and Laboratory Methods. '-^261 



Goldhorn (1901) fixed air-dried blood preparations for fifteen seconds in 

 pure methylic alcohol or in pyroxylic spirits, washed in running water, stained 

 for seven or more seconds in yL to i per cent, aqueous solution of eosin (prefer- 

 ably Griibler's yellowish ivasserloeslkh), washed and again stained for ten 

 seconds in a faintly alkaline solution of his polychrome methylen blue, which is 

 prepared as follows: Dissolve two grams of methylen blue in 300 c. c. of water. 

 Shake the solution well and add four grams of lithium carbonate ; shake again 

 thoroughly, and pour into a large open porcelain dish over a water bath. Allow 

 the boiling water to touch the bottom of the dish. Stir frequently and decant, 

 after fifteen minutes, into a glass-stoppered bottle. Filter paper should be 

 avoided, cotton may be used in the funnel. Set aside for a few days and slightly 

 acidify with a 4 or 5 per cent, solution of acetic acid. Now add enough of a 

 saturated solution of lithium carbonate to render the stain faintly alkaline. In 

 order to know when this point has been reached trial blood smears should be 

 stained until the addition of another trace of lithium carbonate causes the red 

 corpuscles to take some of the blue. With some practice this point is rapidly 

 determined. A stain prepared in this manner does not deteriorate, but on the 

 contrary seems to improve on keeping if kept in well-stopped bottles. To deter- 

 mine the best time of staining, a number of preparations should be left in the 

 dye for a varying number of seconds and the interval giving the desired result 

 fixed upon. 



Nuclei of the megaloblasts stain beautifully, an intranuclear net-work is well 

 shown. Necrosis and polychromatophilia of the red cells are shown. Other 

 nucleated red cells stain well. Polymorphonuclear neutrophiles show their 

 granules and " chromatin-bridges." The lymphocytes stained for a short time 

 frequently show nucleoli in a thick capsule, and basophile granules in the cyto- 

 plasm. The lymphocytes may be stained deeply, hence the value of the stain in 

 lymphatic leukaemia. Blood plates show purplish dots within a bluish body. 



Goldhorn's polychrome methylen blue, improved formula, is now for sale, 

 ready for use. The staining technique is as follows : 



Methyl alcohol, fifteen seconds, wash in running water. 



Eosin, yL per cent, aqueous solution, seven to fifteen seconds, wash as 

 before. 



Goldhorn's polychrome methylen blue, thirty to sixty seconds. Wash thor- 

 oughly ; dry by agitation of the smear in the air, avoiding heat and the use of 

 filter paper. Mount in Canada balsam. 



Should the polychrome dye by exposure to the air become too alkaline — the 

 erythrocytes staining too deeply — add a few drops of a 4 or 5 per cent, solution 

 of acetic acid. The stain does nor deteriorate, but improves on keeping. 



For staining malarial parasites it is recommended that the polychrome dye 

 should act for from 90 to 120 seconds, that the chromatin of the half-grown 

 forms may become well stained. 



Hanna (1901) describes a modification of the Romanowsky-Nocht method, 

 given by Berestneff (in Russian). A 1 per cent, aqueous solution of methylen 

 blue (med. puriss. Hochst), containing 0.3 per cent, of carbonate of soda, is 

 heated for three hours on a water bath and then filtered. One c. c. of this solu- 



