Microbiology

Gram Staining



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On the basis of their reaction to gram stain, bacteria can be divided into two major groups-

a)      Gram positive bacteria

b)      Gram negative bacteria

To understand the difference between gram positive and gram negative bacteria, it is essential to first have the knowledge of gram staining technique. Gram staining is one of the most important and widely used staining technique in microbiology laboratory. It was first introduced by Hans Christian in 1884.  In gram staining, the smear made is first flooded with crystal violet. It imparts purple color to all cells. It is then washed and iodine solution is poured. Both types of bacteria appear purple or dark violet in color. The smear is again washed with alcohol (ethanol or ethanol acetone solution), which acts as a decolorizing agent and removes the purple color from the cell of some species. The alcohol is rinsed off and again stained with counterstain safranine. Finally it is washed off, blotted dry and observed under microscope. The difference in color which is observed after the staining process helps in identification process of types of bacteria that have been isolated from the specimens.

Gram positive bacteria retains crystal violet and hence appear deep violet in color and gram negative bacteria loses the crystal violet and retains safranine and hence appear red in color. In gram staining, an insoluble crystal violet-iodine complex is formed inside the cell. This complex is extracted from the gram negative bacteria but not from gram positive bacteria by decolorizing agent (alcohol). The cell wall of gram positive bacteria consist of thick peptidoglycan layer and have no lipid outer membrane. These peptidoglycan layer become dehydrated by alcohol causing the pores in the walls to close and preventing the insoluble crystal violet-iodine complex from escaping. By contrast, gram negative bacteria has thin peptidoglycan layer with an outer lipid membrane. Alcohol readily penetrates this lipid rich outer membrane and extracts the crystal violet-iodine complex from the cell and these bacteria remains colorless, thus retaining the color of safranine.


Some common examples of gram positive and gram negative bacteria are listed below-

Gram positive

a)      Cocci

Staphylococcus sp. Streptococcus sp. Micrococcus sp.     

b)      Rod

Bacillus sp. Clostridium sp. Corynebacterium sp.  Gram negative

a)      Cocci

Neisseria sp. Moraxella sp.

b)      Rod

E.coli Shigella sp. Pseudomonas sp. Enterobacter sp. Salmonella sp. Proteus sp.

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