106 Methods in Plant Histology 



gradually increased. 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 it is very doubtful whether these possess any real advan- 

 tages. It is not necessary to use a large amount of alcohol: 2 or 3 

 times the volume of the material is sufficient. 



The grades of alcohol may be used several times, but it must be 

 remembered that pollen grains, fungus spores, starch grains, and vari- 

 ous granules are likely to be left in the alcohol, so that, when it is 

 necessary to know the identity of every such structure, only pure 

 alcohols should be used. 



As the alcohols absorb water from the material, they become 

 weaker and weaker. If the various alcohols be poured in a large 

 " waste alcohol" bottle, when a couple of liters has been accumulated, 

 the strength may be determined by testing with an alcoholometer. 

 Then any grade of less strength can be made from this stock. 



The time necessary for each of the stages has not been determined 

 with any certainty. About 4 hours seems to be long enough for 

 each of the grades from 2J to 70 per cent; for 70, 85, and 95, about 

 10 hours each; for absolute alcohol, 12 to 24 hours, changing two 

 or three times. If material is to be kept in alcohol, leave it in 85 per 

 cent, but where labor is no object, it is better to go on and imbed 

 it in paraffin. 



CLEARING 



Let us suppose that the material has been thoroughly dehydrated, 

 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. 



