Editorial: 100 years ago: Giemsa's solution for staining of plasmodia

Once the French physician Alphonse Laveran had identified Plasmodium as the causative agent of malaria (Laveran 1880), diagnosis of this infection had become possible. Laveran had used direct microscopy to detect plasmodia in the blood of patients; later staining with Methylene Blue was introduced but not satisfactory. In St Petersburg in 1891, Dimitri Romanowsky observed in that it was possible to differentially stain nuclei and cytoplasm of the plasmodia in infected erythrocytes (chromatin red and cytoplasm blue) with a mixture of eosin Y and Methylene Blue. To obtain this differential staining, the Methylene Blue solution had to have ‘aged’ or ‘got mouldy’. However, the specific effect was difficult to reproduce because of the ageing of Methylene Blue, and the mixture had to be prepared and tested for each staining. Proper diagnosis of malaria was one of tropical medicine's greatest problems at the beginning of the 20th century. Hence a number of modifications to the staining procedure were proposed, e.g. by W.B. Leishman and J.H. Wright, but none were easy to handle or reproduce. Bernhard Nocht, the chief medical officer of the harbour of Hamburg, had a keen interest in malaria. Experimenting to improve the Romanowsky staining, he found that ageing of Methylene Blue induced formation of a new dye (which he called ‘red from Methylene Blue’) that could stain the chromatin of plasmodia (Nocht 1899). In October 1900, Nocht became director of the newly founded Institut für Schiffs- und Tropenkrankheiten (Institute for Maritime and Tropical Diseases) in Hamburg (renamed Bernhard-Nocht-Institute in 1942). As his first coworker, he appointed Gustav Giemsa (1867–1948), a pharmacist with extensive training in chemistry and bacteriology (Figure 1) who had previously served for 2 years in Dar-es-Salaam, a German colony at the time. Members of the Institute for Maritime and Tropical Diseases in 1922. Gustav Giemsa third from right, Bernhard Nocht fifth from right. When Nocht asked him to further improve the Romanowsky stain, Giemsa investigated the nature of the new dye and succeeded in identifying it as azure B (trimethylthionine). He found that an aqueous solution of the acidic eosin and the basic azure B was unstable, but that dissolving the two dyes with an excess of basic azure B in a glycerol–methanol mixture produced a stable stock which yielded reproducible staining results. Apparently Giemsa was in a hurry to publish the manuscript since he wrote ‘I am unable to report conclusively on the stability of the staining solution since I could only observe it for 20 days’ (Giemsa 1904). In this publication (Figure 2) he also noted that the solution was commercially available under the label ‘Giemsa's solution for Romanowsky's stain’. Giemsa's solution turned out to be very stable and thus a method was discovered that remains – without modification – one of the most widely used procedures in laboratory medicine today. First page of Giemsa's publication (Giemsa 1904).