26 
North Carolina Agricultural Experiment Station 
cineurn and B. sojae, one vascular parasite of cruciferous plants, B. campestre, 
and a storage rot organism of vegetables and root crops, Bacillus carotovorus, 
have been employed in these studies. The cultures were secured from the 
sources noted in a previous paper (4), and have been maintained in stock on 
potato agar. 
Culture media. Plain bouillon, consisting of 1 per cent Armour’s pep¬ 
tone, 0.3 per cent Liebig’s beef extract and 0.5 per cent NaCl in distilled water, 
served as the basis of the media employed throughout the work. Twenty per 
cent solutions, in distilled water, of the carbohydrates were sterilized at 10 
pounds for ten minutes. Addition of carbohydrate to bouillon was made with 
aseptic precautions. The carbohydrates were of the purity known as “bacto 
sugars” prepared by the Digestive Ferments Company. Prepared media were 
always incubated for twenty-four to forty-eight hours previous to inoculation 
to insure sterility. Strong solutions of HC1 and NaOH have been employed 
in adjustment of reaction. No media were heated after adjustment of reac¬ 
tion or after the addition of carbohydrate. 
pH Determinations. Clark and Lubs (9) standard buffer solutions 
which differed by increments of 0.2 pH were prepared. These were checked 
electrometrically and were found to vary from their assigned value by a few 
points at most in hundredths place. Tubes containing the medium to be tested 
were compared colorimetrically with these standards containing the appro¬ 
priate indicators. By the use of the method of superposition in a comparator 
block, it was possible to make readings correct to within 0.1 pH. A consider¬ 
able number of cultures of each organism w T as employed in each series. Changes 
in reaction w T ere followed by readings at definite consecutive intervals. Each 
consecutive reading w r as made with a different culture. 
EXPERIMENTAL 
It seemed advisable in the beginning to obtain information as to the optimum 
initial pH concentration for growth in bouillon and to the character and rate 
of change of reaction in this medium, and in broths of each of the two compo¬ 
nent materials, beef extract and peptone, before giving consideration to the 
fermentations in carbohydrate bouillons. 
Experiment I~—The Influence of Hydrogen Ion Concentration on the Rato 
of Cell Multiplication in Bouillon 
Cultures adjusted to the reactions shown in Table 1 were grown in flasks of 
250 cc. capacity filled to one-half their capacities for the initial volume of bouil¬ 
lon. To avoid the lag phase, transfers from stock agar w^ere grown for 24 
hours in tubes of bouillon of the same composition with a pH value of 7.2 and 
inoculations were then made from these cultures. A temperature of 23°-25°C. 
was maintained. The changes in growth w T ere followed by removing at two 
hour intervals, under aseptic conditions, a one cc.. sample after having rotated 
the flasks briskly to thoroughly mix the contents. This sample w^as diluted 
and plated in duplicate in potato dextrose agar, composed of 2 per.cent shredded 
agar made up with watery extract from boiled potatoes plus 2 per cent dex¬ 
trose. These plates were counted after an incubation period of four days. 
