FURTHER BIOCHEMICAL METHODS VI4.-I] 



distinguishing between the members of the colon group, is acetyl- 

 methyl-carbinol (CHs'CO-CHOII-CHa). The Voges-Proskauer test is 

 described in Leaflet V (p. V44-2O) . A method for accurately detecting this 

 compound, originally i)roposed l)y Lemoigne (191 '3) has been im- 

 proved and described in detail by Kluyver, et al (19'-2.5). The pro- 

 cedure depends u])on oxidation to diacetyl (CHsCOCOCHs), dis- 

 tillation and precipitation in the form of nickel dimethylglyoxime, 

 which shows as characteristic reddish crystals. Stahly and Werkman 

 (1936) show that approximately 84% of the acetyl-methyl-carbinol 

 may be thus determined. 



2,3-Butiilcne Glycol. A further common by-product in the case of 

 organisms of the groups that frequently show the presence of acetyl- 

 methyl-carbinol is 2,3-butylene glycol. A method of determining this, 

 depending upon oxidation to acetaldehyde and subsequent titration 

 of the HCl formed by the reaction between the acetaldehyde and 

 hydroxylamine hydrochloride, has been developed by Brockmann 

 and Werkman (1933). The following method is a modification of that 

 of Brockmann and Werkman. 



Sugar interferes in the alkaline distillation, and if present must be 

 removed prior to analysis. This is accomplished by the CuS04-lime 

 method of Hewitt. (Hewitt, 1932). The liquor to which has been 

 added the copper-lime reagent is brought to definite volume and 

 centrifuged. The supernatant is decanted and filtered. This method 

 also removes citric acid. 



An aliquot of the sample is made alkaline to phenolphthalein and 

 anhydrous Na2S04 added. (10 g. Na2S04 for 50 ml. aliquot). The 

 solution is directly distilled (in a Kjeldahl flask of convenient volume) 

 to saturation (20 ml.), and 14 volumes (280 ml.) removed by steam 

 distillation. The distillate is made up to definite volume, and an 

 aliquot, containing not more than 0.6 mM of glycol, removed, and 

 6 ml. of a potassium periodate reagent (5.75 g. KIO4 dissolved in 

 100 ml. 3.6 N H2SO4) is added. 



Distill into 10 ml. fresh 1% NaHSOa, with the end of the adapter 

 beneath the surface of the NaHSOs solution. Destroy excess bisulfite 

 by adding 0.25 A^ I2, with starch indicator. Destroy aldehyde-bisulfite 

 complex by adding excess NaHCOs, (0.5-1 g.) and titrate the 

 liberated bisulfite with weak (0.()5A0 I2, using starch indicator 

 (Friedemann and Graeser, 1933). Compute the amount of 2,3- 

 butylene glycol from the equation: ml. of I2X normality of l2^4 = 

 cone, of butylene glycol in millimols; (i.e. 1 ml. 0.05 A' 12 = 0.00112 g. 

 butylene glycol). 



If acetyl-methyl-carbinol is present, an abnormally high glycol 

 value results. Acetyl-methyl-carbinol must be determined in the 

 distillate, and one-half the value obtained subtracted from the un- 

 corrected glycol value. (Stahly and Werkman, 1936). 



Interfering Reactions. Many of the methods of analysis may result 

 in serious error, owing to their lack of specificity. Each type of fermen- 

 tation requires a careful selection of methods. The following are a 

 few examples of interference. 



