Romanowsky stain

The value of Romanowsky staining lies in its ability to produce a wide range of hues, allowing cellular components to be easily differentiated. This phenomenon is referred to as the Romanowsky effect, or more generally as metachromasia.[7]

Romanowsky effect

Blood film stained with Giemsa showing Plasmodium (center of image), the parasite that causes malaria infections.

In 1891 Romanowsky[8][9][10] developed a stain using a mixture of eosin (typically eosin Y) and aged solutions of methylene blue that formed hues unattributable to the staining components alone: distinctive shades of purple in the chromatin of the cell nucleus and within granules in the cytoplasm of some white blood cells. This became known as the Romanowsky or Romanowsky-Giemsa effect.[11][12][6][13][4] Eosin and pure methylene blue alone (or in combination) do not produce the Romanowsky effect,[13][4] and the active stains which produce the effect are now considered to be azure B and eosin.[14][3][13]

Dmitri Leonidovich Romanowsky (1861-1921).

Although debate exists as to who deserves credit for this general staining method, popular usage has attributed it to Dmitri Leonidovich Romanowsky.[14][16][19]

In the 1870s Paul Ehrlich used a mixture of acidic and basic dyes including acid fuchsin (acid dye) and methylene blue (basic dye) to examine blood films.[25][26][27][28][16] In 1888 Cheslav Ivanovich Chenzinsky used methylene blue, but substituted the acid fuchsin used by Ehrlich with eosin.[14][27][28] Chenzinsky's stain combination was able to stain the malaria parasite (a member of the genus Plasmodium).[28][19] Neither Ehrlich's or Chenzinsky's stains produced the Romanowsky effect as the methylene blue they used was not polychromed.[16]

Dmitri Romanowsky in 1890 published preliminary findings of his blood stain (a combination of aged methylene blue and eosin), including the results when applied to malaria infected blood.[6] This use of polychromed methylene blue differentiated Romanowsky's stain (and the subsequent formulations) from those of Ehrlich and Chenzinsky, which lacked the purple hue associated with the Romanowsky effect.[16] Romanowsky's 1890 publication did not include a description of how he modified his methylene blue solution,[6][16] but in his 1891 doctoral thesis he described methylene blue best as used after mold began forming on the surface.[6][16] Other than the use of an aged methylene blue solution, Romanowsky's stain was based on Chenzinsky's stain technique.[16] Romanowsky's use of his method to study the malaria parasite has been attributed to the continued interest in his staining method.[26]

Ernst Malachowsky

Ernst Malachowsky has been credited with independently observing the same stain combination as Dmitri Romanowsky in 1891,[6][13] although he has also been credited with being the first to do so.[16][19] Malachowsky was the first to use a deliberately polychromed methylene blue solution,[15] which Malachowsky accomplished by the addition of borax to the staining mixture.[16] Malachowsky is reported to have demonstrated the stain on June 15, 1890, and in the same year to have published a paper "describing his public demonstration".[19] Both the Romanowsky and Malachowsky methods were able to stain the nucleus and cytoplasm of the malaria parasite, when until this point the stains used had only colored the cytoplasm.[19]

Gustav Giemsa.

In 1899, Louis Leopold Jenner developed a more stable version of the methylene blue and eosin stain by collecting the precipitate that forms in water-based mixtures and redissolving it in methanol.[28][15][4] Romanowsky-type stains prepared from the collected precipitates are sometimes known as eosinates.[4] Besides increasing the stability of the stain, the use of methanol in Jenner's stain had the effect of fixing the blood samples,[4] although Jenner's version of the stain does not produce the Romanowsky effect.[28][19][15]

Richard May and Ludwig Grünwald in 1892 published a version of the stain (now known as the May–Grünwald stain) which is similar to the version proposed by Jenner in 1899, and likewise does not produce the Romanowsky effect.[28][19][15]

In 1901, both Karl Reuter and William Leishman[22] developed stains that combined Louis Jenner's use of alcohol as the solvent and Malachowsky's use of polychromed methylene blue.[19][15][4] Reuter's stain differed from Jenner's in using ethyl alcohol instead of methanol, and Leishman's differed from Jenner's by using eosin B instead of eosin Y.[19][4]

James Homer Wright in 1902 published[18] a method using heat to polychrome the methylene blue, which he combined with eosin Y. This technique is known as Wright's stain.[19][20]

Gustav Giemsa's name has also become associated with the stain as he is credited with publishing a useful formulation and protocol in 1902.[13][6][26] Giemsa attempted to use combinations of pure dyes rather than polychromed methylene blue solutions which are highly variable in composition.[20][19][15] Giemsa sold the rights to produce his stain, but never fully published details on how he produced it,[19] although it is thought that he used a combination of azure B and methylene blue.[15] Giemsa published a number of modifications of his stains between 1902 and 1934. In 1904[29] he suggested adding glycerin to his stain, along with the methanol, to increase its stability.[23][19]

Giemsa stain powders produced in Germany were widely used in the United States until the interruption of the supply during World War I, which caused increased utilization of James Homer Wright's method for polychroming methylene blue.[19][1]

• Liu's stain
• Malaria antigen detection tests
• Papanicolaou stain
• Staining (biology)