34 STAINING METHODS. 



the process by the use of heat. To obtain good thin sections, the 

 material, cut in small cubes, must be very thoroughly hardened in 

 absolute alcohol. The piece selected for cutting may be attached to 

 a cork by the use of melted glycerin jelly, which is hardened by 

 placing the cork and attached piece of tissue in alcohol. This an- 

 swers for well-hardened pieces of liver, kidney, , etc., but the hollow 

 viscera and tissues of loose structure will require embedding in 

 paraffin or celloidin. Any well-made sledge microtome will answer 

 for cutting the sections, if the knife is properly sharpened. The sec- 

 tions should, of course, be cut under alcohol, and they can scarcely 

 be too thin when the object is to ^demonstrate the presence or ab- 

 sence of bacteria. Very thin sections ma} T be cut dry by embedding 

 in paraffin having a melting point of 50 C. In this case the knife 

 is set at a right angle to the material to be cut, and the sections 

 are spread out upon and attached to the glass slide for staining. 



One of the most useful solutions for staining tissues is Lofflers 

 alkaline solution of methylene blue (No. 4). A freshly-prepared so- 

 lution will stain sections in four or five minutes. Superfluous color 

 is removed by immersing the sections in diluted alcohol or in a one- 

 half-per-cent solution of acetic acid for a few seconds. The sectiofis 

 are dehydrated in absolute alcohol, cleared up with oil of cedar, and 

 mounted in a drop of cedar oil 'for examination, or. in balsam if 

 they are to be preserved. 



Gram's method may be used as directed for cover-glass prepara- 

 tions, the sections being first stained in aniline-gentian-violet solu- 

 tion (No. 1), then washed in water, or in aniline water as recently 

 (1892) recommended by Botkin, then decolorized in the iodine solu- 

 tion (see page 29). The sections when decolorized are again washed 

 in water, dehydrated in absolute alcohol, cleared in cedar oil, and 

 mounted in balsam. 



Weigert's Method, This is a modification of Gram's method in 

 which the sections are dehydrated by the use of aniline oil. The 

 stained section, after having been washed, is transferred to a clean 

 glass slide, the excess of water is removed by the use of filtering 

 paper, and the iodine solution is placed upon it in sufficient quantity 

 to cover the entire section. When sufficiently decolorized this is re- 

 moved in the same way. The section is then dehydrated by placing 

 a few drops of aniline oil upon it, removing this with filtering paper, 

 and repeating the operation once or twice. The aniline oil must 

 then be completely removed by the use of xylol, after which the sec- 

 tion is mounted in balsam. 



Kiihne's Method. The object of this method is to prevent the 

 removal of the color from stained bacteria in sections during the 

 treatment which such sections usually receive before they are ready 



