76 BULLETIX 51, U. S. DEPARTMENT OF AGEICULTUEE. 



These tubes were incubated for two days at 37° C, at the end of 

 which time each dihition 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 dihition higher than the plating. 

 The denomination of the highest dilution showing positive results 

 was rejDorted as the number of gas-producing organisms in the 

 sample. This is the generally accejDted presumptive test for B. coll 

 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 Enclo's medium. From' these plates typical 

 coli-like colonies were selected and examined to ascertain whether 

 they conformed to the definition of B. coll communis as given in 

 the 1905 Keport of the American Public Health Association. For 

 this purpose they were subjected to the following tests, morpholog3% 

 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 clulcite, sucrose, man- 

 nite, and raffinose, in order to classify them' according to the scheme 

 outlined in the 1912 Standard Methods of Water 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 Analysis, American Public Health Association, 

 except that they were made in larger quantities than there specified 

 and were cleared with egg 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 nitrogeii. — Briefly stated, the method consisted in 

 making the egg solution slightly alkaline with sodium carbonate, 

 driving out the ammoniacal nitrogen with a current of air, absorb- 



