198 Methods of Observing Micro-organisms 



to remain for two or three minutes, according to the intensity 

 of the staining desired. A longer period of staining may pro- 

 duce 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) 100.0 grams. 



Solution II: 



Sodium carbonate 0.5 gram. 



Water 100.0 c.c. 



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 maximum precipitation. 

 The exact quantity can only be determined by titration. A pre- 

 cipitate 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 c.c. 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 pour- 

 ing off the stain, twice the number of drops of a i : 100,000 aqueous 

 eosin solution are added. The two fluids gradually mix, trans- 

 fusion currents are formed, and the specimen is allowed to stand 

 for exactly two minutes longer. It is during this time that the 

 staining takes place. A precipitate usually forms upon the 

 surface of the fluid, so that it must not be poured off, but splashed 

 off by dropping distilled water upon it from a height. The dis- 

 tilled water is added until it no longer shows any color, when the 

 specimen is drained, dried, and mounted in balsam. 



The student may also try staining with hematoxylon and 

 eosin, thionin and eosin, methylene-blue and eosin, or any 

 other dyes, some of which sometimes bring out special de- 

 tails of structure. The protozoa do not show the same re- 

 action to Gram's stain that makes it so useful for differ- 

 entiating the bacteria. 



* "Ann. de 1'Inst. Pasteur," 1904, xvm, 761. 



