July 21, 1923 
Bacterial Spot of Lima Bean 
149 
optimum reaction and toleration limits 
Beef-peptone bouillon was adjusted to each of the following reactions 
with sodium hydroxid and hydrochloric acid: +32, +25, +22, +20, 
+ 15, +10, 4-5, +2,0, —5, — 7, —12, and —16. These were uniformly 
inoculated with 48-hour-old broth cultures and incubated at 24 0 C. At 
the end of 1 week there was growth in all tubes between +25 and —7. 
Heavier clouding developed at 4-20 than at 4-10, but the greenish color 
did not appear in the 4 - 20 medium. A heavy precipitate developed at 
o, 4-2, 4-5, and 4-10. At —5 there was only moderate clouding, and 
very slight growth at — 7. The optimum temperature for growth is, 
therefore, 4-10 to 4-20 Puller’s scale. 
toleration of sodium chlorid 
Beef-peptone bouillon titrating 4-15 (Fuller’s scale) and containing 
0.25, 0.5, 1, 1.5, 2, 3, 4, and 5 per cent, respectively, of sodium chlorid 
was uniformly inoculated from 4-10, beef-peptone agar. There was 
heavy clouding in 0.25, 0.5 and 1 per cent after s days, and a thin pellicle 
was formed. The pellicle would come down intact upon agitation. 
There was only slight clouding and a small quantity of ropy precipitate 
in 3 and 4 per cent after 2 days. No clouding was apparent in 5 per cent 
solution. After 8 days the heaviest clouding was manifest in 0.5 and 1 per 
cent. There was a pellicle and an amorphous precipitate. There was 
a ropy precipitate and medium to light clouding in all concentrations 
except 5 per cent. No clouding occurred in 5 per cent solution, but a 
small amount of ropy precipitate developed. 
Temperature Relations 
In 4-10 nutrient broth the thermal death point lies between 49 0 and 
50° C. This was determined by inoculating 5 cc. portions of the broth 
in thin-walled test tubes of 13 mm. diameter, by means of 2 loops of a 
48-hour-old broth culture and by allowing them to incubate one-half 
hour before plunging them into water held at the desired temperature. 
Five inoculated tubes were held for 10 minutes at each temperature in 
two different tests and were then plunged immediately into cold water 
until they were thoroughly cooled. After this they were incubated at 
25 0 C. Good growth took place in all tubes heated up to and including 
49 0 C., but none occurred above that. 
The relative optimum temperature was determined by incubating 3 
inoculated tubes of nutrient broth and potato agar slants at temperatures 
rangingfrom 3 to 39 0 C. at intervals of two or three degrees. This range was 
repeated, and a third series was run at the higher temperatures 25 0 , 30°, 
31°, 33°> 34°, 37°» 3$° C., respectively. Results were uniform in all 
three series. After 3 days the optimum growth occurred at 28° to 30° C. 
in broth, and at 26° to 30° C. on agar. As the period of incubation 
increased, the maximum growth on agar dropped slowly, appearing at 
24-26° C. after 18 days. Below 23° C. the clouding in broth was not so 
strong and the fluorescence was much more marked than at higher 
temperatures. Growth was very slow at 3 0 C., but cultures remained 
viable for some time at that temperature. At 35° C. there was good 
growth after the first 24 hours but the increase was slow thereafter and 
the characteristic color did not develop. Very slight growth occurred 
above 35° C. After being incubated at 37 to 38° C. for 1 week on 
potato agar, the organism was killed. 
