70 PRINCIPLES OF BACTERIOLOGY 



The following are the most commonly used basic aniline stains: 



Violet stains methyl violet, gentian violet, crystal violet. 



Blue stains methylene blue, thionin blue. 



Red stains basic fuchsin, safranin. 



Brown stain Bismarck brown. 



Green stain methyl green. 



Of the above stains the violet and red stains are the most intense 

 in action. It is thus easy to overstain a specimen with them. Of the 

 blue, methylene-blue probably gives the best differentiation of struc- 

 ture and it is difficult to overstain with it. 



These dyes are all more or less crystalline powders, and while some 

 are definite chemical compounds, others are mixtures. For this reason 

 various brands are met with on the market and the exact duplication 

 of stains is not always possible. Dyes should be obtained from reliable 

 houses only; most bacteriologists obtain them from Griibler, of Leipzig. 

 It is advisable to keep on hand not only the important dyes, but also 

 stock solutions from which the staining solutions are made. The stock 

 saturated alcoholic solutions are made by pouring into a bottle enough 

 of the dye in substance to fill them to about one-quarter of their capacity. 

 The bottle should then be filled with alcohol, tightly corked, well shaken, 

 and allowed to stand for twenty-four hours. If at the end of this time 

 all the staining material has been dissolved, more should be added, 

 the bottle being again shaken and allowed to stand for another twenty- 

 four hours. This must be repeated until a permanent sediment of 

 undissolved coloring matter is seen upon the bottom of the bottle. This 

 will then be labelled "saturated alcoholic solution," of whatever dye 

 has been employed. The alcoholic solutions are not themselves em- 

 ployed for staining purposes. The solution for use is made by filling 

 a small bottle three-fourths with distilled water, and then adding the 

 concentrated alcoholic solution of the dye, little by little, until one can 

 just see through the solution. Care must be taken that the color does 

 not become too dense; usually about one part to ten is sufficient. Small 

 wooden cases come prepared for holding about one-half dozen bottles 

 of the staining solutions. This number will answer for all purposes. 



General Observations on the Principles of Staining Bacteria. 



Microchemical Reaction and Staining of the Cell Body. Of special 

 importance in this regard is the resistance which bacteria possess to 

 diluted alkalies. Inasmuch as the majority of animal tissues are dis- 

 solved when treated with alkalies, this method has been adopted for 

 rendering visible unstained bacteria in tissues. As a rule, bacteria 

 are stained yellowish with iodine solution, a few only in consequence 

 of their starchy constituents being stained blue. 



Bacteria may be stained with various dyes of very different chemical 

 composition, such as hsematoxylin and certain plant dyes, etc., but 

 most of these are of little practical value as compared with the basic 

 aniline colors. R. Koch was the first to recognize the affinity of bacteria 



