188 MANUAL OF MICROBIOLOGICAL METHODS 



Technics for Isolated Reactions 



Reactions Involving Oxidation Reduction 



Thunberg technic. The Thunberg technic for estimation of dehydro- 

 genase activity is especially applicable to cell suspensions and to prepara- 

 tions which are too dense for spectrophotometric measurement (see 

 page 190). In some instances, the Thunberg technic, observed instru- 

 mentally in a colorimeter, provides an economical substitute for a 

 spectrophotometer. 



In general, a tube fitted with an outlet for evacuation and a side arm 

 is used. Buffer, substrate, and methylene blue are placed in the tube, 

 and the bacterial suspension introduced into the side arm. The type 

 and concentration of buffer and substrate depend on the nature of the 

 system studied, while the methylene blue is commonly employed in a final 

 concentration of 1/20,000 to 1/60,000 (1.3 X 10-^ M to 4.4 X 10-^ M). 

 Methylene blue in the oxidized state is blue-colored; in the fully reduced 

 state, the dye is white (leuco) . The system is made anaerobic to prevent 

 the autooxidation of the methylene blue by molecular oxygen; this is 

 generally achieved by evacuation using a water aspirator or vacuum 

 pump and then filling with nitrogen. The tube is placed in a constant- 

 temperature bath, the bacterial suspension tipped in after temperature 

 equilibration, and the rate, time, or both, required for dye reduction 

 measured by visual inspection or by the aid of a photoelectric colorimeter. 

 When methylene blue reduction is measured with the unaided eye, a 

 standard (90 per cent reduction) is usually employed. This is prepared 

 as the experimental tubes with two exceptions: adding one-tenth the 

 amount of methylene blue and using a heat-inactivated cell suspension. 

 As a control, a tube without substrate is included so that the rate of 

 endogenous dye reduction may be ascertained; this endogenous rate is 

 often significant, since the organism may contain a considerable quantity 

 of hydrogen donors. A dehydrogenase is considered present when the 

 methylene blue reduction time in the presence of added substrate is less 

 than the endogenous time. A more complete discussion of this method 

 is presented in Umbreit et al. (1944). 



Many other dyes have been substituted for methylene blue in the 

 Thunberg method, including indophenol derivatives and, more recently, 

 triphenyltetrazolium chloride (Kun and Abood, 1949; Gunz, 1949; 

 Smith, 1951). In each case, however, the general principle is the same; 

 i.e., electrons are transferred from the substrate to the acceptor. 



Acid formation. The availability of pH meters presents a quick, simple 

 method for following acid production during the course of a reaction. 

 When weakly buffered or unbuffered media are used, the time course of 



