METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 185 



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 0.1 per 

 cent, aqueous solution of 'yellowish, water-soluble' eosin and mix 

 thoroughly. Collect on a filter the abundant precipitate which imme- 

 diately appears. When the precipitate is dry, dissolve it in methylic 

 alcohol (Merck's 'reagent') in the proportion of 0.1 gm. to 60 c.c. 

 of the alcohol. In order to facilitate solution the precipitate is to be 

 rubbed up with 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 faults. It does not undergo any other spontaneous change than 

 that of concentration by evaporation. 



"A most important fault met with in the working of some samples 

 of this fluid is that it fails to stain the red blood corpuscles a yellow or 

 orange color, but stains them a blue color which cannot readily be 

 removed by washing with water. This fault is due to a defect in the 

 specimen of eosin employed. It can be eliminated by using a proper 

 'yellowish, water-soluble' eosin." 



Method of Staining. (a) Unheated air-dried films 1 are covered 

 with the stain, which is allowed to act for one minute. 



(6) Add an equal volume of distilled water to the stain and allow to 

 stand for three minutes. 



(c) Wash in water for thirty seconds, or until a pink color develops. 



(d) Dry rapidly with filter paper and mount in balsam. 2 

 Giemsa Method. 3 Preparation of Stain: 



Azur II (eosin) 3.0 grams 



Azur II 0.8 grams 



Glycerin, C. P 250 c.c. 



Neutral absolute methyl alcohol 250 c.c. 



The dyes are dissolved in the glycerin at 60 C.; the alcohol, warmed 

 to 40 C., is then added, thoroughly mixed by shaking, and allowed 

 to cool slowly to room temperature, then filtered. Immediately before 



1 Films more than a few hours old do not stain as well as fresh ones. 



2 The balsam must be neutral in reaction. 



3 Giemsa, Cent. f. Bakt., 1904, xxxvii, 308. 



