26 FIXATION PROCEDURES 



hundred or more times longer than with freezing fixation, so 

 that artefacts have much longer to develop. Secondly, chemical 

 fixatives are inherently rather violent reagents and inevitably 

 break up most of the adsorption systems of cells, and so they 

 temporarily render many molecules more diffusible than is 

 normal. Consequently, diffusion artefacts must be expected, 

 even with large molecules, when chemical fixation is used. 



It cannot be too strongly emphasized that, although freeze- 

 drying presents such a great advantage over chemical fixation, 

 artefacts may nevertheless arise with some types of molecules 

 during the fixation process. Also small molecules such as in- 

 organic ions and sugars may diffuse to a significant degree in the 

 wax used for embedding. This must be a subject for future 

 investigation. 



Until the degree of diffusion during fixation and infiltration 

 is ascertained, it will not be possible to tell whether the locali- 

 zation of small molecules is reliable, even when freeze-drying, 

 followed by embedding in wax, is used as the basis of technique. 

 Thus most of the results obtained by microincineration have in- 

 volved specimens infiltrated in wax. Whether the distribution 

 of sodium, potassium, etc., found by this technique is correct 

 cannot be determined until it is known what artefacts develop 

 during fixation, infiltration, and embedding in wax. 



Dr. L. G. Bell has also found that artefacts may arise when 

 gels are subjected to the freeze-drying technique. Although 

 one knows that, when examined with a microscope, a gelatin gel 

 is homogeneous, a section of gelatin gel prepared by the freeze- 

 drying technique is not homogeneous. It appears that when 

 weak gels are dried and forced into wax, cracking and contrac- 

 tion occur so as to give rise to a somewhat fibrous material. The 

 fibres are, however, artefacts. 



From time to time it has been questioned whether the fact 

 that, after a specimen has been dried, it must normally be sub- 

 jected to a fixing agent does not deprive one of a great deal of 

 the advantage which is gained from the great speed with which 

 diffusion is restricted by cooling at room temperatures. How- 

 ever, there is probably very little loss in efficiency. Once a 

 specimen has been effectively dehydrated, most of the molecules 

 of molecular weight greater than, say, 1000, are bound into a 

 solid unit which will break down only if the specimen is passed 

 into a polar solvent; e.g., the protein molecules and the nucleic 



