88 BACTERIOLOGY. 



Aniline oil, carbolic acid, and some other chemicals, when added 

 to the aniline dyes, have the property of acting in the manner 

 of mordants, in some way fixing the colour in the bacteria, so that 

 they are not so readily acted upon by decolorising agents. 



Liiffler's Solution. — Potash intensifies the staining power, and 

 Koch and Loffler have both used it with methylene blue. Loffler's 

 solution consists of 30 grammes of methylene blue in 100 grammes 

 of 1 in 10,000 solution of potash. It may be used with advantage 

 for almost all kinds of bacteria. 



Gram's Method. — With a solution of gentian -violet the whole 

 film on the cover-glass is at first stained violet. By immersing the 

 cover-glass in a solution of iodine in iodide of potassium the stain 

 is fixed in the bacilli, but not in any debris, pus colls, or tissue 

 elements present in the film. Consequently by transferring the 

 cover-glass to alcohol the bacilli alone remain stained, the violet 

 colour being merely changed to blue. By employing a contrast 

 colour, such as eosin, a double staining is obtained. In some 

 bacteria the sheath is by this method differentiated from the 

 protoplasmic contents. 



The stock solution of gentian-violet is prepared by shaking up 

 1 cc. of pure aniline with twenty parts of distilled water, and 

 filtering the emulsion. Half a gramme of the best finely powdered 

 gentian- violet is dissolved in the clear filtrate, and the solution filtered 

 before use. 



The details of the method will now be described. In the first 

 place, it is much better to employ the aniUne-gentian-violet solution 

 quite freshly prepared, and the following useful method is invariably 

 used by the author : Place four or five drops of pure aniline in 

 a test-tube, fill it three-quarters full with distilled water, close the 

 mouth of the tube with the thumb, and shake it up thoroughly. 

 Filter the emulsion twice, and pour the filtrate into a watch-glass 

 or glass capsule. To the perfectly clear aniline water thus obtained 

 add drop by drop a concentrated alcoholic solution of gentian-violet 

 till precipitation commences. Cover-glasses must be left in this 

 solution about ten minutes, transferred to iodine-potassic-iodide 

 solution until in itwo or three minutes the film becomes uniformly 

 brown, and then rinsed in alcohol. The process of decolorisation may 

 be hastened by dipping the cover-glass in clove-oil and returning it 

 again to alcohol. The cover-glass is once more immersed in clove-oil, 

 then dried by gently pressing between two layers of filter-paper, 

 and finally mounted in Canada balsam. 



