Feb. 3, 1923 
Bacterial Leafspot of Geranium 
367 
Methylene blue. —Reduction of methylene blue takes place in 7 to 
11 days, according to temperature. Tests for the reduction were made 
in milk colored to a robin’s egg blue. The reduction begins from the 
bottom of the tube and goes upward. In 3 days it is white at the bot¬ 
tom of the tube for one-eighth of the liquid. The rest of the liquid is 
a slightly lighter blue than the control. No coagulation occurs. In 11 
days there is entire reduction in some tubes. When entirely reduced 
the milk coagulates. Those tubes with a slight blue at the surface are 
not coagulated. In 15 days the blue in all tubes is wholly reduced. 
Temperature 20° to 22 0 C. When tubes of methylene blue milk are 
inoculated and placed at 27 0 to 28° reduction occurs throughout in 6 
days. (With the Lewis organism the tests for the reduction of methy¬ 
lene blue were negative.) 
THERMAL RELATIONS 
Thermal death point. —The thermal death point lies between 51 0 
and 51.5 0 C. Transfers were made from well-clouded 24-hour-old cul¬ 
tures and tested many times. The beef bouillon titrating +13 to +17 
on Fuller’s scale (P H 6.8 to 7.0) was held in thin-walled test tubes and 
after transfers were made was kept and heated for 10 minutes at con¬ 
stant temperature in a water bath. Growth occurred at all temperatures 
tried (48°, 48.5°, 49 0 , 49.5 0 , 50°, 50.5 0 , 51 0 ) except 51.5 0 . Once in 
six tubes there was no growth at 50°, but in another six tubes all grew 
at 50.5 0 . In one out of two tests growth took place at 51 0 . None at 
51.5 0 . (The thermal death point of the Lewis organism is 48.5 0 .) 
Optimum temperature. —The optimum"temperature is around 27 0 C. 
Tests were made with temperatures from io° to 40°. The organism 
clouds +15 bouillon in 24 hours from 23 0 to 28°, but in 48 hours the 
growth is heavier at 26° to 28°. (Agrees with the Lewis organism.) 
Minimum temperature. —Growth occurs at i° C. In one test with 
the organism grown in +15 beef bouillon there was a trace of clouding 
in 16 days. The temperature ranged from — i° to 1.75 0 . In a sec¬ 
ond test in which the thermostat ranged from o.6° to 1.25 0 —never over 
1.25 0 and usually under i°—growth was visible in 49 days. It was 
never more than a faint clouding. (The Lewis organism grows in both 
beef agar and beef bouillon at o°.) 
Maximum temperature. —Growth occurs at 35 0 C, but it is very 
feeble. There is none at all at 36° to 37 0 . (Very little difference from 
the Lewis organism.) 
TEST FOR ANAEROBISM 
The organism will not grow in an atmosphere deprived of oxygen. 
Agar and bouillon transfers were placed in a specially devised jar from 
which the oxygen was removed as follows: A solution of 35 gm. of potas¬ 
sium hydroxid dissolved in 350 cc. of water was poured over 40 gm. of 
pyrogallic acid. The bottle for this solution had previously been adjusted 
in a jar where the transfers were. The mixture was poured in the bottle 
and its top left uncovered; but the jar was covered, then another cover 
inserted in a bed of mercury was placed over the whole. A tube of 
methylene blue milk and litmus milk had been placed in the jar with the 
cultures as controls on the presence of oxygen. 
