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



in ordinary cylindrical staining pots having a capacity of 175 cc, 

 the medium consisting of twenty-four to forty-eight hour hay in- 

 fusion plus a small quantity of sterilized chopped hay. This is 

 the second step. In these staining pots the organisms are al- 

 lowed to multiply rapidly for a period of from two to three days 

 giving rise to what we call 'rich cultures.' The first conjugation 

 test is then made. This, the third step in the process, consists 

 in transferring with a pipette used exclusively for one pure line, 

 a thousand or more Paramecium from the staining pot to a Syra- 

 cuse dish and to this collection a little fresh hay infusion is also 

 added which seems to give a stimulus for further, but limited, 

 cell division. The Syracuse dishes are numbered to correspond 

 with the pure line used, and stacked away for daily observations. 

 On the second day there are from 2500 to 3500 Paramecium in 

 each Syracuse dish. All of the pure lines are tested simultan- 

 eously and the fourth step in the process consists in the daily 

 search amongst these thousands of Paramecium for conjugating 

 pairs. For these examinations we use a Zeiss binocular micro- 

 scope with lens combination of no. 2 oculars, and an A2 double 

 objective, giving a magnification of about 24 diameters. In 

 making the examinations the numbers on the Syracuse dish are 

 kept hidden and are looked at only when the record of the exam- 

 inations is made; in this way all of the pure lines are subjected to 

 the same careful scrutiny and if conjugating pairs are observed, 

 the number counted is recorded. The condition of the organism 

 is also recorded by symbols and the minimum and maximum tem- 

 peratures for 24 hours are taken dail . These daily examinations 

 last for a period of from four or five, to ten days, or until the 

 minus sign appears on two or more consecutive records (p. 502). 



Three such tests at least are made for each rich staining pot 

 culture, the first one after two to three days in the rich culture, 

 the second after from five to eight days, the third after eight to 

 ten days, and finally the staining pots themselves are examined 

 for at least four different periods, to see if conjugations are in 

 progress there. Approximately 12,000 different individuals from 

 each pure line are thus watched for a period of several days and 

 if conjugations occur there is little chance of their being over- 



