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TutorialBeginner
Disaggregate bacteria
After ~16 hours of incubation, the overnight culture is at a high cell concentration and we can use it to demonstrate BactoBox® measurements on concentrated bacterial suspensions.
Vortex thoroughly and vigorously
BactoBox is a flow cytometer and works best when cells are present in a single-cell suspension.
Cells stick together and you need thorough disaggregation to prepare a single-cell suspension.
One minute of vortexing may sound excessive, but trust us. You need it!
Step by step
1
Get a sample of the culture
Make figure-eight swirls to suspend the culture
Use a serological pipette to aspirate 2 mL culture.
Transfer to a 5 or 15 mL centrifuge vial.
2
Disaggregate clumps of bacteria
Vortex the sample 1 minute at max speed.
It can be tricky to get the vortexing technique right. Pay attention to the below details.
Good vortexing technique
Choose a mixer platform that is compatible with your vial. Some platforms can accommodate multiple vial types and sizes.
Use a gentle grip on the vial cap while vortexing the tube. Start with a firm 3-finger hold on the cap and switch to a gentle press with one finger once the motion starts.
The left panel demonstrates proper vortexing technique: The cap is initially held with three fingers to start the motion, after which a single finger applies light downward pressure while allowing the tube to vortex freely.
The center panel demonstrates improper technique: The vial is held too tightly using a central grip. The liquid bounces slightly, but the vortex does not form.
The right panel demonstrates improper technique: A 50 mL vortex mixer attachment is used for the 15 mL tube. The vortex does not start even though the grip is correct.
Demonstration of correct (✔) and incorrect (✘) vortexing technique on a diluent vial. Note that the sampling vial with the overnight culture will have a different appearance; nonetheless the technique is the same.
Summary
You retrieved a sample of the overnight culture and used vortex mixing to disaggregate clumps of bacteria. Proceed to Dilute and measure to determine cell concentration with BactoBox®.
