20 FIXATION 



It is necessary at the outset to distinguish between preservation 

 and fixation. 



A small piece of tissue that has been cut out of an organism will 

 generally cease to retain its structure at the microscopical level 

 quite soon, unless special precautions are taken to keep the cells 

 alive. The main potential causes of damage are evaporation, 

 osmotic sw^elling or shrinkage, attack by bacteria or moulds, and 

 autolysis. 



Autolysis is the self-digestion of cells by enzymes that are 

 always present wdthin them and presumably synthesize proto- 

 plasmic proteins during life. On the cessation of normal vital 

 activity their action is reversed, at any rate in the sum-total of its 

 effect. These enzymes are known collectively as kathepsin. Two 

 of them are proteinases, similar to pepsin and trypsin: these shorten 

 the protein chains to peptides. Two others are an aminopeptidase 

 and a carboxypeptidase, capable of pulling the terminal amino- 

 acids off the newly-exposed ends of the peptide molecules. As a 

 result there is a general dissolution of the proteins, which are 

 rendered no longer capable of coagulation by heat or chem- 

 ical agents. The rate of autolysis can be measured by finding 

 what proportion of the protein has become non-coagulable in 



a given time. The process is slow at 6° C but still occurs even 

 ato°.i«2 



To preserve a piece of tissue, then, it is necessary to place it 

 in a fluid that will neither shrink nor swell it, nor dissolve or 

 distort its constituent parts; will kill bacteria and moulds; and 

 will render kathepsin inactive. A fluid that will do this is a pre- 

 servative. 



It would be very convenient to have fluids in which separate 

 cells or teased fragments of tissues might be indefinitely pre- 

 served and in which they could be examined microscopically at any 

 time. Although it has been customary for centuries to preserve 

 organisms and their parts in suitable fluids for subsequent study 

 with the naked eye or hand-lens, not many cytologists or histo- 

 logists have sought to elaborate fluids of this kind that would serve 

 their needs. The best-known of the few such fluids that exist are 

 Petit's (usually know^n as the fluid of Ripart and Petit ^^^) and 

 Amann's,^' '^ especially the latter's lactophenol. Amann called his 

 fluids Beohachtiingsmedien, to indicate that they not only prevented 

 the decay of tissues, but were media in which cells might remain 

 during microscopical examination. It is desirable that research 



