34 STAINING METHODS. 



Saturated aqueous solution of alum, . .10 c.c. 



Saturated alcoholic solution of gentian -violet, . . 1 c.c. 



B. 



Tannicacid, .....- 1 g- 

 Distilled water, 10 c.c. 



"The solutions should be made with cold water, and immediately after 

 mixing the stain is ready for use, 



" The cover slip is to be carefully cleaned, the grease being burned off m a 

 flame, and after it has cooled the bacteria are spread upon it, well diluted in 

 water, care being taken to exclude culture medium. After the preparation 

 has been thoroughly dried in the air it should be held over the name with the 

 fingers (the preparation need not be fixed) as Loftier has directed. After- 

 ward the stain is gradually poured on the slip and heated gently, bringing 

 the fluid almost to a boil ; the slip covered with the hot stain should then be 

 laid aside for one minute, then washed in water and mounted. 



** If the filtered stain is used, a second stain of aniline water containing 

 gentian-violet had better be used, which should be applied but a moment and 

 thru washed off, thus leaving a clean field, showing only bacteria lightly 

 stained, with their flagella still more lightly colored." 



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 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 

 t >r 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- 

 lution \\ill stain sections in four or five minutes. Superfluous color 

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

 half-|K'r-(vnt solution of acetic acid for a few seconds. The sections 



