340 A MANUAL OF BACTERIOLOGY 



(2) While still warm from the heat fixation flood with filtered 

 carbol-fuchsin for half a minute. Again warm for a few second 

 over the flame without actual boiling. Allow it to stand and stain 

 for seven minutes. 



(3) Wash thoroughly in running water, and then decolorise in 

 either of the following solutions : 



(a) In Pappenheim's solution. 1 Place the preparation in a wide- 

 mouthed bottle containing the solution for not less than four, and 

 not longer than twelve, hours. Wash, dry, and mount. Tubercle 

 bacilli are the only organisms stained red. 



(6) In Pappenheim's solution without methylene-blue. Proceed as 

 in (a) ; wash in water and counter-stain for a minute in weak 

 aqueous methylene-blue solution. The tubercle bacilli are biilliantly 

 red. 



(c) In 25 per cent, sulphuric acid. Pour on a few drops of the 

 acid and allow it to act for half a minute. Pour off, and then place 

 the preparation in a wide-mouthed bottle containing the acid for 

 not less than sixteen hours and not more than twenty-four hours. 

 Wash thoroughly, counter-stain with weak aqueous methylene-blue. 

 Tubercle bacilli are the only bacilli which retain the red. 



Acid-fast bacilli in milk and butter. Numerous acid-fast bacilli 

 have been obtained from milk and butter. They usually grow 

 freely and quickly on agar and on gelatin without liquefaction, 

 sometimes as a creamy layer, sometimes as a dry, crinkled film, 

 which may be pigmented (yellow, orange, pale brown or brick red). 

 Some are pathogenic to guinea-pigs by massive intra-peritoneal 

 inoculation only, producing a plastic peritonitis, but not nodules 

 in the organs. In culture, the bacilli are acid-fast and occasionally 

 resemble B. tuberculosis, but are generally thicker. (See Petri 

 Arb. a. d. Kais. Gesundheitsamte, xiv, 1897 ; Rabinowitsch, Zeitschr. 

 f. Hyg., xxvi, 1897 ; Grassberger, Munch, med. Woch., 1899, Nos. 11 

 and 12 ; Tobler, ibid, xxxvi ; Swithinbank and Newman, Bacteri- 

 ology of Milk [Murray, 1903].) 



Grass bacilli and mist bacillus. Moeller isolated from a grass 

 (Phleum arvense) an acid-fast bacillus which he termed the Timothy- 

 grass bacillus ; other grasses also yield acid-fast bacilli (Grass 

 Bacillus II). They grow readily on culture media, and are not so 

 acid-fast as the tubercle bacillus. The Mist bacillus was isolated 

 from dung, and is considered by Pettersson to be identical with 

 the Timothy-grass bacillus. (See Moeller, Deutsch. med. Woch., 



1 Pappenheim's solution consists of one part of corallin (rosolic acid) 

 in 100 parts of absolute alcohol, to which methylene-blue is added to 

 saturation ; 20 parts of glycerin are then added. 



