APPENDIX 269 



2 minutes over free flame, constantly stirring. Restore loss 

 by evaporation. Filter through absorbent cotton and cotton 

 flannel, passing the liquid through until clear. Titrate and 

 record final reaction. Tube, using lo c.c. in each tube. 

 Sterilize. 



SUGAR BROTHS 



Sugar broths shall be prepared in the same general manner 

 as the standard nutrient broth, with the addition of i 

 per cent of dextrose, lactose, saccharose or other sugar, 

 just before sterilizing. The removal of muscle-sugar by 

 inoculation with B. coli is unnecessary if small amounts 

 of gas are disregarded. 



If, however, test-tubes of small dimensions are used and 

 the presence of any gas, however small, is taken to indicate 

 gas formation the removal of muscle sugar is necessary. 



The reaction of sugar broths shall be neutral to phenol- 

 phthalein. 



Sterilization may be done in streaming steam, as usual, 

 or in the autoclave at 120° C. (15 pounds pressure) for 

 15 minutes. Sterilization in the autoclave seems to be 

 preferable to that in flowing steam. 



For the routine work of testing samples in water for 

 B. coli, especially large volumes of water are to be mixed 

 with broths of such strength that the resulting mixture 

 will be one of normal strength. Dextrose broth made 

 with Liebig's Beef Extract is not equal in effectiveness 

 to that made of fresh beef extract and should not be sub- 

 stituted for the latter. 



LACTOSE BILE 



The lactose bile medium consists of sterilized undiluted 

 fresh ox gall (or a 10 per cent solution of dry fresh ox gall) 

 to which has been added i per cent of peptone and i per 

 cent of lactose. The addition of peptone is important. 



