# Contributor timeline: the methylene blue and eosin blood stain

Standalone reference document for the article "Who developed the purple blood stain?"
(`/en/articles/who-developed-the-purple-blood-stain/`). Every dated entry names the source
it rests on. Where an entry comes from an older publication that was read through a modern
review rather than in the original, the source column says so.

## How to read the source column

- **direct** - the publication was read in full text for this article.
- **via review** - the publication is known through the body text and reference list of a
  modern review that was read in full. The original was not inspected.

Two reviews carry almost the whole of this timeline and were both read in full:

- Krafts KP, Hempelmann E, Oleksyn BJ. "The color purple: from royalty to laboratory, with
  apologies to Malachowski." *Biotechnic & Histochemistry* 2011;86(1):7-35.
  doi:10.3109/10520295.2010.515490. [PMID 21235291](https://pubmed.ncbi.nlm.nih.gov/21235291/)
  (cited below as **Krafts 2011a**, direct)
- Krafts K, Hempelmann E, Oleksyn BJ. "In search of the malarial parasite: biographical
  sketches of the blood stain contributors." *Parasitology Research* 2011;109(3):521-529.
  doi:10.1007/s00436-011-2475-4. [PMID 21660627](https://pubmed.ncbi.nlm.nih.gov/21660627/)
  (cited below as **Krafts 2011b**, direct)

