58 PRESERVING AND PREPARING PATHOLOGICAL SPECIMENS. 



Methods of Staining. 



Sections of hardened tissues may be stained for microscopical study 

 in a variety of ways, but for routine work the double staining with 

 hsematoxylin and eosiu is most generally useful, and is applicable to 

 nearly all cases. 



H^MATOXYLIN solution (Delafield's) is prepared as follows: To 100 

 c.c. of saturated solution of ammonia alum add 1 gram of hsematoxyliu 

 crystals dissolved in 6 c.c. of ninety-five-per-cent alcohol. This solution 

 is exposed to the light for a week, the color meanwhile changing from a 

 dirty red to a deep bluish-purple color. 1 Then 25 c.c. each of glycerin 

 and wood naphtha are added. This mixture is allowed to stand for a day 

 or two and is then filtered, and the filtration is repeated at intervals until 

 a sediment no longer forms. 



The solution is now ready for staining, the best results being obtained 

 by diluting the fluid with from ten to twenty times its bulk of water. 

 The sections are immersed in the fluid, and allowed to remain until they 

 have acquired a distinct purple color which persists after rinsing in 

 water. They are now placed for a moment in a dilute alcoholic solution 

 of eosin, and then mounted in glycerin which has been colored lightly 

 with an alcoholic solution of eosin. In this way the nuclei of the cells 

 will be stained of a purple color, while the cell bodies, and to a certain 

 extent the intercellular substance, will be colored a light rose-red. 



If specimens are to be mounted in Canada balsam, they are stained 

 with hsematoxylin as before, and the eosin staining is done by tinging, 

 with a saturated alcoholic solution of eosin, the alcohol with which the 

 final dehydration of the specimen is accomplished. A similar result 

 may be obtained by tinging the oil of cloves or origanum with which the 

 clearing of the sections is effected. 



Iron Hcematoxylin (Heidenhain's). Sections are soaked for an hour 

 in a two-per-cent solution of ammonia sulphate of iron, then rinsed with 

 water and put for an hour in a one-half-per-ceut aqueous solution of 

 hsematoxylin (prepared by heating) ; again rinsed and put again in the 

 iron solution, in which the color gradually fades. The section must be 

 watched during the process of the differentiation which takes place in 

 the iron solution, and when this is accomplished to a proper extent the 

 section is thoroughly washed in running water and mounted in the usual 

 way. This method is especially valuable for the study of nuclear struc- 

 tures, the color of these ranging from blue to black, depending upon the 

 length of time of immersion in the stain and the grade of differentiation. 



By the use of this method, micro-organisms may be stained black, and 

 in this condition are, as Learning has shown, well fitted for the purposes 

 of photomicrography. 



1 The time required for this " ripening " of the solution may be spared by the use of 

 hsematin in the place of hsematoxylin. 



