V orticellidae 133 



moist chambers, platinum loop, non-absorbent cotton, alfalfa hay, wheat 

 kernels, glass-distilled water, spring water, La Motte buffer mixtures of 

 known pH, agar-slant cultures of the bacterium Achromobacter liquefa- 

 ciens. From the materials listed above prepare the following: 



Standard liquid nutrient: 2 grams alfalfa hay, 3 grams wheat kernels, 

 100 cc. glass-distilled water. Boil 5 minutes, pour off the liquid, filter it, 

 restore to the original volume (100 cc.) by adding glass-distilled water, 

 sterilize under 15 pounds' steam pressure for 10 minutes. 



Activating liquid, solution 1 : 5 cc. sterile standard liquid nutrient, 10 

 cc. filtered sterile spring water, 1 loopful Achromobacter liquejaciens. 

 Approximate pH value 6.2. 



Activating liquid, solution II: Dilute 2 parts of a freshly prepared 

 activating liquid solution I to 50 parts by adding glass-distilled water; 

 to 5 parts of a La Motte buffer mixture of known pH (best results ob- 

 tained when buffers are in the range pH 6.2 to 6.8 inclusive) add 3 parts 

 of the dilute activating liquid. Thus activating liquids may be pre- 

 pared in the pH range of the buffers. 



Method: Prepare a cyst culture by obtaining 50 or more organisms 

 in a Columbia culture dish ; then the glass cover for the dish should be 

 sealed in place with petrolatum and the vessel set aside in a moist cham- 

 ber until starvation and lack of oxygen induces encystment. Activate 

 the cysts by removing the old culture fluid from the dish containing the 

 cysts; wash cysts in three changes of distilled water and cover them by 

 adding either solution I or solution II. At room temperatures of 20 

 to 24 C, excystment begins within 30 to 55 minutes after activation. 

 Conjugation begins approximately 14 hours after activation, reaches 

 its maximum intensity at the end of 24 hours and begins to decline at 

 the end of 36 hours. The duration of conjugation epidemics may be pro- 

 longed for a variable period of 12 to 36 hours by removing all except a 

 few drops of the liquid from the culture dish and adding fresh activating 

 liquid; best results are obtained when this change is made at the time 

 when the conjugation epidemic begins to subside. The method is 

 invariably successful for Vorticella microstoma, V. convallaria, and V. 

 nebulijera var. similis. The excystment technique is a modification 

 of the one described by Barker and Taylor (1933). 



References 

 For the culture of Vorticella see also pp. 60, 134, and 136. 



Bibliography 



Barker, H. A., and Taylor, C. V. 1933. Studies on the excystment of Colpoda 



cucullus. Physiol. Zool. 6:127. 

 La Motte. 1933. The A. B. C. of pH control. Baltimore. 