## Timeline

| Date | Person | Contribution | Source |
| --- | --- | --- | --- |
| 1856 | William Henry Perkin, aged 18 | First commercial synthetic dye, mauveine, found while trying to make quinine from aniline. Made synthetic dyes available to medicine. | Perkin's own account summarised in Krafts 2011a (via review) |
| 1858 | Joseph von Gerlach | Book on carmine staining. Turned histological staining from scattered experiment into a method. Gerlach is treated in the literature as the father of modern staining technique. | Gerlach 1858, via review |
| 1865 | Wilhelm Heinrich Erb (1840-1921) | First application of a synthetic dye to blood. Used picric acid; some red cells showed a reticular pattern. | Erb 1865, via review |
| 1875 | Adolf von Baeyer | Eosin synthesised and named from Eos, the Greek for the flush of dawn. | Baeyer 1875, via review |
| 1876 | Heinrich Caro (1834-1910) and Adolf von Baeyer | Methylene blue developed at BASF. Caro was director of the BASF laboratory. | Krafts 2011a, Krafts 2011b |
| 1877-1880 | Paul Ehrlich (1854-1915) | Classified aniline dyes as acid or basic; found acid dyes stain red cells, eosinophil granules and eosinophilic cytoplasm, basic dyes stain nuclei, mast cell granules and lymphocyte cytoplasm. | Ehrlich 1880, via review |
| 1880 | Paul Ehrlich | The *neutralen Farbkörper*, a neutral stain. Saturated aqueous methylene blue poured into saturated aqueous acid fuchsin until the supernatant cleared, then more acid fuchsin until the precipitate redissolved. Red cells and eosinophil granules red, nuclei deep blue, lymphocyte cytoplasm pale blue. Dark violet granules took up both components, which is the origin of the word *neutrophil*. Made the first differential white cell count possible. | Ehrlich 1880; protocol in Krafts 2011a (via review) |
| 1882 | Robert Koch | Used methylene blue to demonstrate the tubercle bacillus. | Koch 1882, via review |
| 1883 | Paul Ehrlich | Improved Koch's methylene blue method for tubercle bacilli. | Ehrlich 1883, via review |
| 1885 | Heinrich August Bernthsen | Showed that alkaline conditions demethylate methylene blue, giving methylene azure and methylene violet. The chemical basis of what the stain makers called ripening. | Bernthsen 1885, via review |
| 1888 | Czesław Chęciński (1851-1916) | Replaced Ehrlich's acid fuchsin with eosin. The first methylene blue plus eosin blood stain, and the prototype for everything after it. Visualised the malaria parasite (blue daisy-like forms, now *Plasmodium malariae*; sickle forms, now *P. falciparum* gametocytes) but not its nucleus. Published method: saturated aqueous methylene blue diluted 1:2 with water; eosin at 0.5 g in 60 g alcohol and 40 g water. | Chęciński 1888, thesis 1889, quoted in Krafts 2011a (via review) |
| 1889 | Ernst Malachowski (1857-1934) | In one preparation, saw the plasmodial nucleus stain differently from the cytoplasm. Could not reproduce the effect. | Krafts 2011a, Krafts 2011b |
| 1890 | Albert Plehn | Same two dyes with a higher proportion of methylene blue and less alcohol; 2:1 then 3:1. Added potassium hydroxide, probably to neutralise the acidity of the more concentrated methylene blue. Rose red cells, deep blue nuclei, rose eosinophil granules. Wider colour range, still no purple. | Plehn 1890a, 1890b; recipe in Krafts 2011a (via review) |
| 15 June 1890 | Ernst Malachowski | First public demonstration of the purple stain, produced by alkalinising the methylene blue with borax. Chromatin of the plasmodial nucleus clearly visible, nucleus distinguishable from cytoplasm. Staining took 24 hours; he described the results as easily reproducible. | Malachowski 1890; demonstrated in Krafts 2011a, Krafts 2011b |
| August 1891 | Ernst Malachowski | Published the technique, including the borax instruction, and stated the implication for the parasite's nature. This is the last article he wrote on blood staining. | Malachowski 1891, via review |
| June 1891 | Dmitri Romanowsky (1861-1921) | Thesis submitted in St Petersburg. | Romanowsky 1891, thesis; Krafts 2011a (via review) |
| September 1891 | Dmitri Romanowsky | Published his purple method. His best results came from a stock methylene blue solution that had stood for over two months with mould growing on it. Not reproducible by others. | Romanowsky 1891; Krafts 2011a, Krafts 2011b |
| 1892 | Paul G. Unna | Showed that carbonates, especially potassium carbonate, accelerate ripening of methylene blue, and that the product could be extracted with ether. "Unna's polychrome methylene blue". | Unna 1892, via review |
| 1898 | Bernhard Nocht (1857-1945) | Concluded that the purple had to come from a substance in the methylene blue that was not methylene blue, and built his blue solution from Unna's polychromed methylene blue. | Nocht 1898, via review |
