274 XXI. BACTERIA YEAST. 



added, and afterwards, instead of an alcoholic solution of fuchsin, 

 4 to 5 grams of the solid dye, whether fuchsin, methyl violet, 

 or methylene blue, is added, and the solution thoroughly shaken. 



Staining in the Tissues. Only in the most exceptional cases 

 can it be recommended to examine sections from fresh tissues 

 for bacteria. It is much more advantageous, and usually indeed 

 necessary, to previously harden such tissues. For this hardening 

 absolute alcohol is best, since the use of other hardening media 

 makes the success of the subsequent staining less certain. Pieces 

 of tissue to be hardened in alcohol should not be larger than a 

 hazel-nut, and should lie for at least three days in a relatively 

 large quantity of absolute alcohol or strongest methylated spirits. 

 Not all bacteria can be equally readily stained. Staining, how- 

 ever, almost invariably results from the use of a strong alkaline 

 solution of methylene blue, which may be considered the best all- 

 round stain for bacteria. This solution is prepared if 30 c.c. con- 

 centrated alcoholic solution of methylene blue is added to 100 c.c. 

 potash solution of strength 1 : 1000 ; it does not keep long. Stain- 

 ing is completed in a few minutes, and the sections are then 

 washed in 0*5 per cent, acetic acid, dehydrated in alcohol, cleared 

 in oil of cedar, and preserved in Canada balsam. 



If it is desired to have only certain bacteria in the section 

 stained, the rest, as well as the tissue, can be decolorised. In 

 general the bacteria resist decolorising better than the tissue 

 elements, but show, on the other hand, varying degrees of tena- 

 city in colour-retention. For such "isolation," that is, the staining 

 of particular ' bacteria, Gram's method is most commonly used. 

 This is specially noteworthy for the fact that it brings about the 

 decolorising of the nuclei in the cell-tissues without altering the 

 colour of most bacteria. The sections are first stained with 

 aniline-water-methyl-violet, or aniline-water-fuchsin, freshly pre- 

 pared exactly as described above, i.e., 5 c.c. pure aniline oil added 

 to about 100 c.c. distilled water, thoroughly shaken, and filtered 

 through a previously-damped filter. To this clear aniline water 

 11 c.c. of a concentrated alcoholic solution of methyl violet (better 

 than the gentian violet which was at first recommended) are 

 added, it is again filtered through a damped filter, and finally 10 

 c.c. absolute alcohol added. This solution keeps for about a 

 fortnight. Sections stained with this fluid are transferred either 

 direct, or after slight rinsing in alcohol, to an iodine solution 

 which contains 2 parts potassium iodide and 1 part iodine to 300 



