CHAPTER VIII 

 THE FREEZING METHOD 



1. Use a piece of spleen which has been properly fixed and 

 later preserved in 70 per cent, alcohol. Transfer it through 50 

 and 35 per cent, alcohol successively to water, and wash it for 12 

 hours in running water. 



2. Place it into a gum and syrup mass for 24 hours (a saturated 

 solution of loaf sugar in 30 c.c. of distilled water, added to 50 c.c. 

 of gum mucilage. Prepare a supply of gum mucilage by dissolving 

 60 grams of best gum acacia in 90 c.c. of distilled water). 



3. Examine the freezing microtome carefully (Fig. 33). 



4. Remove the gum and syrup mixture from the outside of 

 the tissue with a cloth, put a little gum mucilage (not gum and 

 syrup) on the freezing disk of the microtome, and place the tissue 

 in it in such a way that longitudinal sections through the hilum 

 may be cut. Surround the object with gum mucilage and set the 

 freezing apparatus to going. If carbon dioxide is used, open the 

 valve very cautiously, and let only a small quantity of the gas 

 escape. 



NOTE. Carbon dioxide is commonly used for charging soda water 

 and beer. It may be purchased in iron cylinders containing about 20 

 pounds of the liquified gas. The cylinder, when empty, is exchanged 

 for a charged one, so that the purchaser pays only for the contents. The 

 Bardeen microtome (Fig. 33) may be screwed directly upon the carbon- 

 dioxide cylinder when the latter is in a horizontal position, or, if desired, 

 the cylinder may be placed vertically and the microtome attached by 

 means of an L-shaped piece of heavy tubing. This microtome has the 

 advantage over the common forms of freezing microtomes of wasting less 

 gas and of greater freedom from clogging. It is advantageous to have 

 an extra long handle to the key which is used for opening the escape 

 valve of the carbon-dioxide cylinder. 



5. As soon as the gum is frozen, continue to add more until the 

 tissue is completely covered and frozen. 



6. Work the microtome screw with one hand and plane off 

 sections (15 to 20 microns thick) with the other. The well- 



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