Bacterial Growth Quantification Protocol

Briefly, after vortexing the complete cultured test tube, serial dilutions were prepared by mixing 100 μL from the broth with 900 μL 0.9% sodium chloride (NaCl) in water. Dilutions of 10³ CFU/mL (“dilution X”) and 10² CFU/mL (“dilution Y”) were prepared and plated (100 μL per plate) on agar plates with 5% sheep blood (BD™ Columbia Agar with 5% Sheep Blood, Becton Dickinson GmbH, Heidelberg, Germany) in duplicates (Figure 1). For the growth control (no biomaterial, only pathogen) only dilution Y (10² dilution) was plated in duplicate (Figure 1). The plates were placed on a shaking plate (IKA® KS4000 IC, IKA®-Werke GmbH & Co. KG Staufen, Germany) at 37°C and were cultured for 16–18 h before colonies were counted. The loss of broth for plating per time point (100 μL per test tube), was not replenished, since the loss of bacteria was expected to be negligible.
After 16–18 h, the agar plates were photographed and CFUs were counted using OpenCFU 3.9.0 for MSSA, MRSA, E. coli and E. faecalis (Geissmann, 2013 (link)). The CFU for P. aeruginosa were counted manually, since these colonies could not be detected by OpenCFU software due to low contrast of the colonies on the blood agar plate.