VI42-12 MANUAL OF METHODS FOR PURE CULTURE STUDY 



Acetyl-methyl-carbinol is oxidized by CuSOi in the determination 

 of reducing sugars (cf. Stahly and Werkman, 1936, and Langlykke 

 and Peterson, 1937, for correction factors). 



Approximately 5% of the lactic acid volatilizes during steam dis- 

 tillation of the volatile acids. When lactic acid is present in large 

 amounts, the volatile acids should be neutralized, evaporated to a 

 small volume (25-50 ml.), acidified with H2SO4, and again steam 

 distilled. This procedure eliminates most of the lactic acid from the 

 distillate. Thirty per cent of pyruvic acid volatilizes; usually three dis- 

 tillations are necessary to eliminate this acid from the distillate. 

 It is, perhaps, better to determine the volatilized pyruvic acid by 

 eerie sulfate oxidation (Fromageot and Desnuelle, 1935) or by the 

 iodoform reaction (Wendel, 1932) and apply a correction for this acid. 



Acetone is usually determined by the iodoform reaction. Any other 

 neutral volatile compound which gives the iodoform reaction will, 

 of course, interfere with this method, particularly acetyl-methyl- 

 carbinol, nearly 60% of which volatilizes during a half volume dis- 

 tillation. The acetyl-methyl-carbinol in the distillate can be deter- 

 mined as nickel dimethyl-glyoximate and a correction applied, or 

 the procedure of Langlykke and Peterson (1937) may be used. 



Acetyl-methyl-carbinol and 2,3-butylene glycol interfere in the 

 determination of lactic acid. They may be removed by alkaline 

 steam distillation (14 volumes) from a solution saturated with 

 Na2S04. The lactic acid is determined on the residue of distillation. 

 When sugars are present, alkaline distillation causes caramelization 

 and consequently, interference with both the glycol and lactic acid 

 determinations. Separation of the glycol from the sugar and lactic 

 acid may be accomplished by extraction of an alkaline solution with 

 ether continuously for 72 hours. The glycol is recovered in the extract. 

 The interference of sugar may also be avoided, without extraction, by 

 removing the sugar by copper-lime treatment (Hewitt, 1932) and 

 then making an alkaline distillation. 



Determination of Dehydrogenases 

 The determination of the presence of a specific dehydrogenase may 

 be made by the Thunberg technique (methylene blue reduction). 

 There are many modifications of this procedure (e.g. Hopkins and 

 Dixon, 1922; Yudkin, 1933). These modifications are concerned 

 with methods of obtaining anaerobic conditions and amounts of 

 reactants. 



The essential points of the procedure are: 



1. To have a glass tube with a side arm or hollow stopper in which 

 anaerobic conditions can be maintained. 



2. A constant temperature water-bath. 



3. An adequate buffer. 



4. An accurate control. 



Anaerobic conditions may be obtained by vacuum, vacuum fol- 

 lowed by oxygen-free nitrogen, or by oxygen-free nitrogen alone. If 

 the latter is employed, the apparatus should be arranged to allow 

 bubbling of nitrogen through the reagents for a few minutes. 



