REACTIONS OF FIXATIVES WITH TISSUES AND CELLS 71 



revealed in these ways may be hard to trace to their sources. An 

 estimate of the amount of nucleic acids lost by cells on fixation 

 can be obtained by measurement of the optical density of parts of 

 the cell before and after fixation. ^*^ This method depends on the 

 opacity of nucleic acids to ultra-violet light of wave-length 

 265 m[i. A photomicrograph may be taken with this light before 

 and after fixation, and the density of the images compared. 

 Allowance must be made for change in volume of the part 

 measured. Results with cultured fibroblasts from the chick embryo 

 suggest that 4% neutral formaldehyde may reduce the amount of 

 nucleic acids (DNA -j- RNA) in the nucleus by between 10% and 

 35%. It would be interesting to have comparable figures for all 

 primary (unmixed) fixatives. 



In order to investigate fixative as opposed to preservative action, 

 it is necessary to examine cells that have been through the com- 

 plete routine of microtechnique, and to compare these with living 

 cells. Buchsbaum ^* in Chicago has worked in this way with cul- 

 tured amphibian macrophages. He studied them in life by phase- 

 contrast microscopy, and then fixed, washed, dyed, and mounted 

 the same individual cells, making photographic records at every 

 stage. The results showed that the changes caused by fixation 

 were more fundamental, in this particular case, than any that re- 

 sulted from subsequent treatment; but embedding was omitted, 

 and this is often a destructive process. 



All the studies of this kind so far mentioned are open to one 

 major objection. The cells were separate from one another, and the 

 fixative came directly up against them at its full concentration. 

 In ordinary microscopical preparations the superficial cells are 

 often damaged while those lying below the surface are well fixed 

 (p. 28). Unless it so happens that information is particularly 

 required about separate or superficial cells, the test should be 

 designed in such a w^ay that its result does not depend on the 

 response of such cells, and it should involve the whole of the after- 

 treatment that is to be used in practice. Since dyeing does not 

 ordinarily introduce serious distortions, any dye or dyes that will 

 reveal the structure of the object clearly may be used; but for a 

 full test of any fixative it would be necessary to try all the usual 

 embedding and mounting media. 



Ideally each primary (unmixed) fixative should be tried with 

 each embedding and mounting medium, and each fixing/embed- 

 ding/mounting method should be assessed from the point of view 



