Freezing at −80°C (no cryoprotectant): bacteria samples

Bacterial samples were diluted in 900 μL of 1 × PBS. One hundred microliters of the same concentrations used in the ZIRC method (10⁴ and 10⁸ CFU/mL) were pipetted into 2 mL cryogenic vials (No. 430659; Corning), and they were then put directly in a −80°C freezer. There was no cryoprotectant or freezing medium added to these samples. This procedure was conducted in triplicate for each dilution.

• Low concentration samples (10⁴ CFU/mL):

o 100 μL of the 10⁴ CFU/mL dilution was placed into 2 mL cryogenic vials (done in triplicate).

• High concentration samples (10⁸ CFU/mL):

o 100 μL of the 10⁸ CFU/mL broth was placed into 2 mL cryogenic vials (done in triplicate).

• Final volume frozen = 100 μL/sample.

• Final freezing temperature = −80°C.