46 FIXATION 



increased. It is obvious that fixation cannot involve increase in 

 solubility, and there are therefore many 'denaturing agents', such 

 as urea, that could not possibly be fixatives. It is easier to study 

 denaturation, however, if the product be soluble; and for this 

 reason much of our knowledge of the process, admirably sum- 

 marized in several reviews, ^^' ^^' ^^^' *^^ is not directly applicable 

 to the problems of microtechnique. For similar reasons most of the 

 study of denaturation has been devoted to proteins in the form of 

 sols, yet gels are more interesting to the biologist, because proto- 

 plasm is essentially a soft gel; and gelled proteins can also be 

 denatured. 



We may broadly distinguish additive from non-additive fixation. 

 In the former, the fixative molecule or a considerable part of it 

 adds itself to the protein; in the latter it does not. The word 'de- 

 naturation' was formerly held to imply that the change involved 

 was non-additive, but this usage is not quite general today. It has 

 even been said that any chemical change in a protein, not involving 

 disintegration into amino-acids, is denaturation.^^^ In this book, 

 however, the word will be used to mean a non-additive change in 

 a protein, causing it to become less capable of remaining in 

 intimate relation with water as a sol or gel, and more reactive. The 

 resulting loss of solubility ordinarily manifests itself in coagulation, 

 if the protein be a sol ; a gel is rendered harder and opaque. 



Polypeptides, in the sense of short chains of amino-acids, cannot 

 be denatured: the process occurs with the very long chains of 

 amino-acids that constitute proteins. 



The chief non-additive or denaturing fixatives are these: 

 methanol, ethanol, acetone, nitric acid, hydrochloric acid. 



If a protein sol be mixed with a denaturing fixative, the reaction 

 is usually so quick that coagulation appears to be instantaneous. 

 Careful experiment has shown, however, that there are in fact 

 three stages. First, reactivity is increased; then flocculation 

 follows, but the flocculus is soluble in weak acids or alkalis; 

 finally the flocculus hardens into a coagulum, only soluble by 

 proteolysis. Some so-called denaturing agents, such as urea, only 

 cause the first change; and it is for that reason that they are not 

 usable as fixatives. 



Denaturation may be brought about in many different w^ays; for 

 instance, by subjection to very high pressure, extension in ex- 

 tremely thin films, or exposure to ultrasonic waves or ultra-violet 



