88 MICROSCOPIC EXAMINATION OF BACTERIA. 



solution. Wash, counter-stain, and mount. Another method 

 is first to immerse the film for two minutes in chloroform and 

 then in 5 per cent, chromic acid solution for 1 to 2 minutes. 

 When stained after the method of staining the tubercle bacil- 

 lus the result is not very satisfactory. The decolorizing agent 

 should not be so strong ; a 3 per cent, solution of the acid is 

 sufficient. 



Ajezky first places the film, before fixing, in a hot 0.5 per 

 cent, hydrochloric acid solution for three to four minutes, 

 heating it gently. Decolorize with 45 per cent, sulphuric 

 acid. Counter-stain with methylene-blue or malachite-green 

 and mount. 



Neisser's method : Float the preparation on an anilin-water 

 fuchsin solution for one hour, heating it constantly to near 

 the boiling-point. Wash in water, decolorize with a 25 per 

 cent, solution of hydrochloric acid, counter- stain, and 

 mount. 



Fiocca's method : To 20 c.c. of a 10 per cent, ammonia solu- 

 tion add from 10 to 20 drops of an alkaline solution of any 

 of the anilin dyes. Heat until steam is given off, then place 

 the film in the hot staining solution for from 5 to 15 minutes. 

 Decolorize in a 25 per cent, solution of nitric or sulphuric 

 acid ; wash in water, counter-stain with malachite-green, vesu- 

 vin, or safranin, and mount. 



All these methods stain the spore red, and the parent germ 

 takes on the color of the contrast-stain. None of these 

 methods is entirely satisfactory, and spore-staining still re- 

 mains a discouraging procedure. Many attempts must be 

 made before even a measure of success is attained. 



CAPSULES : When bacteria are grown artificially on culture- 

 media, capsules usually do not appear. For the demonstration 

 of the capsules the fresh, germ-containing material must be 

 used. The sputum from a case of lobar pneumonia is most 

 suitable. 



Johne's method : The film is stained in a warm 2 per cent, 

 solution of gentian-violet for a few minutes and decolorized 

 in a 2 per cent, solution of acetic acid. After washing in 

 water the specimen is mounted in water. Canada balsam 

 shrinks the capsule. For permanent specimens the cover- 



