THE NUCLEI OF UROLEPTUS MOBILIS 297 



I. TECHNIQUE 



A. Culture methods 



The first attempts to cultivate Uroleptus on hay infusion 

 failed. The organisms were gradually accustomed to the stand- 

 ard hay infusion diet by placing individuals on successive days 

 in the medimn in which they were found, diluted each day with 

 increasing proportions of fresh standard infusion. In all of these 

 attempts, after the organisms were finally accustomed to fresh 

 twenty-four-hour hay infusion, their vitality was soon lost and 

 death resulted. Fresh hay infusion was then discarded, and 

 boiled flour water, twenty-four hours old, was substituted. To 

 make this, 150 mg. of white flour is boiled for ten minutes in 100 

 cc. of spring water and allowed to stand exposed to the air for 

 twenty-four hours. 



With this medium it was found that the organisms would live 

 and would divide about once in three days. Later, a more sat- 

 isfactory medium was obtained by mixing two parts of this 

 flour water, two parts of spring water, and one part of old hay 

 infusion. This improved medium was used for nearly three 

 months, the individuals dividing approximately once a day. 

 Finally, a still better medium was obtained by boiling 100 mg. 

 of chopped hay with 130 mg. of flour in 100 cc. of spring water 

 for ten minutes, and diluting this, when twenty-four hours old, 

 with an equal part of fresh spring water. With this medium 

 made fresh every day, the organisms divide from one to three 

 times per day, giving abundant material for the study of various 

 vital activities. 



As in previous culture work, a single individual is transffrrod 

 to about 200 mg. of the culture medium contained in a flat, 40- 

 mm. square, ground glass, hollowed dish, 8 mm. in thickness. 

 On the following day the number of individuals is counted and 

 a record is made of the number of divisions that have taken place. 

 A single individual from these is then isolated and transfers, 'd to 

 fresh culture medium made the day before. This procedure is 

 followed daily, the records furnishing data for comparison of the 

 states of vitality of the race that is not allowed to conjugate. 



