116 METHODS IN PLANT HISTOLOGY 



no advantages, unless the mixture is very complete before it reaches 

 the material. If the drops fall near the material, the liquid is in a con- 

 stant turmoil. 



In passing through the graded series, 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 it is wise to pour 

 back through a filter each time, thus keeping the alcohols clean. 



As the alcohols absorb water from the material, they become weaker 

 and weaker. If the various alcohols be poured in a large "waste alco- 

 hol" bottle, when a couple of liters have 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. We recommend three grades a day, morning, 

 noon, and evening, for the first six grades; for the 30, 40, and 50, 

 change twice a day, morning and evening; 85, at least 24 hours and 

 better 48 hours, for this is the best grade in which to complete the 

 hardening which will make the material able to withstand the subse- 

 quent processes; 95, overnight or 24 hours; absolute, 24 hours, chang- 

 ing two or three times. Material may be left in any of the grades over- 

 night, or 24 hours. If it 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. 



Various clearing agents are in use. Xylol is the most generally em- 

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

 cedar oil, clove oil, turpentine, and chloroform are used for the same 



