12 THE MATURATION OF THE EGG OF THE MOUSE. 



1903). Moreover, in cooperation with Professor Castle, the junior writer 

 obtained in 1904 by the above-described method a litter of three rats, 

 which have been used for breeding purposes in Dr. Castle's experiments. 

 Similar breeding experiments with mice are too few to be of any value ; 

 but eggs of mice artificially inseminated when compared with those of 

 mice naturally impregnated appear normal in every respect. 1 



In all, 149 mice have been artificially inseminated, but as only 85 

 have been studied in detail the rest unfortunately can not be included 

 here. 31 of the 85 have furnished eggs which contained spermatozoa 

 or pronuclei. A further discussion will be found on page 20. 



Only sound mice, white, hybrid, and black, have been used for 

 study. They have been killed at all hours of the day and night during 

 the first 40 hours after parturition. While at first chloroform was used, 

 it was found to be quite as humane and quicker to stun them and then 

 break their necks by pinching them quickly with the thumb and fore- 

 finger just behind the head. 



The ovaries with the oviducts attached were immediately removed 

 and fixed for from 20 to 60 minutes in the following modification of 

 Zenker's fluid : 2 per cent corrosive sublimate, 2 per cent potassium 

 bichromate, 10 per cent glacial acetic acid. The fluid was made up in 

 two separate solutions: (A) 4 per cent bichromate, (B) 4 per cent (aque- 

 ous sol.) sublimate and 20 per cent acetic acid. When desired for use, 

 equal portions of A and B were mixed. After fixation the ovaries and 

 oviducts were washed in several changes of warm water until fairly 

 white, i.e., from 12 to 24 hours; then left in 70 per cent alcohol contain- 

 ing iodine for from 12 to 24 hours; quickly dehydrated, cleared in xylol, 

 and embedded in paraffin. This process gives clear fixation of ovarian 

 eggs without shrinkage of eggs or nuclei and without destroying the 

 finer st.ucture. Various other mixtures, with and without osmic acid, 

 have not given satisfactory results. 



The whole ovary and oviduct were cut into sections 8 micra thick, as 

 thinner sections divide the nuclei and spindles into too many parts. 

 The sections were affixed to slides with albumen, being spread by the 

 water method, and were stained by one or the other of these three methods : 



(1) with iron hematoxylin followed by either Congo red or orange G, 



(2) with Bohmer's hematoxylin and Congo red, or (3) with Mallory's 

 (1905) phosphotungstic-acid hematoxylin. The first gives clear out- 

 lines, but does not show the structure of chromosomes well. Bohmer's 

 dye when used for 24 hours or more gives excellent results. Mallory's, 

 when used in just the right way, is the best of any of the stains tried. 

 The method employed for Mallory's stain was as follows: From water 

 the sections were put into a constantly agitated solution of 0.25 per cent 



1 Since the above was written the junior writer has obtained two litters of 

 perfectly healthy mice by artificial insemination performed about 24 and 30 hours, 

 respectively, after parturition. 



