Journal of Applied Microscopy. 333 



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

 extremely important consideration, for the least trace of moisture, a trace so 

 slight as to be almost imaginary, is nevertheless sufficient to make a preparation 

 poor or indifferent when it might have been excellent. 



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. Therefore transfer 

 from water to 35 per cent, alcohol, which should act for six to twenty-four hours. 

 Then use 50 per cent, for a similar period. Material may now be placed in 

 70 per cent, alcohol, where it may remain until ready for use, since 70 per cent, 

 alcohol is a good preservative. Various devices, like constant drips and osmotic 

 apparatus, have been proposed to secure a more gradual transfer. Whether these 

 have any real advantages still remains to be proved. The writer has taken well 

 fixed fern prothallia through the series 35 per cent., 50 per cent., 70 per cent., 

 without the slightest plasmolysis. Such things as fern prothallia, filamentous 

 algai, etc., can be watched under the microscope as the transfer is made, and 

 if plasmolysis results the series of alcohols may be made closer, e. g., 10 percent., 

 20 per cent., 30 per cent., etc. It is said that material left for some time in 70 

 per cent, alcohol will shrink in spite of good killing and fixing, and it is also 

 claimed that its capacity for staining is diminished. Some recommend that glyc- 

 erine be added to the alcohol, others prefer to complete the dehydrating process 

 and leave the material in an essential oil, wdiile still others would imbed it and 

 keep it in paraffin. The last is doubtless best of all, but requires such an 

 immense amount of labor that it is impracticable for general purposes. Nearly 

 all of our own material, which is not needed for immediate use, is in 70 per cent, 

 alcohol, unless, of course, the material has been put into formalin or some such 

 reagent which kills, fixes, and preserves all at once. 



After the 70 per cent, alcohol, use 85 per cent., 95 per cent., and 100 per 

 cent, successively, allowing six to twenty-four hours for each. The 70 per cent, 

 would probably complete all the hardening which is necessary, but the other 

 three must be used to complete the removal of water. 



Up to this point the processes are exactly the same, whether the material is to 

 be imbedded in paraffin or celloidin. 



4. Clearing. — Let us suppose that the material has been thoroughly dehy- 

 drated so that not the slightest trace of water remains. If the supposition 

 chances to be contrary to fact, all the work which has preceded, as well as 

 all which is to follow, is only an idle waste of time. The purpose of a clearing 

 agent is to make the tissues transparent, but clearing agents also replace the 

 alcohol. At this stage the latter process is the essential one, the clearing which 

 accompanies it being incidental. The clearing, however, is very convenient, since 

 it shows that the alcohol has been replaced and that the material is ready for the 

 next step. 



Various clearing agents are in use. Xylol is the most generally employed, 

 and for most purposes it seems to be the best. Bergamot oil, cedar oil, clove 

 oil, turpentine, and chloroform are all necessary for special purposes. 



