VI42-6 MANUAL OF METHODS FOR PURE CULTURE STUDY 



tion of the added carbon compound can occur. Often the poor growth 

 may be due to the lack of necessary inorganic salts or to some un- 

 known organic factor in the peptone which is required by many 

 bacteria. Probably the most satisfactory way to supply the latter fac- 

 tor is thru the use of yeast extract. (See yeast extract broth, p. Uu-5, 

 Leaflet II.) This furnishes a satisfactory basal medium in studying 

 propionic acid bacteria, streptococci or lactobacilli. In the case of 

 some microaerophiles better growth may be secured by employing a 

 semisolid agar as a basal medium (see p. iii4-5). Some bacteria, on 

 the other hand, fail to grow on standard broth or agar because of the 

 presence of too much organic matter. For them the ammonium 

 phosphate medium (liquid or agar) given on page iiii-15 will often give 

 satisfactory results. This synthetic medium must be used with a little 

 caution, however, as it is poorly buffered and quite a high final H-ion 

 concentration (e.g., pH 5) may not necessarily mean acid production 

 from the carbohydrate (see discussion three paragraphs below). 



It is often necessary to prevent an appreciable rise in H-ion con- 

 centration. This is ordinarily accomplished by adding an excess of 

 sterilized CaCOa to each culture tube or flask, or by suitable buffering 

 of the medium. 



If calcium carbonate is used, it should be a fine powder so as to 

 provide great surface for neutralization of the acids formed. In addi- 

 tion the carbonate should be suspended throughout the medium by 

 adequate agitation, otherwise calcium carbonate is not a very effec- 

 tive neutralizing agent. 



Analytical Methods. In a study of fermentation, the following deter- 

 minations are commonly made: Final H-ion concentration, residual 

 sugar, kinds and quantities of organic acids, neutral solvents, carbon 

 dioxide. The choice as to which of these determinations to make and 

 sometimes as to what methods to employ must often depend on the 

 organism or group of organisms under investigation. In a complete 

 study it is necessary to account for the carbon originally present in 

 the substrate (usually a carbohydrate). This carbon should theoreti- 

 cally be accounted for among the products of fermentation. Likewise 

 the state of oxidation of the products should equal that of the sub- 

 strate, indicated by the redox index. The use of the redox index is 

 extremely useful in careful fermentation studies as a measure of the 

 accuracy of results. For a discussion see Johnson, Peterson and Fred 

 (1931). 



Final H-ion Concentration. This may be determined colorimetrically 

 or electrometrically according to the accuracy desired and the appli- 

 cability of the method to the conditions of the experiment. The color- 

 imetric method is given in Leaflet IX. Standard texts, like Clark's 

 "The Determination of Hydrogen Ions" 3rd Ed., should be consulted 

 for the electrometric method; the use of the glass electrode has 

 recently found marked favor (see Leaflet IX, p. iXi5-7.) 



In interpreting results, the buffer content of the medium must be 

 taken into consideration. The final reaction is the resultant of various 

 factors including the following: production of fatty acids, of COo, of 

 ammonia (or other basic substances) from nitrogenous matter pres- 

 ent; withdrawal of either cation or anion from mineral salts with con- 



