Optical density (OD) measurement is applied universally to estimate cell numbers of microorganisms growing in liquid cultures. It is a fast and reliable method but is based on the assumption that the bacteria grow as single cells of equal size and that the cells are dispersed evenly in the liquid culture. When grown in such liquid cultures, the human pathogen Staphylococcus aureus is characterized by its aggregation of single cells into clusters of variable size. Here, we show that aggregation during growth in the laboratory standard medium tryptic soy broth (TSB) is common among clinical and laboratory S. aureus isolates and that aggregation may introduce significant bias when applying standard enumeration methods on S. aureus growing in laboratory batch cultures.

We provide a simple and efficient sonication procedure, which can be applied prior to optical density measurements to give an accurate estimate of cellular numbers in liquid cultures of S. aureus regardless of the aggregation level of the given strain. We further show that the sonication procedure is applicable for accurate determination of cell numbers using agar plate counting of aggregating strains.