TISSUES 77 



of these vital-staining methods is somewhat ambiguous. Investigations of the 

 oxidation-reduction conditions of tissues have been reported by Aubel, Aubertin, 

 Deloyer, Mauriac, Pincussen, Redslob, Reiss, Roclie, Seitz, VeUinger, Vies, Wurmser. 

 and others. 



Suspensions of tissues reduce many oxidation-reduction dyes but Harden and 

 Norris (1914, 1915) found that yeast cells and muscle tissue no longer reduced methy- 

 lene blue when they had been thoroughly washed ; the reducing ability was restored 

 by adding various organic compounds. Harden and Zilva (1915) extended the 

 observation to B. coli. Thunberg's (1921) vacuum-tube technique depends on these 

 observations. Washed tissues or cells are incubated in an evacuated tube with 

 methylene blue. If the washing has been effective there is no reduction of the 

 methylene blue, but when a suitable " metabolite " is added, the methylene blue 

 becomes colourless showing reduction. The methylene blue, in the presence of 

 appropriate enzyme systems of the cells and tissues, effects oxidation or dehydro- 

 genation of the metabolite by acting as hydrogen acceptor. Thus succinic acid 

 may be oxidised to fumaric acid by methylene blue in the presence of washed tissues 

 or cells : — 



Succinic acid + methylene blue -j- enzyme -^ Fumaric acid -|- leuco-methylene blue. 



A considerable volume of work on these lines has been published by Quastel, 

 Stephenson, Whetham, Wooldridge and others using suspensions of washed B. coli. 

 These washed bacteria were originally described as " resting bacteria " but the 

 variable damage caused by the washing process led Kendall, Friedemann and Ishikawa 

 (1930) to the conclusion : — " Finally it must be admitted that the procedure for 

 obtaining ' resting ' bacteria is not quantitative. Attempts to correlate chemical 

 activity with density of suspension were not successful." This and the tendency of 

 the organism to proliferate (Sandiford and Wooldridge, 1931) has led to caution 

 in use of the term " resting " bacteria. The toxic effect of basic dyes on enzymes 

 (Quastel and Wheatley, 1931) also affects the conclusions reached. 



In many of the experiments quoted above the extraneous and unknown effect 

 of methylene blue or other dye is included and Clark and his collaborators (1926) 

 have performed similar experiments in the absence of dyes and observed the 

 oxidation-reduction potentials established. In this way the capacity-effect of the 

 dye and other possible effects are ob\dated. 



When pieces of fresh tissue, such as minced liver, or cell suspensions, e.g., yeast 

 cells, were transferred to buffer solutions kept under anaerobic conditions the oxida- 

 tion-reduction potential fell to a level of about E^ —0-2 volt, which indicates an 

 intense reducing condition. In the presence of a dye, such as methylene blue, the 

 fall in potential was slower as the oxidised form of the dye had first to be reduced 

 to the leuco-form before the potential could fall below the potential corresponding 

 to the oxidised form of the dye. 



When the tissue or cells had been repeatedly washed before suspension in buffer 

 solutions the electrode potential became ill-defined, and the more complete the 

 washing the more indefinite was the potential, but when " metabolites " such as 

 sodium succinate were added the characteristic drift of potential towards a negative 

 (reducing) level was observed. In this case again the addition of dyes, such as 

 methylene blue, in the oxidised condition, retarded the establishment of reducing 



