CHROMATIN, ANIMAL CHROMOSOMES, NUCLEOLI 265 



chromo-formol-osmic-acetic mixtures, which have become standard 

 for specific types of tissues or for a given stage of mitosis or 

 meiosis. Witli regard to the cliromosomes it must be realised 

 that they are comjjlex structures which vary both in their 

 physical and chemical state in the several phases of mitosis, 

 and a fixative which, for example, gives very satisfactory 

 metaphase plates may preserve the more disperse phases very 

 poorly. In selecting the method to be followed, some thought 

 should be given to the type of study it is desired to make. When 

 the counting of chromosomes is a primary consideration, a 

 certain amount of shrinkage is not detrimental and may be a 

 positive advantage, when high numbers are dealt with. For 

 such studies picro-formol-acetic, or chromo-aceto-osmic mixtures 

 should be tried first, or if unavoidably dense membranes must be 

 penetrated, such as a thin layer of chitin, then Carnoy's fluid 

 or some one of the mercuric fixatives such as Gilson's or 

 Petrunkevitch's is recommended. But when one wishes to study 

 the internal structure of chromosomes, shrinkage is to be avoided, 

 as far as possible, and certain pre-fixation treatments may be 

 extremely valuable. In the sections following the reader will find 

 detailed information about the various methods which are con- 

 sidered, by the writers, as the best for a particular type of tissue 

 or phase of mitosis for animals and plants. 



626. Hot and Cold Fixation. Nuclear fixatives are commonly 

 employed at room temperature, but some workers advocate the 

 use of warm or hot fluids and others believe the best results are 

 obtained by keeping the vial of fixative (previously chilled by ice) 

 on ice while the material is being fixed. Cold Flemming's solution 

 is recommended by Ezra Allen {Anat. Rec, x, 1915, p. 16), and 

 Hance {Anat. Rec, xii, 1917, p. 371) for the preservation of 

 mammalian chromosomes. The fixative which is generallv used 

 warm, at 37°-38° C, is Allen's modification of Bouin's fluid. 

 When hot fixatives are used, the period of fixation is reduced. It 

 is possible that the chilling alters the physical state of the proto- 

 plasm, allowing the fixative to penetrate before the chromosomes 

 have clumped, and that heat accelerates the penetration of the 

 reagents. In any event, we feel that the matter will repay 

 investigation and that cytologists will do well to try both hot and 

 cold methods. 



627. Stains. The iron alum haematoxylin method of Heidenhain 

 has always been and is now the chromosome stain par 

 excellence. Most cytologists, perhaps, prefer the short method, 

 i.e., mordanting from a few minutes up to an hour or two and 

 staining a correspondingly short time. At present, possibly the 

 next most popular stain is gentian violet, especially the method 

 of Newton (§ 1364) or of La Cour (§ 1364), but it should be noted 

 that this stain will fade after certain types of fixation. Feulgen's 



