2o8 Appendix. 



nutrient broth just before sterilizing. Except in the case 

 of dextrose broth it is important that the muscle-sugar in 

 the meat infusion be removed by inoculating with B. coli. 

 . The reaction of sugar broths shall be neutral to phenol- 

 phthalein. 



Sterilization shall be done in streaming steam in the case 

 of all sugar broths to prevent inversion of the sugar. 



For the routine work of testing samples of water for 

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

 with broths of such strength as to make the resulting 

 mixture one of normal strength. Liebig's Beef Extract 

 may be substituted for beef infusion in the preparation 

 of dextrose broth only: three grams of the beef extract 

 for each liter of broth. 



NUTRIENT GELATIN AND AGAR. 



Nutrient gelatin and agar shall be prepared as follows : 



Gelatin. Agar. 



1. Boil 15 g. thread agar in 500 c.c. 



water for half an hour and make up 

 weight to 500 g. or digest for 10 

 minutes in the autoclave at 110° C. 

 Let this cool to about 60° C. 



2. Infuse 500 g. lean meat 24 Infuse 500 g. lean meat 24 hours 

 hours with 1000 c.c. of dis- with 500 c.c. of distilled water in 

 tilled water in refrigerator. refrigerator. 



3. Make up any loss by evaporation. 



4. Strain infusion through cotton flannel. 



5. , Weigh filtered infusion. 



6. Add one per cent Witte's pep- Add two per cent of Witte's pep- 

 ton and 10 per cent gold ton. 



label sheet gelatin. 



