Measure a dense culture

TUT-2: Your first BactoBox® measurement on an overnight culture with high bacterial concentration

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 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.

A typical overnight culture must be diluted 1:10,000 to hit the right concentration.

Use two consecutive 1:100 dilution steps to prepare a 1:10,000 dilution.

A 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. 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

3. Prepare sample

4. Dilute and measure

5. Summary

Summary

After this introduction you can proceed to Get things ready.