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The Gram stain, discovered by Dr. Hans Christian Gram in 1884, is the most useful differential stain used to aid in identifying bacteria. It divides bacterial cells into two major groups, Gram positive and Gram negative, which makes it an essential tool for classification and differentiation of bacteria. The Gram stain is a differential stain that requires two stains in the procedure: a primary stain and a counterstain.  The primary stain is crystal violet, which is followed by an iodine solution. The iodine is called a mordant, which is a substance (often a metallic component) that combines with a dye to form an insoluble colored compound.  The insoluble precipitate is called the crystal violet-iodine complex.

The mordant iodine is used to intensify the primary stain. Gram positive bacteria retain the primary stain in their cell walls.  A decolorizing step occurs between the application of the primary stain and the counterstain. It removes the primary stain and decolorizes Gram negative bacteria.  After decolorizing with 95% ethanol, a counterstain of safranin is applied to the smear.

Depending on the components of the cell wall, bacteria will retain the primary stain during decolorizing or lose the primary stain and take up the counterstain.  Organisms that resist decolorizing and retain the crystal violet-iodine complex appear purple or dark blue under the microscope and are called Gram positive (G+).  Conversely, bacteria that decolorize, or give up the crystal violet- iodine complex will accept the safranin counterstain and appear red.  They are Gram negative (G-) bacteria. 

The Gram stain allows for recognition of the shape and pattern or arrangement of bacteria. The shapes are cocci  (Sing: coccus; round), bacilli (sing: bacillus; rod-shaped), and spirilla (spiral). Based on how the bacteria divides during replication, different patterns or arrangements may be produced.The cellular morphology, or shapes, of the individual cells and their arrangement in pairs, chains, or clusters are useful in the identification of the bacteria.

The arrangements or patterns found in cocci are:

a.   diplococci pairs

b.  streptococci chains

c.   staphylococci grape-like clusters

d.   tetrads four in a square

e.   sarcina eight in a cube

Bacilli vary in size from very long oblongs to short rods. They may even have squared-off ends, or one end larger than another, forming a club. The patterns or arrangements they form are:

a. diplobacilli

b.  streptobacilli

Spiral bacteria usually occur singly. Some are very tightly coiled; whereas others are long and slightly curved or only curved at one end.