Crosscheck CFU and cells

In the below step by step instructions we demonstrate how to compare the real-time BactoBox® measurements to the final plate count results.

Schematic for the crosscheck workflow: A sample is vortexed and subjected to 1:100 dilutions for BactoBox® measurements and 1:10 dilutions for plate counts. Three dilution series are done for each method. Each dilution series is measured once with each technique. This is an example. The actual cell concentration of your sample may require different dilutions.

Disaggregate thoroughly

For in vivo experiments, the bacterial culture will is often harvested by centrifugation and resuspended in 1× PBS to remove the growth medium components. Disaggregate carefully to convert the centrifugation pellet into a single-cell suspension.

Always disaggregate - especially after centrifugation

Bacteria tend to clump. Especially when centrifugation is used to harvest the culture into a neat pellet. Vortex thoroughly to get a single-cell suspension. This is needed for reliable BactoBox® and plate count results.

Step by step

1

Prepare the sample

Vortex the resuspended pellet thoroughly 1 min at max speed. Use a suitable mixer platform and ensure you see a proper vortex forming in the tube

2

Dilute the sample for BactoBox® measurements

Make a 1;10 000 dilution. See Hit the right concentration for more information.

A 1:10,000 dilution is done by two steps. First a 1:100 is done. Second an additional 1:100 dilution of the first vial.

BactoBox® diluent is not suited for plate counts

Use a proper dilution medium like buffered peptone water for plate counts. Don't use the BactoBox® diluent for this purpose.

3

Measure on BactoBox®

First run the 1;10,000 dilution dilution on BactoBox®.

If the result is <30,000 cells/mL then run the 1:100 dilution.

Add your data to a table like the example below. You can download our example and use it for your calculations.

crosscheck-excel-template.xlsx

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4

Do two additional BactoBox® measurements

Make a fresh dilution series to reach the dilution that worked in step 4. Then measure.

Repeat this step for a total of 3 BactoBox® repeats as shown in the example.

5

Prepare plate counts in triplicates

Dilute and plate to determine CFUs. Prepare three separate dilution series to include the variability involved with dilution, mixing, and plating. Incubate at optimal growth conditions.

Plate smarter

Use the cell concentrations determined via BactoBox® analyses to calculate suitable dilutions for plate counts. See the workflow Plate smarter for more information.

6

Determine CFUs

Count colonies on the plates that contain between ~25–250 colonies. Add the CFU results to the table.

7

Compare results

Use the excel template or an AI tool to determine the arithmetic mean, standard deviation, coefficient of variation (CV), and log difference between methods.

A log difference of 0 corresponds to a perfect agreement between the methods. BactoBox®-to-CFU log difference is typically within ±0.3, this corresponds to a two-fold difference. In the below calculation example the log difference is 0.06.

Summary

With this step by step guide you know the essentials to compare BactoBox® and CFU methods.

Contact us if the BactoBox® results fall outside the acceptable range

Contact us if the BactoBox® measurements did not meet your specifications. Together we can explore solutions to improve the agreements between results. Often the root-cause is insufficient sample workup.

Run your measurements via Access

BactoBox® works well as a stand-alone device. That said you can elevate your BactoBox® experience by running your measurements via Access. For more information, see Access.