450 APPENDIX 



MAKING AND STAINING OF FROZEN SECTIONS 



The various types of ether freezing microtomes are not very satisfactory when 

 only used occasionally. With the general introduction of cylinders containing 

 compressed carbon dioxide, which is used for aerating waters, we have at hand a 

 practical and convenient method of making frozen sections. 



The instrument makers furnish a freezing microtome of the Bardeen type whicl 

 can be attached directly to the cylinder by a revolving clamp nut. 



It is necessary to have a stand to support the iron cylinder in a horizontal posi- 

 tion. The tissue, which may be taken at operation for immediate diagnosis, or 

 which preferably has been fixed in formalin for twelve to eighteen hours is immedi- 

 ately placed in water. If the tissues have been in alcohol it will require hours of 

 washing before they can be frozen. The piece of tissue which is to be frozen should 

 not be more than one-fifth of an inch thick. Having placed the piece of tissue on 

 the freezing box of the microtome we turn the valve* of the cylinder to allow the 

 gradual escape of gas. When frozen solid, we elevate the freezing box holding the 

 frozen tissue, by revolving a graduated disk with the left hand. In the right hand 

 we firmly grasp a well-sharpened blade of a carpenter's plane mounted in a wooden 

 handle. This is held at an angle of 45 degrees to the polished ways of the micro- 

 tome. By alternate shoving and withdrawing of the blade, held rigidly, we accumu- 

 late on the blade a number of sections. Then dip the blade in a vessel of water to 

 detach the sections which float in the water. Keep repeating the process until 

 numerous satisfactory sections are obtained. Handles for holding the Gillette 

 razor blades are good substitutes for the carpenter's plane. 



These sections may be picked up with a strip of cigarette paper and applied to a 

 clean slide upon which a very small loopful of albumin fixative has been smeared 

 out with 30% alcohol. The piece of cigarette paper with the section underneath 

 is then firmly smoothed out upon the slide with filter-paper. The piece of cigarette 

 paper is then carefully stripped off and the section remains attached to the slide. 

 By careful heating over a very small flame, until the vapor just arises, the section 

 is fixed to the slide and we can then stain the section in any way that may be desired. 



NOTE. The procedures for carrying the tissues through celloidin are not given 

 as it requires perfect condition of the entire cutting surface of the microtome knife 

 and a considerable time for the passage through reagents and celloidin. It is more 

 suitable as a method when sections for class work are to be prepared. 



B MOUNTING AND PRESERVATION OF PATHOLOGICAL SPECIMENS 

 AND ANIMAL PARASITES 



To Mount Small Round Worms. Wash the hook, whip, or filarial worm in salt 

 solution, then drop in 70% alcohol containing 5% of glycerine; the glycerine-alcohol 

 mixture being at a temperature of 6oC. When cool, pour into Petri dishes and 

 allow the alcohol to evaporate in the 37C. incubator. 



Mount in glycerine jelly, preferably in a concave slide, and ring the preparation 

 with gold size. The following is the formula for Kaiser's glycerine jelly: Soak one 

 part of gelatin in six parts of distilled water for two hours. Then add seven parts 

 of glycerine. To the mixture add i% of carbolic acid, warm for fifteen minutes, 

 with constant stirring, and then filter through cotton. 



