332 CALIFORNIA ACADEMY OF SCIENCES. [Proc. 3D Ser. 



paraffin, the use of which was found necessary to obtain 

 good ribbons. Sections 3-5 /a. thick were cut and fixed 

 on the slide by the water-albumen method. 



Flemming's triple stain was used, with some modifica- 

 tions. The length of time for each stain was ascertained 

 separately. It was finally found advisable not to use the 

 safranin solution usually employed, but to substitute for it 

 Babes' safranin A, consisting of a mixture of equal parts 

 of a saturated alcoholic solution of safranin and a saturated 

 aqueous solution of safranin, because the former had a 

 tendency, when old, to give a muddy appearance to the 

 cytoplasm. 



In the case of orange G, even weak solutions took out too 

 much blue. By substituting for this a solution of iodine 

 and potassium iodide of the same strength and composition 

 as the fixing-fluid mentioned under number 36, much better 

 results were obtained. In fact the potassium iodide 

 iodine seemed to fix the blue in the fibers, so that absolute 

 alcohol and clove-oil could be relied upon to wash out the 

 superfluous blue. 



The staining process was as follows: After dissolving the 

 paraffin in xylol and removing all traces of the latter by a 

 double washing in 95 per cent, alcohol, the slides were 

 placed in the safranin for five minutes and then decolorized 

 in absolute alcohol to which o.i per cent, hydrochloric acid 

 had been added. After washing thoroughly in water to 

 remove all traces of the acid, the slides were placed for 

 exactly five minutes in a concentrated aqueous solution of 

 gentian violet, and then washed off in water; after which 

 they were immersed for twenty seconds in the solution of 

 iodine and potassium iodide above referred to. This 

 appeared to fix the blue in an admirable way, so that after 

 dehydrating in absolute alcohol for one second, the prepa- 

 rations, in clove-oil, could be watched under the microscope 

 until the cytoplasm was perfectly clear, the fibers a dark 

 blue, and the granular zone brown-violet. The nucleolus 

 was a bright red as well as the chromatin, except in the 

 earlier stages, when the chromatin stained blue. 



