OXIDATION -REDUCTION 



183 



OXIDATION-REDUCTION 



E'o represents the potential at any given 

 pH of a system in which the ratio of oxi- 

 dant to reductant is unity. 



In order to get the indicator dyes into 

 single cells the microinjection technique 

 of Chambers is used. Chambers recom- 

 mends dilute aqueous solutions of the 

 basic dyes, i.e., 0.05% to 0.1%, and in- 

 jects successive small doses. Needham, 

 J. and D. M., Proc. Roy. Soc. B, 1926, 

 99, 173-199 ; 383-397 used 1% solution 

 since weaker solutions of particular 

 dyes could not be seen under the micro- 

 scope when injected into cells. 



The determinations are carried out 

 aerobically (cells maintained in a micro 

 drop in water-saturated air at atmos- 

 pheric pressure) andanaerobically (cells 

 held in an atmosphere of purified process 

 nitrogen saturated with water). 



For example, under aerobiosis, if all 

 the indicators down to and including 

 methylene blue are reduced at pH 7.0 

 by cells of a particular type; and if 

 ethyl capri blue is only partially re- 

 duced (and the rest of the indicators not 

 reduced), the reducing intensity of the 

 aerobic cell is approximately —0.072 

 volts at pH 7.0. The same procedure is 

 followed for cells anaerobically. 



To detect the presence of the indicator 

 after decolorization by the cell proto- 

 plasm, reoxidation of the reductant can 

 be accomplished by injecting dilute po- 

 tassium ferri cyanide or of potassium 

 dichromate in the anaerobic state, or by 

 exposure to air in the anaerobic state. 

 The recovery of color on oxidation is a 

 necessary control demonstrating that 

 the indicator has been reversibly re- 

 duced and not reversibly destroyed. 



It is also essential to bring the cell 

 interior into contact both with oxidant 

 and reductant of the indicator. This 

 is necessary to determine whether the 

 indicator, which would shift to the 

 potential of the electromotive system 

 present, is behaving in a truly rever- 

 sible manner. 



The aqueous solutions of the acid 

 dyes, e.g. the various indophenols give 

 the most clear cut results. Upon in- 

 jection they rapidly diffuse throughout 

 the cell before being reduced. The 

 experimental evidence indicates that 

 the speed of reduction of the indicator 

 dyes decreases as the potential of the in- 

 dicator approaches that of the cell. 



In the immersion method slices of 

 tissue are bathed in solutions of the in- 

 dicator dyes. Here it is not only neces- 

 sary to distinguish between penetrating 

 and nonpenetrating indicators but also 

 to watch for differences in the rapidity 

 with which cells and certain cell inclu- 

 sions are stained by the various in- 

 dicators. For example, indicators con- 

 taining the sulfonated radicals do not 



readily penetrate cells, while the non- 

 sulfonated more or less rapidly pene- 



Fildes, P., Brit. J. Exp. Path., 1929, 

 10, 151-175 measured the oxidation- 

 reduction potential of the subcutaneous 

 tissue fluid of the guinea pig, and also 

 its effect on infection. Guinea pigs 

 were inoculated with indicator dyes 

 (0.01%) in both the reduced and oxi- 

 dized states and he observed whether 

 change had occurred. The injections 

 were made superficially so that the im- 

 mediate effect could be seen through the 

 shaved skin. The oxidized form of 

 methylene blue remained a strong blue, 

 and the reduced dye assumed a distinct 

 blue color. This indicated that the sub- 

 cutaneous tissue maintained an oxida- 

 tion-reduction potential on the positive 

 side of reduced methylene blue. 



Then "indophenol 1" (naphthol-2 so- 

 dium sulphonate indo 2, 6 dibromo- 

 phenol) in both states was injected and 

 the animals examined. After 40 min- 

 utes the oxidized and reduced forms of 

 the dye were at about the same intensity 

 of blue. Therefore it was concluded 

 that the Eh^ of the subcutaneous tissue 

 was positive to that of reduced indo- 

 phenol 1. The rate of oxidation was 

 slower here than in the case of methy- 

 lene blue, because the difference in Eh 

 of the tissues and the reduction point 

 of the dye was less. 



Eh = E'o- 0.062 log 



100 -a 



(at 37°C. 



where Eo' is a constant characteristic 

 of the particular system and a = % 

 reduction. 



Finally the dye indicator, "indophe- 

 nol 2" (phenolindophenol 2, 6 dibromo- 

 phenol) was injected. The reduced 

 form of the dye remained colorless while 

 the oxidized form faded from 20 to 80 

 minutes. Addition of ferricyanide 

 failed to restore all the reduced dye, so 

 the results were complicated by decom- 

 position of the dye in the tissues. It 

 was concluded that the Eh of the tissue 

 fluids is positive to the zone of complete 

 reduction of indophenol 1. 



The oxidation-reduction potential of 

 the ciliary body was determined (Frie- 

 denwald, J. S. and Stieher, R. D., Arch. 

 Ophth., 1938, 20, 761-786) by introduc- 

 ing indicator dyes into the stroma or 

 epithelium of ciliary body under aerobic 

 and anaerobic conditions. After equi- 

 librium had been reached, the degree 

 of bleaching was observed microscopi- 

 cally. Then an oxidizing agent was 

 added, such as ferricyanide, and re- 

 covery of the color was noted. The 

 ratio of intensity of color before and 

 after oxidation with ferricyanide gave 

 the potential in the system since it 



