552 Transactions of the Society. 



hamiotoxylin, and thionin, particular attention being paid to the 

 differentiation process. 



One or two modifications were made regarding the strength of 

 the ha?matoxylin solution, and length of time occupied in staining. 



The use of thionin in my hands until late has, owing to its 

 rather great tendency to fade, been somewhat disappointing. 

 Although a very fine and powerful stain for this class of work, 

 perhaps quite equal in its capacity to iron-heematoxylin for 

 bringing out fine detail, the difficulty of maintaining the critical 

 point of differentiation owing to fading has made me more than 

 once think of giving up using it. 



However, by continually trying different methods, I have 

 found the following give the most satisfactory result, and up to 

 the present hold the stain without any signs of depreciation. 



The chief point to lay stress upon in the use of thionin as a 

 stain, is to employ solutions after staining that are perfectly free, 

 or as near free as possible, from acid reaction. Unfortunately, 

 most of the essential oils used for clearing, are on the acid side, or 

 very soon become so by keeping, and more often than not also, 

 balsams are found in like condition and overlooked in this respect. 

 I find cedar oil the most satisfactory to use and very nearly acid- 

 free ; but as the durabilitv of thionin and the delicate staining with 

 it is so easily affected by acid, it is as well to subject sections that 

 have been stained by it to as short an immersion in cedar oil as 

 possible, a time just sufficient to effect the proper clearing only, 

 and mount direct in a perfectly neutral or natural balsam. 



What I have been doing lately is to mount direct from absolute 

 alcohol into natural or neutral balsam (Griibler), a process re- 

 quiring much care. This latter although giving (as far as I am 

 able to say) a permanent staining result, is not nearly so satis- 

 factory regarding clearness of definition, which, however, for the 

 examining of the finest details in connection with this subject of 

 mitosis must be studied in preference to the former. 



Coming to the other important part, the one of observation, of 

 the mitotic figures ; it will be easily observed upon examining the 

 nuclei of many of the above-mentioned cells in their quiescent 

 state, that the nucleoli stand out the most clearly and densely 

 stained, next, the chromatin granules, least of all the linin net- 

 work. 



At a stage very soon after the first appearance of commence- 

 ment of the spireme, the nucleoli take the stain less deeply in the 

 central portion, the periphery remaining unchanged or very slightly 

 affected. 



As the development of the spireme proceeds, the staining power 

 of the whole nucleolus diminishes to some extent, and signs of a 

 granular appearance take place in the periphery, and a much more 

 intimate association with the chromatin granules or thread is 



