52 GEORGE ALFRED BAITSELL 



isolated on a depression slide in some of the culture medium and 

 placed in the moist chamber. When it had produced four individ- 

 uals by division, these were isolated and thus the four main lines 

 of a culture were started. These four lines were examined daily, 

 the number of divisions observed and recorded and one animal 

 isolated from each to continue the main lines. The slides from 

 which the isolation took place were saved as stock in order to re- 

 plenish the main lines in case any of the individuals isolated were 

 lost through accident. In this work three days stock was kept 

 which, with the main lines, made in all sixteen slides under obser- 

 vation in a culture. For isolation, capillary pipets were made use 

 of, a sepaj-ate one being reserved for each culture. In all the iso- 

 lation work the greatest care was taken to prevent contamination 

 of the cultures. All pipets and slides that were used were care- 

 fully sterilized immediately before using. A dissecting micro- 

 scope with a 10 multiple lens was used in all the isolation work. 

 Permanent preparations of the individuals from the cultures were 

 made from time to time. The method used was that of Calkins 

 ('02a) and Woodruff ('05). ^ 



Two culture media were employed ; a ' constant ' medium which 

 consisted of a 0.025 per cent, solution of Liebig's extract of meat, 

 and a hay infusion medium. There are at least two essential con- 

 ditions for a 'constant' culture medium. It must contain the 

 elements necessary for the maintenance of protoplasm and it must 

 be a medium in which bacteria will develop readily, in order to pro- 

 vide food for the animals. A solution of beef extract fulfils these 

 requirements and previous work by other investigators having 

 shown that strong solutions of beef extract will artificially stimu- 



2 This method is given by Woodruff as follows: "The specimen to be preserved 

 is isolated by means of a fine-pointed pipet on a clean depression slide (which is 

 kept just for this purpose) with as little of the culture medium as possible. To 

 this is added three or four drops of bichlorid of mercury in saturated solution with 

 5 per cent, of glacial acetic acid. After about five minutes the specimen is trans- 

 ferred to another slide and a few drops of 75 per cent alcohol is added. A slide is 

 now smeared with a trace of egg albumin and the specimen is taken from the 75 

 per cent alcohol and gently spurted on to the albumin. After a short time, when the 

 alcohol has coagulated the albumin, the slide with the specimen adhering to it is 

 transferred to a jar of 75 per cent, alcohol and thereafter treated by the ordinary 

 slide method." 



