266 CHROMATIN, ANIMAL CHROMOSOMES, NUCLEOLI 



stain (§ 623) gives excellent results and should be more generally 

 employed. And there are. of course, many other nuclear stains 

 which are satisfactory when skilfully used. 



628. Fixation of Mammalian Chromosomes. The material 

 studied is generally either the testis of the adult or tissue taken 

 from a very young embryo (the amnion gives excellent figures). 

 In either case it is absolutely essential that fresh material be used, 

 since even a delay of one or two minutes may cause a clumping 

 of the chroinosomes. 



If the testis is to be preserved, no anaesthetic should be used. 

 Either the animal is castrated quickly, or it can be stunned or 

 killed by a blow on the head and the testis quickly removed. Some 

 workers prefer to slice the testis into thin layers a millimetre or 

 two thick and preserve these, others cut the testis into small 

 pieces and then tease these directly in the fixing agent. Experience 

 has indicated (see, for example, Winiwarter and Oguma, Arch, 

 d. Biol., xxxvi, 1926, p. 102) that it is inadvisible to place 

 mammalian testis in physiological salt, or in Ringer's solution, 

 in order to separate the tubules of the testis. Mammals vary 

 greatly in the ease with which the tubules may be separated, the 

 mouse and rat and some other rodents being the least difficult 

 in this respect. In working with germinal tissue, due regard 

 must be given to the breeding season of the form and only healthy 

 and properly nourished animals should be used. 



When preserving embryos, if the amnion is to be studied, this is 

 simply exposed unbroken and the embryo is dropped into the pre- 

 serving fluid. After washing, small sections of the amnion are stained 

 with iron haematoxyhn and mounted in toto in damar or balsam, after 

 clearing with one of the vegetable oils (oil of cedar is excellent). 



If one wishes to study dividing cells in mesodermal or nervous tissue 

 (in vertebrates the former is better),, it is well to chop up the embryo 

 before preservation because good chromosome plates are not found, as 

 a rule, more than a few cell layers from the surface of the tissue. 



Three general methods have been employed for the fixation of 

 mammalian chromosomes, all of which have given excellent 

 figures. 



H. DE Winiwarter {Arch. d. Biol., xxvii, 1912, p. 91) and 

 some others have used Flemming's solution, in which the amount 

 of acetic acid has been reduced. Hance {Anat. Rec, xii, 1917, 

 p. 371) adds a little urea (about 0-5 per cent.) to Flemming's 

 solution, which is chilled on ice. The tissue is placed in this cold 

 fluid and kept there for about twenty-four hours. (The tempera- 

 ture of the fixative on ice registers about 4° to 5° C.) More 

 recently, Oguma and Kihara {Arch. d. Biol., xxxiii, 1923, p. 493) 

 have fixed thin slices of human testis for one minute in Carnoy's 

 solution (the 6:3:1 mixture), and then transferred them to 

 strong Flemming's solution for twenty-four hours. 



