FIXING AND HARDENING ■ 23 



be artifacts. In the past, to these objections, the answer has 

 been made that with a wide variety of agents we obtain essentially 

 the same fixation image and this constancy must rest on a physical 

 basis, whether or not there is an optical differentiation in the 

 living tissue. To this may now be added some observations of the 

 late Dr. Belar. He showed that in the spermatocytes of the 

 grasshopper, Stenobothrus , the chromosomes of the living cells 

 are visible at all stages of meiosis, while in another species, 

 Dissoteira, the nucleus appears empty and the chromosomes are 

 first seen after fixation {Protoplasma, ix, 1930, p. 209). In the 

 paper cited, and in many others, Belar gives photomicrographs of 

 the same cells, in the living state, with the chromosomes clearly 

 visible, after fixation, when the chromosomes may still be seen 

 clearly, and after fixation and staining. A comparison of these 

 figures will convince anyone that after fixation and staining the 

 condensed chromosomes have essentially the same form as in the 

 living state. It seems safe to say, therefore, that as far as such 

 gross morphological details are concerned, our nuclear fixatives 

 may be relied on to give valid images of the living clu"omosomes. 

 It should be pointed out, however, that for the finer details of 

 nuclear organisation, for which the living material gives us little 

 evidence, we cannot be so certain of our grounds. 



From the practical standpoint, checking fixation images in 

 living material is often difficult, and the method is limited in its 

 application, and it may be asked by what criterion may we judge 

 the quality of fixation. Unfortunately, there is no objective 

 standard recognised and the matter becomes subjective. The 

 careful investigator will try out a number of fixatives on new 

 material, in order to eliminate any features which result from the 

 use of a single reagent. 



34. The Practice of Fixation. See that the structures are perfectly 

 living at the instant of fixation, otherwise you will 07ily fix pathological 

 states or post-inortem states. 



Some observers have made special observations on the effect of delay 

 in fixation ; J. Thornton Carter {Phil. Trans. Roy. Sac, Series B, 

 vol. ccviii, 1917) has made some interesting experiments on the finely 

 granular ameloblasts in the developing teeth of the pike. He noticed 

 that the cytoplasm gave evidence of marked changes unless fixed within 

 three minutes of " death " ; these changes were manifested by the 

 behaviour of the cytoplasmic granules to stains ; the selectivity of the 

 latter was progressively altered as the rapid post-mortem changes were 

 set in action. 



Fixation is generally performed by immersion of the objects in 

 the fixing liquid. In this case, everything should be done to 

 facilitate the rapid penetration of the fixing agent. To this end 

 let the structures be divided into the smallest portions that can 

 conveniently be employed, and if entire organs or organisms are 



