102 Phylum Protozoa 



stock cultures it should be diluted with one or two volumes of distilled 



water. 



New stock cultures should be prepared every three or four days. 

 Excessive bacterial activity and an accumulation of metabolic waste are 

 inimical to the growth of Didinium, and may lead to the production of 

 structural abnormalities, or to encystment, conjugation, or death. 



Pure lines of Didinium may be maintained with no great difficulty in 

 depression slides kept in moist chambers. The procedure is similar to 

 that employed with stock cultures, but owing to the high rate of reproduc- 

 tion and consequent accumulation of waste, transfers should be made 

 daily to fresh slides. 



A seemingly inherent predisposition toward conjugation sometimes 

 leads to difficulty in the culture of pure lines, and a high mortality among 

 exconjugants sometimes leads to the loss of the stock cultures, if con- 

 jugation assumes epidemic proportions. Fortunately, the tendency to 

 conjugate may be suppressed in most cases by keeping the cultures at a 

 temperature which never exceeds 2 1° C. On the other hand, conjugation 

 may often be induced by increasing the temperature from 2i°C. to28°C. 

 Some races rarely conjugate; others conjugate frequently, and it is some- 

 times advisable to discard the latter. Conjugants are distinctly smaller 

 than vegetative individuals, and the death of the exconjugants is due in 

 some instances to their inability to ingest large Paramecia. A diet of 

 smaller Paramecia may enable them to survive. 



Didinium encysts readily as a result of absence of food, accumulation 

 of metabolic waste, or excessive bacterial growth. Of the three factors, 

 absence of food is most effective in inducing encystment, and it is the 

 factor that is most easily controlled experimentally if it is desirable to 

 obtain cysts. Exhaustion of the food supply leads commonly to the 

 encystment of the animals within eight to twelve hours. While the cysts 

 in my experience do not withstand desiccation, they will remain viable 

 for five or six years in water, and it is often convenient to keep Didinium 

 from year to year in the encysted condition. To store cysts for future 

 use, stock cultures may be made up in vials instead of watch glasses. 

 When the Paramecia are all consumed, many of the Didinia will encyst 

 on the sides and bottom of the vial, or on pieces of hay, if these are pres- 

 ent. Didinium usually encysts against a solid object, the cysts being 

 cemented to the object by the gelatinous ectocyst. The vials should be 

 only half filled with fluid, so that some air will be present when they are 

 finally stoppered and stored away. The cysts may be activated later by 

 replacing the fluid in the vials with hay infusion from a vigorous Para- 

 mecium culture. 



All Paramecia used in the culture of Didinium should be well-fed speci- 

 mens. Much of the difficulty which has been experienced in culturing pure 



