132 THE AMEEICAN MONTHLY [June, 



present. Plate cultures in gelatine were then made. One small plat- 

 inum wire loop (holding from gL to ^L c. c.) of the fermenting urine 

 was added to a tube containing about lo c. c. of liquid gelatine. After 

 thoroughly mixing, from 2 to 4 loops of the first gelatine were added to 

 a second tube of gelatine. The gelatine was poured on sterile plates. 

 (These were carefully protected from external contamination by means 

 of flamed bell-jars. Sterile " double-dishes" were also used.) They 

 were kept at a temperature varying from 65° to 75° F. The number 

 of loops taken for the first tube of gelatine depended upon the number 

 of germs seen in the microscopical examination. When the germs were 

 exceedingly numerous the first dilution was made in a. tube containing 

 about 7 c. c. of sterile distilled water. The first plate usually developed 

 innumerable colonies after 24 to 48 hours. The second plate as a rule 

 developed from 50 to 300 colonies. As soon as they wxre sufficiently 

 developed they were examined both macroscofically and microscopi- 

 cally and a subculture made in sterilized urine and gelatine from a 

 colony of each apparently different form. The plate cultures were re- 

 peated after the urine had reached its maximum degree of alkalinity, 

 and again after it had been standing for from 2 to 3 weeks. In a few 

 cases parallel plates in agar were made, but no germ was found to de- 

 velop in this medium that did not grow on the gelatine. The subcul- 

 tures were carefully examined, and all those in which the urine culture 

 became alkaline were retained for more careful study. By this process 

 I have isolated from urine about 12 forms of bacteria, mostly micrococci, 

 of which five, on account of their property of producing ammoniacal 

 fermentation in urine, will receive a somewhat detailed description. Of 

 the remaining forms, one was a streptococcus which was present in 

 every specimen of urine examined, two were bacilli, and the remainder 

 were micrococci. The bacilli were very rarely found. 



Culture Media Employed in Testing the Reaction of These 

 Germs. — For a preliminary examination of these germs the ordinary 

 culture media were employed, but in order to test their power to 

 transform urea into carbonate of ammonia, media containing that sub- 

 stance were necessarily resorted to. Of those sterilized, normal urine 

 seemed to be the most practical. Other media, such as acid-beef-in- 

 fusion-peptone and gelatine plus urea, were also employed. Of the 

 ordinary media, nutritive gelatine was the most important from a dif- 

 ferential standpoint. 



(i) Ste7'il urine. — This was prepared by distributing fresh acid urine 

 in sterile culture tubes, about 10 c. c. in each tube. As the urea is 

 broken up by a high temperature the tubes were placed in a large 

 water-bath and heated to a temperature of 65° C. for two hours each 

 day for four consecutive darjs. Their sterility was tested by allowing 

 them to stand for several days in an incubator at a temperature of 36°. 

 C. The urine remained in every instance clear and acid in reaction. 



(2) Acid-bouillon phis urea. — This was prepared by macerating 

 100 grams of finely chopped beef (freed from fat) in 200 c. c. of distilled 

 water. After standing for from 18 to 24 hours in a cool place it was 

 strained through a coarse linen, and to the simple beef-infusion \ per 

 cent, sodium chloride and i per cent, peptone were added. It was then 

 boiled for thirty minutes, cooled, filtered, and \ per cent, urea added. 

 The liquid was then distributed in sterile culture tubes, and sterilized 



