THE PARAFFIN METHOD 115 



gauze. With shears, cut a piece about 8 cm. square; bend it into a 

 cyhnder; solder along the edge; and solder in a circular piece for a bot- 

 tom. Stand several of these in a dish about 6 cm. deep; let a gentle 

 stream of water come in at the bottom, and wash for 24 hours. 



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 solu- 

 tion. 



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 en- 

 tirely for these purposes. It completes the hardening and at the same 

 time dehydrates, that is, it replaces the water in the material, an ex- 

 tremely important consideration, for the least trace of moisture inter- 

 feres 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. Twenty 

 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 protoplast 

 from the cell wall — was avoided, the series was thought to be suffi- 

 ciently 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: 2|, 5, 7|, 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 2| per cent. It will be noted that the series begins 

 with very close grades and that the intervals are gradually in- 

 creased. The claim is that, by beginning with very weak alcohols in 

 close grades, more perfect dehydration can be secured at the end of 

 the series. Various devices, like constant drip and osmotic apparatus, 

 have been proposed to secure a more gradual transfer; but these have 



