Improved colony forming unit recovery from aggregating lactobacilli via mild probe sonication

Colony forming unit (CFU)-based enumeration of culturable bacterial populations is essential for microbiological research, probiotic quality control, and regulatory compliance. However, this is compromised in highly aggregative lactobacilli strains because individual viable cells within an aggregate often coalesce to produce a single colony. This study evaluated physical and chemical strategies to enhance CFU recovery by dispersing bacterial aggregates while preserving bacterial membrane integrity. Probe sonication, bath ultrasonication, and treatments with sodium chloride (NaCl), lithium chloride (LiCl), and guanidine hydrochloride (GHCl) were compared, and cell population dynamics were further assessed by flow cytometry. Probe sonication efficiently dispersed aggregates and significantly increased CFU recovery without compromising cell-membrane integrity, whereas bath ultrasonication showed limited efficacy. Chemical treatments were either ineffective or caused substantial losses in membrane integrity, particularly with LiCl and GHCl. These findings demonstrate that controlled probe sonication is a simple and efficient pre-plating step that substantially improves CFU recovery in highly aggregative lactobacilli strains. Combining disaggregation with complementary culture-independent methods may provide a more reliable framework for probiotic viability assessment in both research and industrial applications.