14 MARGARET C. FERGUSON 



differ materially from those generally employed in cytological 

 work. The fixing fluids tested were chrome-osomo-acetic acid 

 solution, chrome-acetic acid solution, corrosive sublimate in 

 aqueous solution, absolute alcohol, and Carnoy's fluid. The 

 first two were tried with variations in concentration and in length 

 of time. The chrome-osomo-acetic acid solution giving by far 

 the best results, the other fixers were entirely discarded. It 

 was made up according to the following formula : 



Chromic acid crystals 1.3 gms. 



Osmic acid (in glass bulb) 5 gms. 



Glacial acetic acid 83 c.c. 



Distilled water 160.0 c.c. 



ii 



This solution used in one half strength and allowed to act for 

 about 15 hours proved to be most excellent for fixing the pro- 

 thallium at the time when it consists of a wall layer of proto- 

 plasm containing numerous free nuclei. For the development 

 of the pollen-grain and fertilization stages, it was most satisfac- 

 tory when undiluted, and allowed to act for about 24 hours. If 

 the fluid blackened at all, it was poured off after 2 or more 

 hours and fresh added. 



After fixing, the material was washed in running water from 

 2 to 12 hours, but as a rule specimens were not kept in the 

 running water longer than 6 hours. The very convenient piece 

 of apparatus described by Durand ('99) was used for this process. 

 Subsequent to washing, material was dehydrated in 8 grades of 

 alcohol beginning with 15^ and ending with the absolute. It 

 was not allowed to stand in the lower grades for more than 6 

 hours, and was rarely kept in the absolute alcohol longer than 

 that time ; the latter was changed 3 times, once about every 2 

 hours, to insure perfect dehydration in as short a time as pos- 

 sible. After material had been in 85^ alcohol for 12 hours, it 

 was decolorized in a 35^> solution of hydrogen peroxide, made 

 up in 95^ alcohol, for 24 hours. While material was always 

 bleached in toto, it was frequently found necessary to decolor- 

 ize again on the slide. After dehydration, material was brought 

 gradually, through ascending grades, into pure cedar oil, xylol 

 or chloroform. The best results were obtained with the cedar 

 oil and it was far more commonly used than the others. If it 



