\-(:)L. 12 (1953) GROWTH AND PHAGE PRODUCTION OF B. megatherium iv 



43 



Fig. 2. Effect of addition of phage 

 on the rate of production of 

 phage by megatherium 899a. Cell 

 suspension prepared as in Fig. i 

 and 10 ml placed in each of 2 

 tubes, 8 X lo^ "T" phage/ml 

 added to one tube and both tubes 

 shaken at 34°. 



appears in the equation as the catalyst, but does not act as the catalyst when added from 

 an outside source, it follows that the P/'ml formed 

 during the course of the reaction must be proportional 

 to the catalyst, although it is not the catalyst itself. 



The conclusion that the ratio of PjB, RNA/B 

 and protein/5 is constant during the log growth period 

 is only approximately correct in the experiment just 

 described, since this period exists over a small part of 

 the curve. Theoretically, a true "steady state" exists 

 only when the composition of the medium remains 

 constant. This condition can be strictly fulfilled by the 

 addition of more medium at a constant rate exactly 

 equal to that required to maintain a constant cell 

 concentration. It can be approximated by working in 

 the range of very low cell concentrations or by repe- 

 ated dilution of the growing culture. (Very dilute cell 

 suspensions are impractical owing to the difficulty of 

 collecting enough cells for analysis.) 



Preliminary experiments showed that the culture 

 remains in log growth if the cell concentration is 

 maintained between i-io^ml and about S-io^ml by 

 repeated dilution. The growth is the same if the cehs 

 are centrifuged and suspended in fresh media, or if the suspension is simply diluted 

 1/5 in fresh media. 



The results of an experiment in which resting cells were grown in 5 % peptone and 

 diluted 1/2 as soon as they reached a concen- 

 tration of S-io'/ml is shown in Fig. 3. The 

 phage growth rate is 6-9 per h for the first 2.5 

 h, while the cell growth rate gradually increa- 

 ses from 1.3 to 1.7 per h where it remains 

 constant. The cells are now in log growth. As 

 soon as this occurs the phage rate also drops 

 to 1.7 so that, from this point on, the ratio of 

 phage/cells is constant. The results are still 

 somewhat irregular, as only one plaque count 

 was made on each sample, and the cells did 

 not reach the log growth until near the end of 

 the experiment. 



The experiment shown in Fig. 4 was 

 started by allowing a suspension containing 

 about i-io^ Bjml to stand at 25° for 18 h. 

 The cells grow very slowly during this time, 

 owing to lack of oxygen, but pass through 

 most of the lag phase. The tube was then shaken at 34°. The free P/ml and cells/ml of 

 the suspension and the RNA and protein of the washed cells were determined at inter- 

 vals. As soon as the cell concentration reached 5- loVml, the suspension was diluted 1/5 

 with fresh 5% peptone, and this cycle repeated 3 times. 

 References p. 50. 



ICf' 



E icf 

 a. 



03 Iff 



i^^lK^^'^ 



3.5 40 

 Hours 



Fi 



3. Changes in P/ml and B/ml in 

 culture of 899a, kept in log growth by 

 repeated dilution. Cell suspension prepared 

 as in Fig. i and shaken at 34°. Diluted 

 1/2 with fresh 5% peptone when Bjml = 

 8 • lo'and every half hour thereafter. 



