Method IV 33 



The dehydrating, clearing, and imbedding should be done as 

 quickly as possible. 



3. Dehydrate by transferring the pieces from the washing 

 fluid to 10-12 volumes of 70 per cent, alcohol for 15 minutes. 



Replace 70 per cent, with 95 per cent, alcohol, 20 minutes. 

 Replace 95 per cent, with an equal volume of absolute alcohol 

 for 30-40 minutes. 



Agitate frequently in each grade. 



4. Clear with chloroform. The small pieces of tissue should 

 remain in 10-15 volumes of chloroform for 30-50 minutes. 

 Usually the tissue is cleared soon after it sinks to the bottom of 

 the vessel. 



5. Imbed in paraffin, {a) Transfer from pure chloroform to 

 4-5 volumes of a mixture of equal parts chloroform and hard 

 paraffin (melting point about 58° C.) for 20-30 minutes. 



((^) The tissue may now be removed from the dark -box and 

 transferred to melted hard paraffin in the paraffin bath-pan, 

 where it must remain 30-40 minutes. 



(c) Imbed and cool paraffin as in I, 4, (c) and (d), p. 16. 



6. Make sections 10-15 /* or thinner, according to the tissue 

 and the object in view. For the study of neurofibrils in large 

 nerve-cells the sections should be thick. For section-cutting 

 see I, 5, p. 17. 



7. Fasten sections to the slide by the albumen water method 

 (I, 6, p. 17). Use as little albumen fixative as possible, since 

 ■the film has a tendency to stain. 



8. Dissolve off parafifin by placing the slide in chloroform for 

 10-15 minutes. 



9. Transfer from chloroform to absolute alcohol for ib 

 minutes. 



10. From absolute alcohol the slides are passed successively 

 through the decreasing grades of alcohol (95 per cent., 70 per 

 cent., and 50 per cent.), about 3 minutes in each, down to distilled 

 water. In the water it is recommended that the slides either 

 remain for at least 6 hours, or that they be simply well rinsed (2 

 or 3 minutes) in the water. 



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