MUCH GRANULES 407 



seconds. He believed that the reason why the bacilli were resistant to ordinary- 

 stains was that they were surrounded by a capsule which was permeable only to 

 alkalies. Ziehl (1882) showed that this conception was wrong ; the bacilli could be 

 stained quite satisfactorily by a dye of acid reaction. The stain he advocated was 

 a 2 per cent, alcoholic methyl violet solution in carbolic acid water. Later Ziehl 

 employed carbol-fuchsin. Neelsen increased the strength of phenol in the stain, 

 and the Ziehl-Neelsen method is the one that is now usually employed. 



It consists in covering the film with carbol-fuchsin (basic fuchsin 1 part, absolute 

 alcohol 10 parts, and 5 per cent, phenol in water 100 parts), and gently heating till the 

 steam rises ; the heating is continued for 5 to 15 minutes, the water lost by evaporation 

 being replaced by fresh stain. The film is then washed thoroughly in water, and treated 

 with a 15-20 per cent, solution of a mineral acid. If the film is from a pure culture, the 

 effect of the acid will be merely to dissolve the excess stain ; but if a film of tu))erculous 

 pus or a section of tuberculous tissue is being treated, the acid will turn the preparation 

 yellow, indicating that the stain has been removed from the tissue ceUs, or from other 

 organisms that may be present. The treatment with acid is continued for 5 to 10 minutes 

 as a rule, tiU subsequent washing with water causes no more than a faint pink tinge to 

 reappear. The film is then thoroughly washed in running water to remove all the acid. 

 It is counterstained with a 1 per cent, aqueous solution of methylene blue for 5 minutes, 

 after which it is washed and dried in the usual way. By this method the acid-fast bacilli 

 are coloured red, while the tissue cells and all other organisms are coloured blue. Some 

 workers prefer a yeUow counterstain — usually 1 per cent, picric acid. The success of 

 this method deijends partly upon the heat employed, which renders the waxy material 

 in the tubercle bacillus more permeable to aqueous dyes, and partly on the phenol, which 

 acts as a mordant. 



Numerous other methods of staining have been described (Spengler 1907, 

 Herman 1908, Mori 1911, Bozzelli 1914, Schulte-Tigges 1920, Kieffer 1921, Shoub 

 1923, Pottenger 1942). 



The property of acid-fastness appears to be due to the presence in the bacilli 

 of unsaponifiable wax (Anderson 1932). This substance was apparently first 

 isolated by Aronson in 1898. It was referred to as one of the higher alcohols by 

 Bulloch and Macleod (1904), and was termed " mykol " by Tamura (1913). The 

 larger the amount of cbloroform-soluble wax, the greater is the resistance of the 

 bacilli to decolorization (Darzine 1932). The tubercle bacilli contain more of this 

 substance than the saprophytic acid-fast bacilli (Table 25), and are therefore 

 usually more strongly acid-fast. The degree of acid-fastness, however, is dependent 

 on a number of factors, and no reliance should be placed on it in determining the 

 particular type of organism under investigation. Not all workers are agreed on 

 tbe simple chemical explanation of acid-fastness just given. Sordelli and Arena 

 (1934), for example, state that it is a property of intact bacilli, and believe that it 

 depends on the existence of a semi-permeable membrane around the organisms 

 which allows fuchsin to diffuse in but does not allow acid fuchsin to diffuse out. 



In formol-fixed tissue sections the bacilli often stain very poorly with Ziehl- 

 Neelsen. According to Fielding (1934), this is due to the development of an acid 

 reaction following autolysis of the tissues, and can be overcome by fixing in a 

 weakly alkaline solution of formol, or by staining with alkaline fuchsin. 



Much's granules. — Much (1907) brought evidence to show that under certain conditions 

 the bacilli might be present in the tissues in the form of non-acid-fast granules. Starting 

 from the ol)servation that in the Perlsucht nodules of cattle, and in cold abscesses of man. 



