STAINING BACTERIA 515 



evaporation go on in a -warm room for twenty minutes, then fix the 

 film of saprophytes by means of fairly strong osmic acid vapour ; 

 float the cover with the surface of bacteria downwards on a vessel 

 of solution of violet of methyl-anilin for an hour or less, drain the 

 edge of the cover-glasses on blotting-paper, and mount in glycerin. 



(2) Now take drops of the fluid from the several vessels and in 

 a moist growing cell examine the living forms, and compare these 

 with your dried and stained preparations. 



(3) By another method, which will apply also to the bacillus of 

 tuberculosis, a layer of sputum or of putrefactive fluid may be spread 

 as before upon a cover-glass, dried in an air-oven at about 100 F., 

 and then passed three times, moderately slowly, through the flame 

 of a spirit-lamp, so as to thoroughly ' fix ' the preparation by 

 coagulating its albumen. Mix 1 c.c. of concentrated solution of 

 methylen-blue in alcohol, O2 c.c. of 10 per cent, solution of potash, 

 and 200 c.c. of distilled water. On to this float the cover with its 

 surface of bacteria downwards and leave for twenty-four hours ; the 

 film will be coloured blue ; place a few drops of a solution of vesuvin 

 all over the film, which drives out the methylen-blue from all but 

 the bacteria. Finish with alcohol and oil of cloves, and mount in 

 balsam. 



For the same purpose Professor Heneage Gibbes gives a method 

 which has proved of great value. Take of rosanilin hydrochloride 

 2 grms., methylen-blue 1 grm. ; rub them up in a glass mortar. Then 

 dissolve anilin oil, 3 c.c., in rectified spirit, 15 c.c. ; add the spirit 

 slowly to the stains until all is dissolved, then slowly add distilled 

 water, 15 c.c. Keep in a stoppered bottle. 



In the usual way dry the sputum, &c., on a cover-glass and fix in 

 a flame as a few drops of the stain are poured into a test-tube and 

 warmed. As soon as steam rises pour into a watch-glass and float 

 the cover-glass on the warm stain ; allow it to remain four or five 

 minutes ; or if we do not heat the stain but use it cold, let it remain 

 for at least half an hour. Wash in methylated spirit until no 

 colour comes off ; drain, and then dry in an air-oven, and mount in 

 balsam. 



Staining Bacteria in Tissues (Lofner's solution). To 100 parts 

 of solution of caustic potash of 1 : 10,000 add 30 parts of saturated 

 alcoholic solution of methylen-blue. Filter. Stain section for one 

 or two hours, wash out with acetic acid of \ per cent., followed by 

 water. Dehydrate with absolute alcohol, clear with cedar oil, and 

 mount in balsam. 



A process of differential staining of bacillus tuberculosis which 

 was devised by MM. Pittion and Roux was presented recently (1889) 

 to the Societe de Medecine de Lyon, and has met with high com- 

 mendation. It requires three solutions : 



A. Ten parts of fuchsin dissolved in 100 parts of absolute alcohol. 



B. Three parts of liquid ammonia dissolved in 100 parts of distilled 

 water. 



C. Alcohol 50 parts, water 30 parts, nitric acid 20 parts, anilin- 

 green to saturation. In preparing this solution dissolve the green 

 in the alcohol, add the water, and lastly the acid. 



L2 



