470 THE TUBERCLE BACILLUS GROUP 
tages of the serum agar or glycerin agar media ordinarily used for 
cultural purposes, but it is also adapted for the direct culti^'ation of 
tubercle bacilli from sputum or tubercular material. Sputum or pus is 
shaken with at least an equal volume of normal XaOH, and incubated 
with occasional shaking for one or two hours. The XaOH is cau- 
tiously neutralized with normal HCl (using litmus paper as an indi- 
cator) and centrifugalized. The sediment is smeared upon the surface 
of the Petroff medium. It is usually desirable to make three smear- 
ings, after sixty, ninety and one hundred and twenty minutes respec- 
tively. 
The Peiroff Medium: Infuse 500 gm. of lean meat, finely minced, 
with 500 cc. of a 15 per cent glycerin solution. Keep in the ice-box 
for twenty-four hours. Filter through cloth. Add 1 per cent by 
volume of a 1 per cent alcoholic solution of gentian violet, and mix 
with sterile whole eggs in equal proportions. Solidify in a slanting 
position in test-tubes, using a serum inspissator, at 80° (\ for three 
successive days, one hour each day. 
For bovine tubercle bacilli, the glycerin is omitted. 
The coagulated serum or the e^g medium may be used for subcultures. 
Glycerin agar or glycerin potato is also suitable for this purpose. It 
is essential to protect the cultures from evaporation and to incubate 
them in a slanting position. This is best accomplished by sealing the 
slant cultures after they are made, either with paraffin or with corks 
which ha\e been charred to kill off moulds or other organisms. Then 
covered with lead foil. A pin-hole should be made to permit of renewal 
of oxygen. Tubercle bacilli grow fairly readily on the surface of 
glycerin broth after they have become accustomed to artificial media. 
A fresh thin film from egg medium floated on the surface of the broth 
is the best method of obtaining the growth in this medium. The 
organisms must be floated on the surface of the broth, otherwise growth 
does not take place. If the growth sinks to the bottom all development 
ceases. Tubercle bacilli do not grow readily in gelatin or other artificial 
media not containing glycerin or proteins derived from blood serum 
or egg. Recently considerable attention has been paid to the filter- 
ability of tuberculous material and of tubercle bacilli through the 
coarser grades of stone filters.^ 
Cultures of tubercle bacilli which have been grown on artificial 
media for some time may be gradually accustomed to develop in media 
of simple composition. Proskauer and Beck- grew the organism upon 
the Uschinsky medium to which glycerin was added: Wherry^ and 
Lowenstein'* have employed media in which ammonium salts were 
the only source of nitrogen. Kendall, Day and Walker^ have corro- 
borated these results. Tuberculin appears to be produced even in 
these simple media. Long*' has found that tubercle bacilli of typical 
morphology and virulence grow well upon media containing the am- 
1 See Hauduroy: Presse med., 1926, 34, 227. ^ ztschr. f. Hyg., 1894. 18, 128. 
3 Jour. Infec. Dis., 1913, 13, 144; Centralbl. f. Bakteriol., orig., 1913, 70, 115. 
* Centralbl. f. Bakteriol., orig., 1913, 68, 591. 
s Jour. Infec. Dis.. 1914. 15, 428. « Am. Rev. Tuberc, 1919, 3, 86. 
