100 APPLIED BACTERIOLOGY 



fluous colour is removed by immersing the sections in 

 diluted alcohol, or in a 0'5 per cent, solution of acetic acid 

 for a few seconds. The sections are now dehydrated in 

 absolute alcohol, cleared in xylol or oil of cloves, transferred 

 to the slip, blotted, and mounted as usual. 



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

 the removal of the colour 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 : 10 c.c. of a saturated alcoholic solution of methy- 

 lene blue is added to 100 c.c. of 5 per cent, solution of 

 carbolic acid (phenol). The sections are placed in this 

 solution for thirty minutes, then washed in water, and 

 decolourised in very dilute hydrochloric acid (2 drops strong 

 acid in 100 c.c. water). This decolourising operation must 

 be very carefully conducted, as very thin sections will only 

 require to be immersed for two or three seconds, after 

 which the sections are at once transferred to an alkaline 

 solution prepared as follows : 10 drops of a saturated solu- 

 tion of lithium carbonate in 10 c.c. of water. The sections 

 are then washed in water for a few minutes, dehydrated in 

 absolute alcohol, which Kiihne colours with a little methy- 

 lene blue. The sections are now placed in aniline oil, 

 which also contains a little dissolved methylene blue.* 

 The sections are now washed in colourless aniline, then in 

 xylol, and lastly mounted, as usual, in balsam. 



Ziehl-Neelsen Method. — This important method is used for 

 the diagnosis of tubercle and leprosy. These are the only 

 two organisms ordinarily met with which withstand the 



* The anilLae oil blue solution is prepared by shaking an excess of 

 methylene blue with colourless aniline, when, after standing with 

 frequent agitation, the coloured oil is filtered off. 



