APPENDIX 445 



Next take a paper box (made of stiff writing-paper folded over a square of wood) 

 and fill with the melted paraffin. As quickly as possible drop in the piece of tissue 

 taken out of the paraffin bath with heated forceps and, so soon as the paraffin begins 

 to solidify on the surface, place the paper box in ice water. When paraffin is rapidly 

 cooled, crystallization is less. 



The Acetone Method. Take the tissues out of the 70% alcohol and place in ace- 

 tone. After remaining in acetone for one or two hours, the tissues should be trans- 

 ferred to fresh acetone for an equal length of time. Dry calcium chloride in the bot- 

 tom of the acetone bottles keeps it dehydrated. They should then be placed in 

 xylol for about one-half hour and then embedded in paraffin as directed above. 



The Chloroform Method. The procedure may be the same as in the method of 

 passing through alcohols to xylol, substituting chloroform for xylol and then trans- 

 ferring to paraffin. 



Where absolute alcohol is not obtainable, very satisfactory results may be 

 obtained by transferring tp a mixture of 95% alcohol and chloroform after immer- 

 sion in 95% alcohol. Then going from the alcohol-chloroform mixture to pure 

 chloroform, thence to paraffin. 



Rapid paraffin imbedding methods. 



When a piece of tissue is not more than Y inch square and ^ inch thick, it is 

 very easy to run it through in three to six hours. Thus: 



10% Formalin (in 37C. incubator), i hour. 



70% Alcohol (in 37C. incubator), i hour. 



95% Alcohol (in 37C. incubator), i hour. 



Absolute alcohol (in 37C. incubator), H hour. 



Xylol (in 37C. incubator), V?. hour. 



Paraffin (in 55C. incubator), ^ to 2 hours. 



Method of Lubarsch. In this excellent method small pieces of tissue not more 

 than }/$ inch thick are placed in a wide test-tube containing 10% formalin for 10 to 

 15 minutes, changing the fluid twice. Transfer to 95% alcohol 10 minutes changing 

 alcohol once. Absolute alcohol, for 10 minutes changing twice. Pure aniline oil 

 until tissues are transparent, 15 to 30 minutes. Xylol, changing two to three times 

 or until the xylol is no longer yellow, 10 to 20 minutes. Imbed in paraffin for 20 

 minutes to i hour. During the entire process keep the test-tube in a water bath 

 or incubator at 5oC. It is necessary to have a good microtome. The best is that 

 of Minot. Very satisfactory sections can be cut with the various types of student 

 microtomes, costing from twelve to twenty dollars. 



(In using a hand microtome, a razor with a flat edge is necessary. After expe- 

 rience, sections thin enough for histological but not for bacteriological examination 

 can be made.) 



If the piece of tissue is properly dehydrated and imbedded, thin sections (3 to 

 ioju) should be easily obtained, provided the knife be sharp. One advantage about 

 the paraffin method is that it is only necessary to have a small part of the blade in 

 proper condition. With celloidin the entire cutting edge must be perfect. Having 

 cut the sections, they should be dropped on the surface of a bowl of warm water 

 (45C.). This causes the section to flatten out evenly. 



Decakification.This is best accomplished by fixing in 10% formalin for twenty- 



