W. F. LOOMIS 351 



of pH, pOo, pNH;5 and pCO^. In essence, the method consists of 

 setting these variables in the water of uncrowded cultures twice a 

 day. These twice-daily water changes are usually carried out thirty 

 minutes and five hours after the cultures have been given their 

 daily feeding of brine shrimp. In addition, the closed culture vessels 

 are left on a shaking machine that shakes them strongly every 

 twenty minutes (Fig. 3). Continuous mixing mechanisms, such as 

 tissue culture roller tubes have been found to be either damaging 

 to Hydra or not strong enough to break up the halo zone. 



Figure 5 illustrates the method used in setting the pCOo of a 

 series of Hydra cultures. Between one and ten Hydra are placed 

 in a 30 ml. Pyrex weighing bottle with an interchangeable ground 

 glass stopper within which are placed 25 ml. of culture solution 

 which leaves 5 ml. of air space. Into such vessels are injected 1-10 

 ml. BVC culture solution whose pCO^. has been set at 10% atm. by 

 bubbling it for ten minutes with the gas from a Matheson tank of 

 compressed air containing 10% COo. This bubbling is carried out 

 about once a week, for the CO^-enriched BVC solution can be stored 

 in 100 ml. syringes as shown in Figure 5 since CO^ does not escape 

 from solutions stored in this fashion. Figure 5 also demonstrates 

 the method of filling such 100 ml. syringes from the bottom of a 

 500 ml. graduate in which the bubbling is carried out. 



The daily routine, therefore, consists of filling a 30 ml. dispens- 

 ing syringe from the 100 ml. syringe-reservoir and injecting 0, 1, 2, 

 3, 4 ml. etc. of this solution through a long needle into the bottom 

 of half filled culture vessels and then bringing their total fluid 

 content to 25 ml. When these vessels are shaken, the gas and water 

 phases equilibrate and then remain constant. The actual level of 

 pCOo in the various cultures is determined the next morning by the 

 direct method described above. 



The great solubility of NH-.. makes pNH;, easy to adjust for all 

 that is necessary is to add varying amounts of NH4OH to aliquots 

 of the culture solution whose pH has been set with a bufl^er system. 

 Culture solutions containing various concentrations of NH4OH 

 are thus prepared before the start of an experiment and then stored 

 in capped gallon jugs. Whatever buffer is desired is also included 

 in such solutions, provision being made for the change of pH that 



