APPENDIX 373 



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

 than 1/5 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 50 C. It is preferable 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 $12 to $20. 



(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 

 io/0 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 

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



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

 four hours, then placing a small piece of the bone (not exceeding one-half inch square 

 and one-fifth of an inch thick) in concentrated sulphurous acid. 



This decalcifies in about 2 to 7 days. Wash thoroughly in alkaline water 

 and then in tap water. Pass through alcohols and xylol and imbed and section as 

 before described. 



To Stain Sections. It is first necessary to affix the section to a slide or cover-glass. 

 To attach the section firmly to the slide, so that it will not become detached in 

 subsequent treatment, pick up a section on a strip of cigarette paper. 



A sheet of cigarette paper is cut into about five pieces (1/2X1 1/2 inches). 

 Inserting the strip of cigarette paper under the section, it is easily lifted up out of 

 the water. Then apply the slip of cigarette paper, section downward, to a perfectly 

 clean slide. Blot with a piece of filter-paper, then strip off the piece of filter-paper 

 leaving the section smoothly applied to the slide. Next place in the 37 C. incubator 

 for twelve to twenty-four hours and the section will be found to be so firmly attached 

 that it will not be dislodged by subsequent treatment. 



For Immediate Diagnosis. Take a loopful of albumin fixative (white of fresh 

 egg, 50 c.c.; glycerin, 50 c.c.; sodium salicylate, i gram) and deposit it on a cover- 

 glass. Now take up a loopful of 30% alcohol (i drop of 95% alcohol and two drops 

 of water) and applying it over the albumin fixative, smear out the mixture uniformly 

 over the cover-glass. 



2. Pick up a section on a strip of cigarette paper and apply it to the prepared sur- 

 face on the cover-glass. Blot with gentle pressure with a piece of filter-paper over 

 the strip of cigarette paper, and strip off this latter, leaving the section attached to 

 the cover-glass. 



3. Now, turning the flame of the Bunsen burner down very low or with a small 

 alcohol flame, we hold the cover-glass in a Stewart's forceps, section side up, over 

 the flame and slowly lower it until the paraffin is observed to melt. This shows a 



