470 G. N. CALKINS AND L. H. GREGORY 



is attained for the first time. In order to see if further division 

 of these four would lead to other variations, each was carefully 

 followed and the progeny isolated after each of the next three 

 divisions, eight lines finally resulting from each of the four 

 original cells. 



A. Permanent cultures 



These original thirty-two cells raised to this stage by daily iso- 

 lation in watch glasses, were placed in separate vials and allowed 

 to multiply, the culture fluid (twenty-four hours hay) being 

 changed every two or three days at first, later about once a 

 week. These cultures of pure lines may be designated the 'per- 

 manent cultures.' In adding new food medium the contents of 

 the vial amounting to about 2 cc. are poured off, leaving about 

 0.25 cc. on the bottom, fresh medium then is added to make up 

 2 cc. By this process the Paramecia are kept in good condition 

 and are not over-fed and conjugation rarely occurs. (In April and 

 March two pairs were found in the J 1 line) . In order to safe- 

 guard the cultures against accidents, three complete sets of per- 

 manent cultures are carried along at the same time. Many of the 

 pure lines have been saved by this precaution, for notwithstand- 

 ing the utmost care with them the entire population of a vial is 

 found dead from time to time, the causes of such mortality being 

 '.some slight difference in density or of bacterial flora. In all 

 tests made from permanent cultures all three sets are used. 



B. Petri dish cultures 



In addition to the permanent cultures exposed to daylight in 

 corked vials, other cultures of the pure lines have been kept in a 

 dark cabinet in Petri dishes. Such cultures, however, do not 

 flourish as well as those in the vials — multiplication is less rapid 

 and the organisms require much more frequent change of med- 

 ium. These cultures have not been used in making the various 

 tests. 



