INTRODUCTION TO FIXATION 29 



and mounted in Canada balsam, their volume is only 34% of 

 what it was when they were alive; yet formaldehyde is superior 

 to the six other primary fixatives in this respect. Ethanol is the 

 worst of all: the final volume is only 19% of the original. 



The addition of 'indiff'erent' or non-fixative salts to fixatives 

 often improves the results obtained. A saline solution may be 

 prepared, having about the same osmotic pressure as the body- 

 fluids of the organism from which tissue is to be taken, and the 

 fixative substance dissolved in this instead of distilled water. It 

 follows that the complete solution is hypertonic. This would be 

 thought likely to cause great shrinkage of the cells, but in fact 

 shrinkage is reduced by the presence of the indifferent salt, and 

 indeed that is precisely why it is used. The way in which it acts is 

 obscure. ^^ The use of an indifferent salt is particularly helpful 

 when the fixative is chromium trioxide or formaldehyde. 



To make a general study of the artifacts caused by fixation, 

 separate cells are often examined in a suitable saline solution 

 while still alive, and a fixative is then allowed to run under the 

 coverslip. The immediate effects are thus easily observed. The 

 classical work of this kind was done by dark-ground micro- 

 scopy.^" The introduction of phase-contrast microscopy resulted 

 in a renewal of interest in work of this kind.'*'' ^^' ^^' The method 

 is valuable but open to two objections. First, it is clearly a test 

 of preservation, not of fixation. Secondly, it shows only how 

 fixatives affect cells that come into immediate contact with them. 

 Many valuable fixatives damage the most superficial cells of a 

 piece of tissue but give good results at a little depth below the 

 surface. When separate cells are examined, they all react like 

 superficial cells, and the fixative may be wrongly condemned. 



To overcome these objections, small pieces of suitable tissue 

 may be fixed, embedded, sectioned, dyed, and mounted, and the 

 resulting preparation carefully compared with what can be seen 

 in living cells from the same material. It is best to choose tissues 

 that are readily available but difficult to fix well. The testes of 

 various animals (mammals, urodeles, insects) are suitable. ^°' ^^ 

 Embedding in paraffin is particularly likely to cause distortion, 

 and this medium should therefore be chosen for a rigorous test. 