| 1899 | Bernhard Nocht | Extracted the polychrome solution with chloroform. The residue, *Rot aus Methylenblau*, was the part that produced the purple. | Nocht 1899, via review |
| 1899 | Louis Leopold Jenner (1866-1904) | Introduced absolute methanol as the solvent for the methylene blue plus eosin precipitate. Stabilised the solution and removed the need for heat fixation of blood films. He did not use polychromed methylene blue, so his stain produced no purple. | Jenner 1899, via review |
| 1901 | Michaelis | Identified *Rot aus Methylenblau* as azure I. | Michaelis 1901, via review |
| 1901 | William Boog Leishman (1865-1926); Karl Reuter | Independently combined eosin with polychromed methylene blue, filtered off the precipitate and redissolved it in alcohol. Leishman used methanol and eosin B; Reuter used ethanol and stressed the need for an absolutely pure solvent. Stable stain, purple preserved. Leishman used his own stain in 1903 to discover *Leishmania donovani*. | Leishman 1901, Reuter 1901, via review |
| 1902 | Richard May and Ludwig Grünwald | Published a method virtually identical to Jenner's. Like Jenner, they used unmodified methylene blue, so no purple. | May and Grünwald 1902, via review |
| 1902 | James Homer Wright (1870-1928) | Simplified the preparation of polychromed methylene blue by heating it for one hour in flowing steam instead of holding it at 65 °C for 12 hours and then standing for 10 days. Claimed the method would not fail to give the promised result "even in the hands of beginners in hematology". | Wright 1902, quoted in Krafts 2011a |
| 1902 | Berthold Carl Gustav Giemsa (1867-1948) | Replaced randomly polychromed methylene blue with measured quantities of known dyes. Called his thiazin *methylene azure*; identified Nocht's residue as a mixture of methylene azure and methylene violet and held that methylene violet harms staining. Best results at 5 mol eosin to 4.4 mol azure. Later replaced azure I with azure II, giving blue rather than grey leukocyte cytoplasm. Never published how he prepared the pure azure; sold it to Dr Grübler's laboratory, which marketed it as azure I. | Giemsa 1902a, 1902b; Krafts 2011a (via review) |
| 1904 | Gustav Giemsa | Recommended powdered azure II and eosin in equal weights of glycerin and methanol, a combination that stayed stable in tropical conditions. | Giemsa 1904, via review |
| 1914-1918 | - | Blockade cut the supply of Grübler's dyes outside Germany. | Conn 1980, via review |
| 1939-1945 | - | German dyes became unobtainable altogether, and demand for stain rose with the number of wartime field hospitals. | Lillie 1969; Conn 1980, via review |
| 1940-1943 | Ralph Dougall Lillie, Maurice Alexander Roe, Aimee Wilcox | United States Public Health Service team investigating why American substitutes were inferior. Found that both azure B and methylene blue were needed. Compared azures A, B, C and IV, toluidine blue and thionine with eosin; all the azures gave the polychromatic effect, best with azure B; faint with toluidine blue, none with thionine. Azure B replaced the unavailable azure I. | Roe, Lillie and Wilcox 1940; Lillie 1943, via review |
| 1969 | Ralph Dougall Lillie | Drew the distinction between biological stains, made to specifications that fit diagnostic work, and non-biological stains such as textile dyes, made to looser ones. Both kinds of product carried the same name. | Lillie 1969, quoted in F03 and in Krafts 2011a |
| 1978 | Ralph Dougall Lillie | Proposed that the family of stains should be called Malachowski stains, since he introduced deliberate alkali polychroming of the methylene blue component. | Lillie 1978, quoted in Krafts 2011a |
| 1993 | Kristine Krafts | Review of the whole lineage, listing the names in sequence from Ehrlich to Wilcox. | Krafts 1993, via review |
| 2010 | A. Bezkurov | Argued the priority question for Romanowsky. Cited by Krafts and colleagues as the opposing position. | Bezkurov 2010, via review |
| 2011 | Kristine Krafts, Ernst Hempelmann, Barbara J. Oleksyn | Reconstructed Malachowski's life from archive documents in Poznań and Wrocław and from family papers held at the Leo Baeck Institute in New York. Concluded that Malachowski produced the purple deliberately and first, and called the attribution to Romanowsky "a travesty". | Krafts 2011a, Krafts 2011b |
| 2011 | Comment published in the same journal | A comment on the long paper appeared at *Biotechnic & Histochemistry* 2011;86(5):366, doi:10.3109/10520295.2011.582885. It is listed here as a record only. The comment itself was not read for this timeline, so it is not characterised. | Comment record, not read: doi:10.3109/10520295.2011.582885 |

