APPENDIX. 123 



and incubated for twenty-four hours at 37 C, whereby 

 the muscle sugar is removed by fermentation. 



From this fermented infusion, broth is made in the 

 ordinary way, 2 per cent of the desired sugar added, and 

 the medium tubed and sterilized. All sugar containing 

 media should be sterilized with great care to avoid break- 

 ing down the sugar, hence it is advisable to use the 

 discontinuous method, heating to ioo° C. for twenty 

 minutes on three successive days, rather than to give a 

 single heating at the very high temperature. There is, 

 however, but little action at 105 C. 



It is the custom in some laboratories to prepare broth 

 and sugar solutions separately, and mix the two when a 

 sugar test is to be made. 



MILK. 



Fresh milk in a tall jar is placed in the refrigerator over 

 night, to allow the cream to rise. The skim-milk is then 

 siphoned off from below the cream, tubed, and sterilized 

 at uo° C. for fifteen minutes, or on three successive days 

 at 100° C. for twenty minutes. 



LITMUS MILK. 



This is prepared as above, with the addition- of \ c.c. 

 blue litmus solution to give a faintly alkaline reaction. 



If sterilized at a high temperature, reduction of the 

 litmus may take place with loss of color, but oxidation 

 will follow on cooling and exposure to air. 



