CHROMATIN, ANIMAL CHROMOSOMES, NUCLEOLI 263 



If it were possible to expose naked chromosomes directly to the 

 action of fixatives, we could probably develop one fluid which 

 would preserve all types of chromosomes well, at a given phase of 

 mitosis or meiosis. But in practice, the intervening cellular 

 structures, which must be penetrated before the chromosomes are 

 affected, force us to greatly modify our technique for various types 

 of animal and plant tissue. While practically all of the nuclear 

 fixatives have been developed empirically, and we know very 

 little regarding either the chemistry or physics of fixation, within 

 the past decade we have gained some insight into a few factors 

 which affect the penetration of reagents, and the effect of hydrogen 

 ion concentration. And a brief summary of the facts may serve 

 to explain, to the reader, why we have in use, at the present time, 

 a number of fixatives which contain the same ingredients and 

 differ only slightly in the proportions of the reagents. 



ZiRKLE (Protoplasma , iv, 1928, p. 201) has shown that the fixation 

 image is largely determined by the reagent which first reaches the cell 

 structure. As an example, tissue fixed in a mixtire of formol and acetic 

 acid may give the " acid " fixation image in the cortical layers, where 

 no mitoclionchia will be found, while deeper within, the mitochondria 

 (fixed first by the formalin) show with appropriate stains. The penetra- 

 tion of a given reagent is dependent upon its concentration (Crozier, 

 Joiir. Gen. Physiol., v, 1922, p. 65). Different tissues may affect the 

 concentration of a reagent in different ways, not only by the physical 

 density of cell parts, and by simple dilution, but also by some sort of 

 chemical union between the tissue and the reagent. In these respects 

 no two fixative agents may be just alike. Another factor which may 

 greatly disturb the normal fixation image is the nature of the substances 

 which are dissolved by the fixative. Thus, Zirkle has shown that the 

 tannic acid present in some plant tissues may affect the action of 

 reagents to a marked degree. 



Zirkle has made a number of studies on the effect of hydrogen ion 

 concentration on fixation. Using the same ingredients but changing 

 the pU of the fluids, may give the most diverse fixation images. See 

 Zirkle, Protoplasma, x, 1934, p. 31, for literature. 



In the light of the foregoing discussion it may be said that there 

 are two cardinal points in making preparation for chromosome 

 studies. The first is that the material to be preserved must be 

 absolutely fresh, a condition especially important for animal 

 tissues, where a few minutes' delay may profoundly affect the 

 whole nuclear structure before the fixative is applied. The 

 second is that, no matter what fixation is employed, the cells to be 

 fixed must be brought into as close contact with the fixative as 

 possible. One well fixed cell is, in general, more useful than 

 thousands which are not, and it is a waste of time to preserve 

 large hunks of tissue for chromosome study. In dense tissues, 

 such as the testes of many vertebrates, it is rare to find well 

 preserved chromosomes more than four or five cell layers from the 

 surface. Other things being equal, the more finely divided the 



