Growth of S. pneumoniae INS-E611 during Phase 2 in supplemented THB. One-mL bacterial suspension produced by Tube 5 (Phase 1) after 12 h of incubation was inoculated into 9-mL fresh broth (Tube 6, 1:10 dilution) and diluted further into Tube 7 (1:100) and Tube 8 (1:1000). Incubation under 5% CO2 atmosphere and pH adjustment every hour followed for 5–6 hours, with simultaneous plating for CFU counting. The graph illustrates the time to reach maximum growth for each inoculum size before triggering autolysis: 4, 5 and 7 hours for Tubes 6, 7 and 8, respectively. With this method, very similar maximum growths were attained under different inoculum sizes. Experiments with each tube were done 1–3 times.