156 MASS. EXPERIMENT STATION BULLETIN 167. 



extract was prepared from flask a bj^ straining through two layers of 

 cheesecloth and filtering through Swedish filter paper. This extract will 

 be designated as "original' in Table I. The remaining flasks were placed 

 in a water bath at 40° C. and kept for forty minutes with frequent stirring. 

 Flask b was then removed, and the extract was prepared in the same man- 

 ner as the "original." Flasks c, d, e and / were brought to the boiling 

 point. Flask c was removed at the end of ten minutes, d at the end of 

 thirty minutes, e at the end of forty-five minutes and / at the end of sixty 

 minutes. Each extract was prepared after cai'eful counterweighing. The 

 foregoing extracts were used for further study. After twenty-four hours 

 incubation at 37° C. group B was treated identically as group A. 



Medium II. — In this medium Liebig's meat extract was used instead 

 of fresh lean beef, and three separate lots were prepared, namelj^ A, B and 

 C. Lot A was prepared by taking 2 grams of Liebig's meat extract secured 

 from one jar; B, the same amount of the meat extract from a second jar; 

 and C, the same amount, from a third jar. The object of preparing lots 

 A, B and C in Medium II. was to measure any difference which might 

 exist among the containers of Liebig's meat extract. The preparation of 

 these lots from this point is identical. To each, 200 cubic centimeters of 

 distilled water were added and the whole shaken for thirty minutes. At 

 the end of this time, 30 cubic centuneters of the mixture were removed 

 from each flask. The rest was then immersed in the water bath at 40° C. 

 for forty-five minutes. Again 30 cubic centimeters of the mixture were 

 taken out from each. The remainder was then brought to the boiling 

 point and held for ten, forty-five and sixty minutes, 30 cubic centimeters 

 of the mixture being removed at the end of each period. After each oper- 

 ation the flask was carefully counterweighed. 



Medium III. — ■ In this medium two separate lots, namely, A and B, 

 were prepared. Ten grams of Witte's peptone obtained from each of two 

 different bottles were used, respectively, for lots A and B. By this means 

 it was hoped to note any difference existing in Witte's peptone taken from 

 different containers. The preparation of A and B from this point on is 

 identical. After 5 grams of NaCl had been added to each, a paste was 

 made with 50 cubic centimeters distilled water. The whole was then 

 made up to one liter. This was shaken for thirtj'' minutes and treated in 

 the same manner as Medium II., with these exceptions: 100 cubic centi- 

 meters were heated in an autoclave for thirty minutes under 15 pounds' 

 pressure; 300 cubic centimeters were subjected to fractional sterilization 

 for fifteen minutes on three successive days. 



Medium IV. — In this medium three separate lots, namel}', A, B and 

 C, were made up at three different times from the same materials. These 

 different lots were prepared and treated alike, as follows : 10 grams Liebig's 

 meat extract were dissolved in 500 cubic centimeters distilled water. This 

 was thoroughly shaken for thirty minutes. At the same time, 10 grams of 

 Witte's peptone and 5 grams NaCl were mixed separately into a smooth 

 paste with 200 cubic centuneters distilled water, and the volume was 



