MEDIA MAKING AND APPARATUS 267; 



neutralizing it). Autoclave: when cool, inoculate with a pure 

 culture of B. coli and incubate 24 hours at 37 C. If gas collects 

 in the closed arm as the result of growth, a considerable quantity of 

 sugar is still present. If no gas collects, but a growth occurs in 

 the closed arm, a trace of sugar is still present. If the closed arm 

 remains free of growth, all the sugar has been removed. 



FURTHER STEPS COMMON TO ALL MEDIA, 



6. Adjust to the phenolphthalein 1.5% (normal) acid point 

 (except that broths containing added sugars should be adjusted to 

 the phenolphthalein neutral point). See below. 



7. Heat over boiling water for 30 minutes (i. e., the medium 

 itself should be at or about ioo° C. for just 30 minutes). 



8. Restore loss by evaporation to double original weight of 

 filtrate (Step 2; i. e., to 2,000 grams) ; check and, if necessary, ad- 

 just reaction. 



9. Boil over a free flame for just five minutes : counting the 

 five minutes from the beginning of actual boiling. Stir constantly 

 to prevent burning, especially in making nutrient gelatin of any 

 variety. 



10. Restore loss by evaporation; check and adjust reaction. 



11. Filter, tube, and sterilize. 



Note : In all cases, the weight of the medium before filtering, 

 i. e., at the completion of Step 10, should be double the weight of the 

 filtrate obtained in Step 2. 



Note : About 5 c.c. of agar, gelatin or liquid media should 

 be placed in each test tube, except that for all media to be used in 

 plating, 10 c.c.'s (exactly) should be used. 



Note : The Committee recommends alternatively, the addition 

 of sugars and litmus just before tubing and sterilization in Step n,, 

 instead of in Step 5 as above. 



