BOUILLON MEDIA 45 
Hasseltine (1917) has confirmed these results and has emphasized 
the importance of this question in the differentiation of members 
of the B. colt group. The work of these investigators emphasizes the 
necessity of using some other method then heating for sterilizing media 
containing disaccharides and trisaccharides. 
Trypsin Broth (Gordon et al). Take some fresh bullock’s heart 
free from fat and vessels, mince the meat very finely and weigh. To 
each 3 kilo add 1 liter of water and make faintly alkaline to litmus 
with 20 per cent KOH solution. Heat this slowly to 75° to 80° C. 
for five minutes. Cool to 37° C., and add 1 per cent of liquor trypsine 
comp., and keep it at 37° C. for 23 to 3 hours. When trypsinizing is 
finished, test for peptone with copper sulphate and KOH, then render 
slightly acid with glacial acetic acid, and bring slowly to the boil for 
one-fourth hour. Leave over night in a cool place, and siphon off the 
clear liquid in the morning. Make faintly alkaline to litmus, and 
sterilize in an autoclave at 118° C. for one hour on each of two days 
(if not to be used at once). 
Levene (1918) and Burling and Levene (1918) have stated that 
0.5 per cent lactose broth should be used in the presumptive test for 
coli bacteria in water analysis. With the higher carbohydrate con- 
tent of the broth, the limiting hydrogen ion concentration is reached 
more quickly. 
Nitrate Broth. This is a special broth containing nitrite-free 
nitrate. It may be prepared in different ways. Standard methods for 
the Examination of Water and Sewage, 1912, recommended a medium 
of the following composition: 
Tap water.... 0.0.0... eee cee ee eee 1000 c.c. 
Witte’s peptone..... 2.2... cee ee eee ee eee 1 gm. 
Potassium nitrate (nitrite free).............. 0.2 gm. 
Another formula is as follows: 
Distilled water... 2.0.2.0... ccc ce cece eeeaee 1000 c.c. 
Leibig’s meat extract..... 2.0.0.0... eee eee 3 gms. 
Potassium nitrate c.p. KNO3............... 10 gms. 
The following has been used with some success: 
Distilled water..... 2. .....0 fee eee ee eee eee 1000 c.c. 
Leibig’s meat extract...... ....-.--.--2205. 3 gms. 
Witte’s peptone... . 1.02... eee eee eee ee 10 gms. 
Potassium nitrate, KNO3......... pines eens 3 gms. 
