384 LIBBIE H. IIYMAN. 



2. Method of Determination of Carbon Dioxide Production. 

 For this purpose the simple method first suggested by Haas ('16) 

 was employed. A set of standard tubes containing phenol red 

 solutions covering the range of hydrogen ion concentration from 

 pH 6.8 to 8.4 must be at hand. These are now sold by dealers 

 in chemical supplies. For the experiments tubes of the same 

 dimensions are necessary. The animals to be tested are placed 

 in such tubes and those to be compared must be of the same 

 weight. Phenol red in powder form is added to sea-water until 

 the density of color is the same as that in the tubes of the standard 

 set. An equal volume of this water is then added to each of the 

 tubes containing the animals and the tubes then sealed with 

 paraffin. If the inside of the tube above the water is wiped 

 dry, paraffin will adhere to it firmly and the melted paraffin 

 can be then added directly onto the surface of the water. The 

 sealed tubes are placed in a water bath at constant temperature 

 and the changes in tint due to the production of carbon dioxide 

 by the organisms are recorded in terms of pH by comparison 

 with the standard tubes. There is of course some error (probably 

 about .2 pH) in working with sea-water unless the standard 

 sets have been especially prepared for such work. This error 

 is of no consequence in comparative work. 



j. Material and General Procedure. Only freshly collected 

 sponges were employed and these were used as soon as brought 

 in by the collectors. Only the cleanest and most perfect speci- 

 mens were used; those selected were placed in a dish of clean 

 sea-water and repeatedly squirted about with a pipette to free 

 them as far as possible from foreign materials clinging to their 

 surfaces. Unfortunately in the case of sponges it is not possible 

 to determine by inspection whether the specimens are in good 

 physiological condition or not. The selected specimens were 

 placed on a glass plate, osculum and base cut off and discarded, 

 and the body then cut into two nearly equal halves. Tin-so 

 pieces were then placed in the tubes for the respiration tests. 



It was of course necessary to weigh the pieces. They were 

 gently rolled about on hard filter paper until they no longer 

 wet the paper, then transferred to small weighing bottles, pre- 

 viously weighed, and weighed to the fourth place. In the case 

 of the oxygen consumption tests, the pieces were weighed after 

 the conclusion of the experiments. For the carbon dioxide pro- 



