STAINING METHODS. 35 



lution will stain sections in four or five minutes. Superfluous color 

 is removed by immersing the sections in diluted alcohol or in a one- 

 half -per-cent solution of acetic acid for a few seconds. The sections 

 are dehydrated in absolute alcohol, cleared up with oil of cedar, and 

 mounted in a drop of cedar oil for examination, or in balsam if 

 they are to be preserved. 



Grain's method may be used as directed for cover-glass prepara- 

 tions, the sections being first stained in aniline-gentian-violet solu- 

 tion (No. 1), then washed in water, or in aniline water as recently 

 (1892) recommended by Botkin, then decolorized in the iodine solu- 

 tion (see page 29). The sections when decolorized are again washed 

 in water, dehydrated in absolute alcohol, cleared in cedar oil, and 

 mounted in balsam. 



Weigert's Method. This is a modification of Gram's method in 

 which the sections are dehydrated by the use of aniline oil. The 

 stained section, after having been washed, is transferred to a clean 

 glass slide, the excess of water is removed by the use of filtering 

 paper, and the iodine solution is placed upon it in sufficient quantity 

 to cover the entire section. When sufficiently decolorized this is re- 

 moved in the same way. The section is then dehydrated by placing 

 a few drops of aniline oil upon it, removing this with filtering paper, 

 and repeating the operation once or twice. The aniline oil must 

 then be completely removed by the use of xylol, after which the sec- 

 tion is mounted in balsam. 



Kiihne's Method. The object of this method is to prevent the removal 

 of the color from stained bacteria in sections during the treatment which 

 such sections usually receive before they are ready for mounting i.e., 

 during the washing and dehydrating processes usually employed. For 

 staining, Kiihne prefers a methylene-blue solution prepared as follows: 

 Methylene blue, 1.5 parts; absolute alcohol, ten par-ts ; triturate in a watch 

 glass and add gradually one hundred parts of a solution of carbolic acid 

 containing five parts in one hundred of water. The section is placed in this 

 solution for about half an hour, then washed in water and decolorized in a 

 weak solution of hydrochloric acid ten drops to five hundred grammes of 

 water. This part of the operation must be conducted very carefully, and 

 usually thin sections will only require to be dipped in the acid solution for an 

 instant, after which they must be at once immersed in a solution of lithium 

 eight drops of a saturated solution of carbonate of lithium in ten grammes 

 of water. They are then allowed to remain in a bath of distilled water for 

 a few minutes, after which they are dipped into absolute alcohol, which 

 Kiihne colors by the addition of methyl en e blue. The sections are then 

 placed in aniline oil which contains a little methylene blue in solution, 

 where they are dehydrated without the color being extracted from the stained 

 bacteria present. The aniline-oil blue solution is prepared by adding an ex- 

 cess of dry methylene blue to a small quantity of clarified aniline oil. The 

 undissolved pigment settles to the bottom, and a few drops of the colored 

 solution are added to a little aniline oil in a watch glass to make the colored 

 dehydrating bath. The section is next washed out in pure aniline oil not 

 colored after which every trace of aniline oil is to be removed by the use 

 of xylol. The section is cleared up in turpentine and mounted in balsam. 



