let it sit for one minute. After one minute rinse the remaining stain off the slide. The next step is to grams iodine for one minute. The Gram’s iodine fixes the crystal violet to the bacterial cell wall. After the Gram’s iodine rinse the sample with acetone or alcohol and rinse with water. The alcohol acts to decolorize the sample in the case of a Gram-negative bacteria, however its important to be careful because if the alcohol remains on the sample for too long, it may decolorize Gram-positive cells. The last step is to add the secondary stain called the safranin and let sit for one minute. After one minute, wash the stain off and let dry.

Gram staining is an important step in identify cultures and often the first step in the process in differentiating whether the pathogen is gram-negative or gram-positive.