FIXATION PROCEDURES 25 



perature falls in the specimen, that significant diffusion cannot 

 occur after liquid-air temperatures have been reached, even 

 with a rapidly diffusing substance such as ethyl alcohol. 



It thus appears that freeze-drying, or rather freezing in cool 

 isopentane, produces an effective fixation in a time which is of 

 the order of 1 percent of the time required for the effective 

 action of a chemical fixative, when a specimen of about 2 milli- 

 meters in thickness is considered. In making these calculations, 

 it has been assumed that the fixative moves into the specimen 

 by thermal diffusion only. In some cases the rate of penetra- 

 tion of a chemical fixing agent may be somewhat increased or 

 reduced by shrinking, cracking, or swelling of the specimen. 

 But it is highly improbable that the time for effective fixation 

 by chemical substances falls much below 100 times that required 

 for effective fixation by cooling, even with the maximum rate of 

 penetration which may be achieved when an accidental flow of 

 fixative is procured by cracking. 



In theory it is possible that serious diffusion artefacts may 

 develop during fixation even with the freeze-drying technique. 

 This is certain to be the case with small molecules and small 

 ions, and it is improbable with substances of high molecular 

 weight. Substances of low molecular weight tend to be uni- 

 formly distributed in cells by thermal agitation. Opposed to 

 the effect of thermal agitation are adsorption and other more 

 complicated activities of the cell. The normal distribution of 

 a low-molecular-weight substance represents a compromise be- 

 tween these tendencies. As the temperature falls the dispersing 

 effect of thermal agitation declines correspondingly and the 

 relative importance of adsorption forces increases; since the 

 diffusion time for small molecules is bound to be small com- 

 pared with the cooling time, this abnormal dominance of ab- 

 sorption forces must inevitably produce redistribution of small 

 molecules. Diffusion artefacts of this type cannot be avoided. 

 On the other hand, with high-molecular-weight molecules ad- 

 sorption is normally much greater than for small molecules, and 

 the diffusion time is much larger. Consequently, with freeze- 

 drying, diffusion artefacts are not likely to occur with molecules 

 of high molecular weight. But with chemical fixation the bal- 

 ance is thrown heavily in favour of diffusion artefacts in two 

 ways. First, the action time of chemical fixation is about a 



