350 THE BIOLOGY OF HYDRA : 1961 



4) pH 



Generally speaking, Hydra differentiate sexually above pH 7, 

 the optimum being about pH 8. Since a pCO^, of 0.5%— 1% atm. is also 

 required, the original pH of the unused culture solution must either 

 be about pH 9 in weakly buffered solutions such as BVC, or else 

 about pH 8 when strongly buffered with sodium bicarbonate, tris 

 (hydroxmethyl) aminomethane, or Versene, which is a buffer as 

 well as a chelating agent since it is an organic amine. In addition, 

 we have seen that Hydra produce their own buffer — NH4HCO;i — 

 in sufficient amounts to be very important. For example, the water 

 from a dense culture of Hydra may contain as much as 5 mg./l. 

 NH,, (i.e. 10 mg./l. NH4OH). At pH 8, this would be almost entirely 

 in the form of ammonium bicarbonate, this concentration of ammo- 

 nia having served to neutralize COo that otherwise would have 

 created a pCOo of 0.80% atm. Since this newly formed ammonium 

 bicarbonate now serves as so much extra bicarbonate, it is clear 

 that the liberation of NH-; during digestion affects the pH, the 

 bicarbonate concentration and both the pNH:j and the pCO:-. Since 

 all determinations of pCO.. from pH depend on knowing the bicar- 

 bonate concentration ( Henderson-Hasselbalch equation), it follows 

 that all such measurements are suspect in crowded cultures since 

 these can spontaneously increase their bicarbonate concentration 

 through this mechanism. The direct method of measuring pCOo 

 described above, of course, is not subject to this error. 



The powerful effect of buffer concentration is seen in the fact 

 that for an entire year we failed to produce any sexual Hydra when 

 they were grown in 70 mg. T. CaCL.; 350 mg/1. NaCl; and 10 mg./l. 

 NaHC03 (12). When the NaHCO., was increased tenfold to 100 

 mg. 1. (14), almost every culture in the laboratory turned sexual 

 ( 21 ) . In this connection, it is interesting that Dr. Park never observed 

 any sexual Hydra over a period of six years while using an unbuf- 

 fered culture solution composed of 0.4 mg. 1. KCl; 10 mg./l. NaCl; 

 and 4.8 mg. T. CaCL. We have confirmed her observations and also 

 found that Hydra rapidly turn sexual when 100 mg./l. NaHCOa 

 is added to her solution. 



I would now like to describe in some detail a convenient method 

 of growing Hydra (and other hydroids) under constant conditions 



