Staining Protozoa 167 



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 loo cc. of the filtered mixture add 500 

 cc. of a 0.1 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 cc. 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 spontaneous 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 to remain for two 

 or three minutes, according to the intensity of tlae staining desired. 

 A longer period of staining may produce a precipitate. 



3. Wash the preparation in water for thirty seconds or until the thinner 

 . portions of the preparation become yellow or pink in color. 



4. Dry and mount in balsam. 



Films more than an hour old do not stain so well as fresh ones. Old films 

 show bluish instead of pink erythrocytes.- 



Marino's stain* is extremely delicate and gives still more beautiful results 

 where parasites are present. It is an azur-eosin combination, prepared as 

 follows: 



Solution I: 



Methylene-blue (medicinal) . . ., 0.5 gram 



Azur II 0.5 " 



Water (distilled) . . . ; 100. o cc. 



Solution II: 



Sodium carbonate. . . 0.5 gram 



Water ....'/. 100. o cc. 



Pour the two solutions together and stand the mixture in the thermostat 

 for forty-eight hours at 37°C.; then add 0.2 per cent, aqueous solution of 

 eosin ("yellowish aqueous eosin"). The quantity of this solution must 

 be varied according to the blue dyes employed, so as to secure the maxi- 

 mum precipitation. The exact quantity can only be determined by 

 titration. A precipitate now forms in the course of twenty-four hours. 

 This is caught upon a filter-paper and dried. 



The precipitate, dissolved in methylic alcohol, in the proportion of 0.04 gm. 

 of the powder to 20 cc. of the methylic alcohol, forms the stain. 



Method. — The stain is dropped upon the spread so as to cover it, the number 

 of drops being counted. It is permitted to act for exactly three minutes 



■ for purposes of fixation, then, without pouring off the stain, twice the 

 number of drops of a I :ioo,ooo aqueous eosinsolution are added, t The 



* "Ann. de ITnst. Pasteur," 1904, xviii, 761. 



t Marino used a r : 20,000 aqueous solution of eosin, but the 1 : 100,000 

 solution is less apt to cause objectionable precipitation of the dye and- gives 

 equally good results. 



