190 MANUAL OF MICROBIOLOGICAL METHODS 



required to oxidize one molecule of diphosphopyridine nucleotide. Since 

 the oxidation of reduced diphosphopyridine nucleotide by ferricyanide is 

 a relatively slow process in low concentrations of the latter, ferricyanide 

 is added to the reaction mixture in an amount at least twice that required 

 for oxidation of the substrate present. 



A slight difference in stoichiometry exists for the case of simultaneous 

 acid production and diphosphopyridine nucleotide reduction. 



CH3CHO + DPN+ + H2O ^ CH3COO- + DPNH + 2H+ 



Here, three molecules of CO2 are produced per molecule of substrate oxi- 

 dized when measured by the ferricyanide manometric method. 



Spectrophotometric measurements. The course of a given reaction is 

 often followed spectrophotometrically, advantage being taken of the fact 

 that the change in concentration of one of the products or reactants may 

 be observed as a change in light absorption at a given wavelength. The 

 classic examples of this technic are reactions which involve di- or tri- 

 phosphopyridine nucleotide. These nucleotides exhibit an absorption 

 peak at 340 m/i when in the reduced state, while the oxidized forms do not 

 absorb an appreciable amount of light at that wavelength. Therefore, 

 during the course of a reaction which results in reduction of pj^ridine 

 nucleotide, light absorption at 340 mju increases. The measurement may 

 consist of readings taken at definite intervals after initiation of the reac- 

 tion, in which case the time course of the reaction is plotted, or readings 

 may be taken at the beginning and end of the reaction, in which case only 

 the extent of the reaction is determined. 



Other applications also involve changes in optical density (absorption) 

 at a particular wavelength of light, such as the decrease in absorption at 

 265 mjjL during the deamination of adenosine-5-phosphate or the increase 

 in absorption at 220 mju during formation of certain keto compounds. 



The method obviously requires previous knowledge of the spectrum of 

 the compound to be measured. In addition, the extinction coefficient 

 for the compound at the particular wavelength employed must be known 

 if the measurement is to be completely quantitative. 



Although several types are available, the most commonly used spectro- 

 photometer is the Beckman model DU. A complete instruction manual 

 is supplied with this instrument. 



Nitrogen Metabolism 



Proteins. Proteins are estimated quantitatively by the methods pre- 

 viously described (see pages 179-180). The results of total nitrogen 

 determination (pages 173-174) are converted to protein values when 



