What is Gram staining?  What is its significance?  What is the procedure of the Gram stain?

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Gram staining is a chemical procedure, named after Hans Christen Gram who developed it.  Gram staining is performed in-vitro and is used to identify a bacterial organism based on the amount of peptidoglycan present in its cell wall. The result classifies bacteria as either gram-positive, gram-negative or gram-indeterminate.  A stain, crystal violet (CV), is applied to a sample of bacteria.   After the staining process is complete, gram-positive bacteria retain the stain and are purple, whereas gram-negative bacteria do not, the purple is washed away.  Gram-indeterminate bacteria do not yield any color with this test and therefore cannot be classified as gram-positive or gram-negative.

The Gram staining procedure is:

1. Apply the crystal violet (CV) stain to a bacteria sample 

2. Add Iodine (binds with CV, forming a CV-I compound)

3. Add decolorizer (acetone or alcohol).  Gram-positive will remain purple, gram-negative will not.

4. Apply counterstain (gives gram-negative bacteria a pinkish red color).

Gram staining is not as reliable as other methods used to determine bacterial identity, but  it does produce fast results.

The information gained from Gram staining is significant in medicine.  It reveals important information about the cell wall of bacteria which is used to determine treatment protocol, for example what antibiotic would be effective in treating the bacteria.

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Gram staining is a visualization method and a general cataloging technique for bacteria.  A majority of bacteria can be divided into two very general classes, Gram positive and Gram negative, depending on how they react to the Gram staining procedure.

The procedure was invented by and named after Danish biologist and doctor Hans Christian Gram.  He created the technique while working as a bacteriologist in Germany in the late 19th century.

Gram positive bacteria are stained a dark purple or blue color.  Gram negative bacteria are stained a pink or light red color due to the counterstain used in the procedure.  This staining procedure allows for bacteria present in a sample to be readily viewed by the human eye under a microscope.  It also allows for different types of bacteria to be distinguished from one another by the different coloring.  The difference between Gram positive and negative staining is the result of the peptidoglycan layer surrounding the bacterial cell membrane.  Peptidoglycan is basically a polymer of proteins and sugar molecules that surround the cell membrane.  It is often referred to as the cell wall.  Gram positive bacteria tend to have thick cell walls that readily trap the stain.  Gram negative bacteria have a thinner cell wall that is surrounded by an additional liposaccharide membrane.

The Gram staining procedure is as follows.  First, the cells are washed with an aqueous solution of crystal violet stain.  Crystal violet is an organic dye that is a positively charged ion so it readily dissolves in water.  The thick cell wall of Gram positive bacteria readily absorbs the dye since it makes electrostatic interactions with the cell wall.  Next, the cells are washed with an iodine solution that forms a highly colored complex with crystal violet, thereby trapping the dye molecules in the cell wall.  Next, the cells are rinsed with a solvent like ethanol or acetone.  This rinse removes the outer membrane of Gram negative bacteria and any dye that has been trapped by it.  Finally, a counterstain is applied (safranin is a commonly used counterstain).  This colorizes the newly exposed cell wall of Gram negative bacteria with a lighter, different color since the two stains are different. 

It should be noted that not all bacteria fall nicely into the Gram positive and negative categories.  Some types of bacteria do not stain at all and are called Gram indeterminate bacteria.  Other bacteria give a mix of colors and are called Gram variable.  In the age of highly powered microscopes and DNA analysis Gram staining is a bit of an anachronism, but it has stood the test of time and is still a commonly used microbiology tool today.

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