W. F. LOOMIS 353 



tip of the needle slipped under the ground glass stopper of the cul- 

 ture vessel. Since the air space within the vessel is only 5 ml., it is 

 clear that emptying the 100 ml. syringe into this space flushes it with 

 twenty times its own volume and so sets the air phase to whatever 

 percentage of oxygen one desires. 



Summarizing then, buffer and ammonia concentration are 

 arranged at the start of an experiment by preparing sufficient 

 amounts of appropriate culture solution to last for the duration of 

 the experiment. Then pCOo-enriched water is injected at each 

 water change from a syringe and the air space blown out with 

 No-diluted air. The vessels are then left on a shaking machine that 

 agitates them every twenty minutes. 



Proof that pH, pO., pNH., and pCO. do not change is obtained 

 by analysis. For this, the pH and pCOo are first determined and 

 the results plotted on semi-log paper as in Figure 6. After this it is 

 an easy matter to follow the cultures with a daily pH which yields 

 their pCOo as taken from the logarithmic calibration curve. 



The above system has been gradually evolved over several years. 

 With its aid, we can now analyze and control most of the seemingly- 

 magic variables that affect this system. How can the mere change 

 of one Pyrex vessel for another completely alter the results (Table 

 2)? A second rinse? Leaving the cultures over the weekend? Chang- 

 ing the bicarbonate concentration? Aerating the cultures? These 

 and many other operational variables are reflected in the changed 

 values found in our four feedback variables. Some of them are listed 

 in Table 3 where they are correlated with Rachevsky's four zones 

 as well as with their equivalents in the analogy of the man in the 

 little woolly sweater. 



Perhaps further work will show that sexual differentiation can 

 be chemically induced by appropriate levels of pH, pOo, pNH^ 

 and pCO^, i.e. that it is just a matter of finding correct dosage 

 levels. Alternatively, it may be that further feedback xariables are 

 involved that will need identification, analytic quantitation and 

 artificial application before Hydra will differentiate sexually in a 

 no-feedback system. To date we have run experiments that appar- 

 ently eliminate: carbon monoxide, ethylene, carbonic anhydrase, 

 biotin, folic acid, lactic acid, as well as the possibility that a diurnal 



