418 REDUCTION OF METHYLENE BLUE 



ing the oxygen content in gaseous mixtures by use of the indicator 

 methylene blue. The procedure differs somewhat from the indicator 

 method recently described by Osterhout,^ where organisms are placed 

 in a hemocyanin solution (blue) and the time necessary for them to 

 use up practically all of the oxygen is indicated by reduction to the 

 colorless condition of the hemocyanin. 



In using the milk-methylene blue method it is only necessary to 

 shake a milk-acetaldehyde-methylene blue mixture with the gas to be 

 analyzed and determine how long it takes for the blue color to disap- 

 pear. The end-point can be quite accurately determined by com- 

 parison with a similar tube of milk containing no methylene blue. A 

 control determination of the time necessary for decolorization of 

 milk-acetaldehyde-methylene blue mixture shaken with air must be 

 made under the same conditions. As air contains 21 per cent oxygen, 

 if it takes 60 minutes to decolorize with air and 40 minutes to decolor- 

 ize with the unknown gas, the latter must contain go or f of 21 = 14 

 per cent of oxygen. Carbon dioxide in the gas up to 5 per cent does 

 not afTect the reducing action of milk. 



The rate of decolorization of methylene blue by milk can be in- 

 creased by raising the temperature or increasing the concentration of 

 the reducing enz3rme. This is easily done by evaporating the milk 

 in vacuo to | to | its volume. The rate is roughly proportional to the 

 concentration of the milk. Increase in temperature has the same 

 marked accelerating action as on all chemical reactions, and it is 

 important to maintain the temperature constant in all comparative 

 work. 



The reducing enzyme is unstable and cannot be preserved for 

 any length of time by adding toluene, chloroform, or thymol to the 

 milk. The addition of 2 per cent NaF to milk will prevent the growth 

 of bacteria without affecting its reducing powers during a period of 

 2 months. Some samples of canned evaporated milk which I examined 

 did not exhibit a reducing action. Since colloidal platinum and formic 

 acid reduce methylene blue rapidly, imitating the milk-aldehyde 

 reducing action,^ it is likely that a protected platinum solution 



^ Osterhout, W. J. V., /. Gen. Physiol, 1918-19, i, 167. 



^ Bredig, G., and Sommer, F., Z. physik. Chcm., 1910, Ixx, 34. 



