In bioleaching field, shake flask is a common experimental device that has been used in numerous research works to cultivate microorganisms. However, none of these studies has considered if the agitation and aeration conditions applied to these cultures were sufficient to avoid O2 transfer limitations and to promote efficient solid particles dispersion, two key mechanisms in bioleaching experiments. Our study aimed at assessing the influence of the design of shake flask experiments on the growth of At. ferrooxidans using elemental sulfur (S0) as substrate. Cultures of At. ferrooxidans in 0Km medium with 1% sulfur were incubated at 30°C for 10 days in duplicates. Different culture volumes (V, 75 to 150 mL), agitation rates (N, 101 to 350 rpm) and orbit diameters (D0, 19 to 25 mm) were used to vary the gas-liquid mass transfer coefficient (kLa, from 8 to 41 h-1) and the power dissipation per unit of volume (P0/V, from 21 to 982 W/m3) from one culture to another. Daily monitoring of the cultures included the measurement of pH, dissolved oxygen concentration and biomass (concentration of bacteria in the liquid phase). Dissolved sulfate concentration was also measured by IEC at the beginning and the end of the culture. At. ferrooxidans growth (characterized by µmax) was improved with the increase of kLa and P_0/V while S0-biooxidation rate was increased. Moreover, whatever the experimental conditions, dissolved oxygen concentration was always above 85% of saturation, indicating that there was no limitation of the gas-liquid mass transfer in the range of kLa used for the study. Increasing P0/V for a given value of kLa improved the growth rate while it remained the same for a fixed P0/V and different kLa. As an example, for a kLa equal to 16 h-1, µmax increased from 0.028 to 0.035 h-1 when P0/V increased from 34 to 198 W/m3. On the contrary, for P0/V equal to 200 W/m3, µmax remained close to 0.035 h-1 when kLa was increased from 16 to 23 h-1. It was also observed that µmax reached a plateau when P0/V was above 600 W/m3. Interestingly, at the plateau, the agitation rate was higher than the theoretical critical rate N_C required to achieve a complete suspension of the sulfur particles in the liquid, which was not the case for the other cultures. These results show that microbial growth of At. ferrooxidans on S0 was mainly influenced by particles dispersion and not by gas-liquid mass transfer. In this case, P_0/V and N_C are two key factors that must be considered in the design of the experiments. The study confirms that operating conditions such as N, Do and V, which influence the aeration and homogenization of the culture in shake flask, must be carefully selected to avoid limiting bacterial growth.