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Misses M. Stephenson and M. D. Whetham. 



immediately replaces the empty one, and is closed by stopper D. Mercury 

 (kept under 5 per cent, formalin) is then introduced by means of a sterile 

 curved pipette to form a seal and prevent contamination of the tube whilst 

 allowing sufficient play to the glass rod. The pieces of growth can be ground 

 in the sand and a fairly even emulsion formed. When this is complete, the 

 club and seal are withdrawn with the stopper D and the cotton-wool plug is 

 replaced in the test-tube, after about 25 c.c. of sterile saline have been 

 introduced and mixed with the emulsion. To avoid sowing a contaminated 

 culture, the diluted organism was then incubated for 24 hours and sown on 

 to slopes of tryptic agar, which medium favours the rapid growth of con- 

 taminating organisms. After 24 hours' incubation, the growth on these 

 slopes was examined by the Ziel-Neelsen method of staining. If satisfactory, 

 the original emulsion was sown by means of sterile pipettes, 1 c.c. to each 

 Roux bottle. We have employed this method for a large number of experi- 

 ments and have only once suffered from contamination, which was detected 

 on the slopes before the bottles were sown. 



The bottles thus sown were incubated at 37°, and sample bottles were 

 withdrawn at intervals in order to analyse both the medium and the organism 

 grown upon it. It will be seen that the success of the experiment depends 

 on introducing into each bottle an equal amount of the emulsified culture. 

 The analysis of duplicate Roux bottles withdrawn simultaneously shows 

 agreement within the limits of accuracy of the analytical methods used, as 

 will be shown later. 



Two methods are most frequently used to estimate the amount of growth 

 of any organism : (1) plating out and counting colonies ; (2) separation of the 

 growth by centrifuging, washing, drying, and weighing direct. The former 

 was impossible in our experiments owing to the agglutinated character of the 

 growth ; the latter was equally unsuitable, since the growth was always mixed 

 with the powdered calcium carbonate employed to control the reaction of the 

 medium. In our experiments, therefore, we regarded the protein nitrogen 

 synthesized as a measure of the growth of the organism. This protein nitrogen 

 was precipitated by colloidal iron and estimated by Kjeldahl's method. The 

 other characteristic of the organism to be examined was its fat content. The 

 lipoid material of the bacillus may be conveniently divided into two : 

 fraction A is that extracted from the dried organism by purified ether ; this 

 fraction contains no phosphorus. From the residue after extraction of A is 

 obtained fraction B, by treatment with boiling alcohol and a subsequent 

 second extraction with ether ; B contains phosphorus. For convenience 

 fraction A has been termed the " fat " fraction, B the " phosphatide " fraction, 

 and the sum of the two " total lipoid." 



