Studies in Fat Metabolism of Timothy Grass Bacillus. 263 



the iayer of chalk on the bottom, which consequently fails in its function. 

 For this or other reasons connected with aeration, the growth of the organism 

 slows down and comes to a standstill long before 

 the. organic constituent of the medium is used up. 

 (2) The withdrawal of samples by means of delivery 

 tubes plugged with cotton-wool proved completely 

 unsuccessful. When the medium was shaken in 

 order to distribute the growth evenly before taking 

 a sample, the scum formed clots or flakes which 

 tended to float, making it impossible to blow out a 

 fair sample of medium through the delivery tube. 



The method finally adopted was identical with 

 that first employed, viz., culture in Roux bottles. 



'/i Mercury 

 seal 



■ 



_4 



The bottles, each containing about 5 grm. of calcium 

 carbonate, were plugged in the usual way and baked 

 for an hour at 130°-140°. Exactly 150 c.c. of medium 

 were then introduced into each bottle, and the set 

 of bottles was steam-sterilized on each of three 

 successive days. 



The culture was first sown on glycerol potato 

 slopes and incubated for four to seven days. A 

 number of test-tubes (6 inch by 1 inch) containing a 

 small quantity of clean sand, together with about 

 5 c.c. of saline, were plugged and autoclaved. Into 

 one of these the growth from the potato slopes was 

 removed by a platinum loop. The growth from one 

 potato slope was sufficient for two or three Eoux 

 bottles. When sufficient growth had been removed 

 to sow the required number of bottles, it was 

 emulsified. 



The emulsification of this organism presents 

 difficulties. The apparatus shown in fig. 1 was 

 employed. A is a solid glass club, the handle of 

 which passes through tubes B and C. These are 

 arranged to form a mercury seal. The rubber 

 stopper D is fitted into a test-tube of the same size 



as those containing the sterile sand and saline, and the whole is wrapped in 

 paper and sterilized in an oven at 120°-130° for an hour. Stopper D is then 

 removed from the empty tube. At the same moment the cotton-wool plug is 

 removed from the tube containing the organism, sand and saline, and this tube 



