Measure a dense culture

This tutorial is for your second bacterial measurement on BactoBox®. It is slightly more advanced than the first tutorial Do a simple measurement but still simple and predictable with short hands-on time. You will make an overnight culture and learn how to dilute to a suitable working range.

The success criterion for this tutorial is to get a BactoBox® measurement within 30,000 to 5,000,000 cells/mL. You can complete this tutorial as a 🎒Beginner to BactoBox® measurements.

BactoBox® skill level	Time to complete (E. coli)	Hands-on time	Requirements
🎒 Beginner	⏳ 17 hours	⏱️ 0.5 hours	7.6a ≥ v2026.02a Escherichia coli WDCM 00013 Vitroids™

Soluble E. coli Vitroids™ disc

For this tutorial we recommend Escherichia coli WDCM 00013 Vitroids™ ATCC^® 25922 from Sigma/Merck. Lead time is usually 1-5 days. Price is ~200 USD including shipping. 10 vials are included in each shipment. Concentration per disc is stated as 50,000-150,000 CFU mean value range.

A soluble Vitroid™ disc containing 50,000–150,000 CFU of E. coli ATCC® 25922 .

Alternative: Use an in-house E.coli glycerol stock instead of a Vitroids™ disc

You will get the best experience if you procure Escherichia coli WDCM 00013 Vitroids^™ from Sigma/Merck. Alterantively, you can use an in-house E. coli glycerol stock to prepare an overnight culture. See more details in Inoculate LB medium.

Alternative: Use an in-house E.coli cryo stock instead of a Vitroids™ disc

You will get the best experience if you procure Escherichia coli WDCM 00013 Vitroids^™ from Sigma/Merck. Alternatively, you can use an in-house E. coli glycerol stock to prepare an overnight culture. See more details in Inoculate LB medium.

Hit the right concentration

An E. coli shake flask overnight culture typically has ~1×10¹⁰ cells/mL, i.e. 10,000,000,000 cells/mL. Max concentration for a BactoBox® measurement is 5×10⁶ total/mL, i.e. 5,000,000 total/mL.

The bacterial culture must be diluted 1:10,000 to hit the right concentration. In principle you could make a ~1:10,000 dilution by transferring 1 µL of sample to 10 mL (10,000 µL) diluent. But this would be imprecise because it is difficult to reproducibly transfer 1 µL with a pipette.

The bacterial culture must be diluted 1:10,000 to hit the right concentration. This is done by two consecutive 1:100 dilution steps.

Instead we use two consecutive 1:100 dilution steps to prepare a 1:10,000 dilution.

Instead we use two consecutive 1:100 dilution steps to prepare a 1:10,000 dilution.

In principle, the 1:100,000 dilution will also work, but it requires more dilution steps and the time-to-result is twice as long.

In principle, the 1:100,000 dilution will also work, but it requires more dilution steps and the time-to-result is twice as long.

Dilution series for an overnight culture. Two consecutive 1:100 dilutions are used for a 1:10,000 dilution. The dashed green lines show the BactoBox® working range from 30,000–5,000,000 total/mL. The green columns are within the working range. The orange columns are outside the working range.

Dilution series for an overnight culture. Two consecutive 1:100 dilutions are used for a 1:10,000 dilution. The dashed green lines show the BactoBox® working range from 30,000–5,000,000 total/mL. The green columns are within the working range. The orange columns are outside the working range.

An 1:10,000 dilution is made by two consecutive 1:100 dilutions.

Save time and reagents by using 1:100 dilutions instead of 1:10 dilutions

BactoBox® has a ~100-fold dynamic range and you save time and reagents by using 1:100 dilutions. These dilutions are done by transferring 101 µL culture to 10 mL diluent.

The 1:100 dilutions are seldom used in microbiology, where 1:10 is standard. The 1:10 dilutions are needed for plate counts where the dynamic range is only 10-fold (25–250 colonies are typically counted on an agar plate).

Save time and reagents by using 1:100 dilutions instead of 1:10 dilutions

BactoBox® has a ~100-fold dynamic range. You save time and reagents by using 1:100 dilutions.

This is different from the 1:10 dilution series used in microbiology. The 1:10 dilutions are needed for plate counts where the dynamic range is only 10-fold (25–250 colonies are typically counted on an agar plate).

Overview

In this tutorial we will demonstrate how to solubilize the disc and get the bacteria ready for BactoBox® analyses. The overall steps in the tutorial are given below.

1. Get things ready

2. Inoculate LB medium using either a Vitroids™ disc or an in-house glycerol stock
3. Disaggregate bacteria

4. Dilute and measure

5. Summary

Summary

After this introduction you can proceed to Get things ready.