FIXATION PROCEDURES 23 



ing preserves most of the proteins and other substances present 

 in the cell in such a normal condition that they readily dissolve 

 in many polar substances. A fixation procedure may be ap- 

 plied after drying and before embedding, or it may be applied 

 to individual sections after embedding in wax. We are at 

 present experimenting with the possibility of fixatives which 

 can be used as vapors to act directly upon the specimen after 

 it has been dried. Gases such as formaldehyde, carbon sub- 

 oxide, hydrochloric acid, and osmic acid have potentialities for 

 such a procedure. Most of our work, however, has involved 

 material which has been embedded and sectioned after freeze- 

 drying. The sections are then flattened either by using a non- 

 aqueous solvent such as alcohol, or by flattening the section on 

 mercury. Then, after the wax has been removed from the sec- 

 tion, the latter can be subjected to the fixing action of alcohol 

 and other anhydrous solutions which may contain acids, bases, 

 heavy metals, etc., as desired. As a result of such procedures, 

 the specimens become sufficiently insoluble in polar solvents. 



It is of interest to calculate the time relationships in freeze- 

 drying and in fixation by chemical agents. Studies of cell per- 

 meability show that when cells 2 microns in thickness are placed 

 in a solution of a solvent such as ethyl alcohol, the concentra- 

 tion of alcohol inside the cell reaches about 50 percent of that 

 of the outside in about 10 seconds. Thus, if individual cells 

 of this size were brought into direct contact with alcohol, the 

 concentration of alcohol would reach about 50 percent in about 

 10 seconds and fixation would become effective in about 10-20 

 seconds. But most of the material with which we deal is in 

 tissue sections which are much thicker than 2 microns. For a 

 thin slice of tissue 2 millimeters in diameter placed in alcohol, 

 the time required for fixation to become effective would be ap- 

 proximately yiOOO times longer, i.e., on the average with this 

 material it would require roughly 330 seconds or more before 

 fixation becomes really effective with a chemical fixative. When 

 more slowly penetrating materials, such as heavy metals, are 

 used as fixatives, the time for effective fixation may be con- 

 siderably increased. 



The time taken for effective fixation to be accomplished by 

 the technique described above, using isopentane, can be roughly 



