1889.] MICROSCOPICAL JOURNAL. 161 



BACTERIOLOGY.* 



Staining Reagents. — The stains employed in bacteriological studies, 

 with, perhaps, a few exceptions, are the aniline dyes introduced by 

 Weigert. Flugge (" Handbuch der Hygiene," Pt. I, Fermente und 

 Microparasites, p. 287), on the authority of Ehrlich, classified the ani- 

 line colors into two distinct groups, each of which has very distinct 

 chemical and physiological characteristics, the acid and the basic dyes. 



In the first group the coloring matter acts as an acid in combining 

 with bases to form salts, although it does not necessarily give an acid 

 reaction, nor is it necessarily in the form of a free acid. Among the 

 more important are eosin, picric acid, aniline black, and purpurin. 



To the second group, the basic dyes, belong by far the greater num- 

 ber of those which are used in staining bacteria. These are principally 

 fuchsin, methyl violet, methylene blue, Bismarck brown, and gentian 

 violet. These basic colors are sold as salts and not as free bases, whilst 

 fuchsin, for example, may be obtained as an acid salt, as chloride, or 

 acetate of rosaniline. 



With these nucleus and germ-tinting reagents both nuclei and bacteria 

 can be stained red, brown, blue, or violet, according to the stain em- 

 ployed, for, as a rule, micro-organisms react to staining fluids very 

 much as do the nuclei of cells. This holds good in so far that most 

 nuclear stains will impart a similar tint to the micro-organism as they 

 do to the nuclei themselves. Beyond this, however, it is found that the 

 micro-organisms hold the coloring matter more tenaciously than do the 

 cell nuclei, and that the nuclei maybe decolorized by the use of certain 

 reagents, such as acetic, nitric, or hydrochloric acid. 



The first experiments on staining germs were made with carmine and 

 haematoxylin, and Koch and others were successful in staining not only 

 the bodies of bacteria, but also, in certain cases, in demonstrating the 

 flagella. These reagents are now, however, superseded by the aniline, 

 colors, principally the basic series. 



Weigert's gentian violet and picro-carmine method demonstrates the 

 affinity of the basic aniline colors. The sections of tissue containing 

 bacteria are first placed in gentian violet (saturated alcoholic solution 

 of gentian violet, 11 cc, aniline water, ioocc, absolute alcohol, 10 cc.) 

 for several minutes, then washed in alcohol, transferred to water, and 

 afterwards to Weigert's solution of picro-carmine for half an hour. 



They are further washed, first in water, then in alcohol, are clarified 

 with clove oil and mounted in balsam. By this method the nuclei are 

 stained red and the bacteria violet ; the carmine has replaced the gen- 

 tian violet in the nuclei, but has had no effect upon the violet taken up 

 by the bacteria. In a similar manner a solution of iodine and iodide 

 of potassium does not remove the basic colors from certain micro-organ- 

 isms, but it rapidly decolorizes nuclei and other tissues. It is upon this 

 fact that Gram's method is founded. 



Weigert's Picro-carmine. t — Add aqua ammonia, 4 grams, to car- 

 mine, 2 grams, and allow it to stand for twenty-four hours in a damp 

 place, and then add 200 grams of picric acid. Allow the whole to re- 



* Conducted by V. A. Moore. 



t Dolley ; Technology of DacLeria Investigation, p. 251. 



