The present report describes improved chromatographic procedures which are capable of separating and quantitating complex mixtures of acidic fermentation by-products produced by anaerobic bacteria grown in two glucosecontaining media. These methods are reliable and are sensitive, being able to detect as low as 0.5 μmoles of each by-product. Sample preparation has been simplified, and the methylation conditions have been optimized. It is also indicated in this investigation that each culture produced different patterns of by-products in each medium, indicating that the types and quantities of by-products produced in one medium cannot be used as a basis for characterization of these same cultures when grown in a different medium. Finally, it is shown that cultures can be characterized by the distinctive molar proportions of by-products they produce within each medium.