184 MANUAL OF MICROBIOLOGICAL METHODS 



quantitatively by the products formed. In some instances, particularly 

 during aerobic processes, part of the substrate disappearing is assimilated 

 by the organisms while at the same time the rest of the substrate is broken 

 down to various products (Clifton, 1946, 1951). Use of a growth medium 

 during substrate dissimilation permits maximum assimilation and the 

 elaboration of adaptive metabolic mechanisms, each with resultant effects 

 upon the nature and quantity of end products. Thus, use of washed cell 

 suspensions is desirable, employing a medium containing only the sub- 

 strate to be dissimilated plus those other substances required for the 

 process: phosphate, buffer, metallic ions, etc. Care must be exercised in 

 sterilizing the reaction medium; heat-labile compounds are sterilized by 

 filtration, usually of a concentrated solution, and added aseptically to 

 the rest of the reaction medium. Sterilization of the reaction mixture is 

 unnecessary if resting cell suspensions are employed, since short-term dis- 

 similation (0.5-3 hr) is generally adequate. 



In order to obtain a quantitative accounting of the dissimilation proc- 

 ess or a ''balance," it is necessary to determine the gases formed, used, or 

 both, as well as the dissolved products. An atmosphere of defined com- 

 position must therefore be employed in contact with the reacting solution. 

 Both these objectives may be realized by shaking the solution in a closed 

 system (e.g., Warburg apparatus) or by bubbling nitrogen continuously 

 through the solution (provided nitrogen fixation does not occur), the 

 emitted gases being passed through a suitable absorption train. At the 

 end of the chosen period of time, the reaction is stopped by the addition 

 of enough nonvolatile mineral acid to bring the pH to 2-3; this also 

 releases bound CO 2 which is included in the gas measurements. The 

 solution is then freed of cells and proteinaceous material by treatment 

 with a suitable agent (barium or zinc hydroxide or trichloracetic acid) 

 and centrifugation, and the clarified solution analyzed. A small sample 

 of the reaction mixture may be removed just before the reaction is 

 stopped to check for bacteriological purity ; this is necessary for reactions 

 conducted in culture media and which proceed for long periods of 

 incubation. 



Owing to space limitations, it is not possible to describe here in detail 

 the methods employed in dissimilatory products analysis. Reference is 

 made to the following books: Johnson et al. (1949), McNair (1947), 

 Neish (1952), and Umbreit et al. (1949), which contain detailed descrip- 

 tions of individual methods and apparatus. Sensitive micromethods 

 have been described and continue to appear which are particularly useful 

 for analysis of Warburg flask contents, eliminating the need in many cases 

 for more cumbersome equipment (Black, 1949; Bueding and Yale, 1951; 

 Kennedy and Barker, 1951; Neish, 1952). The methods described by 

 Neish (1952) are most generally applicable to the problems discussed 



