424 HISTOLOGICAL TECHNIQUE. 



The anilin-water is prepared by shaking a few drops of anilin 

 with distilled water, allowing the mixture to stand for about ten 

 minutes, and then filtering through well-moistened filter-paper. 

 The filtrate should contain no globules of the anilin. In order to 

 avoid this the filtration should be stopped before all the watery 

 part of the mixture has run through the paper, otherwise oily drops 

 of anilin will follow. 



Precipitates are likely to occur in this gentian-violet solution 

 when it is first prepared. After twenty-four hours these are less 

 abundant. The solution deteriorates soon after that time, and 

 should not be used more than a week after its preparation. 



B. Stirling's formula : 



Gentian-violet, 5 grams. 



Alcohol, 10 cc. 

 Anilin, 2 cc. 



Distilled water, 88 cc. 



This solution keeps better than the preceding. Both must be 

 filtered carefully through moistened filter-paper immediately before 

 being used. 



10. Gram's Solution. This is a differentiating agent used in con- 

 nection with anilin-gentian-violet : 



Iodine, 1 gram. 



Potassium iodide, 2 grams. 



Distilled water, 300 cc. 



The specimens are first overstained with the gentian- violet solu- 

 tion. They are then washed in water and placed in Gram's solution 

 for from three to five minutes. While in this solution they turn a 

 brown color, and the combination between the coloring-matter and 

 some of the elements of the specimen is loosened. The specimen 

 is then transferred to 95 per cent, alcohol, in which it remains until 

 no more color is given off. If sufficient color has not been removed, 

 the treatment with Gram's solution and alcohol may be repeated. 

 After this differentiation the specimen may be dehydrated, cleared, 

 and mounted ; or a contrast-stain may be used before those manipu- 

 lations. This is a useful method for staining bacteria in sections 

 of tissue when the species of bacteria are such as resist the decolor- 

 izing action of the iodine. In this respect different species of bac- 



