34 STAINING METHODS. 



A. THE MORDANT. 



Tannic acid, ten-per-cent aqueous solution, filtered, . 10 c.c. 



Corrosive sublimate, saturated aqueous solution, . 5 c.c. 



Alum, saturated aqueous solution, . . . . 5 c.c. 



Carbol-fuchsin solution, 1 . . . . . 5 c.c. 



''Mix thoroughly. A precipitate forms, which must be allowed to de- 

 posit, either by centrifugalizing or simply by allowing to stand. Remove 

 the clear fluid with a pipette and transfer to a clean bottle. The mordant 

 keeps well for one or two weeks. 



B. THE STAIN. 



Alum, saturated watery solution, . . . .10 c.c. 



Gentian violet, saturated alcoholic solution, . . 2 c.c. 



" The stain should not be more than two or three days old when used. It 

 may be substituted in the mordant in place of the carbol fuchsin. The film 

 having been prepared as above described, pour over it as much of the mor- 

 dant as the cover glass will hold. Heat gently over a flame till steam begins 

 to rise, allow to steam for about a minute, and then wash well in a stream 

 of running water for about two minutes. Then dry carefully over the flame, 

 and when thoroughly dry pour on some of the stain. Heat as before, allow- 

 ing to steam for about a minute, wash well in water, dry and mount in a 

 drop of xylol balsam ? ' (Muir and Ritchie). 



METHODS OF STAINING BACTERIA IN TISSUES. The solutions re- 

 commended for staining cover-glass preparations are also used in 

 staining bacteria in thin sections of the various organs, in which 

 they are found in certain infectious diseases; but, in general, a 

 longer time is required to stain sections, and it is best not to hasten 

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

 material, cut in small cubes, must bs 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 may 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 Loffler's 

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



1 Basic fuchsin, 1 part; absolute alcohol, 10 parts; solution of carbolic acid 

 (1 : 20), 100 parts. 



