298 GAEY N. CALKINS 



After an individual is transferred to fresh medium, the re- 

 maining individuals are placed in a Syracuse dish containing 

 about 10 cc. of the fresh culture medimn. Here they multiply 

 in large nmnbers, constituting the 'stock' material, the source 

 of dividing and conjugating forms. 



B. Total preparations 



In handling the material for fixing and staining, the following 

 methods have been used. A dividing individual, or a conju- 

 gating pair, is drawn up in a capillary pipette and deposited on 

 a clean slide with a minimum of water. The object is then cov- 

 ered with a couple of drops of killing fluid. After three to five 

 minutes, the object is drawn into a second capillary pipette 

 (used only for the killing agent) and transferred to a watch-glass 

 containing 95 per cent alcohol. A clean, thin cover-glass is then 

 prepared by smearing one surface with egg albmnen. The 

 specimen is then drawn up in a third capillary pipette from the 

 alcohol and spurted on the smeared side of the cover-glass. The 

 accompanying alcohol coagulates the albumen which holds the 

 object during subsequent treatment. Care must be taken to 

 prevent drying of the specimen when the alcohol evaporates; 

 this is accomplished by flooding the smeared surface with the 

 stain to be used, and setting the cover-glass in a moist chamber. 

 To dehydrate, after staining, the cover-glass is placed, with for- 

 ceps, into salt cellars or Syracuse dishes containing successive 

 grades of alcohol. It is well to use at least two dishes of abso- 

 lute alcohol to prepare the object for xylol. After clearing in 

 xylol the unsmeared surface of the cover-glass is carefully wiped 

 with a dry cloth, and the object is finally mounted in balsam. 



For study of the nuclear structures, I have found that, for 

 killing, a saturated aqueous solution of bichloride of mercury, 

 with a trace of acetic acid, gives excellent results when followed 

 by the iron-haematoxylin stain, and this method was mainly 

 used in the present investigation. All stages were confirmed 

 on raaterial fixed in Flemming's fluid, Bouin's fluid, and Schau- 

 dinn's fluid. 



