For the last 150 years, bacteria have primarily been investigated in liquid bacth cultures (LBC). Contrary to most expectations, these cultures are not a homogeneous mixture of single-celled bacteria as free-floating bacterial aggregates eventually develop in most LBC. These aggregates share characteristics with biofilms such as increased antibiotic tolerance. We have investigated how aggregates develop and what influences this development in LBC of Pseudomonas aeruginosa We focused on how the method of inoculation impacted aggregation by assessing aggregate frequency and size using confocal laser scanning microscopy. Four traditional methods of initiating an overnight bacterial culture were investigated comprising inoculation directly from frozen culture, using agar grown cells, or using cells grown in liquid. We discovered a direct link between the inoculation method and the size and frequency of biofilm aggregates in a LBC, with inoculation directly from a plate resulting in the most numerous and largest aggregates. These large aggregates had an overall impact on the cultures' subsequent tolerance towards tobramycin indicating that the inoculation method has a profound impact on antibiotic tolerance. We also observed a mechanism where pre-formed aggregates recruited single cells from the surrounding culture in a "snowball effect", building up aggregated biomass in the culture. This recruitment was found to rely heavily on the exopolysaccharide Psl. Additionally, we found that both Escherichia coli and Staphylococcus aureus also produce aggregates in LBC. Our results stress the importance of inoculation consistency throughout experiments and the substantial impact aggregate development in LBC has on the output of microbiological experiments.IMPORTANCE Liquid pure cultures are fundamental to the field of microbiological research. These cultures are normally thought of as a homogeneous mix of single cell bacteria. The present study shows how this is not always true. Bacteria may aggregate in these liquid cultures. The aggregation can be induced by the method chosen for inoculation. The presence of aggregates can significantly change the outcome of experiments by altering the phenotype of the cultures. The study found a mechanism where pre-formed aggregates are able to recruit surrounding single cells in a form of snowball effect creating more and larger aggregates in the culture. Once formed, these aggregates are hard to remove. Aggregates in liquid cultures may be an immense unseen challenge for microbiologists.