Re-appearance of the Nucleolus. By E. J. Sheppard. 599 



mander that I have worked upon. I do not think that sections of 

 plants, rootlets, etc., require nearly such long baths ; 12 to 24 hours 

 in my opinion is plenty. These periods of immersion can and 

 should be modified if fixatives other than those mentioned or their 

 like be used. 



A point with regard to the differentiation process which should 

 not be forgotten, is that the strength of the iron solution for this 

 purpose should be weak ; i.e., whatever be the strength of the 

 mordanting solution used (a 3 p.c. I find about the best), it should 

 be diluted five to ten times its volume with water, according to 

 the rapidity it has for extracting the hematoxylin ; this can be 

 easily ascertained by working a test section. 



The extraction should not be too slow nor too quick : roughly 

 from 15 to 20 minutes should be occupied in differentiating a sec- 

 tion or series fixed to the slide. Vegetable sections will take 

 slightly less time than this. 



Not too much importance, however, must be put upon timing 

 results in this all-important part of the process, as the only really 

 reliable way is to repeatedly stop the process by washing for about 

 half a minute and very carefully examine under the Microscope 

 with the aid of a £-in. lens until you are satisfied the best point 

 has been reached. 



All other treatment remains the same, as previously described 

 in my other paper. 



Now regarding my observations made with a view to ascer- 

 taining how and when the nucleolus makes its re-appearance ; the 

 Master stage is the one which calls for most careful study and 

 observation. 



The first thing to be noticed is, that soon after the formation of 

 the diaster and the arrival of daughter-chromosomes at or near the 

 poles, the chromosomes begin to link up to form the dispirem. 



At or about this time, and before the diasters have lost very 

 much of their characteristic shape, a looping in the chromatin may 

 be observed as shown (Plate XIX. figs. E and B.). The loops 

 may be one or more in number in each half of the diaster, i.e. 

 in each future daughter-nucleus. 



It is in these loops that the nucleoli are at some time going to 

 make their appearance. 



It does not always follow that these loops are apparent at one 

 and the same time in each half of the diaster, and I am not pre- 

 pared to say that the number of loops that may be seen is indicative 

 of the number of nucleoli that are going to be present in each 

 daughter -nucleus, for there often appear more loops than there are 

 likely to be nucleoli present. 



From the first appearance of the loops the whole area of 

 inclusion is stained, but with varying degrees of density ; this may 

 be influenced to some extent by the compactness of the chro- 



