METHODS OF PHYSIOLOGICAL ANALYSIS 6i 



Winkler method and the respirometer and microrespirometer methods. 

 The Winkler method, as used for aquatic respiration, consists in deter- 

 mination of the amount of oxygen dissolved in water, before and after a 

 period of respiration, by the material concerned in a closed container with- 

 out air. The procedure consists in the addition of the Winkler reagent to 

 a known volume of water and titration. With the respirometer methods 

 the quantity of oxygen consumed is determined from decrease in gas vol- 

 ume measured in the manometer connected with the respiratory changer, 

 the carbon dioxide produced being removed. When the material is 

 aquatic, it is usually placed in a small volume of water in the respiratory 

 chamber, and provision is made for maintenance of equilibrium of oxygen 

 in air and water in the chamber, usually by some type of shaker. Constant 

 temperature is maintained. 



The Winkler method, as used by Hyman and others with various in- 

 vertebrates,' has been criticized on the ground that absorption of iodine 

 by substances in the water, particularly organic substances, such as slime 

 or tissue fluids, may result in incorrect oxygen values.^ 



Carbon dioxide production can be determined or estimated in various 

 ways. The first data on differences along the polar axis were obtained 

 with planarian material by Tashiro with the "biometer" (Child, 1913a), 

 but most of the later work along this line has been done with colorimetric 

 methods. A method of comparative estimation used with Dugesia 

 dorotocephala (Robbins and Child, 1920) and with Corymorpha (Child and 

 Hyman, 1926) is based on color change of a nontoxic acid-alkali indicator 

 (phenolsulphonephthalein) in solution in the water containing the pieces, 

 equal weights of animal material and equal volumes of solution being used 

 for comparison. The rate of change in hydrogen-ion concentration, as in- 

 dicated by change in color of the indicator solution or the time required to 

 attain a certain color as compared with standard colors of known pH, 

 serves for comparison of different lots of material. In another colorimetric 

 method^ the material, in a small volume of water, if aquatic and unable to 

 stand exposure to saturated air, gives off CO^, into the water; it passes 

 from the water into the air of the respiratory chamber and from the air 

 into the indicator solution. Equilibrium between air and indicator solu- 

 tion is maintained by continued to-and-fro movement less rapid than the 



' Hyman, 19166, 1919a, b, c, d, e, 1920a, c, 19236, 1925, 1926a, 1932a; Hyman and Galigher, 

 1921; Child and Hyman, 1926; Lund, 1928. 



^ See Appendix I, pp. 729-31. 



3 Parker, 1925, 1929; Watanabe, 1931; Watanabe and Child, 1933. 



