The Paraffin Method 105 



are washed out with water; aqueous solutions of corrosive sublimate 

 are also washed out with water; but alcoholic solutions should be 

 washed out with alcohol of about the same strength as the fixing 

 agent; picric acid, or fixing agents with picric acid as an ingredient, 

 must not be washed out with water, but with alcohol, whether the 

 picric acid be in aqueous or alcoholic solution. When washing with 

 water, running water is best, and where this is not convenient the 

 water should at least be changed frequently. The washing-out 

 process usually requires about 24 hours. 



HARDENING AND DEHYDRATING 



After the material has been washed, it is necessary to continue 

 the hardening and also to remove the water. Alcohol is used almost 

 entirely for these purposes. It completes the hardening and at the 

 same time dehydrates, that is, it replaces the water in the material, 

 an extremely important consideration, for the least trace of moisture 

 is likely to interfere seriously with the infiltration of the paraffin. 



The process of hardening and dehydrating must be gradual; 

 if the material should be transferred directly from water to absolute 

 alcohol, the hardening and dehydrating would be brought about 

 in a very short time, but the violent osmosis would cause a ruinous 

 contraction of the more delicate parts. In recent years, cytologists 

 have been making the dehydration process more and more gradual. 

 Ten years ago most workers began the dehydration process with 

 35 per cent alcohol and used the series 35, 50, 70, 85, 95, and 100 per 

 cent alcohol. Some placed an intermediate grade between water and 

 35 per cent alcohol. If plasmolysis the tearing away of the proto- 

 plast from the cell wall was avoided, the series was thought to be 

 sufficiently gradual; but a series which may avoid plasmolysis may 

 not be adequate if one is to study the finer details of cell structure. 

 The following series is recommended: 1\ , 5, 7J, 10, 15, 20, 30, 40, 50, 

 70, 85, 95, and 100 per cent. There is no particular virtue in the 

 fractions: it is convenient to make 10 per cent alcohol, dilute with 

 an equal volume of water for the 5 per cent, and dilute the 5 per cent 

 with an equal volume for the 2J per cent. It will be noted that the 

 series begins with very close grades and that the intervals are 



