Parameciidae 113 



bacteria, by their own efforts, can not reach the top in so short a time. 

 Usually, however, one such washing does not free the Protozoa from 

 adherent or ingested bacteria, and in most cases it is necessary to repeat 

 the procedure once or twice, each time sucking up beneath the fresh 

 column of sterile water in a fresh pipette about 2 inches of fluid from the 

 top of the previous pipette. In some cases it is advisable to leave the first 

 or second pipette for 18 to 24 hours to give the surface migrants a chance 

 to evacuate the remains of ingested micro-organisms and to multiply to 

 some extent. This procedure is followed by another rapid washing. 

 Finally a drop of the surface fluid is inoculated into a tube of sterile 

 medium (see Part II). In this manner the ciliates Trichoda pur a, three 

 strains of the thermal ciliate Saprophilus ovijormis from Hot Springs, 

 Virginia, a large undetermined ciliate, Paramecium caudatum and P. 

 multimicronucleatum, and the flagellates Chilomonas Paramecium, Para- 

 polytoma satura, and an undetermined monad from the intestine of the 

 fly, Lucilia caesar, were all freed of bacteria, as shown by consistently 

 negative findings in stained films and aerobic and anaerobic cultures on 

 routine laboratory media at both room and incubator temperatures. The 

 upward migration here involved is a genuine geotropic reaction, as it 

 occurs in pipettes held in the dark and in those sealed without any air 

 space at the top. 



This same pipette method may be used for positively geotropic Proto- 

 zoa. The pipette is nearly filled as before with sterile water, the tip is 

 sealed and then a small amount of the contaminated culture is layered on 

 top of the sterile liquid. After a suitable time the end of the pipette is 

 cut off and one or two drops either inoculated into culture medium or 

 placed at the top of another water column for a second washing. In this 

 way an undetermined free-living monad was freed of bacteria after two 

 washings, each consuming about 30 minutes. With either negatively 

 or positively geotropic organisms the addition of killed yeast cells (see 

 Part II) to that part of the water column toward which the Protozoa 

 were to migrate, accelerated their migration. 



Migration through V-Tubes. In the second method, V-shaped tubes 

 are used, one arm of the "V" being 12 cm. long with an inside diameter 

 of 28 mm., the other 9 cm. long with an inside diameter of 8 mm. The 

 tube, after sterilization, is filled with 15 cc. of melted semi-solid medium 

 (see Part II) . When this has set to a soft gel the contaminated Protozoa 

 are introduced by means of a long fine capillary through the small arm 

 into the bottom of the large one. The tube is permitted to stand for a 

 length of time dependent of the Protozoa concerned, and then samples 

 are taken from the surface of the medium in the large arm. One such 

 treatment frequently suffices. A modification of this technique was used 

 to separate Spirillum undulans from other bacteria. A loopful of con- 



