416 REDUCTION OF METHYLENE BLUE 



This paper deals with the reduction of methylene blue by milk and 

 acetaldehyde under varying partial pressures of oxygen. The study 

 was undertaken through the observation that milk will reduce oxy- 

 luciferin to luciferin, the light-producing substance of luminous ani- 

 mals, or methylene blue to leucomethylene blue, only if the milk and 

 reducible material stand for some time.^ The reduction does not oc- 

 cur in either case if the mixtures are continually shaken with air. 

 It appears that time is necessary for using up the dissolved oxygen 

 present. If the oxygen is removed from a milk-acetaldehyde-methyl- 

 ene blue mixture by exhaustion with an air pump, decolorization (re- 

 duction) occurs almost immediately. Without exhaustion of the air 

 such a mixture must remain undisturbed for over an hour at room 

 temperature before reduction occurs. 



Preliminary experiments showed that the amount of reducing en- 

 zyme varied in different samples of milk. The addition of 1 cc. M 

 acetaldehyde to 10 cc. of one sample of milk gave the quickest reducing 

 action. Either more or less aldehyde increased the time. Methylene 

 blue was added till a blue color in the milk was marked (0.1 cc. 0.01 

 M ( = 0.319 per cent) methylene blue was used). With less methylene 

 blue slightly less time is required. The standard mixture was there- 

 fore 10 cc. of milk and 1 cc. m acetaldehyde -f 0.1 cc. 0.01 m methylene 

 blue. If shaken with air (21 per cent oxygen) and set aside in a test- 

 tube at 20°C., the blue color in one particular case completely disap- 

 peared, except at the surface in contact with air, in 43 minutes. If 

 shaken with air containing only half the normal amount of oxygen, 

 decolorization occurs in 23 minutes, about one-half the time. The 

 times for decolorization have been determined for other concentra- 

 tions of oxygen and are given in Fig. 1 where times for decolorization 

 are plotted along ordinates and oxygen concentrations along the 

 abscissae. 



The reduction in concentration of oxygen was accomphshed by 

 partially exhausting the air over the milk-acetaldehyde-methylene 

 blue mixture in a large test-tube with an air pump, at the same time 

 shaking the mixture vigorously. Tubes which have been brought 

 into equilibrium with air in partial vacua can be removed to air under 



5 Harvey, E. N., /. Gen. Physiol., 1918-19, i, 133. 



