\V. F. LOOMIS 345 



syringes to protect the samples from equilibrating with the gases 

 in the atmosphere. Furthermore, the tip of the needle of a syringe 

 may be placed at the exact point from which it is desired to take 

 the sample. 



Our method of determining pOo has been described in detail 

 elsewhere (13, 15). Basically, it consists of drawing 0.5 ml. of 

 leuco indigo cannine reagent into a tuberculin syringe followed 

 by 0.5 ml. of the water sample to be tested. After mixing, the red 

 color that develops is measured at 586 m/x by placing the intact 

 syringe within the light path of a Beckman spectrophotometer, thus 

 avoiding all contamination of the reagent with atmospheric oxygen. 



NH:i is determined in our laboratory by mixing 0.5 ml. of water 

 sample with 0.5 ml. of Nessler solution that has been diluted one 

 to live. The resulting color in the syringe is measured at 480 uifi 

 by the method described above for oxygen. 



pCOo is measured directly by a method that has been published 

 elsewhere ( 17 ) . As originally described, this method required modi- 

 fication of a Henderson-Haldane apparatus, but this has since been 

 found unnecessary, the standard apparatus (New York Laboratory 

 Supply Co. 44250) being found sufficiently accurate for all prac- 

 tical purposes. One analysis takes about three minutes. It consists 

 of 1 ) filling a 20 ml. syringe with 10 ml. of water sample and 10 ml. 

 of air; 2) shaking the half-filled syringe for thirty seconds so as to 

 enrich the gas phase with the CO2 dissolved in the water phase; 

 and 3) measuring the percentage CO2 in tlie aii* phase volumetric- 

 ally in the Henderson-Haldane apparatus by measuring its 

 percentage shrinkage after exposure to NaOH. 



Using these four methods, pH, p02, and pNHg, pCOo may be 

 determined in any given culture in less than ten minutes. Three of 

 the methods require only 0.5 ml. water samples while even the 

 fourth (the pCOo analysis) may be scaled down to 0.5 ml. if a 

 Scholander burette is used in place of a Henderson-Haldane appa- 

 ratus.'^ Alternatively, halo zone water may be prepared in large 

 amounts by growing many Hydra in a closed vessel that is placed 

 on the shaking machine described above so that the micro and 

 macroenvironments are mixed every twenty minutes. Sexual dif- 



^Personal communication from Dr. Leonard Muscatine. 



