656 LABORATORY EXERCISES 



over ether. The ether vapor quickly dissolves the celloidin to cause the sections 

 to adhere firmly to the slide on removal from the chamber. After the removal of 

 the celloidin, the sections can be stained with appropriate stains. For mounting 

 in Canada balsam, celloidin sections may be cleared with a mixture of 3 parts xylol 

 and I part phenol. 



Parafin Method. — ^The fixing and dehydrating of material for imbedding in 

 paraffin is performed in a manner similar to that for work with celloidin up to the 

 dehydration in absolute alcohol. The following schedule should be followed 

 subsequently. 



Transfer from absolute alcohol to pure xylol, allowing at least two hours in each 

 of the following three mixtures. ?^ alcohol + J^ xylol; H alcohol + J^ xylol; 

 5:4 xylol + yi alcohol, xylol. Add to the mixture of paraffin dissolved cold in xylol. 

 Place in melted paraffin in the bath, kept at SS°C., two to twenty-four hours as 

 convenient. Imbed in paper capsules, or in small shallow glass dishes. Section 

 with rotary microtome; about 6 to 10^ is a good thickness. 



See Lesson 43 for details of cutting frozen section by the microtome and the 

 method of freezing each section. Lesson 43 may be introduced here. 



Fastening of Sections to Slide. — After cutting, fasten section to slide by using 

 Meyer's albumen, or by the process of drying on the slide after treatment with tepid 

 water to remove the wrinkles. 



Dissolve off paraffin in xylol. 



Pass down through 100 per cent., 95 per cent., 85 per cent., 70 per Cent., 50 per 

 cent., 30 per cent., alcohol, thirty seconds each. 



Delafield's hjematoxylin, fifteen minutes. 



Rinse in water five minutes. 



Pass up through 30 per cent., 50 per cent., 70 per cent., 95 per cent., and absolute 

 alcohol. 



Put in xylol at least one minute. 



Mount in balsam. 



Note. — All of the material obtained in the inoculation experiments should be 

 studied microscopically. The above methods of fixing, imbedding, sectioning and 

 staining are applicable in all of this work. 



If time permits, all of the organisms inoculated Ln the plants should be recovered 

 and in pure culture by the methods outlined in Lesson 22. Direct inoculation of 

 media in plugged test-tubes can be used. A reinoculation of the recovered organisms 

 is desirable, if time permits the class to undertake such additional work. 



LESSON 43 



Freezing of Material and Culling. — Freezing Microtome. — The material may be 

 imbedded in a thick solution of gum arable which is frozen on a metal plate cooled 

 to the freezing temperature by conducting under the plate a mixture of ice water 

 and salt. This is accomplished by filling a glass vessel full of a mixture of ice and 

 salt and conducting the water from the jar by a tube {A) through metal a box (fi) 

 on which the sections are placed in the mucilage. 



