76 BULLETIN 51^ U. S. DEPARTMENT OF AGRICULTUEE. 
These tubes were incubated for two days at 37° C, at the end of 
which time each dilution showing gas was recorded as positive. 
The fermentation tests were in every case started with the 1 to 10 
dihition and carried at least one dilution higher than the plating. 
The denomination of the highest dilution showing positive results 
was reported as the number of gas-producing organisms in the 
sample. This is the generally accepted presumptive test for B. coVi 
group. 
FURTHER EXAMINATION OF THE GAS-PRODUCING ORGANISMS. 
As far as time permitted, one of the higher dilutions from each 
sample showing gas production was plated qualitatively either on 
litmus lactose agar or Endo's medium. From' these plates typical 
coli-like colonies were selected and examined to ascertain whether 
they conformed to the definition of B. coll comiminis as given in 
the 1905 Eeport of the American Public Health Association. For 
this purpose they were subjected to the following tests, morphology. 
Gram stain, motility, liquefaction of gelatin, coagulation of milk, 
production of indol from peptone solution, reduction of nitrates to 
nitrites, fermentation of lactose, and fermentation of dextrose. 
They were then tested for gas production in dulcite, sucrose, man- 
nite, and raffinose, in order to classify them according to the scheme 
outlined in the 1912 Standard Methods of AVater Analysis, American 
Public Health Association. 
CULTURE MEDIA USED. 
The nutrient agar, gelatin, and broth were made from fresh beef 
practically in accordance with the directions given in Standard 
Methods of Water Analj^sis, American Public Health Association, 
except that they were made in larger quantities than there specified 
and were cleared with ^gg white and filtered through paper. 
The lactose bile salt medium was prepared by dissolving 10 grams 
of peptone, 5 grams of bile salt (commercial sodium taurocholate), 
and 5 grams of sodium chlorid in 1 liter of distilled water, filtering 
and adding 10 grams of lactose. 
The sugar broths were prepared by adding 1 per cent of the sugar 
to neutral sugar-free nutrient broth, made from fresh beef. 
The milk was fresh, separator skimmed. It was used both with 
and without litmus. 
ANALYTICAL METHODS USED IN THE "EGG INVESTIGATION" DURING 
THE SUMMER OF 1912, AT OMAHA, NEBR. 
Ammoniacal nitrogen. — Briefly stated, the method consisted in 
making the Qgg solution slightly alkaline with sodium carbonate, 
driving out the ammoniacal nitrogen with a current of air, absorb- 
