348 THE BIOLOGY OF HYDRA : 1961 



variable in that stagnant-but-not-ciowded cultures would turn sexual 

 just as would crowded-but-not-stagnant ones. Indeed this last obser- 

 vation explained why cultures of Hydra placed in an ice-box for 

 several weeks sometimes turned sexual, a method often advocated 

 by earlier workers who believed that they were mimicking the nat- 

 ural drop in temperature found in ponds in the fall of the year when 

 Hydra often spontaneously turn sexual. Our observations suggested 

 that it was the stagnation rather than the lowered temperature that 

 induced sexuality, for we observed other experimental cultures turn 

 sexual at 20°, 25° and 30° (14). 



Analysis of over thirty spontaneously sexual cultures showed 

 that the pO^ was uniformly reduced from the 21% atm. of fully 

 aerated water to about 15% atm. (70% saturation with air). When 

 Hydra were grown in BVC solution whose pOo had been artificially 

 lowered this amount (by aeration with a 15% O^— 85% N^. gas mix- 

 ture), no sexual differentiation took place. Closer analysis (Table 2) 

 revealed that reduced pO^ and sexual differentiation occurred simul- 

 taneously but not proportionately and it was concluded that lowered 

 pOo was not the sole inductive stimulus if indeed it was not merely 

 an unimportant accompanying factor (16). 



2) pCO, 



Shortly after finding that lowered oxygen tension could not 

 substitute for partial anaerobiosis, we began to investigate the 

 possibility that an increase in the partial pressure — or pCOo — of 

 carbon dioxide gas dissolved in the culture solution might be the 

 inductive stimulus. This possibility was difficult to investigate at 

 first because no accurate means of measuring pCO^. existed. As with 

 oxygen tension therefore, it was first necessaiy to develop an accu- 

 rate and convenient determination, and as soon as this was available 

 ( 17 ) it was found that spontaneously sexual cultures routinely 

 showed an increase in pCO^ from the 0.03% atm. of fully aerated 

 water to around 0.60% atm. Indeed pCOo levels as high as 1.2% atm. 

 were found in crowded cultures exposed to 100% oxygen rather than 

 air, and a record level of 1.43% atm. was found to occur naturally 

 in the hypolinmion of a neighboring fresh water pond in April ( 23 ) . 



