Giemsa

GIEMSA, Gustav (Gustav Giemsa, born 1867), chemist, known for his work in dyes and chemotherapeutic agents, quinine, etc. Head of the chemical department at the Hamburg Tropical Institute. In wide laboratory circles, he is mainly known for introducing a modification of the Romanowsky method for staining blood elements and blood parasites. Giemsa staining. The Giemsa name is given to the azure-eosin solution (Azur-Eosinfarbung fur die Romanowsky-Farbung n. Giemsa). The development of this method began with the discovery of Romanowsky (St. Petersburg, 1891) that in a mixture of methylene blue and eosin, the protoplasm of protozoa is stained in blue shades, and nuclei in red, whereas when used separately, methylene blue (basic dye) stains nuclei blue, and eosin (acidic dye) stains protoplasm pink. Nocht (1899) pointed out that this paradoxical phenomenon may be connected with the fact that in old, as well as specially prepared alkaline solutions of methylene blue, a new substance ('Rot aus Methylenblau') is formed, which gives these solutions a reddish tint, and they acquire the property of staining nuclei in reddish colors. This substance was isolated and named azure (Bernthsen, 1885). Its exact composition was later clarified (Kettmann, 1906) (see Azure). For the preparation of staining solutions according to the Romanowsky principle, a number of methods were proposed. All of them differed in instability and inconsistency of results. Giemsa (1905) was the first to prepare a completely stable solution that gave consistent results. Method G.: 3.0 Azur II-Eosin and 0.8 Azur II are dried in a desiccator over H₂SO₄, crushed, sieved through the finest silk sieve, and dissolved in 250 cubic cm of chemically pure glycerin (Merck) at 60°. To this is added 250 cubic cm of methyl alcohol (Kahlbaum) heated to 60°, shaken, and after 24 hours filtered. There are also other methods for preparing azure-eosin mixtures. Some authors (Pantoni, 1916) prepare a mixture of methylene blue, azure, and eosin, approaching the Nocht method. Application method. Fixed blood smears are stained with Giemsa solution (1-2 drops per 1 cubic cm of distilled water). The results of staining depend on the properties of the water. Weakly alkaline water (pH 6.4-6.8) is usually required. To determine the suitability of the water, a crystal of hematoxylin or a few drops of its alcoholic solution is added. The water is suitable if no pink coloration occurs within 2 minutes: otherwise it is acidic and must be alkalized with a 1% solution of Natr. carbon. Staining results: erythrocytes are stained in pink-purplish tones, nuclei of leukocytes, lymphocytes, and monocytes, as well as azurophilic granules, in various shades of reddish-purple, nuclei of blood parasites in purplish-red. The protoplasm of lymphocytes and monocytes in blue-gray-blue tones of different shades. The so-called neutrophilic granulation is stained in reddish-purple shades. Eosinophilic and pseudo-eosinophilic-from yellowish to copper-rose, basophilic-in violet-blue shades. In a more acidic medium (pH about 5.6-5.8), eosin shades become brighter, in a more alkaline one (pH about 7.0) blue and violet tones are enhanced. Blood smears are best preserved in an unmounted state. Staining with G. solution is also applied to sections, which after staining are passed from water through mixtures of acetone and xylene (water→acetone→1/2 acetone+1/2 xylene→1/3 acetone+2/3 xylene→xylene), and mounted in neutral Canadian balsam, cedar oil, or vaseline oil. Giemsa solution is widely used in microbiological techniques, facilitating in many cases the detection of fine cytological details, for example, capsules and metachromatic granules in bacteria. Staining by G. is one of the basic methods in the study of the morphology of protozoan blood parasites-malarial plasmodia, trypanosomes, etc. This method is often used for staining Tr. pallidum, which after ordinary alcohol fixation takes on a characteristic delicate pink color. After fixation with formalin, G. solution gives Tr. pallidum more violet shades. Staining by G. is often combined with other stains, for example, with eosin-methylene blue of May-Grünwald (Pappenheim's method). The theoretical basis of the action of the Giemsa-Romanowsky azure-eosin mixture, and especially the cause of the characteristic purplish-red staining of protozoan nuclei (the so-called 'Romanowsky effect'), have not yet been finally clarified. Unna (Unna, 1922) believes that eosin mordants the nuclei and thus serves as a link between their crystals and azure. Giemsa himself (1922) considers that the role of mordant is played by azure, which prepares the nuclei for subsequent staining with eosin. Ready-made G. solutions are sold by various laboratories. The best known are: in Germany Grubler [Grubler (Hollborn) Leipzig], and in the USSR-Institute of Pure Chemical Reagents (IREA, Moscow). In addition, Grubler also produces tablets, which are dissolved in methyl alcohol with glycerin. Prices: Giemsa (solution) 100 cubic cm-2.70 Mark; in tab. sice, (each tablet gives 50.0 cubic cm of solution)-10 tablets-13.30 Mark. French authors (Tribondeau and Dubreuil, 1917) prepare a similar mixture ('Azeo'=Azureosinat), dissolving 1% each of Azure I and eosin in a mixture consisting of 75 parts alcohol and 25 parts glycerin. After a few days, an azure solution (2 parts to 8 parts of the mixture) is added to the obtained mixture.