54 MIORO-ORGANISMS IN WATER 



a ver)^ small flame, care being taken to move it up and 

 down in a vertical direction above the flame until bubbles 

 begin to appear on tlie surface ; it is then allowed to 

 cool for one minute, when it is again heated as before 

 until bubbles rise, and then subsequently cooled. This 

 is repeated about five times. The cover-glass is then 

 removed from the hot solution and placed in a dish with 

 a o per cent, solution of alcoholic hydrochloric acid 

 (100 parts of absolute alcohol to three of hydrochloric 

 acid), the preparation-side of the cover-glass upwards. 

 It should remain in this solution for one minute, and 

 is then washed with water as usual and coloured with 

 methylene blue. If gentian violet is used instead of 

 fuchsine, the bacilli must be stained after the decolor- 

 ation with Bismarck brown. 



Fiocca ^ recommends the following method for stain- 

 ing spores, which he states is not only very successful,, 

 but very expeditious : About 20 c.c. of a 10 per cent, 

 ammonia solution is poured into a dish, to which is 

 added ten to twenty drops of an alcoholic solution of 

 the aniline dye employed. The whole is heated until 

 steam begins to rise, when the ordinary cover-glass pre- 

 parations are introduced. On an average three to fiA^e 

 minutes' immersion is suflicient ; in the case of very 

 obstinate spores, such as those of anthrax, ten to fifteen 

 minutes is necessary. The cover-glass is removed and 

 quickly placed in a decolorising solution, such as a 

 20 per cent, solution of either sulphuric acid or nitric 

 acid. The preparation is then washed with water and 

 stained with an aqueous solution of some colour in 

 contrast to that used for the spores. 



Staining of flagella. — The fiagella, or organs of 

 locomotion, which are attached to some bacilli and 



^ 'Ueber eine neue Methode der Sporenfarbiing,' Centralhlatt fur- 

 BaJderiologie, vol. xiv., 1893, p. 8. 



