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In microscopy, negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible. This contrasts with positive staining, in which the actual specimen is stained.
Gram stain (Gram staining or Gram's method), is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. It may also be used to diagnose a fungal infection. [1] The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884. [2]
The negatively charged cell wall of many microorganisms attracts the positively charged chromophore which causes the specimen to absorb the stain giving it the color of the stain being used. Positive staining is more commonly used than negative staining in microbiology. The different types of positive staining are listed below. [1]
Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. [1] Their defining characteristic is that their cell envelope consists of a thin peptidoglycan cell wall sandwiched between an inner ( cytoplasmic ) membrane and an outer ...
One commonly recognizable use of differential staining is the Gram stain. Gram staining uses two dyes: Crystal violet and Fuchsin or Safranin (the counterstain) to differentiate between Gram-positive bacteria (large Peptidoglycan layer on outer surface of cell) and Gram-negative bacteria. Acid-fast stains are also differential stains.
Other bacteria are commonly identified with a microscope by staining them with Gram stain. However, the mycolic acid in the cell wall of M. tuberculosis does not absorb the stain. Instead, acid-fast stains such as Ziehl–Neelsen stain , or fluorescent stains such as auramine are used. [ 4 ]
In Gram staining, crystal violet stains only Gram-positive bacteria, and safranin counterstain is applied which stains all cells, allowing the identification of Gram-negative bacteria as well. An alternative method uses dilute carbofluozide. Counterstains are sometimes used to separate animals from organic detritus in microbiology studies.
Furthermore, negative staining with nigrosin can reveal some microorganisms that cannot be stained by regular methods. Nigrosin WS is used in tests for viability because living cells exclude the dye, but it enters dead cells. [5] Nigrosin has minor application in industrial evaporation as a light-absorbing water-soluble pigment. [6]