Plate harvest with E. coli

1. Streak a LB or TSA plate from a freeze stock of E. coli and grow overnight (37 ℃)

2. Pick an approximately 3 mm colony with a 1 μL inoculation loop and snap the plastic in a sterile 9 mL Flip Top buffered peptone water tube.

3. Add 3 mm glass beads until the liquid is at the 1 mL level in the tube.

4. Vortex aggressively to prepare a single-cell solution (1 min, Max RPM). Make sure you see the vortex/cyclone appearing in the tube. If possible, inspect sample by microscopy to verify single-cell solution.

5. Prepare 1:100 dilution by transferring 101 μL of harvest solution to 10 mL diluent in an empty vial. Vortex (15 sec., Max RPM).

6. BactoBox® measurement

   1. Run the Clean program with a fresh disinfection vial

   2. Measure the diluted sample with BactoBox®. Minimum volume is 4 mL. 10 mL is better as this reduces any carry-over from the previous sample.

   3. Note down results as shown in the table below. For the final result, remember to multiply with the dilution (in this case multiply the results by ×100)

7. If technical replicates are needed perform these starting at step 5 to include variation from the pipetting step.

Measurement ID	Sample description	Dilution	Cells/mL	Total/mL