VI4 2-8 MANUAL OF METHODS FOR PURE CULTURE STUDY 



acids. In this case the total volatile acid in the distillate may be 

 determined by titration, and the acetic acid calculated by difference. 

 The formic acid may be determined by oxidation with HgCl2 and 

 the resulting HgCl weighed (Auerbach and Zeglin, 192l2). The 

 Duclaux distillation method as modified by Gillespie and Walters 

 (1917), or Virtanen and Pulkki (1928), or the partition method of 

 Osburn, Wood and Werkman (1933), (1936), may be used for quanti- 

 tative determination of more complex mixtures. 



The partition method is applicable to the quantitative estimation 

 of mixtures of formic, acetic, propionic and butyric acids, and the 

 qualitative detection of other acids. The basis of the method is the 

 characteristic distribution of an acid between water and an immiscible 

 solvent, such as ethyl ether, when the two are vigorously shaken 

 together. 



Pyruvic Acid. A qualitative test is finding increasing use. The test 

 is not absolutely specific for pyruvic acid but under the conditions 

 used in bacteriology is of qualitative significance. 



Pyruvic acid may be determined qualitatively by a blue color pro- 

 duced with Na nitroprusside (Simon and Piaux, 1924). Two milli- 

 liters of the solution containing pyruvic acid are saturated with 

 (NH4)2S04, 4 drops of a 2% nitroprusside solution are added, plus 

 1 ml. cone. NH4OH. After a few minutes, a blue color is produced, 

 specific for pyruvic acid (and acetophenone). Other ketone com- 

 pounds, such as acetone, acetoacetic ester, acetoacetic acid, creati- 

 nine, and glutathione give color reactions varying from orange to 

 red to purple. 



Pyruvic acid may be determined quantitatively either by the re- 

 action with eerie sulfate, or salicylaldehyde. With the eerie sulfate 

 method, pyruvic acid is oxidized to acetic acid and COo. The CO2 re- 

 leased can be determined manometrically, or the excess of Ce++++ 

 can be determined titrimetrically with FeS04. Lactic acid will inter- 

 fere when present in large amounts. The reaction is specific for alpha 

 keto acids. (Fromageot and Desnuelle, 1935.) 



Pyruvic acid may be determined colorimetrically by reaction with 

 salicylaldehyde plus strong KOH. Oxalacetic acid will not interfere. 

 (Straub, 1936.) Less than 0.1 mg. of pyruvic acid can be determined 

 accurately by this method. Acetaldehyde and acetone also give a 

 color reaction. 



Succinic Acid may be precipitated as the silver salt and weighed, 

 or the silver of the salt may be determined volumetrically (Moyle, 

 1924.) The acid may also be determined quantitatively by the use of 

 an enzyme obtained from beef heart. Succinic acid is extracted from 

 solution with ether and determined by measuring the oxygen neces- 

 sary for oxidation of succinate to fumarate in the presence of the 

 enzyme. (Gozsy, 1935). One mole of O2 taken up is equivalent to two 

 moles of succinic acid. The preparation of the enzyme is described by 

 Weil-Malhcrbe (1937) and Krebs (1937). Potter and Elvehjem (1936) 

 describe a simple mechanical modification to replace grinding with 

 sand. 



Substances which will be oxidized by this enzyme preparation are 



