344 THE BIOLOGY OF HYDRA : 1961 



Since the pCO- of the macroenvironment had been artificially 

 increased in this negative experiment, it was concluded that high 

 pCO^ was not the sole factor needed to induce sexual differentiation 

 in Hydra littoralis ( 19 ) . The nature of the postulated second factor 

 is still unknown; it does not appear to be simply lowered pOo or 

 pH, or simply increased pNHg either, or all four factors combined, 

 at least in any combination yet tried. Perhaps a fifth feedback factor 

 exists, or even a sixth, but certainly some combination of known 

 circumstances should be able to be brought together in the macro- 

 environment such that even shaken Hydra are exposed to condi- 

 tions equivalent to that found within the halo zone of stagnant 

 Hydra. 



Before proceeding further, it is perhaps instructive to mention 

 that a powerful group effect exists within this 1957 experiment, 

 i.e. no sexual forms appear if one, rather than ten, Hydra are placed 

 in each vessel. Here is an example of the crowding-effect referred 

 to above in which several halo zones overlap to mutually reinforce 

 one another." 



In contrast, single Hydra mature sexually when 0.1% agar is 

 added to the BVC solution in which they are grown as in Figure 1, 

 the viscosity being thus raised sufficiently to stop all thermal cur- 

 rents and hence allow extra large halo zones to form around even 

 single Hydra. Perhaps it is for this very reason that Puck's sludgy- 

 agar method enables single cells to grow in tissue culture when 

 otherwise groups of fifty to one hundred cells are needed as inocula 

 to obtain growth ( 24 ) . 



With the realization that feedback factors associated with halo 

 zone anaerobiosis were active in this system, it became important 

 to develop quantitative means of measuring them. Rapid micro- 

 methods were consequently devised for pOo, pNHg and pCOo, a 

 Beckman micro glass electrode (Beckman 290-31 or 290-80) being 

 already available for determining the pH of unaerated 0.5 ml. 

 samples of water. All four methods are carried out in hypodermic 



-Heisenberg's principle that the act of observing something alters the thing observed 

 enters here, for high levels of pCOs were first tried on ten Hydra so as to be statistic- 

 ally significant. Only later did it become clear that tlie ten Hydra affected each other 

 in a positive group effect so as to turn sexual when a lone Hydra would not, even 

 though he was exposed to as high pCOo as were the ten. 



