Staining Protozoa 197 



If the covers are to be stained, they can most conveniently 

 be held in the Stewart forceps. If the slides are used, they 

 can be held in the fingers. 



The stain most useful is that of Romanowsky. It has 

 many modifications, of which the most used and best known 

 are Giemsa's, Jenner's, Leishmann's, Wright's, and Marino's. 

 These stains can be bought either in solution or in tablet 

 form ready for solution. 



Those most highly to be recommended are Wright's and 

 Marino's. 



Wright's Blood-stain. This is a modification of Leishmann's stain, 

 to which it is to be preferred because it can be made in a few 

 hours instead of eleven days. It combines the methylene-blue- 

 eosin combination of Romanowsky with the methyl -alcohol 

 fixation of Jenner. 



It is prepared as follows:* 



''To a 0.5 per cent, aqueous solution of sodium bicarbonate add 

 methylene-blue (B. X. or "medicinally pure") in the proportion 

 of i gm. of the dye to 100 c.c. of the solution. Heat the mixture in 

 a steam sterilizer at 100 C. for one full hour, counting the time 

 after the sterilizer has become thoroughly heated. The mixture 

 is to be contained in a flask of such size and shape that it forms 

 a layer not more than 6 cm. deep. After heating, the mixture is 

 allowed to cool, placing the flask in cold water if desired, and is 

 then filtered, to remove the precipitate which has formed in it. 

 It should, when cold, have a deep purple-red color when viewed, 

 in a thin layer, by transmitted yellowish artificial light. It 

 does not show this color while it is warm. To each 100 c.c. 

 of the filtered mixture add 500 c.c. of a o.i per cent, aqueous 

 solution of "yellowish, water-soluble" eosin and mix thoroughly. 

 Collect the abundant precipitate which immediately appears 

 on a filter. When the precipitate is dry, dissolve it in methylic 

 alcohol (Merck's "reagent") in the proportion of o.i gr. to 

 60 c.c. of the alcohol. In order to facilitate the solution the 

 precipitate is to be rubbed up with the alcohol in a porcelain 

 dish or mortar with a spatula or pestle. 



"This alcoholic solution of the precipitate is the staining fluid. It 

 should be kept in a well-stoppered bottle because of the volatility 

 of the alcohol. If it becomes too concentrated by evaporation, 

 and thus stains too deeply or forms a precipitate on the blood- 

 smear, the addition of a suitable quantity of methylic alcohol will 

 quickly correct such fault. It does not undergo any other spon- 

 taneous change than that of concentration by evaporation." 



Method of Staining. The blood-films are permitted to dry in the air 

 (not heated) : 



1. Cover the film with a noted quantity of the staining fluid by 



means of a medicine dropper. 



2. After one minute add to the staining fluid the same quantity of 



distilled water by means of the medicine dropper, and allow it 



*Mallory and Wright, "Pathological Technique," 1911, p. 364. 



