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A simple staining method for bacteria that is usually successful, even when the positive staining methods fail, is to use a negative stain. This can be achieved by smearing the sample onto the slide and then applying nigrosin (a black synthetic dye) or India ink (an aqueous suspension of carbon particles).
Under the microscope, the India ink stain is used for easy visualization of the capsule in cerebral spinal fluid. [10] The particles of ink pigment do not enter the capsule that surrounds the spherical yeast cell, resulting in a zone of clearance or "halo" around the cells. This allows for quick and easy identification of C. neoformans.
Because most capsules are so tightly packed, they are difficult to stain because most standard stains cannot penetrate the capsule. To visualize encapsulated bacteria using a microscope, a sample is treated with a dark stain, such as India ink. The structure of the capsule prevents the stain from penetrating the cell.
India ink (British English: Indian ink; [1] also Chinese ink) is a simple black or coloured ink once widely used for writing and printing and now more commonly used for drawing and outlining, especially when inking comic books and comic strips. India ink is also used in medical applications.
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.
India ink of the CSF is a traditional microscopic method of diagnosis, [35] although the sensitivity is poor in early infection, and may miss 15–20% of patients with culture-positive cryptococcal meningitis. [36]
India ink was invented in China, [11] [12] though materials were often traded from India, hence the name. [11] [12] The traditional Chinese method of making the ink was to grind a mixture of hide glue, carbon black, lampblack, and bone black pigment with a pestle and mortar, then pour it into a ceramic dish to dry. [11]
An advantage of using this method, rather than regular positive stains like methylene blue or carbol fuchsin, is that prior fixation by heat or alcohol is not needed, so the organisms are seen in more lifelike shapes. Furthermore, negative staining with nigrosin can reveal some microorganisms that cannot be stained by regular methods.