TUBERCLE BACILLUS 469 
and non-acid-fast, but may regain their acid-fastness.' When they 
are non-acid-fast they do not appear to niidtiply. The exact signifi- 
cance of these granules (Much granules) is as yet undetermined; 
whether they are identical with the "splinters" described by Spengler- 
is problematical. The "splinters" are usually colored red with fuchsin, 
and they frequently appear in tubercle bacilli that do not stain uni- 
formly, appearing as rows of red, acid-fast granules. According to 
Spengler they may be found in sputum or other tuberculous material 
as heaps of small granules, even if the bacilli themselves cannot be 
demonstrated. 
Isolation and Culture. It is difficult to cultivate the tubercle bacillus 
directly from lesions upon artificial media and it is even more diffi- 
cult to obtain pure cultures directly from sputum, feces, or lung 
cavities where tubercle bacilli are growing in the presence of other 
organisms which develop much more rapidly on artificial media. The 
initial growth on artificial media is the most difficult to obtain. Either 
coagulated dog's serum^ or the Dorset egg medium^ is well adapted for 
this purpose. Tissue containing tubercles is removed from the animal 
with aseptic precautions to sterile Petri dishes. The tissue is minced 
somewhat and then distributed over the slanted surface of either ihe 
serum or the egg medium. At the end of a week or ten days the bits 
of tissue are moved around to fresh surface areas; at the end of two 
to four weeks the tubercle bacilli appear as minute gray nodules 
which gradually spread, forming eventually a wrinkled dull gray- 
yellow growth covering the greater part of the medium. Subcul- 
tures from the original culture grow better on artificial media than 
the original culture, although even subcultures grow very slowly. 
Wherry and Erviu'^ state that a partial pressure of CO2 amounting to at 
least 17 mm. is necessary to induce luxuriant growth of tubercle l)acilli 
in artificial cultural media. Xovy*^ has shown that the most luxuriant 
growths are obtained when freshly inoculated slants of the proper kind 
are sealed with wax, through which a pin-hole is made after solidification. 
Petroff ^ has devised a medium which not only possesses the advan- 
1 Much stain. 
Saturated alcoholic solution of methyl violet 10 cc. 
Two per cent phenol solution (aqueous) 90 cc. 
Procedure: 
1. Fix smears and stain with the carbol methyl violet stain for twenty-four hours. 
2. Remove stain and treat with Gram iodide stain for fifteen minutes. 
3. Wash one minute with 5 per cent nitric acid solution. 
4. Decolorize with alcohol-acetone solution (equal parts) until no more stain can 
be washed out. 
5. Counterstain with 1 per cent safranin (aqueous) 
6. Wash dry and examine. 
Much granules appear as blue-black bodies on a pink background. 
2 Deutsch. med. Wchnschr., 1907, 33, 337. 
3 Coagulated at 75° C; Theobald Smith: Jour. Exp. Med., 1898, 3, 647; Trans. Assn. 
Am. Phys., 1898, 13, 417. 
* Bureau of Animal Industry, Annual Report, 1902, p. 574. 
5 Jour. Infec. Dis., 1918, 22, 194. « Ibid., 1925, 36, 168. 
" Jour. Exp. Med., 1915, 21, 38. 
