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LB Lennox medium is commonly used for bacterial cell culture, but the amount of biomass obtained while sufficient for molecular biology and biochemical studies, is insufficient for applied microbiology research. Thus, supplementation of LB Lennox with glucose is commonly used in delivering more nutrients for biomass formation. However, an optimal concentration of glucose exists for each bacterial species beyond which biomass formation would be reduced due to the low culture broth’s pH and acetate accumulation that arise from the metabolism of excess glucose. Hence, addition of a phosphate buffer system in LB Lennox medium might moderate pH fluctuation detrimental to biomass formation. Finally, while LB Lennox medium is rich in amino acids, supplementation with an easily assimilable nitrogen source may augment cell growth. Using Escherichia coli DH5α (ATCC 53868) as model organism, this study aimed to understand the effect of glucose and ammonium chloride supplementation on growth performance of the bacterium at 37 oC, and whether a high capacity (89 mM phosphate) buffer system could help reduce pH fluctuation and improve biomass formation. Experiment results revealed that a high capacity phosphate buffer (89 mM phosphate) enabled higher optical density to be obtained in LB Lennox medium supplemented with 2 g/L and 6 g/L glucose. Specifically, maximal optical density of 6.0 was obtained in buffered LB Lennox with 2 g/L glucose compared to 5.0 in unbuffered LB Lennox with 2 g/L glucose. Similarly, 6.8 was the maximal optical density of E. coli DH5α grown in buffered LB Lennox medium with 6 g/L glucose, while 2.8 was obtained in the unbuffered LB Lennox medium with the same glucose concentration. For comparison, the maximal optical density for E. coli DH5α grown in unbuffered LB Lennox medium was 3.4. pH fluctuation was significantly reduced with incorporation of a high capacity phosphate buffer system in LB Lennox medium and correlated with higher biomass formation even for buffered LB Lennox medium without glucose supplementation (maximal optical density of 3.7). On the other hand, supplementation of unbuffered LB Lennox medium with 1 g/L and 4 g/L ammonium chloride did not affect growth performance and pH profile; thereby, indicating that the medium was not deficient in nitrogen. Finally, diauxic lag phase was not observed in growth of E. coli DH5α in LB Lennox medium supplemented with 2 g/L glucose, with and without a phosphate buffer, which indicated that 2 g/L glucose supplementation did not significantly alter the carbon and nitrogen balance of the medium. Collectively, high capacity phosphate buffer system helped moderate pH fluctuations; thereby, enabling greater biomass formation in LB Lennox medium with 2 g/L and 6 g/L of glucose supplementation. Supplementing LB Lennox medium with ammonium chloride did not alter growth performance and pH profile, which highlighted that the medium is not deficient in nitrogen.