FURTHER BIOCHEMICAL METHODS vi4,-7 



sequent freeing of acid or base. Accordingly, direct comparisons be- 

 tween results in different basal media should not be made. 



Residual Sugar. Determination of sugar in cultures and in uninocu- 

 lated controls may be made by the method of Shaffer and Hartmann 

 (1921) or its modification by Stiles, Peterson, and Fred (1926). Both 

 are iodometric modifications of the Fehling procedure. In using this 

 analytical method it is important that the medium contain only a 

 little more sugar than the bacteria can use. The method has its great- 

 est accuracy only within certain limits, so it is important that wher- 

 ever possible the amount of reducing sugar in the aliquot lie within 

 those limits. Accordingly, preliminary determinations with varying 

 percentages of sugar are often necessary before deciding on the most 

 suitable concentration or the most satisfactory volume to employ 

 for an aliquot. 



It is understood that the method is not as accurate in media con- 

 taining beef broth as in solutions that are free from it. It cannot be 

 used in the presence of nitrites; but these may first be removed by 

 heating in the presence of urea and acid. 



Quantity of Acid Produced {Titratable Acidity). Titration of an ali- 

 quot sample of a culture with standard alkali to an arbitrarily chosen 

 end-point (usually phenolphthalein or phenol red) is often employed 

 (after deduction of corresponding blank titration value) as a measure 

 of the quantity of acid products present. The sample may be boiled 

 before titration if it is desired to exclude COifrom the determination. 

 The results are most directly expressed in terms of normal acid, or as 

 milliliters of N /lO acid per 100 ml. of culture. They are sometimes ex- 

 pressed presumptively in terms of the predominant organic acid (e.g., 

 lactic acid) assumed to be produced by the bacteria. 



Nature of Acids Produced. To neutralize the acids produced, an 

 excess of CaCOa may be added to the medium (see p. VI42-6). Or if it 

 is not desirable to have carbonate present an indicator may be added 

 and sterile NaOH introduced aseptically from time to time from a 

 container sterilized with the culture flask. Incubation should con- 

 tinue to completion. 



The acids most frequently present are: (1) the volatile fatty acids, 

 formic, acetic, propionic and butyric; (2) the non-volatile acids, 

 lactic and succinic. Separation of the volatile acids is ordinarily 

 effected by steam distillation after acidification with H2SO4 to pH 2.0 

 to liberate the acids. It is necessary to collect twelve volumes of dis- 

 tillate; e.g., 300 ml. from 25 ml. of medium, in order to remove the 

 volatile acids quantitatively. The non-volatile acids are recovered 

 from the residue of the steam distillation by continuous extraction 

 with ether for 48 hours. 



Lactic acid may be determined in the extract by oxidation with 

 permanganate to acetaldehyde. The aldehyde is bound in bisulfite 

 and the bound bisulfite determined iodomctrically (Friedemann and 

 Graeser, 1933). The succinic acid may be precipitated as the silver 

 salt and weighed, or the silver of the salt determined volumetrically 

 (Moyle, 1924). 



The volatile fatty acids frequently consist of formic and acetic 



