1889.] MICilOSCOPICAL JOUKNAL. 259 



BACTERIOLOGY.* 



Kiihne's Methylene-Blue Method of Staining Bacteria. — 



This method is especially recominended for staining bacteria in sec- 

 tions of animal tissues, although it is equally applicable to cover-glass 

 preparations made from fresh tissues. The usual differences in the 

 method of staining cover-glass preparations and sections are to be 

 observed. 



The advantages to be derived from this method are found in its be- 

 ing applicable to all known forms of bacteria. It eliminates the use of 

 special stains for certain micro-organisms where only their presence is 

 to be demonstrated. It possesses superior powers of differentiations 

 between bacteria and the tissue elements. The method as given by 

 Dr. Kiihnef is essentially as follows: 



The sections which have been cut by the ordinary method (although 

 Dr. Kiihne recommends the freezing microtome for this purpose) are 

 transferred directly from alcohol to a watch-glass containing carbol- 

 methylene-blue (i). The sections should remain in this staining fluid 

 for about one-half hour. .Some bacteria, such as the bacillus of leprosy, 

 requiring a longer time, one to two hours. If the sections remain in 

 the staining fluid for a much longer period the differentiation between 

 the germs and tissue elements becomes more difficult. 



After staining for the desired length of time, the exact period of 

 which will have to be determined by test experiments for the different 

 germs and tissues, the sections are rinsed in clear water and then 

 placed in acidulated water (2) until they become a pale blue. They 

 are then w'ashed in a weak, watery solution of carbonate of lithium 

 (3) and again placed in clear water. This part of the procedure is 

 very important, and to insure good results should be performed with 

 much care. The time that the sections should remain in the decolor- 

 izing agents varies with their thickness, histological structure, and the 

 intensity of the stain, making it impossible to give any definite rule to 

 be followed. The degree of decolorization can be very nearly determined 

 at any moment by moving the sections about in the fluid by means ot 

 a glass rod. If the section is very thin or if there are other reasons 

 why it should take up very little of the stain a momentary immersion 

 in the acidulated water is sufficient. In all cases whei'e the staining 

 process is completed the sections should have a pale blue color, for if 

 darker the over-stained corpuscles and cell nuclei of the tissue would 

 obscure the bacteria. In cases where it is feared that too much color 

 has been removed in the acid a drop of a saturated WMtery solution ot 

 methylene-blue should be added to the lithium water. 



After the sections have remained in the water for some minutes they 

 are dehvdrated in absolute alcohol in which, in difficult cases, a little 

 methylene-blue may be dissolved, and then transferred to a watch-glass 

 containing methylene-blue aniline oil (4). The sections can be 

 dehydrated in the alchohol without injury to the stained bacteria. The 

 sections are now transferred to pure aniline oil, in which they are 

 rinsed, and then placed in some essential oil, as turpentine, where they 



* This Department is conducted by V. A. Moore. 



t Kiihne, Pralctische Anleitung zum mikroskopischen Nachweis der Bakterien im tierischen 

 Gewebe, p. 15. 



