Aug. 19,1918 
Reaction on Nitrogen-Assimilating Bacteria 
325 
killed quickly in solutions containing small amounts of acid. The differ¬ 
ence in behavior of the bacteria from alfalfa and lupine is evident. The 
latter are more resistant to sulphuric acid than the former. 
Table III .—Effect of sulphuric acid and sodium hydroxid on the reproduction of nitrogen- 
fixing bacteria 
No. 
Normal acid or alkali in 
100 cc. of medium. 
Concentra¬ 
tion of acid 
or alkali. 
Number of bacteria id i cc. of medium two weeks after 
inoculation. 
Alfalfa 1 
(inoculum 
350,000). 
Lupine 21 
(inoculum 
1,570,000). 
Red clover 
13 (inocu¬ 
lum 
1,300,000). 
Soybean 17 
(inoculum 
1,100,000). 
Azotobacter 
I 3 X- 
1 
Neutral. 
Neutral... 
12,300,000 
30,000.000 
17,100,000 
29,100,000 
1,560,000 
2 
0.1 cc. sulphuric acid- 
Nf 1,000 _ 
7,200,000 
21,900,000 
Lost. 
22,300,000 
212,000 
3 
0.2 cc. sulphuric acid- 
N/soo . 
None. 
700 
Lost. 
None. 
None. 
4 
0.5 cc. sulphuric acid 
NI200 . 
None. 
None. 
None. 
None. 
None. 
5 
0.1 cc. sodium hydroxid. 
Nli,000 _ 
11,900,000 
24*300,000 
15,600,000 
21,900,000 
7,120,000 
6 
0.2 cc. sodium hydroxid. 
Nlsoo . 
9,800,000 
26, TOO,OOO 
17,800,000 
22,300,000 
3,820,000 
7 
0. 5 cc. sodium hydroxid. 
Nfzoo . 
Lost. 
11,500,000 
24,600,000 
15,100,000 
None. 
8 
1.0 cc. sodium hydroxid. 
Njioo ___ 
8,600,000 
5*900 
12,600,000 
7,100,000 
None. 
One very striking fact shown in the data of this experiment is the 
narrow limits of growth of Azotobacter. This organism is readily affected 
by small amounts of acid or alkali, the limits of growth are approxi¬ 
mately A/7,000 acid and A/500 alkali. These data are in agreement 
with the results of previous investigators. For instance, it has been 
shown by Christensen and his associates (5, 4 , 5) that the formation of 
Azotobacter film in mannitol cultures inoculated with soil is correlated 
with the reaction of the soil—that is, acid soils fail to show any film. 
From the data of the previous tests no conclusions can be drawn with 
respect to the acid or alkali limit of growth of bacteria except within a 
relatively wide range. Therefore further tests were arranged in such a 
way as to give a series of cultures of varying concentration of acid and 
alkali. Here the difference between the reactions of the cultures was 
less than in former experiments. Instead of counting the total number 
of bacteria at different intervals, the cultures were incubated for 21 days 
and then tested for the presence or absence of living bacteria. The 
turbidity of the culture was noted, the presence of bacteria determined 
as shown by a stained mount, and mannitol-agar slants were inoculated 
with a loop of the cultures. The presence or absence of growth on the 
agar slants was taken as an indication of the presence or absence of living 
bacteria in the culture. 
A comparison of the development of different legume bacteria in 
media of varying reactions is presented in Table IV. The data here 
reported were obtained from a series of separate tests. The greater 
resistance of the lupine bacteria to acidity as compared with the alfalfa 
bacteria is clearly shown by the results presented in this table. Seven 
different strains of the alfalfa organism and four of lupine were studied, 
and in each test these different strains of the alfalfa and lupine organism 
behaved alike. The acid range for alfalfa bacteria is approximately 
