512 W. F. LOOMIS 



It is a curious fact that many people react negatively to the mere 

 mention of carbon dioxide tension, saying, "Oh, we know all about COg. 

 There is nothing new in that." Most of them, of course, do not know 

 all about CO2, or even about the crucial diiferences between free and 

 combined COg. What they do remember is the headache they experienced 

 studying this subject in their graduate student days. Such at least was my 

 experience, and it was only when hard experience in the laboratory forced 

 me to the conclusion that pCOg was the active variable in my experimental 

 system that I finally sat down and attempted to master the subject both 

 theoretically and experimentally. 



This was about 4 years ago. Before that, I had found that hydra 

 differentiated sexually into mature males and females when they were 

 grown in crowded cultures, but did not do so when grown in isolation. 

 Clearly, the question was, "What is in the water of crowded cultures that 

 makes them differentiate along this new pathway, activating these' pre- 

 viously dormant genes ?" Attempts to take crowded water and use it to 

 turn a single hydra sexual were unsuccessful until it was realized that 

 simple aeration could remove the active ingredient. Here then was a clear- 

 cut system with which to study some of the chemical variables that control 

 cellular differentiation. 



Our first finding was that "crowded water" contained less dissolved 

 oxygen than did water in which only single hydra had been grown. This 

 suggested that lowered oxygen tension was the operative variable. Further 

 experiments showed that this was not the case : lowered oxygen tension 

 accompanied sexual differentiation in hydra but did not cause it. Appar- 

 ently some gas accumulated in the water of crowded cultures that induced 

 hydra to differentiate along the sexual pathway rather than along the 

 asexual. What was this differentiation-controlling gas ? Analysis by infra- 

 red spectrophotometry, mass spectrography and gas-liquid partition 

 chromatography showed that water from crowded cultures of hydra 

 contained increased amounts of gaseous CO., but no detectable amounts 

 of any gases other than those known to be in normal air. Since earlier 

 experiments had shown that no amount of bound CO2, such as bicar- 

 bonates and carbonates, could induce sexual differentiation in hydra, it 

 seemed necessary to conclude that gaseous CO., dissolved in water was the 

 mysterious variable involved. Secondary variables such as ammonia might 

 also be operating in the system, but the ability of free COo to affect cellular 

 differentiation seemed inescapable. This conclusion was strengthened by 

 finding [2] that uncrowded hydra could be turned sexual by growing them 

 in fresh culture water that had been artificially enriched with CO2 gas 

 (Table I). This experiment has now been repeated in our laboratory seven 

 times and, so far, has always reproduced the published results. I need 

 hardly say that this is a hard rock of fact in a field of variable and con- 