## Primary publications named above, as cited by the two reviews

These are the works the timeline refers to, given in the form in which the 2011 review lists
them. Only the two 2011 reviews, and Lillie 1969 and 1978 through quotation, were read for
this article.

- Baeyer A. Zur Geschichte des Eosins. *Berichte der deutschen chemischen Gesellschaft* 1875;8:146-148.
- Baker J. The blood dyes. In: *Principles of Biological Microtechnique: a Study of Fixation and Dyeing*, chapter 14, 1st ed. London: Methuen; New York: John Wiley & Sons, 1958:262-273.
- Bernthsen A. Studien in der Methylenblaugruppe. *Liebig's Annalen der Chemie* 1885;230:73-136.
- Chęciński C. Zur Lehre über den Mikroorganismus des Malariafiebers. *Centralblatt für Bakteriologie, Parasitenkunde und Infektionskrankheiten* 1888;15:457-460.
- Chęciński C. *К учению о микроорганизмах малярии* (dissertation). Odessa, 1889.
- Clarke G, Kasten FH. *History of Staining*, 3rd ed. Baltimore: Williams and Wilkins, 1983:113-117.
- Ehrlich P. Methodologische Beiträge zur Physiologie und Pathologie der verschiedenen Formen der Leukocyten. *Zeitschrift für klinische Medizin* 1880;1:553-560.
- Ehrlich P. Neue Methode der Färbung von Tuberkelbacillen. *Berliner klinische Wochenschrift* 1883;20:13.
- Erb W. Zur Entwicklungsgeschichte der rothen Blutkörperchen. *Archiv für pathologische Anatomie* 1865;34:138-194.
- Gerlach J. *Mikroskopische Studien aus dem Gebiete der menschlichen Morphologie*. Erlangen: Ferdinand Enke, 1858.
- Giemsa G. Färbemethoden für Malariaparasiten. *Centralblatt für Bakteriologie* 1902;31:429-430.
- Giemsa G. Färbemethoden für Malariaparasiten. *Centralblatt für Bakteriologie* 1902;32:307-313.
- Giemsa G. Eine Vereinfachung und Vervollkommnung meiner Methylenazur-Methylenblau-Eosin-Färbemethode zur Erzielung der Romanowsky-Nochtschen Chromatinfärbung. *Centralblatt für Bakteriologie* 1904;37:308-311.
- Jenner L. A new preparation for rapidly fixing and staining blood. *Lancet* 1899;1:390-392.
- Koch R. Die Aetiologie der Tuberculose. *Berliner klinische Wochenschrift* 1882;15:221-230.
- Krafts K. The Ehrlich-Chenzinsky-Plehn-Malachowski-Romanowsky-Nocht-Jenner-May-Grünwald-Leishman-Reuter-Wright-Giemsa-Lillie-Roe-Wilcox stain: the mystery unfolds. *Clinics in Laboratory Medicine* 1993;13:759-771.
- Leishman W. A simple and rapid method of producing Romanowsky staining in malarial and other blood films. *British Medical Journal* 1901;2:757-758.
- Lillie RD. *H. J. Conn's Biological Stains*, 8th ed. Baltimore: Williams and Wilkins, 1969:1-9.
- Lillie RD. Romanowsky-Malachowski stains. The so-called Romanowsky stain: Malachowski's 1891 use of alkali polychromed methylene blue for malaria plasmodia. *Stain Technology* 1978;53:23-28.
- Lillie RD. A Giemsa stain of quite constant composition and performance made in the laboratory from eosin and methylene blue. *Public Health Reports* 1943;58:449.
- Malachowski E. Beiträge zur Pathologie des Blutes. *Jahresbericht der Schlesischen Gesellschaft für vaterländische Cultur* 1890;68:31-32.
- Malachowski E. Zur Morphologie des Plasmodium malariae. *Centralblatt für klinische Medicin* 1891;31:601-603.
- May R, Grünwald L. Über Blutfärbungen. *Zentralblatt für innere Medizin* 1902;23:265.
- Michaelis L. Das Methylenblau und seine Zersetzungsprodukte. *Centralblatt für Bakteriologie* 1901;29:763-769.
- Nocht B. Zur Färbung der Malariaparasiten. *Centralblatt für Bakteriologie* 1899;25:764-769.
- Nocht B. Zur Färbung der Malariaparasiten. *Centralblatt für Bakteriologie* 1898;24:839 (cited in the review and in Giemsa 1902 without further detail).
- Plehn A. 1890. Two papers the same year, the first at a methylene blue to eosin ratio of 2:1, the second at 3:1, both with potassium hydroxide (cited in the review without full bibliographic detail).
- Reuter K. Ueber den färbenden Bestandteil der Romanowsky-Nochtschen Malaria-Plasmodienfärbung, seine Reindarstellung und praktische Verwendung. *Centralblatt für Bakteriologie* 1901;30:248-256.
- Roe M, Lillie RD, Wilcox A. American azures in the preparation of satisfactory Giemsa stains for malaria parasites. *Public Health Reports* 1940;55:1272-1278.
- Romanowsky D. 1890, method using unmodified methylene blue and eosin. *Врач* 1890;52:1171-1173.
- Romanowsky D. Zur Frage der Parasitologie und Therapie der Malaria. *St Petersburger medicinische Wochenschrift* 1891;16:297-302, 307-315. Thesis submitted at St Petersburg, June 1891; article published September 1891.
- Unna PG. Ueber die Reifung unserer Farbstoffe. *Zeitschrift für wissenschaftliche Mikroskopie* 1892;8:475-487.
- Wright JH. A rapid method for the differential staining of blood films and malarial parasites. *Journal of Medical Research* 1902;7:138-144.

## People, without the stain

Ernst Malachowski's biography, as reconstructed in Krafts 2011a and 2011b:

- Born 12 March 1857 in Strzelno. Grammar school in Posen, 1867 to 1876. Medicine at the University of Breslau, 1876 to 1881.
- Assistant doctor in internal medicine at All Saints Hospital, Breslau, 1882 to 1887.
- Private practice in Breslau with laboratory work; from 1890 at the Poliklinik Dr Malachowski und Dr Bielschowsky, Gartenstraße.
- Public demonstration of the stain, 15 June 1890. Attended the International Malaria Congress in Berlin, 1890.
- Awarded the title *Sanitätsrat* by Kaiser Wilhelm II in 1909, an honour given to physicians with at least twenty years of practice and an exceptional contribution.
- Died 7 March 1934 in Breslau. The whereabouts of his grave is unknown; it was most likely destroyed.
- Married Hedwig Salomonsohn (1872-1942). Six children: Leo Alfred (1889-1917, killed in the First World War), Rosa (1895-1925, a physician), Eva (1900, deported to Izbica in April 1942), Marie (1902-1999, emigrated to London 1939, served in the British Army, later Canada), Gertrude (1904-1940), Berthe (1907-1995).
- Hedwig Malachowski took her own life in Breslau on 22 April 1942 rather than be deported.
- Marie Malachowski's collection of family documents is held at the Leo Baeck Institute, New York. It is the source of most of what is now known about the family.
- A granddaughter, Gabriele, disappeared during the war and is thought to have died in the camps.