Get things ready

You will need these items.

Hardware

• BactoBox® setup including a flow cell.

• Vortex mixer or alternatively a bead beater like bead genie.

• Rack for holding 5 and 15 mL centrifuge vials.

• Sterile 1 µL Inoculation loops. Preferably made of polystyrene (PS). See below hint.

• P200 or P100 pipette.

• Sterile pipette tips.

• Serological pipettes. Preferably 10 and 2 mL versions.

• Serological pipet controller.

Use polystyrene (PS) inoculation loops

After picking a suitable colony, you will snap the inoculation loop in the capture vial. Choose polystyrene (PS) because they snap easier than more "bendable" materials like polypropylene.

Reagents

• 1 × agar plate with fresh colonies. Preferably same agar type as the liquid growth medium.

• 1 × shake flask with growth medium. E.g. 250 mL flask with max 50 mL growth medium.

• 1 × capture vial: 5 mL centrifuge vial with screw cap containing 4 mL cultivation medium and 1 mL 3 mm glass beads. See below hint box for more information.

• Dilution vials either ready-to-use or freshly prepared using the dilution dispenser.

• Disinfection vials.

How to make capture vials

Prepare capture vials with the optimal growth medium for the bacterium of interest. Remember to add 3 mm glass beads as these greatly aid in disaggregation of cell clumps.

1. Prepare relevant growth medium as recommended by the manufacturer. Often, heating and magnetic stirring is needed to dissolve the powder completely.

2. Let medium cool to room temperature before moving to next step.

3. Filter by 0.2 µm syringe or vacuum filter to remove medium particulates.

4. Place 5 mL centrifuge vial with screw cap in an autoclavable rack.

5. Transfer 4 mL growth medium to each 5 mL vial.

6. Add 3 mm glass beads to each vial until the liquid level has risen to the 5 mL mark.

7. Cap the vials loosely and apply a small piece of autoclave tape to keep the cap in place.

8. Autoclave as recommended by the growth medium supplier.

9. Once vials have reached room temperature, cap the vials tightly.

With these things in hand, you are ready to start the Collect and disperse step.