M K 'ROSCOPIC METHODS 73 



and methylene blue) give negative results. The reason for this is that 

 the iodine compounds with the pararosanilines are fast colors, while 

 those with the rosanilines are unstable. These latter compounds when 

 treated with alcohol break up into their constituents, the iodine is washed 

 out, and the dye substance remaining in the tissues stain them uni- 

 formly, that is, without differentiation. But iodine-pararosaniline com- 

 pounds are not thus broken up and consequently stain those portions 

 of the tissue more or less, according to the affinity which they have 

 for the dye substance. The parts stained by Gram are thus distin- 

 guished from those stained violet, not only quantitatively, but qualita- 

 tively; it is not a gentian violet but an iodine-rosaniline staining which 

 occurs. 



Use of Mordants and Decolorizing Agents. In films of blood and 

 pus, and in tissue sections, the tissue elements may be stained to such 

 an extent as to obscure the bacteria. Hence many methods have been 

 devised to use substances which, while increasing the staining power, 

 tend to fix the stain in the bacteria and further to treat by substances 

 which decolorize the overstained tissue to a greater or less extent while 

 leaving the bacteria stained. The staining capacity of a solution may 

 be increased by (a) the addition of substances such as carbolic acid, 

 aniline oil, or metallic salts, all of which probably act as mordants; (b) 

 by the addition of weak alkaline solutions of caustic potash or ammo- 

 nium carbonate; (c) by the employment of heat; (d) by long duration 

 of the staining process. 



As decolorizing agents we use chiefly mineral acids (hydrochloric, 

 nitric, sulphuric), vegetable acids (acetic), alcohol or a combination 

 of alcohol and acid; also various oils, aniline, clove, etc. 



The acid aniline dyes are represented by eosin, acid fuchsin, and 

 fluorescein. 



Formulae of the Most Commonly Used Stain Combinations. LOEFFLER'S 

 ALKALINE METHYLENE-BLUE SOLUTION. This consists of concen- 

 trated alcoholic solution of methylene blue, 30 c.c.; caustic potash in 

 a 0.01 per cent, solution, 100 c.c. The alkali not only makes the cell 

 more permeable, but also increases the staining power by liberating 

 the free base from the dye. 



KOCH-EHRLICH ANILINE-WATER SOLUTION OF FUCHSIN OR GENTIAN 

 VIOLET is prepared as follows: To 98 c.c. of distilled water add 2 c.c. 

 aniline oil, or more roughly but with equally good results, pour a few cubic 

 centimetres of aniline oil into a test-tube, then add sufficient water to 

 nearly fill it. In either case the mixtures are thoroughly shaken and 

 then filtered into a beaker through moistened filter-paper until the filtrate 

 is perfectly clear. To 75 c.c. of the filtrate add 25 c.c. of the concen- 

 trated alcoholic solution of either fuchsin, methylene blue, or gentian 

 violet, or add the alcoholic solution until the aniline water becomes 

 opaque and a film begins to form on the surface. 



CARBOLIC FUCHSIN, OR ZIEHL'S SOLUTION. Distilled water, 100 c.c.; 

 carbolic acid (crystalline), 5 gm.; alcohol, 10 c.c.; fuchsin, 1 gm.; 

 or it may be prepared by adding to a 5 per cent, watery solution of car- 



