58 TEXT-BOOK OF BACTERIOLOGY. 



that they become useless. To meet this difficulty it is best to per- 

 form the whole staining- process upon the slide. 



If desirable to employ Gram's isolated bacterial stain, place the 

 sections, for about twenty-five minutes, in a thin anilin-water gen- 

 tian-violet solution, and then, for about two and a half or three 

 minutes, in iodide of potassium solution, after which they must be 

 washed in alcohol. If they become quite black in the iodine the 

 stain is now detached by the alcohol and passes off in red clouds 

 from the preparation, which, after about twenty minutes, is bleached. 

 It is well to change the alcohol frequently, and in g-eneral not to be 

 too sparing in materials for Gram's process. If the sections which 

 have been colored by Gram's process are afterward to receive a 

 second contrasting stain, they should be placed, for quite a short 

 time, in a very thin solution of Bismarck-brown. 



Yet vesuvin is a bacterial stain, and on that account less suit- 

 able for these purposes than the pure nucleus stains saffranin and 

 carmine. Picro-carmine is very useful for double staining, accord- 

 ing to Gram. 



The best way is to obtain the nucleus coloring first and then 

 stain the bacteria afterward. The sections pass from the alcohol 

 into a strong solution of picric-acid carmine for about half an hour. 

 The superfluous color is then wholly removed in 50$ alcohol. 



The sections are now rose-colored, and on examination the nu- 

 clei will be found of a dark red color, the bodies of the cells of a 

 light red, and the connective tissue pale yellow. Next place the 

 preparations in anilin-water gentian-violet. 



Into a small dish of aniiin-water pour four or five drops of a 

 concentrated alcoholic solution of gentian-violet till the fluid begins 

 to look opaque, but not till a saturation with coloring matter has 

 taken place i.e., not until the iridescent film appears. Let the sec- 

 tions remain in gentian-violet precisely half an hour, and then bring- 

 them directly, without any previous washing in alcohol, into the 

 sodium solution. After three minutes they must be taken out of the 

 iodine and placed in the alcohol; here the stain is washed out, the 

 red ground color of the tissue becomes more and more distinct, and 

 at length the sections have the same color as before their treat- 

 ment with gentian-violet. 



The results of this process are excellent : the staining is in fact 

 triple; the cells are red ; connective tissue, yellow; bacteria, blue; 

 and the latter stand out from their surroundings with remarkable 

 distinctness. In all staining by Gram's method the precipitates 

 are troublesome, and often settle upon the preparation, in large 

 masses. In order to avoid them, Gunther has proposed a slight 



