FIXATION PROCEDURES 19 



the reaction-product concentration and the concentrations of 

 activators and inhibitors in the immediate vicinity of the 

 enzyme. With the exception of one or two substances, such as 

 cytochrome oxidase and muscle hemoglobin, it must be ad- 

 mitted that we are not as yet in a position to determine the exact 

 degree of activity of any enzyme in a particular site in the cell. 



The use of chemical substances to effect fixation has been 

 disadvantageous in cytochemical work, from a purely chemical 

 point of view. It also has many disadvantages in that it is ex- 

 tremely difficult to detect artefacts due to diffusion and adsorp- 

 tion. In view of this, there can be no doubt but that the 

 method of choice is freeze-drying. However, it must be empha- 

 sized that, unless freeze-drying is carried out with rigid atten- 

 tion to detail, the artefacts which may arise can be even more 

 serious than those found with chemical fixation. The method 

 of freeze-drying was introduced by Altmann in 1890, and 

 greatly developed by Gersh. The principle involved is to so- 

 lidify the material by plunging a small piece into a fluid at a 

 very low temperature. The material is then placed in a 

 chamber which is evacuated, and the water is then pumped off 

 while the specimen is kept frozen solid. When all the water has 

 been removed, the specimen may be embedded in wax and sub- 

 jected to sectioning, etc. 



The apparatus which has been used in the past for this pur- 

 pose has suffered from a number of deficiencies. It has been 

 delicate, containing a great deal of glass tubing; it has not been 

 easily possible to process considerable quantities of material; 

 mechanical refrigeration has considerably restricted the range 

 of drying temperatures which can be used and also is frequently 

 unreliable; and, not least, must be mentioned the fact that such 

 apparatus is expensive to build or buy. Mr. L. G. Bell and I 

 have therefore developed, in collaboration with W. Edwards 

 and Co., a freeze-drying apparatus which is sufficiently robust 

 and inexpensive to be suitable for routine work in almost any 

 laboratory. Figure 1 illustrates the principle of the drying unit. 

 The procedure is to fix the specimen by plunging it into isopen- 

 tane which has been cooled in liquid nitrogen. It is desirable 

 to avoid liquid air as a cooling agent, since, if for any reason 

 the isopentane and liquid air become mixed, a violent explosion 

 will ensue. It is necessary to use intermediate cooling agents 



