The mass cultures were grown for 16-18 hours at 37 C., thus producing 

 cultures that contained a minimum of degenerated cells. Of course, 

 this procedure did not supply the maximum amount of growth from each 

 planting, but it did supply the optimum material for the chemical analysis 

 of the normal organisms. This point seems to have been entirely over- 

 looked in previous investigations on bacterial composition. In these, 

 the chief interest was to procure the maximum amount of material from 

 each planting. No attention was, therefore, paid to the metabolic plane 

 of the organism. Thus Brieger (i) used growth four weeks old, Leach 

 (60) worked with cultures one to two weeks old, and Vaughan (15), 

 stating no definite period, gathered the growth when it had reached the 

 maximum. 



(b) Planting of Cultures: The agar was allowed to cool slowly to 43- 

 45 C., thus insuring a minimum volume of water of condensation in the 

 petri dishes after solidification. This is a necessary precaution since in 

 too moist a chamber the growth becomes saturated with moisture. Such 

 a condition is very unfavorable for the purpose at hand, because (i) it is 

 impossible to remove any significant part of the bacterial masses from 

 the agar surface and (2) the small fraction that is obtained is contami- 

 nated with diffused substance from the medium. 



The depth of the agar layer in the plates was 4mm. 

 Transplants were made from the special seed plates described above 

 by means o a bent smooth glass rod. 



(c) Removal of the growth from the agar: Two possible procedures sug- 

 gested themselves. One method involves the detachment of the growth 

 from the subjacent agar with bent glass rods, simplifying its removal by 

 washing with physiological salt solution and pipetting off the bacterial 

 suspension (15, 16). It is evident that a maximum amount of material 

 may be obtained in this way but it is not optimum material for the present 

 study. Much of the soluble substances in the medium are washed into 

 the salt solution. Then, too, some substances diffuse from the cell 

 bodies into the outer liquid. Such material may be removed by several 

 washings and centrifugations, each time discarding the supernatant 

 fluid. By doing so, however, the diffusible bacterial constituents are 

 continually being drawn upon. 



The other-method, although not as economical regarding the collection 

 of material, is far superior in providing a pure mass consisting only of 

 "intact" bacterial structures. This process is simpler and involves 

 fewer manipulations than the first. Thus, the smoothened end of a 

 microscope slide is held lightly but firmly on the agar surface and rotated 

 slowly clockwise. At the same time the dish is turned slowly counter- 

 clockwise. The growth is thereby gathered on the slide and very easily 

 tapped off into a thoroughly dried shallow glass dish. 



In order to determine definitely the method best suited for our purposes, 

 tests were made on: 



(1) The supernatant NaCl solution after centrifugati on of thebacter- 

 suspension, obtained by removing the growth from the agar with NaCl 

 solution. 



(2) The supernatant fluid after centrifugation of the bacterial suspen- 

 sion in distilled water, obtained as in (i) with distilled water substituted 

 for the NaCl solution. 



(3) The NaCl solution after being applied to a fresh agar surface 

 and manipulated as though growth was actually being removed. 



(4) The distilled water wash under the same conditions as in (3). 



is 



