In the field of applied microbiology, reproducibility and experimental variability are important factors that influence both basic research as well as process development for industrial applications. Experimental reproducibility and accuracy depend not only on culture conditions such as temperature and aeration but also on raw materials and procedures used for media preparation. The M9 minimal medium is one of the most common synthetic media for culturing Escherichia coli and other bacteria. This synthetic medium can be used to observe and evaluate the physiological activity of microbes under minimal nutritional requirements and determine the limiting factor for the desired phenotype. Although one of the advantages using the M9 medium is that its composition can be modulated, it is difficult to control presence of trace components and impurities from the reagents for preparing this medium. Herein, we showed that trace ingredients present in the reagents used for M9 media preparation affect the bacterial physiological activities (e.g., cell growth, substrate consumption, and byproduct formation). Additionally, we systematically identified the trace ingredient that influenced phenotypic differences. Our results showed that the selection of reagents and accuracy during reagent preparation is important for experimental reproducibility in the field of bio-engineering and systems biology focused on the systematic and continuous development of biomolecular systems (e.g., biorefinery, metabolic engineering, and synthetic biology).