SPECIAL STAINS

DEFINITION

Special staining is performed to visualize selected tissue elements, entities and

microorganisms. Based on classical dye staining methods, special stains technique provide
valuable information in the evaluation of numerous abnormal or disease conditions. The
following special stains are of high quality, and they satisfactorily demonstrate (on each day
of use), the tissue components or organisms for which they were designed.33

a) Acid fast organisms

b) Iron

c) Bacteria

d) Elastic tissue

e) Fungi

f) Mucin

g) Connective tissue

h) Myelin

i) Nerve fibers

j) Glycogen

k) Reticulin fibers

l) Amyloids

Historic Review

The birth of histopathology is credited to Johannes Muller (1801-1858). In 1838, Muller,

professor of anatomy, physiology, and pathology at the University of Berlin pioneered the
use of the microscope in pathology and published his book, entitled “On the Nature and
Structure Characteristics of Cancer”, which was the first book on histopathology.
Over the years a wide range of dyes has been used for histological staining methods. Most of
these have been adapted from those used in the textile dyeing industry. The dye with
probably the greatest claim to antiquity is indigo. It is extracted from the Indigofera plant,
and there is evidence of its use to dye cloth by the ancient Egyptians some 3000 years ago.

Coincidentally, the Arabic term for indigo is Annil from which the word Aniline derived. It is
a substance extracted from indigo in the early 19th century. The aniline group of dyes had
become the dominant force in the growth of the textile dyeing industry and histological stain
technology.

In early 1860’s one step staining had been used in which the excess stain was washed off in
alcohol or water before the slide was mounted. In 1867, Schwartz introduced two-dye
sequential staining interspersed with a simple washing stage. The technique was further
refined when in 1869 Bottcher incorporated an alcohol differentiation step.13

Hematoxylin is a natural dye with useful properties and a long history in histopathology. It
was reportedly first used by Wilhelm von Waldeyer in 1863. Hematoxylin is obtained from
the logwood tree, Haematoxylon campechianum, found mainly in Central America.12

Hematoxylin itself is a weak dye, and the different staining solutions are based on its
oxidized form, hematein. Hematein, when combined with a mordant, an oxidizer, and
sometimes a differentiating agent, can be used to identify a wide variety of cellular
components. Solutions prepared from hematein are usually called “hematoxylin.”13

Most commonly used counterstain with hematoxylin is eosin. Eosin had been reported as a
general stain for tissues by workers such as Dreschfeld and Fischer in the 1870s. It is a
synthetic xanthene dye which is commercially available as EosinY, ethyl eosin, and Eosin B
(B stands for bluish). Of these, 0.5 or 1% solution of Eosin Y, in distilled water or alcohol, is
commonly used.14
The hematoxylin and eosin staining techniques were first described in 1875–1878. The actual
combination of these two dyes to form a single method has been variously attributed to
Wissowzky in 1875, Reynaud in 1876, and Busch in 1876-78.15

Later modifications such as Delafield’s hematoxylin (1880), Ehrlich’s hematoxylin (1886),

Heidenhahn’s iron hematoxylin (1896), Harris’s hematoxylin (1900) and Mayer’s
hematoxylin (1903) are used in staining procedures till yet.4