Freeze-dried bacterial products

How to measure on freeze-dried bacterial products with BactoBox®

Bag with freeze-dried bacteria.

Use BactoBox® to find the cells/g concentration in probiotics, live biotherapeutic products (LBPs), postbiotics, etc.

Also note that the bacterial concentration in freeze-dried products can be extremely high and therefore it may be necessary to dilute sample more than 10^-5.

Detailed protocol

1

Prepare your sample

1. Homogenize the dry bacterial product as much as possible. E.g. massage, invert, and stir the powder with a spoon or similar.

2. Weigh approximately 3 g of powder into a Stomacher® bag. Note down the weight.

3. 1st dilution (total 1:10) Add diluent to the Stomacher® bag. Target a 1:10 dilution based on weight. If the initial weight is exactly 3 g, the weight of powder plus medium is 30 g.

4. Homogenize the Stomacher bag® with a Stomacher® Lab Blender at 250 ± 10 RPM for 4 minutes

5. Allow the bacteria to rehydrate (wake up) for 30 minutes at room temperature

Proper resuspension and gentle homogenization is essential

Notice that some media protect cells better than others upon resuspension. Buffered peptone water is a good general-purpose medium applicable to many species of bacteria. However, you may need special media for some cases. For example, lactic acid bacteria need MRS to protect cellular integrity during resuspension.

2

Dilute your sample

Prepare three dilution vials: Take three empty vials and add 10 mL of diluent to each.

2nd dilution (total 10^-3)

Dilute 1:100: Transfer 101 μL from your prepared sample into the first dilution vial. Vortex (15 sec., Max RPM).

3rd dilution (total 10^-5)

Dilute 1:100: Transfer 101 μL from your first dilution vial into your second dilution vial. Vortex.

4th dilution (total 10^-7)

Dilute 1:100: Transfer 101 μL from your second dilution vial into your third dilution vial. Vortex.

3

Measure your sample

Measure with BactoBox®. Start with the least concentrated sample and move towards higher concentrations.

First, clean your BactoBox® setup with a fresh disinfection vial.

Measure on the 10^-7-diluted sample

If there result is above the limit of detection (above 30 000 cells/mL) then you are done! Otherwise, move on.

Measure on the 10^-5-diluted sample

If there result is above the limit of detection (above 30 000 cells/mL) then you are done! Otherwise, move on.

Measure on the 10^-3-diluted sample

Is even the most-concentrated sample below the limit of detection? See Concentrate your sample for next steps.

Multiply the cells/mL concentration with the dilution factor to calculate the cells/mL concentration in your sample stock.